All applications of tungsten heavy alloys as list below:
High Density Tungsten Materials for Instrumentation balance weights
High Density Tungsten Materials for Isotope containers
High Density Tungsten Materials for High Temperature shielding
High Density Tungsten Materials for aerospace defense, ordnance and military
High Density Tungsten Materials for Medical Engineering (CT and diagnostic devices)
High Density Tungsten Materials for Radiation Shielding and or sources holders
Laser cut furnace elements/ Laser cut furnace heat shields application for Furnace Construction
Toll Extrusion /Toll Rotary Forging application for Extrusion and Forging
Tungsten alloy weights/powders application for Medical Engineering (CT and diagnostic devices)
Tungsten heavy alloy Balls/Spheres application for Defense
Tungsten Copper Composite Materials for Heat Exchangers
Tungsten heavy alloy Cubes for defense
Tungsten discs and squares
Tungsten fabricated parts
Tungsten Heavy Metal Balance Weights for Automotive crankshaft, braking system, balance
Tungsten heavy metal boring bars for tooling
Tungsten heavy metal parts for radiation protection and aero part
Tungsten High Density Composites
Tungsten mill products
Tungsten sputtering target materials for Sputtering Technology / PVD (Physical Vapor Deposition)
High Density Tungsten Materials for Tungsten Syringe Shields
Vacuum furnace parts for Furnace Construction
Weights for golf clubs, darts, fishing for Sports goods (Golf clubs, /balls, fishing weights)
High Density Tungsten Materials for Stirrers Mo, Mo/W for Glass melting
High Density Tungsten Materials for Plated and stamped components
Parts for X-ray and electron tubes for Medical Engineering (CT and diagnostic devices)
Hanns CEO/Chinatungten.com
2009/07/08
Typical Applications of Heavy Tungsten Alloys (WHA)(2)
While high density metals are relatively no corrosive, plating Is recommended if used in a corrosive atmosphere. Standard plating procedures for cadmium, nickel, and chromium are used. A copper flash Is sometimes used for better adherence. Plating is suggested if used with aluminum to prevent galvanic reaction,Oxidation starts at approximately 400C (752F), and in high humilities at approximately 66C (150F) showing a blue-black film. To minimize oxidation at high temperatures and high humidity, plating is recommended.MACHINING AND FINISHINGWHAs is similar to machining gray cast iron. A coolant is optional and carbide tools are recommended in most cases.TURNING & BORING — Roughing — use C-2 carbide with cutting depth of .030” to 125’ and .008” to .015 feed, at 200 to 300 SFM. Finishing — .010" to .015" cutting depth and 004" to .010" feed at 250 to 400 SFM.DRILLING— Use high speed steel surface treated drills with plain points. Increased clearance angles and automatic feeds are often used to avoid binding and seizing. Carbide drills will give better tool life. A chlorinated oil Is sometimes used as a coolant.TAPPING — Use high speed steel or carbide, two flute plug spiral point taps. Chlorinated oil or a tapping compound is recommended. GRINDING - Use aluminum oxide or silicon carbide wheels of medium hardness.MILLiNG - Use M-2 high speed steel for light cuts and M-42 high speed steel for deep cuts. Carbide cutter inserts will extend tool life. We recommend when using carbide, feeds .007” to .015” per tooth at speeds of 200 to 400 SFM for roughing, and when finished, feeds .003” to .010 per tooth at speeds of 300 to 700 SFM.High density tungsten alloys are not heat treatable; however, stress relieving is sometimes done on machined parts. We suggest heating at 600F in air for two hours and cool in air or in a protective atmosphere at 900F for 30 minutes.SAWING OR CUTTING — When sawing, use a bi-metai coarse blade at high speeds, or a high speed steel coarse blade at low speeds. Coolant can be used. Material also can be cut using high speed abrasive cutoff wheels.As a result of the materials’ characteristics and especially its low thermal expansion. very close tolerances and fine finishes can be held.JOININGCOPPER BRAZING Is a very good method of joining high density tungsten materials to itself or other materials. Joint strength Is close to that of parent material. One disadvantage is that it should be done in a controlled atmosphere, which is not too practical for most users.DIFFUSION BONDING is an ideal way of joining tungsten alloy material to itself, but It has to be done by the material manufacturer. If parts are finished, there may be some distortion from the process.SILVER SOLDERING for most companies is a practical and efficient method of joining these types of material either to themselves or to steel.SUGGESTIONS: Typically .002 clearance between parts to be joined is required, As the part gets larger, more clearance is required. Parts should be as clean as possible, sand blasting is sometimes used, both parts are fluxed, carefully heated until solder flows. Easy flow 45 is commonly used. A slow uniform cooling is recommended, Uneven cooling could set up stress in the joint and the material.SHRINK FITTING is another good method of joining high density tungsten material to steel. Depending on the size, .0005/.0007 interference fit per side is recommended. The tungsten alloy is chilled In dry ice or nitrogen while the steel is heated. When assembled, a slow cool is necessary, while parts are held by a locating pin or fixture.AVAILABILITY high density tungsten material is stocked in the following sizes:1/8" to 1" dia. in 8" & 12" lengths1 1/8" to 2" dia. in 12" lengths1/4" to 1" square in 8" & 12" lengths1 1/8" to 2" square in 12" lengthsRectangles, discs, and rings are also available.Larger sizes up to 6" dia. or 41/2" square x 30" long, and depending on cross section, longer lengths and sizes can be supplied.
All round bars up to 2" diameter are centerless ground, while all larger diameters are lathe finished.If material is not in stock, delivery is normally 10-14 days, or sooner for emergency requirements.Because we make our own tooling, special shapes and sizes can be made quickly and competitively— finished machined to your requirements or as a near net shaped blank.COPPER TUNGSTEN AND SILVER TUNGSTENRefractory metal composites produced by which are. used for:Resistance Welding Die Inserts and Electrode Facings EDM and ECM ElectrodesElectrical Contacts Semi-Conductor Heat Sinks and Thermal BasesWear Surfaces Requiring High Electrical and Thermal Conductivity
Hanns CEO/Chinatungten.com
All round bars up to 2" diameter are centerless ground, while all larger diameters are lathe finished.If material is not in stock, delivery is normally 10-14 days, or sooner for emergency requirements.Because we make our own tooling, special shapes and sizes can be made quickly and competitively— finished machined to your requirements or as a near net shaped blank.COPPER TUNGSTEN AND SILVER TUNGSTENRefractory metal composites produced by which are. used for:Resistance Welding Die Inserts and Electrode Facings EDM and ECM ElectrodesElectrical Contacts Semi-Conductor Heat Sinks and Thermal BasesWear Surfaces Requiring High Electrical and Thermal Conductivity
Hanns CEO/Chinatungten.com
Typical Applications of Heavy Tungsten Alloys (WHA)(1)
Tungsten Heavy Alloys(WHAs), as High Density Metals are made possible by Powder Metallurgy techniques. The process is a mixture of tungsten powder with nickel, iron, and/or copper and molybdenum powder, compacted and liquid phase sintered, giving a homogeneous structure with no grain direction. The result is a very high density, machinable material with unique physical properties.TYPICAL APPLICATIONSWeights and Counterbalances for aircraft control surfaces and rotor blades, guidance platforms, balancing of flywheels and turbines, vibration damping governors, fuse masses, and weights for self-winding watches. Because of the physical properties of high density metal, it is often used as both a weight and structural member.
CRANKSHAFT BALANCING —Used extensively to balance crankshafts in high performance engines. Individual weights are stocked. See our weight chart for formulas and information to solve balancing problems.RADIATION SHIELDING — Tungsten alloys are used for radioactive source containers, gamma radiography, shields and source holders for oil well logging and industrial instrumentation; for collimators and shielding in cancer therapy machines and syringe protection for radioactive injections, There is no licensing required for tungsten alloy materials. It is stable at high temperatures and 1/3 less material than lead can be used for the same energy absorption effectiveness. high density tungsten alloys are used wherever radioactivity has to be controlled mid directed.ROTATING INERTIA MEMBERS — Material Is used for gyro rotors, fly wheels, and rotating members for governors. Because of its unique physical properties, this material can be rotated at extremely high speeds. ORDNANCE COMPONENTS — In spheres, cubes. and projectile shapes. these materials are used for hypervelocity armor penetrating applications. Properties such as elongation, ultimate tensile strength, arid hardness can be varied by manufacturing technique and additives.BORING BARS AND GRINDING QUILLS — The standard for vibration free machining and grinding has been established by Chatter Free and Super Chatter Free materials. It’s used where rigidity and minimum vibration are critical Heavier cuts, Ionger tool life, end a better finish result when using Chatter Free materials. Tool extensions of up to 9-1 are possible depending on the diameter. Tools run cooler because of the high thermal conductivity, and you can braze directly to material without affecting its physical properties.These materials are often used in place of tungsten carbide boring bars because it has a higher density, is readily machinable, less prone to chipping and breakage, and both material and finishing casts are less. See our technical brochure Chatter Free and Super Chatter Free materials.HIGH TEMPERATURE TOOLING — for die casting and extrusion dies, hot upsetting and electro brazing. has developed a special tungsten alloy for this type application called DIEMETAL. It’s a tungsten, molybdenum, nickel, iron based material, It has good thermal conductivity and maintains excellent physical properties at high working temperatures. It resists errosion and scaling, minimizes soldering and heat checking and basically eliminates the need for heat treatment. See our technical bulletin on DIEMETALDESIGN CONSIDERATIONS -Because High Density Metals are produced by powder metallurgy, parts often can be tooled and produced to net or near net shape, minimizing material and machining costs, Normally, medium to small sized parts can be pressed and sintered to a (plus or minus) 1%, both dimensionally, and by weight. As the part gets larger, this percentage increases, Finish for “as sintered” parts is normally 63 or better. When designing parts to be used "as sintered,” deep spines, re-entrant angles, and feather edges should be avoided.
Hanns CEO/Chinatungten.com
CRANKSHAFT BALANCING —Used extensively to balance crankshafts in high performance engines. Individual weights are stocked. See our weight chart for formulas and information to solve balancing problems.RADIATION SHIELDING — Tungsten alloys are used for radioactive source containers, gamma radiography, shields and source holders for oil well logging and industrial instrumentation; for collimators and shielding in cancer therapy machines and syringe protection for radioactive injections, There is no licensing required for tungsten alloy materials. It is stable at high temperatures and 1/3 less material than lead can be used for the same energy absorption effectiveness. high density tungsten alloys are used wherever radioactivity has to be controlled mid directed.ROTATING INERTIA MEMBERS — Material Is used for gyro rotors, fly wheels, and rotating members for governors. Because of its unique physical properties, this material can be rotated at extremely high speeds. ORDNANCE COMPONENTS — In spheres, cubes. and projectile shapes. these materials are used for hypervelocity armor penetrating applications. Properties such as elongation, ultimate tensile strength, arid hardness can be varied by manufacturing technique and additives.BORING BARS AND GRINDING QUILLS — The standard for vibration free machining and grinding has been established by Chatter Free and Super Chatter Free materials. It’s used where rigidity and minimum vibration are critical Heavier cuts, Ionger tool life, end a better finish result when using Chatter Free materials. Tool extensions of up to 9-1 are possible depending on the diameter. Tools run cooler because of the high thermal conductivity, and you can braze directly to material without affecting its physical properties.These materials are often used in place of tungsten carbide boring bars because it has a higher density, is readily machinable, less prone to chipping and breakage, and both material and finishing casts are less. See our technical brochure Chatter Free and Super Chatter Free materials.HIGH TEMPERATURE TOOLING — for die casting and extrusion dies, hot upsetting and electro brazing. has developed a special tungsten alloy for this type application called DIEMETAL. It’s a tungsten, molybdenum, nickel, iron based material, It has good thermal conductivity and maintains excellent physical properties at high working temperatures. It resists errosion and scaling, minimizes soldering and heat checking and basically eliminates the need for heat treatment. See our technical bulletin on DIEMETALDESIGN CONSIDERATIONS -Because High Density Metals are produced by powder metallurgy, parts often can be tooled and produced to net or near net shape, minimizing material and machining costs, Normally, medium to small sized parts can be pressed and sintered to a (plus or minus) 1%, both dimensionally, and by weight. As the part gets larger, this percentage increases, Finish for “as sintered” parts is normally 63 or better. When designing parts to be used "as sintered,” deep spines, re-entrant angles, and feather edges should be avoided.
Hanns CEO/Chinatungten.com
2009/07/07
History of tungsten,tungsten cemented carbide and tungsten heavy alloy
In 1779 Peter Woulfe examined the mineral now known as wolframite and concluded it must contain a new substance. Scheele, in 1781, found that a new acid could be made from tungsten (a name first applied about 1758 to a mineral now known as scheelite). Scheele and Berman suggested the possibility of obtaining a new metal by reducing this acid. The de Elhuyar brothers found in acid in wolframite in 1783 that was identical to the acid of tungsten (tungstic acid) of Scheele, and in that year they succeeded in obtaining the element by reduction of this acid with charcoal. Tungsten occurs in wolframite, scheelite, huebnertie, and ferberite. Important deposits of tungsten occur in California, Colorado, South Korea, bolivia, Russia, and Portugal. China is reported to have about 75% of the world's tungsten resources. Natural tungsten contains five stable isotopes. Twenty one other unstable isotopes are recognized. The metal is obtained commercially be recucing tungsten oxide with hydrogen or carbon. Pure tungsten is a stelel-gray to tin-white metal. Very pure tungsten can be cut with a hacksaw, and can be forged, spun, drawn, and extruded. The impure metal is brittle and can be worked only with difficulty. Tungsten has the highest melting point of all metals, and at temperatures over 1650C has the highest tensile strength. The metal oxidizes in air and must be protected at elevated temperatures. It has excellent corrosion resistance and is attacked only slightly by most mineral acids. The thermal expansion is about the same as borosilicate glass, which makes the metal useful for glass-to-metal seals. tungsten and its alloys are used extensively for filaments for electric lamps, electron and television tubes, and for metal evaporation work; for electrical contact points for automobile distributors; X-ray targets; windings and heating elements for electrical furnaces; and for numerous spacecraft and high-temperature applications. High-speed tool steels, Hastelloy(R), Stellite(R), and many other alloys contain tungsten. Tungsten carbide is of great importance to the metal-working, mining, and petroleum industries. Calcium and magnesium tungstates are widely used in fluorescent lighting; other salts of tungsten are used in the chemical and tanning industries. Tungsten disulfide is a dry, high-temperature lubricant, stable to 500C. Tungsten bronzes and other tungsten compounds are used in paints.
Hanns CEO/Chinatungten.com
Hanns CEO/Chinatungten.com
2009/06/19
Tungsten Carbide:Our New Market
Tungsten Carbide: Which market shall be developed in 2009
I’ll come to Shandong Province, North-East of China for a week for developing the market for our tungsten carbide products.
My Hometown, Shandong province has large coal well in it’s middle countries such as Yanzhou city, Zhoucheng City and Jining City. Also, Shandong has large oil field In Dongying City, these energy make Shandong to be a large province in China, and you may find many powder stations all over there, and they make Shandong rich and larger in the recent years.
Almost of my old friend there, and they are all warm heart men, I wonder if I can get orders from Shandong, because not the reason of the market itself, but I can not drink too much spirit and wine
For our people in Shandong always drink much and talk the business while drinking.
I have no way to meet these guys even most of them were my friends when we were children.
Gook Lucky, Hanns, you are the “wolfman”(wolfram man), and you have never meet any problem can not stopping you!
Hanns CEO/Chinatungten.com
I’ll come to Shandong Province, North-East of China for a week for developing the market for our tungsten carbide products.
My Hometown, Shandong province has large coal well in it’s middle countries such as Yanzhou city, Zhoucheng City and Jining City. Also, Shandong has large oil field In Dongying City, these energy make Shandong to be a large province in China, and you may find many powder stations all over there, and they make Shandong rich and larger in the recent years.
Almost of my old friend there, and they are all warm heart men, I wonder if I can get orders from Shandong, because not the reason of the market itself, but I can not drink too much spirit and wine
For our people in Shandong always drink much and talk the business while drinking.
I have no way to meet these guys even most of them were my friends when we were children.
Gook Lucky, Hanns, you are the “wolfman”(wolfram man), and you have never meet any problem can not stopping you!
Hanns CEO/Chinatungten.com
2009/06/18
The longest commercial promise
The Longest Commercial Promise: Useful Thousands Years of Our Tungsten Heavy Alloy Paper Weight
You know I’m running a tungsten carbide and alloy factory in China, so I hope do something interesting of tungsten. One day, a nice ideas was born suddenly. Why do I make the most dense paper weight in the world by tungsten heavy alloy which with the density of nearly 19g/cc!
No more time to wait, I ask my engineers to find a mould which we produce bucking bars by it, mixed the highest density powder with Fe, Ni and Cu powder as binder metals, then we press it, pre-sintered and final sintered it by the temperature of 1600 degree. 3 hours latter, we got a large cube and also I ask my staff grounding it smoothly. Ask my friend who running a laser workshop to laser our log and company name and web site add onto it. Finally we have a excellent result. That’s a beautiful art! And that’s pretty paper weight, and I think it may be the first paper weight made from tungsten heavy alloy.
Last week, when I come to Shanhai to meet Ms. Keiko who come from Japan, I send the paper weight to her as a gift, when she asked me “ How it works?”
The answer from me was: one thousand year at least, I promise!
Everyone may find these paper weight at www.paper-weight.cn
Hanns CEO/Chinatungten.com
You know I’m running a tungsten carbide and alloy factory in China, so I hope do something interesting of tungsten. One day, a nice ideas was born suddenly. Why do I make the most dense paper weight in the world by tungsten heavy alloy which with the density of nearly 19g/cc!
No more time to wait, I ask my engineers to find a mould which we produce bucking bars by it, mixed the highest density powder with Fe, Ni and Cu powder as binder metals, then we press it, pre-sintered and final sintered it by the temperature of 1600 degree. 3 hours latter, we got a large cube and also I ask my staff grounding it smoothly. Ask my friend who running a laser workshop to laser our log and company name and web site add onto it. Finally we have a excellent result. That’s a beautiful art! And that’s pretty paper weight, and I think it may be the first paper weight made from tungsten heavy alloy.
Last week, when I come to Shanhai to meet Ms. Keiko who come from Japan, I send the paper weight to her as a gift, when she asked me “ How it works?”
The answer from me was: one thousand year at least, I promise!
Everyone may find these paper weight at www.paper-weight.cn
Hanns CEO/Chinatungten.com
About wolfram/alpha
I'm a man who have done business of tungsten(wolfram) for more than 20 years, I have offered tungsten intermediater products ad ammonium paratungstate and oxide tungsten, final tugnsten as tungsten powder and tungsten cemented carbide,tungsten heavy alloy products thousands tonnes. Then, I have also thousands reason to pay my attention to the institue of wolfram/alpha in the past years.
But, recently, I find the web of www.wolframalpha .com was open as a research engine which offers the researher a specific result but not milllions of the related pages as google, I'm very glad if anyone and/or any tools may give me a right result when i want find some information from the net for saveing time and meantine saveing my health.
Of course I should find something interested as tungsten, tungsten carbide and tungsten heavy alloys.but when I was showed the result of tungsten carbide , I found that useless information,even it was recorrecot info. if someone know tungsten or wolfram, who may find it as my feeling.
I do not know who created the pages and also I do not know if it was Mr. Wolfram's web. when I visit it, it seems just there's nothing but introducing how these guys to develop this web. so, I don't think that a nice way for me and that's realy a not googd idea of this web.
It was just one thing interesting, I think, for I'm a man who do biz of wolfram, and Mr.Wolfram is another man who named wolfram but dose not know wolfram.
That's all
Hanns CEO/Chinatungten.com
But, recently, I find the web of www.wolframalpha .com was open as a research engine which offers the researher a specific result but not milllions of the related pages as google, I'm very glad if anyone and/or any tools may give me a right result when i want find some information from the net for saveing time and meantine saveing my health.
Of course I should find something interested as tungsten, tungsten carbide and tungsten heavy alloys.but when I was showed the result of tungsten carbide , I found that useless information,even it was recorrecot info. if someone know tungsten or wolfram, who may find it as my feeling.
I do not know who created the pages and also I do not know if it was Mr. Wolfram's web. when I visit it, it seems just there's nothing but introducing how these guys to develop this web. so, I don't think that a nice way for me and that's realy a not googd idea of this web.
It was just one thing interesting, I think, for I'm a man who do biz of wolfram, and Mr.Wolfram is another man who named wolfram but dose not know wolfram.
That's all
Hanns CEO/Chinatungten.com
2009/05/12
China puts ceiling on 2009 output of tungsten ore, antimony, rare earth
China puts ceiling on 2009 output of tungsten ore, antimony, rare earth
China said Thursday it would impose a ceiling on the output of mineral resources like tungsten, antimony and rare earth in 2009 amid shrinking demand.
The move is aimed to protect China's reserves of these minerals, the Ministry of Land and Resources said in an online statement.
The country's tungsten ore concentrate output is limited to 68,555 tonnes this year; rare earth ore to 82,320 tonnes, and antimony ore to 90,180 tonnes, said the ministry.
These quotas on output were based on decreasing demand across the world as a result of the ongoing global financial crisis, said the ministry.
The ministry said it would also not take any license applications before June 30, 2010 for exploring the three resources.
China holds 40.5 percent of the world's proven tungsten reserves, and is the world's biggest antimony producer. The nation has a proven rare earth reserve of 52 million tonnes, or about 58 percent of the world's total.
The country started to cap the annual output of tungsten in 2002 and that of rare earth in 2006. It is the first time such restrictions have been placed on antimony.
The ceiling on tungsten ore concentrates (with tungsten trioxide content above 65 percent) last year was 66,850 tonnes, and that on rare earth ore at 87,620 tonnes.
The ministry said such caps are intended to stabilize global demand and supply of these products and ensure their sustainable use.
Hanns CEO/Chinatungten.com
China said Thursday it would impose a ceiling on the output of mineral resources like tungsten, antimony and rare earth in 2009 amid shrinking demand.
The move is aimed to protect China's reserves of these minerals, the Ministry of Land and Resources said in an online statement.
The country's tungsten ore concentrate output is limited to 68,555 tonnes this year; rare earth ore to 82,320 tonnes, and antimony ore to 90,180 tonnes, said the ministry.
These quotas on output were based on decreasing demand across the world as a result of the ongoing global financial crisis, said the ministry.
The ministry said it would also not take any license applications before June 30, 2010 for exploring the three resources.
China holds 40.5 percent of the world's proven tungsten reserves, and is the world's biggest antimony producer. The nation has a proven rare earth reserve of 52 million tonnes, or about 58 percent of the world's total.
The country started to cap the annual output of tungsten in 2002 and that of rare earth in 2006. It is the first time such restrictions have been placed on antimony.
The ceiling on tungsten ore concentrates (with tungsten trioxide content above 65 percent) last year was 66,850 tonnes, and that on rare earth ore at 87,620 tonnes.
The ministry said such caps are intended to stabilize global demand and supply of these products and ensure their sustainable use.
Hanns CEO/Chinatungten.com
2009/05/11
Tungsten Heavy Alloy Bucking Bar and Rivert Gun
Tungsten Heavy Alloy Bucking Bar and Rivert Gun
A rivet gun is a type of tool used to drive rivets. Nearly all rivet guns are pneumatically powered. The rivet gun is used on the manufactured head side of the rivet and a bucking bar is used on the buck-tail side of the rivet. Those rivet guns used to drive rivets in structural steel are quite large while those used in aircraft assembly are easily held in one hand. A rivet gun differs from an air hammer in the precision of the driving force.Rivet guns vary in size and shape and have a variety of handles and grips. Pneumatic rivet guns typically have a regulator which adjusts the amount of air entering the tool. Regulated air entering passes through the throttle valve which is typically controlled by a trigger in the hand grip. When the trigger is squeezed, the throttle valve opens, allowing the pressurized air to flow into the piston. As the piston moves, a port opens allow the air pressure to escape. The piston strikes against the rivet set. The force on the rivet set pushes the rivet into the work and against the buck. The buck deforms the tail of the rivet. The piston is returned to the original position by a spring or the shifting of a valve allowing air to drive the piston back to the starting position.Contents1 Types 1.1 One-shot gun 1.2 Slow-hitting gun 1.3 Fast-hitting gun 1.4 Corner riveter 1.5 Squeeze riveter 2 See also 3 References TypesIn aircraft work, there are several types of rivet guns:One-shot gunThe one-shot gun is designed to drive the rivet in just one blow. It is larger and heavier than other types and is generally used for heavy riveting. Each time the trigger is depressed, the gun strikes one blow. It is rather difficult to control on light-gauge metal. Under general suitable conditions it is the fastest method of riveting.Slow-hitting gunThe slow-hitting gun strikes multiple blows as long as the trigger is held down. The repetition rate is about 2,500 blows-per-minute (bpm). It is easier to control than a one-hit gun. This is probably the most common type of rivet gun in use.Fast-hitting gunThe fast-hitting gun strikes multiple light-weight blows at a high rate as long as the trigger is held down. These are repeated in the range of 2,500 to 5,000 bpm. The fast-hitting gun, sometimes referred to as a vibrator, is generally used with softer rivets.Corner riveterThe corner riveter is a compact rivet gun that can be used in close spaces. The rivet is driven at right-angles to handle by a very short barreled driverSqueeze riveterThis gun is different from the above rivet guns in that the air pressure is used to provide a squeezing action that compresses the rivet from both sides rather than distinct blows. The squeeze riveter can only be used close to the edge because of the limited depth of the anvil. Once properly adjusted, the squeeze riveter will produce very uniform rivet bucks. The stationary (fixed) jaw is placed against the head and the buck is compressed by the action of the gun.See alsoRiveting machines Machine Orbital riveting ReferencesBureau of Naval Personnel - [US] Navy Training Course Aviation Structural Mechanic S 3 & 2 NavPers 10308-A. U.S. Navy Training Publications Center, Memphis, Tennessee, 1966, 380 pages Categories: Hand-held power tools Mechanical hand tools Pneumatic tools(and so on) To get More information , you can visit some products about Telephone Clock Alarm , sugar free lollipops , . The products should be show more here!
----- http://himfryang.blogr.com/stories/2009-05-11-Rivet-gun/
Hanns CEO/Chinatungten.com
A rivet gun is a type of tool used to drive rivets. Nearly all rivet guns are pneumatically powered. The rivet gun is used on the manufactured head side of the rivet and a bucking bar is used on the buck-tail side of the rivet. Those rivet guns used to drive rivets in structural steel are quite large while those used in aircraft assembly are easily held in one hand. A rivet gun differs from an air hammer in the precision of the driving force.Rivet guns vary in size and shape and have a variety of handles and grips. Pneumatic rivet guns typically have a regulator which adjusts the amount of air entering the tool. Regulated air entering passes through the throttle valve which is typically controlled by a trigger in the hand grip. When the trigger is squeezed, the throttle valve opens, allowing the pressurized air to flow into the piston. As the piston moves, a port opens allow the air pressure to escape. The piston strikes against the rivet set. The force on the rivet set pushes the rivet into the work and against the buck. The buck deforms the tail of the rivet. The piston is returned to the original position by a spring or the shifting of a valve allowing air to drive the piston back to the starting position.Contents1 Types 1.1 One-shot gun 1.2 Slow-hitting gun 1.3 Fast-hitting gun 1.4 Corner riveter 1.5 Squeeze riveter 2 See also 3 References TypesIn aircraft work, there are several types of rivet guns:One-shot gunThe one-shot gun is designed to drive the rivet in just one blow. It is larger and heavier than other types and is generally used for heavy riveting. Each time the trigger is depressed, the gun strikes one blow. It is rather difficult to control on light-gauge metal. Under general suitable conditions it is the fastest method of riveting.Slow-hitting gunThe slow-hitting gun strikes multiple blows as long as the trigger is held down. The repetition rate is about 2,500 blows-per-minute (bpm). It is easier to control than a one-hit gun. This is probably the most common type of rivet gun in use.Fast-hitting gunThe fast-hitting gun strikes multiple light-weight blows at a high rate as long as the trigger is held down. These are repeated in the range of 2,500 to 5,000 bpm. The fast-hitting gun, sometimes referred to as a vibrator, is generally used with softer rivets.Corner riveterThe corner riveter is a compact rivet gun that can be used in close spaces. The rivet is driven at right-angles to handle by a very short barreled driverSqueeze riveterThis gun is different from the above rivet guns in that the air pressure is used to provide a squeezing action that compresses the rivet from both sides rather than distinct blows. The squeeze riveter can only be used close to the edge because of the limited depth of the anvil. Once properly adjusted, the squeeze riveter will produce very uniform rivet bucks. The stationary (fixed) jaw is placed against the head and the buck is compressed by the action of the gun.See alsoRiveting machines Machine Orbital riveting ReferencesBureau of Naval Personnel - [US] Navy Training Course Aviation Structural Mechanic S 3 & 2 NavPers 10308-A. U.S. Navy Training Publications Center, Memphis, Tennessee, 1966, 380 pages Categories: Hand-held power tools Mechanical hand tools Pneumatic tools(and so on) To get More information , you can visit some products about Telephone Clock Alarm , sugar free lollipops , . The products should be show more here!
----- http://himfryang.blogr.com/stories/2009-05-11-Rivet-gun/
Hanns CEO/Chinatungten.com
2009/05/10
More tungsten alloy and carbide machined products
More tungsten alloy and carbide machined products
What’s the meaning of the new Chinese Central Government’s policy of “Stop Release New Licence of Developing Tungsten and/or Rare Earth Mines”?
According to the reported of CTIA, the Chinese Central Government(CCG) has just announced a new policy of developing tungsten and rare earth mine in 2009-2010, it is reported that none can get the new licence for developing such stagitic nonferrous metals such as tungsten, rare earth from 2009 to 2010. the reason given were envoirement protection, the more and more domestic applications and the out of control of opening and developing of these important materials in the past decades sharp econominc developing.
But, according managers of the majior nonferrous metals company and experts of tungsten and rare earth in China, in fact, the total sum of output and tungsten and rare earth in 2009 maybe less than the plan and total export licence of 2009, the reason is has been down sharply from the last half 0f 2008, some companies who have licences and deal these products in domestic and outside, just exported 20%-50% these commodities inn the first quarter 2009, and there’s no more purchase order for the next quaoter. Then, their views is it may be better in the end of this year, or if the global economic come to better later than it is expected, the market of these nonferrous metals’ prices and output may rise also later than expected by the end of 2009.
Why the CCG announced this new policy at this situation?
I think the reason may 1) CCG would like to let the world know, the cheap markets on the stagitic metals from China will never come in the future; 2) The CCG will control the export of nonferrous metals as W & RE, for there’s a large demand in the domestic, 3) CCG would like show it’s strict policy for the enviorment although all over the world have been in the the large financial crisis, 4) CCG has the weapons itself in the resource “war” in the new century, not the cannon, not the rokets and not the Chinese, but these metals which may controlled by CCG.
The new policy may let more and more Chinese companies who produce and export tungsten ore and/or tungsten intermediate products reduce less it’s production and offer more and more tungsten final products and then, these countries and companies who use the raw materials as tungsten intermediate products as ammonium paratungstate, metatungstate, tungsten acid, tungsten oxide and trioxide to buy tungsten final products and the machined tungsten carbide and alloy parts in the near future.
Then, the new policy just show us a policy was all these companies have to think about the processing and trade stagtic of using tungsten as raw materials.
Hanns CEO/Chinatungten.com
What’s the meaning of the new Chinese Central Government’s policy of “Stop Release New Licence of Developing Tungsten and/or Rare Earth Mines”?
According to the reported of CTIA, the Chinese Central Government(CCG) has just announced a new policy of developing tungsten and rare earth mine in 2009-2010, it is reported that none can get the new licence for developing such stagitic nonferrous metals such as tungsten, rare earth from 2009 to 2010. the reason given were envoirement protection, the more and more domestic applications and the out of control of opening and developing of these important materials in the past decades sharp econominc developing.
But, according managers of the majior nonferrous metals company and experts of tungsten and rare earth in China, in fact, the total sum of output and tungsten and rare earth in 2009 maybe less than the plan and total export licence of 2009, the reason is has been down sharply from the last half 0f 2008, some companies who have licences and deal these products in domestic and outside, just exported 20%-50% these commodities inn the first quarter 2009, and there’s no more purchase order for the next quaoter. Then, their views is it may be better in the end of this year, or if the global economic come to better later than it is expected, the market of these nonferrous metals’ prices and output may rise also later than expected by the end of 2009.
Why the CCG announced this new policy at this situation?
I think the reason may 1) CCG would like to let the world know, the cheap markets on the stagitic metals from China will never come in the future; 2) The CCG will control the export of nonferrous metals as W & RE, for there’s a large demand in the domestic, 3) CCG would like show it’s strict policy for the enviorment although all over the world have been in the the large financial crisis, 4) CCG has the weapons itself in the resource “war” in the new century, not the cannon, not the rokets and not the Chinese, but these metals which may controlled by CCG.
The new policy may let more and more Chinese companies who produce and export tungsten ore and/or tungsten intermediate products reduce less it’s production and offer more and more tungsten final products and then, these countries and companies who use the raw materials as tungsten intermediate products as ammonium paratungstate, metatungstate, tungsten acid, tungsten oxide and trioxide to buy tungsten final products and the machined tungsten carbide and alloy parts in the near future.
Then, the new policy just show us a policy was all these companies have to think about the processing and trade stagtic of using tungsten as raw materials.
Hanns CEO/Chinatungten.com
2009/05/08
Japan Goes Prospecting for Tungsten & Rare Metals
Japan goes prospecting for rare metalsBy Hisane Masaki TOKYO - In addition to oil, natural gas and uranium, resource-poor Japan is now revving up its drive to secure rare metals, which are used in a wide range of high-technology products, including digital home appliances, high-grade steel, and hybrid and fuel-cell cars. Highly alarmed by soaring prices on robust global demand and amid increased export restrictions by some producing countries, the Ministry of Economy, Trade and Industry (METI) mapped out a
new comprehensive strategy recently for ensuring stable supplies of rare metals, especially tungsten, cobalt, vanadium, molybdenum, indium, platinum and rare earth, in the medium and long terms. For Japan, a major importer of these rare metals, ensuring their stable supplies has emerged as an all-important policy task to maintain and strengthen the international competitiveness of its industries. The strategy calls for, among other things, beefing up state stockpiles of rare metals in terms of both volume and scope, promoting the recycling of scraps, developing alternative materials, extending official development assistance (ODA) for the development of new mines, and pumping public funds into efforts to help domestic private firms land mining interests abroad. The strategy also calls for strengthened relations with producing countries through such foreign-policy tools as free-trade agreements (FTAs). Meanwhile, private Japanese companies are aggressively looking for chances to step up their rare-metal exploration projects. Last week, for example, major trading house Sumitomo Corp announced that it has invested about 3 billion yen (US$24.4 million) to acquire an 8.7% stake in Augusta Resource Corp, a Canadian metals-exploration company. Augusta is carrying out a feasibility study at the Rosemont copper project about 50 kilometers southeast of Tucson, Arizona, which holds deposits of copper, silver and molybdenum - a rare metal used mainly as an additive to create specialty steel products. Sumitomo said in a statement that it aims to build up a long-term partnership with Augusta for the development of the Rosemont project. Global rush amid rising pricesGlobal competition is intensifying for non-ferrous metals, including copper and lead, as well as for such energy resources as oil, natural gas and uranium. Prices have risen sharply in recent years on increased global demand, led by red-hot consumption in China. The surge in prices in international markets has been fueled by the inflow of speculative funds. Japan has even seen a bizarre series of theft cases recently in which copper and electric wires, bronze fire bells, faucets, manhole covers and incense burners have been stolen from streets, rice fields and cemeteries across the country. It is widely suspected that high international metal prices and special procurement demand in China ahead of next year's Summer Olympic Games in Beijing are behind the thefts. Spikes in prices for non-ferrous metals are not limited to such base metals as copper, lead and aluminum. Prices for most rare metals, which are widely used as raw materials for high-tech products, have also jumped several-fold in recent years. Indium, for example, was sold at prices 8.5 times as high this March as in March 2002. Indium is used in such products as LCD (liquid crystal display) televisions. Prices for platinum, which is used as a catalyst in fuel cells and catalytic converters for automobiles, also increased 2.4-fold during the same five-year period. Prices for tungsten, which is used to make light-bulb filaments and increase the hardness and strength of steel, rose 4.7-fold during the same period. Among other rare metals, prices for nickel, cobalt, vanadium, molybdenum and manganese also surged 7.1-fold, 4.4-fold, 6.2-fold, 6.0-fold and 2.1-fold, respectively. Changes in supply-demand structureLying behind the sharp surge in prices for non-ferrous metals, including rare metals, are changes in the supply-demand structure for them. New major consuming nations have emerged, most notably China and India, the world's two most populous countries, whose economies are growing at a breakneck pace. On the supply side, meanwhile, a small number of powerful resource majors, such as Anglo American, Rio Tinto and BHP Billiton, now dominate the global markets for non-ferrous metals, wielding great influence over supplies and prices. In the 1990s, for example, seven resource majors accounted for only about 30% of global copper-ore production. But the percentage has increased to 50%. The world has also seen a rising tide of "resource nationalism" in many producing countries recently amid steep rises in prices for various resources, from oil, natural gas and uranium to non-ferrous metals. Despite being a major producer of non-ferrous metals, China has become a net importer of some metals, such as lead, zinc and nickel, as the country gobbles them up to feed its runaway economy. To meet sharply growing demand at home, China has taken export-restraint measures, such as lowered tax rebates, increased export taxes and stricter export quotas, for some rare metals, including tungsten and rare earth, since last year. China has also made aggressive forays into various parts of the world in pursuit of resource interests. China's particular focus on Africa has drawn global attention recently. China is increasingly reliant on the continent for raw materials. Africa supplies one-third of China's oil, with Angola, Sudan and Nigeria being major suppliers. China also gets bauxite from Guinea, copper from Zambia, uranium from Namibia and rare metals from Congo. Japan's heavy dependence on importsThe output of rare metals is small and production areas are disproportionately located. China produces about 90% of tungsten and rare earth. China is also the world’s largest producer of indium, accounting for more than 30% of global total. South Africa produces about 80% of platinum. Japan is a major consumer of rare metals. The world's second-biggest economy accounts for about 60% of global indium consumption. Japan's share of global consumption is high for other rare metals as well, at 20% for platinum, 14% for nickel and tungsten, 25% for cobalt, 17% for molybdenum, 11% for vanadium and 24% for rare earth. Japan imports almost all of the rare .
Japan goes prospecting for rare metalsBy Hisane Masaki metals it consumes. China supplies 90% of Japan's rare-earth imports, 79% of tungsten imports and 70% of indium imports. South Africa supplies 81% of Japan's platinum imports and 49% of vanadium imports. Japan purchases 44% of its nickel imports from Indonesia, 30% of cobalt imports from Finland and 45% of molybdenum imports from Chile. Raising state reserves of rare metalsIncreasingly concerned about global supply shortages, METI
compiled a comprehensive strategy this month for ensuring stable supplies of rare metals in the medium and long terms. It was the result of the first review of a government policy on rare metals in more than 20 years. The new strategy calls for, among other things, increased state reserves of some rare metals. There has been increasing pressure on the government from domestic industries to boost such stockpiles. In fiscal 1983, Japan began stockpiling seven types of rare metals - nickel, tungsten, cobalt, molybdenum, manganese, vanadium and chromium. As of the end of March this year, Japan had reserves of these rare metals equivalent to 34.8 days of domestic demand - 24.4-day stocks controlled by the state and 10.4-day reserves kept by the private sector - compared with the target of 60 days. The government-affiliated Japan Oil, Gas and Metals National Corp (JOGMEC) manages the state-controlled reserves at a warehouse in Takahagi, Ibaraki prefecture. Under the new strategy, the government will increase the state reserves of vanadium, tungsten, cobalt and molybdenum and will also consider expanding the scope of state stockpiles to include indium, platinum and rare earth. The strategy also calls for promoting the recycling of scraps and developing alternative materials. METI specifically plans to commission domestic non-ferrous-metal makers and universities this summer to develop alternative materials in hopes of putting them into practical use in five years' time. Resource diplomacy and public fundsThe new strategy urges the government to step up its diplomacy aimed at securing new supply sources and also dissuading producing nations from taking export-restrictive measures. Tokyo believes that export restrictions should be introduced only as an exception under the international trade rules set by the World Trade Organization. The strategy calls for increased Japanese support for mining development in foreign countries through the extension of ODA money. It also includes pumping public funds into efforts to help domestic private firms acquire mining interests abroad. The envisaged public funds will come from such government-affiliated organizations as JOGMEC, Japan Bank for International Cooperation (JBIC) and Nippon Export and Investment Insurance (NEXI). Even before the strategy was adopted, Japan had already begun to place a greater emphasis on securing non-ferrous metals, including rare metals, as well as crude oil, natural gas and uranium. In February, for example, Mongolian President Nambaryn Enkhbayar visited Tokyo and agreed with Japanese Prime Minister Shinzo Abe to promote cooperation between the two countries on the development of mineral resources, including rare metals. To implement the agreement, the two countries are expected soon to launch a joint committee of government officials and private-sector people. Mongolia is rich in a variety of minerals, especially coal and copper, although these remain largely unexploited. During a tour of resource-rich Central Asia by the METI chief, Akira Amari, at the end of April, JOGMEC signed cooperation agreements with Kazakhstan and Uzbekistan for the development of mineral resources, including rare metals. FTA as a foreign policy toolJapan has already placed priority on concluding FTAs with resource-rich countries, as well as neighboring Asian countries, as a way of beefing up relations with them and thereby ensuring stable supplies of oil, natural gas and other resources. On Monday, Japan signed an FTA with Brunei, an oil-and-gas-rich member of the Association of Southeast Asian Nations (ASEAN). The signing was made during a meeting in Tokyo between Abe and Sultan of Brunei Hassanal Bolkiah. Brunei is the seventh country with which Japan has signed an FTA, after Singapore, Mexico, Malaysia, the Philippines, Chile and Thailand. The FTAs with Singapore, Mexico and Malaysia have already taken effect. Japan is also expected to ink an FTA with Indonesia in August. Japan is also negotiating FTAs with the 10-member ASEAN as a whole, the oil-rich Gulf Cooperation Council, Vietnam, South Korea, India, Australia and Switzerland. Japan is also eyeing South Africa as a potential FTA partner. The Japan-Brunei FTA, which is expected to take effect this year, will eliminate import tariffs on 99.9% of bilateral trade within 10 years. In addition to eliminating tariffs, the FTA is aimed at ensuring stable supplies of oil and natural gas to Japan from the Southeast Asian country. Japan imports almost all of its oil and gas. Japan exported 11.5 billion yen's worth of products to Brunei in 2005, with automobiles and auto parts accounting for 71% of the total. Meanwhile, Japan imported 252.5 billion yen's worth from Brunei, more than 99% of which were liquefied natural gas and crude oil. The Japan-Brunei FTA incorporates an energy clause, under which Brunei will notify Japan in advance of any emergency measures that would restrict exports of natural gas and crude oil. Brunei will also hold discussions on any such measures with Japan and respect existing export contracts. For Japan, getting such a clause concerning resource supplies included in FTAs is a top-priority goal in negotiating such trade deals with resource-rich countries. Hisane Masaki is a Tokyo-based journalist, commentator and scholar on international politics and economics. Masaki's e-mail address is yiu45535@nifty.com . (Copyright 2007 Asia Times Online Ltd. All rights reserved. Please contact us about sales, syndication and republishing.)
Hanns CEO/Chinatungten.http://www.chinatungsten.comcom
new comprehensive strategy recently for ensuring stable supplies of rare metals, especially tungsten, cobalt, vanadium, molybdenum, indium, platinum and rare earth, in the medium and long terms. For Japan, a major importer of these rare metals, ensuring their stable supplies has emerged as an all-important policy task to maintain and strengthen the international competitiveness of its industries. The strategy calls for, among other things, beefing up state stockpiles of rare metals in terms of both volume and scope, promoting the recycling of scraps, developing alternative materials, extending official development assistance (ODA) for the development of new mines, and pumping public funds into efforts to help domestic private firms land mining interests abroad. The strategy also calls for strengthened relations with producing countries through such foreign-policy tools as free-trade agreements (FTAs). Meanwhile, private Japanese companies are aggressively looking for chances to step up their rare-metal exploration projects. Last week, for example, major trading house Sumitomo Corp announced that it has invested about 3 billion yen (US$24.4 million) to acquire an 8.7% stake in Augusta Resource Corp, a Canadian metals-exploration company. Augusta is carrying out a feasibility study at the Rosemont copper project about 50 kilometers southeast of Tucson, Arizona, which holds deposits of copper, silver and molybdenum - a rare metal used mainly as an additive to create specialty steel products. Sumitomo said in a statement that it aims to build up a long-term partnership with Augusta for the development of the Rosemont project. Global rush amid rising pricesGlobal competition is intensifying for non-ferrous metals, including copper and lead, as well as for such energy resources as oil, natural gas and uranium. Prices have risen sharply in recent years on increased global demand, led by red-hot consumption in China. The surge in prices in international markets has been fueled by the inflow of speculative funds. Japan has even seen a bizarre series of theft cases recently in which copper and electric wires, bronze fire bells, faucets, manhole covers and incense burners have been stolen from streets, rice fields and cemeteries across the country. It is widely suspected that high international metal prices and special procurement demand in China ahead of next year's Summer Olympic Games in Beijing are behind the thefts. Spikes in prices for non-ferrous metals are not limited to such base metals as copper, lead and aluminum. Prices for most rare metals, which are widely used as raw materials for high-tech products, have also jumped several-fold in recent years. Indium, for example, was sold at prices 8.5 times as high this March as in March 2002. Indium is used in such products as LCD (liquid crystal display) televisions. Prices for platinum, which is used as a catalyst in fuel cells and catalytic converters for automobiles, also increased 2.4-fold during the same five-year period. Prices for tungsten, which is used to make light-bulb filaments and increase the hardness and strength of steel, rose 4.7-fold during the same period. Among other rare metals, prices for nickel, cobalt, vanadium, molybdenum and manganese also surged 7.1-fold, 4.4-fold, 6.2-fold, 6.0-fold and 2.1-fold, respectively. Changes in supply-demand structureLying behind the sharp surge in prices for non-ferrous metals, including rare metals, are changes in the supply-demand structure for them. New major consuming nations have emerged, most notably China and India, the world's two most populous countries, whose economies are growing at a breakneck pace. On the supply side, meanwhile, a small number of powerful resource majors, such as Anglo American, Rio Tinto and BHP Billiton, now dominate the global markets for non-ferrous metals, wielding great influence over supplies and prices. In the 1990s, for example, seven resource majors accounted for only about 30% of global copper-ore production. But the percentage has increased to 50%. The world has also seen a rising tide of "resource nationalism" in many producing countries recently amid steep rises in prices for various resources, from oil, natural gas and uranium to non-ferrous metals. Despite being a major producer of non-ferrous metals, China has become a net importer of some metals, such as lead, zinc and nickel, as the country gobbles them up to feed its runaway economy. To meet sharply growing demand at home, China has taken export-restraint measures, such as lowered tax rebates, increased export taxes and stricter export quotas, for some rare metals, including tungsten and rare earth, since last year. China has also made aggressive forays into various parts of the world in pursuit of resource interests. China's particular focus on Africa has drawn global attention recently. China is increasingly reliant on the continent for raw materials. Africa supplies one-third of China's oil, with Angola, Sudan and Nigeria being major suppliers. China also gets bauxite from Guinea, copper from Zambia, uranium from Namibia and rare metals from Congo. Japan's heavy dependence on importsThe output of rare metals is small and production areas are disproportionately located. China produces about 90% of tungsten and rare earth. China is also the world’s largest producer of indium, accounting for more than 30% of global total. South Africa produces about 80% of platinum. Japan is a major consumer of rare metals. The world's second-biggest economy accounts for about 60% of global indium consumption. Japan's share of global consumption is high for other rare metals as well, at 20% for platinum, 14% for nickel and tungsten, 25% for cobalt, 17% for molybdenum, 11% for vanadium and 24% for rare earth. Japan imports almost all of the rare .
Japan goes prospecting for rare metalsBy Hisane Masaki metals it consumes. China supplies 90% of Japan's rare-earth imports, 79% of tungsten imports and 70% of indium imports. South Africa supplies 81% of Japan's platinum imports and 49% of vanadium imports. Japan purchases 44% of its nickel imports from Indonesia, 30% of cobalt imports from Finland and 45% of molybdenum imports from Chile. Raising state reserves of rare metalsIncreasingly concerned about global supply shortages, METI
compiled a comprehensive strategy this month for ensuring stable supplies of rare metals in the medium and long terms. It was the result of the first review of a government policy on rare metals in more than 20 years. The new strategy calls for, among other things, increased state reserves of some rare metals. There has been increasing pressure on the government from domestic industries to boost such stockpiles. In fiscal 1983, Japan began stockpiling seven types of rare metals - nickel, tungsten, cobalt, molybdenum, manganese, vanadium and chromium. As of the end of March this year, Japan had reserves of these rare metals equivalent to 34.8 days of domestic demand - 24.4-day stocks controlled by the state and 10.4-day reserves kept by the private sector - compared with the target of 60 days. The government-affiliated Japan Oil, Gas and Metals National Corp (JOGMEC) manages the state-controlled reserves at a warehouse in Takahagi, Ibaraki prefecture. Under the new strategy, the government will increase the state reserves of vanadium, tungsten, cobalt and molybdenum and will also consider expanding the scope of state stockpiles to include indium, platinum and rare earth. The strategy also calls for promoting the recycling of scraps and developing alternative materials. METI specifically plans to commission domestic non-ferrous-metal makers and universities this summer to develop alternative materials in hopes of putting them into practical use in five years' time. Resource diplomacy and public fundsThe new strategy urges the government to step up its diplomacy aimed at securing new supply sources and also dissuading producing nations from taking export-restrictive measures. Tokyo believes that export restrictions should be introduced only as an exception under the international trade rules set by the World Trade Organization. The strategy calls for increased Japanese support for mining development in foreign countries through the extension of ODA money. It also includes pumping public funds into efforts to help domestic private firms acquire mining interests abroad. The envisaged public funds will come from such government-affiliated organizations as JOGMEC, Japan Bank for International Cooperation (JBIC) and Nippon Export and Investment Insurance (NEXI). Even before the strategy was adopted, Japan had already begun to place a greater emphasis on securing non-ferrous metals, including rare metals, as well as crude oil, natural gas and uranium. In February, for example, Mongolian President Nambaryn Enkhbayar visited Tokyo and agreed with Japanese Prime Minister Shinzo Abe to promote cooperation between the two countries on the development of mineral resources, including rare metals. To implement the agreement, the two countries are expected soon to launch a joint committee of government officials and private-sector people. Mongolia is rich in a variety of minerals, especially coal and copper, although these remain largely unexploited. During a tour of resource-rich Central Asia by the METI chief, Akira Amari, at the end of April, JOGMEC signed cooperation agreements with Kazakhstan and Uzbekistan for the development of mineral resources, including rare metals. FTA as a foreign policy toolJapan has already placed priority on concluding FTAs with resource-rich countries, as well as neighboring Asian countries, as a way of beefing up relations with them and thereby ensuring stable supplies of oil, natural gas and other resources. On Monday, Japan signed an FTA with Brunei, an oil-and-gas-rich member of the Association of Southeast Asian Nations (ASEAN). The signing was made during a meeting in Tokyo between Abe and Sultan of Brunei Hassanal Bolkiah. Brunei is the seventh country with which Japan has signed an FTA, after Singapore, Mexico, Malaysia, the Philippines, Chile and Thailand. The FTAs with Singapore, Mexico and Malaysia have already taken effect. Japan is also expected to ink an FTA with Indonesia in August. Japan is also negotiating FTAs with the 10-member ASEAN as a whole, the oil-rich Gulf Cooperation Council, Vietnam, South Korea, India, Australia and Switzerland. Japan is also eyeing South Africa as a potential FTA partner. The Japan-Brunei FTA, which is expected to take effect this year, will eliminate import tariffs on 99.9% of bilateral trade within 10 years. In addition to eliminating tariffs, the FTA is aimed at ensuring stable supplies of oil and natural gas to Japan from the Southeast Asian country. Japan imports almost all of its oil and gas. Japan exported 11.5 billion yen's worth of products to Brunei in 2005, with automobiles and auto parts accounting for 71% of the total. Meanwhile, Japan imported 252.5 billion yen's worth from Brunei, more than 99% of which were liquefied natural gas and crude oil. The Japan-Brunei FTA incorporates an energy clause, under which Brunei will notify Japan in advance of any emergency measures that would restrict exports of natural gas and crude oil. Brunei will also hold discussions on any such measures with Japan and respect existing export contracts. For Japan, getting such a clause concerning resource supplies included in FTAs is a top-priority goal in negotiating such trade deals with resource-rich countries. Hisane Masaki is a Tokyo-based journalist, commentator and scholar on international politics and economics. Masaki's e-mail address is yiu45535@nifty.com . (Copyright 2007 Asia Times Online Ltd. All rights reserved. Please contact us about sales, syndication and republishing.)
Hanns CEO/Chinatungten.http://www.chinatungsten.comcom
2009/05/07
Applications of Tungsten Heavy Alloys(WHA)
Applications of Tungsten Heavy Alloys(WHA)
Tungsten heavy alloys(WNiFe, WNiCu) consist of 85-98% tungsten with balanced commonly nickel and iron or copper. The alloys are made by liquid-phase sintering to give a structure consisting of almost pure tungsten particles in a matrix of the alloy elements. Tungsten Heavy Alloys, with densities between 16.9 and 18.1 g/cm3, represent the heaviest materials generally available to the engineer. It has excellent radiation resistance, thermal and electric conductivities, corrosion resistance and machinable. We can offer tungsten alloys with tungsten contents ranging from 85 to 98% with a range of physical and mechanical properties as well as non-magnetic W-Ni-Cu.
Details Properties of WHA may find at www.tungsten-alloy.com
Applications:
■Tungsten heavy alloy cube for defence, military
■Tungsten heavy alloy radiation shielding, uranium protection
■Tungsten heavy alloy ballast pellets for hunting
■Tungsten heavy alloy balancing weights, counterweight
■Tungsten heavy alloy extrusion dies, die casting components
■Tungsten heavy alloy rivet reaction blocks
■Tungsten heavy alloy bricks for shield wall and yacht weight
■Tungsten heavy alloy bucking bar for light plane’s riveting
■Tungsten heavy alloy for mobile
■Tungsten heavy alloy for crankshaft
■Tungsten heavy alloy for airspace, aircraft & air fighter
■Tungsten heavy alloy cube for clock
■Tungsten heavy alloy petroleum industry, gas & oil well
■Tungsten heavy alloy part and screw for golf club
■Tungsten heavy alloy ball for counterweight
■Tungsten Paperweight in English
■Tungsten heavy alloy bricks
■Tungsten Heavy alloy billets
■Tungsten heavy alloy barrels
■Others as followings
1, Radiation shield, collimator, nuclear shielding, beamstop, PET syringe shield, vial shield, isotope container, FDG container, multi leaf collimator ;
2, Balanced part; tungsten sinker bar, heavy metal boring bar, tuyacht, sailboat, submarine and other vessels crank camshafts, holders for Well Logging, Racing Weights. vibration damping and dynamic balancing. Tungsten sinker bar, Tungsten bucking bar, Tungsten boring bar, Tungsten Sinkers, tungsten alloy counterweights for golf and tungsten alloy dart parts spheres, cubes, and projectile shapes;
3, High-temperature die, Electroheat , brass and copper, Tooling for low-pressure die-casting of aluminium and brass, Hot upsetting dies, Filler rods for die repair, upsetting anvil block, electrical rivet ;
4, Shrapnel head; Penetrators
5, Electrical contact;
6, Balanced ball for missile and plane; golf and tungsten alloy dart parts spheres, cubes, and projectile shapes ;
7, Core for armourpiercing bullet measurement.
Hanns CEO/Chinatungten.com
Tungsten heavy alloys(WNiFe, WNiCu) consist of 85-98% tungsten with balanced commonly nickel and iron or copper. The alloys are made by liquid-phase sintering to give a structure consisting of almost pure tungsten particles in a matrix of the alloy elements. Tungsten Heavy Alloys, with densities between 16.9 and 18.1 g/cm3, represent the heaviest materials generally available to the engineer. It has excellent radiation resistance, thermal and electric conductivities, corrosion resistance and machinable. We can offer tungsten alloys with tungsten contents ranging from 85 to 98% with a range of physical and mechanical properties as well as non-magnetic W-Ni-Cu.
Details Properties of WHA may find at www.tungsten-alloy.com
Applications:
■Tungsten heavy alloy cube for defence, military
■Tungsten heavy alloy radiation shielding, uranium protection
■Tungsten heavy alloy ballast pellets for hunting
■Tungsten heavy alloy balancing weights, counterweight
■Tungsten heavy alloy extrusion dies, die casting components
■Tungsten heavy alloy rivet reaction blocks
■Tungsten heavy alloy bricks for shield wall and yacht weight
■Tungsten heavy alloy bucking bar for light plane’s riveting
■Tungsten heavy alloy for mobile
■Tungsten heavy alloy for crankshaft
■Tungsten heavy alloy for airspace, aircraft & air fighter
■Tungsten heavy alloy cube for clock
■Tungsten heavy alloy petroleum industry, gas & oil well
■Tungsten heavy alloy part and screw for golf club
■Tungsten heavy alloy ball for counterweight
■Tungsten Paperweight in English
■Tungsten heavy alloy bricks
■Tungsten Heavy alloy billets
■Tungsten heavy alloy barrels
■Others as followings
1, Radiation shield, collimator, nuclear shielding, beamstop, PET syringe shield, vial shield, isotope container, FDG container, multi leaf collimator ;
2, Balanced part; tungsten sinker bar, heavy metal boring bar, tuyacht, sailboat, submarine and other vessels crank camshafts, holders for Well Logging, Racing Weights. vibration damping and dynamic balancing. Tungsten sinker bar, Tungsten bucking bar, Tungsten boring bar, Tungsten Sinkers, tungsten alloy counterweights for golf and tungsten alloy dart parts spheres, cubes, and projectile shapes;
3, High-temperature die, Electroheat , brass and copper, Tooling for low-pressure die-casting of aluminium and brass, Hot upsetting dies, Filler rods for die repair, upsetting anvil block, electrical rivet ;
4, Shrapnel head; Penetrators
5, Electrical contact;
6, Balanced ball for missile and plane; golf and tungsten alloy dart parts spheres, cubes, and projectile shapes ;
7, Core for armourpiercing bullet measurement.
Hanns CEO/Chinatungten.com
2009/05/06
No Magic Bullet: Tungsten Alloy Munitions Pose Unforeseen Threat.
No Magic Bullet: Tungsten Alloy Munitions Pose Unforeseen Threat.
by Charles W. Schmidt
In response to concerns about the human and environmental health effects of materials used to produce munitions, countries including the United States have begun replacing some lead- and depleted uranium-based munitions with alternatives made of a tungsten alloy. But this solution may not be the "magic bullet" it was once envisioned to be. Researchers from the Armed Forces Radiobiology Research Institute and the Walter Reed Army Institute of Research now report that weapons-grade tungsten alloy produces aggressive metastatic tumors when surgically implanted into the muscles of rats [EHP 113:729-734]. These findings raise new questions about the possible consequences of tungsten exposure, and undermine the view that tungsten alloy is a nontoxic alternative to depleted uranium and lead.
In the study, male F344 rats were implanted with pellets in each hind leg, an exposure protocol that mimicked shrapnel wounds received in the field. The rats were split into four treatment groups: a negative control implanted with 10 pellets of tantalum (an inert metal), a positive control implanted with 10 pellets of nickel (a known carcinogen), a high-dose group implanted with 10 pellets of tungsten alloy, and a low-dose group implanted with 4 pellets of tungsten alloy and 16 pellets of tantalum. The alloy used in this research was the same as that used in weapons: 91.1% tungsten, 6.0% nickel, and 2.9% cobalt.
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by Charles W. Schmidt
In response to concerns about the human and environmental health effects of materials used to produce munitions, countries including the United States have begun replacing some lead- and depleted uranium-based munitions with alternatives made of a tungsten alloy. But this solution may not be the "magic bullet" it was once envisioned to be. Researchers from the Armed Forces Radiobiology Research Institute and the Walter Reed Army Institute of Research now report that weapons-grade tungsten alloy produces aggressive metastatic tumors when surgically implanted into the muscles of rats [EHP 113:729-734]. These findings raise new questions about the possible consequences of tungsten exposure, and undermine the view that tungsten alloy is a nontoxic alternative to depleted uranium and lead.
In the study, male F344 rats were implanted with pellets in each hind leg, an exposure protocol that mimicked shrapnel wounds received in the field. The rats were split into four treatment groups: a negative control implanted with 10 pellets of tantalum (an inert metal), a positive control implanted with 10 pellets of nickel (a known carcinogen), a high-dose group implanted with 10 pellets of tungsten alloy, and a low-dose group implanted with 4 pellets of tungsten alloy and 16 pellets of tantalum. The alloy used in this research was the same as that used in weapons: 91.1% tungsten, 6.0% nickel, and 2.9% cobalt.
Hanns CEO/Chinatungten।com
Numerical simulation of tungsten alloy in powder injection molding process
Numerical simulation of tungsten alloy in powder injection molding process
The flow behavior of feedstock for the tungsten alloy powder in the mold cavity was approximately described using Hele-Shaw flow model. The math model consisting of momentum equation, consecutive equation and thermo-conduction equation for describing the injection process was established. The equations are solved by the finite element/finite difference hybrid method that means dispersing the feedstock model with finite element method, resolving the model along the depth with finite difference methpd, and tracking the movable boundary with control volume method, then the pressure equation and energy equation can be resolved in turn. The numerical simulation of the injection process and the identification of the process parameters were realized by the Moldflow software. The results indicate that there is low temperature gradient in the cavity while the pressure and shear rate gradient are high at high flow rate. The selection of the flow rate is affected by the structure of the gate. The shear rate and the pressure near the gate can be decreased by properly widening the dimension of the gate. There is a good agreement between the process parameters obtained by the numerical simulation and the actual ones.
Hanns CEO/Chinatungten।com
The flow behavior of feedstock for the tungsten alloy powder in the mold cavity was approximately described using Hele-Shaw flow model. The math model consisting of momentum equation, consecutive equation and thermo-conduction equation for describing the injection process was established. The equations are solved by the finite element/finite difference hybrid method that means dispersing the feedstock model with finite element method, resolving the model along the depth with finite difference methpd, and tracking the movable boundary with control volume method, then the pressure equation and energy equation can be resolved in turn. The numerical simulation of the injection process and the identification of the process parameters were realized by the Moldflow software. The results indicate that there is low temperature gradient in the cavity while the pressure and shear rate gradient are high at high flow rate. The selection of the flow rate is affected by the structure of the gate. The shear rate and the pressure near the gate can be decreased by properly widening the dimension of the gate. There is a good agreement between the process parameters obtained by the numerical simulation and the actual ones.
Hanns CEO/Chinatungten।com
Volatility from copper and tungsten alloys for fusion react or application
Volatility from copper and tungsten alloys for fusion reaktorapplikations
Smolik, G.R.; Neilson, R.M., Jr.; Piet, S.J.Fusion Engineering, 1989. Proceedings., IEEE Thirteenth Symposium onVolume , Issue , 2-6 Oct 1989 Page(s):670 - 673 vol.1Digital Object Identifier 10.1109/FUSION.1989.102308Summary:Accident scenarios for fusion power plants present the potential for release and transport of activated constituents volatized from first-wall and structural materials. The extent of possible mobilization and transport of these activated species (many of which are oxidation driven) is being addressed by the Fusion Safety Program at the Idaho National Engineering Laboratory (INEL). Experimental measurements of volatilization from a copper alloy in air and steam and from a tungsten alloy in air are presented. The major elements released included zinc from the copper alloy and rhenium and tungsten from the tungsten alloy. Volatilization rates of several constituents of these alloys over temperatures ranging from 400 to 1200 are presented. These release rates are recommended for use in accident assessment calculations
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Smolik, G.R.; Neilson, R.M., Jr.; Piet, S.J.Fusion Engineering, 1989. Proceedings., IEEE Thirteenth Symposium onVolume , Issue , 2-6 Oct 1989 Page(s):670 - 673 vol.1Digital Object Identifier 10.1109/FUSION.1989.102308Summary:Accident scenarios for fusion power plants present the potential for release and transport of activated constituents volatized from first-wall and structural materials. The extent of possible mobilization and transport of these activated species (many of which are oxidation driven) is being addressed by the Fusion Safety Program at the Idaho National Engineering Laboratory (INEL). Experimental measurements of volatilization from a copper alloy in air and steam and from a tungsten alloy in air are presented. The major elements released included zinc from the copper alloy and rhenium and tungsten from the tungsten alloy. Volatilization rates of several constituents of these alloys over temperatures ranging from 400 to 1200 are presented. These release rates are recommended for use in accident assessment calculations
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Tungsten Alloy Proves More Is Less
PRLog (Press Release) – Jul 08, 2008 – Recent increases in fuel prices and forecasts for more to come have focussed aircraft owners and operators into doing all they can to minimise their use of fuel. Assisting in the drive for fuel efficiency, lower operating costs and reduced carbon footprint, an unlikely sounding alloy is providing solutions for aircraft manufacturers worldwide. With a density over sixty percent greater than lead, Wolfmet tungsten alloys may be a less-than-obvious choice of material for use in airframes, yet in mid-sized aircraft design they deliver key advantages. Wolfmet’s high density, coupled with good mechanical properties and machining characteristics, allows designers to make significant reductions in the physical size of components. For a given moment about a pivot, Wolfmet alloys therefore allow a lighter balance weight to be sited further from the pivot point. Lower aircraft weight means better fuel efficiency, increased payload availability or greater speed capability. Due to the very high melting point of tungsten (in excess of 3000 °C), Wolfmet alloys cannot be manufactured by traditional casting methods. To overcome this, Wolfmet alloys are manufactured by powder metallurgy techniques. One of the advantages of manufacturing by powder metallurgy methods is that small batch quantities, even single pieces, can be achieved without the severe cost penalties associated with other production methods. M&I Materials has AS 9100 accreditation, and produces Wolfmet materials in accordance with an ISO 9001:2000 approved QA system, including Aerospace Sector Certificate Scheme TS 157-1993. Wolfmet components also meet the United States military MIL-T-21014 specification. M&I Materials Ltd has a state-of-the-art Wolfmet production facility at Trafford Park, Manchester, with an international order book in aerospace, automotive, motor racing, medical and engineering applications. M&I Materials Ltd performs the whole production process, from powder blending through to machining, to achieve unrivalled quality and value in the finished component. Further information is available at www.wolfmet.com.
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Hanns CEO/Chinatungten।com
Reactive Tungsten Alloy For Inert Warheads
http://www.psicorp.com/
Abstract:
Physical Sciences Inc. and ATK Thiokol, Inc. propose to develop novel high-density tungsten-based reactive composites for application to inert kinetic energy munitions. These materials will be inert such that they do not reduce the projectile's Insensitive Munitions compliance. Their critical benefit will be to enhance projectile lethality by depositing a combined kinetic and chemical energy in the target, which is greater than the corresponding kinetic energy deposited by a non-reactive tungsten munition. This lethality enhancement will occur over most of the range of anticipated projectile (1300-5000 fps) and payload pellet (2000-6000 fps) velocities. Novel metallurgical fabrication techniques will be applied in these material developments. In Phase I, we shall fabricate samples of two different tungsten metal-oxidizer systems, and characterize their energetic and mechanical properties.
Benefits:The proposed materials technology development constitutes a potentially great benefit to battlefield scenarios involving penetrator munitions. We anticipate that successful development of these reactive tungsten alloys will allow the Navy to achieve superior inert warhead penetrator designs in the future, incorporating these materials as replacements for conventional components, and as lethality-enhancing projectiles and pellets. The commercial market, originally based in military munitions, could expand to include applications in mining, anti-terrorism, energy exploration, and other industries.
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Abstract:
Physical Sciences Inc. and ATK Thiokol, Inc. propose to develop novel high-density tungsten-based reactive composites for application to inert kinetic energy munitions. These materials will be inert such that they do not reduce the projectile's Insensitive Munitions compliance. Their critical benefit will be to enhance projectile lethality by depositing a combined kinetic and chemical energy in the target, which is greater than the corresponding kinetic energy deposited by a non-reactive tungsten munition. This lethality enhancement will occur over most of the range of anticipated projectile (1300-5000 fps) and payload pellet (2000-6000 fps) velocities. Novel metallurgical fabrication techniques will be applied in these material developments. In Phase I, we shall fabricate samples of two different tungsten metal-oxidizer systems, and characterize their energetic and mechanical properties.
Benefits:The proposed materials technology development constitutes a potentially great benefit to battlefield scenarios involving penetrator munitions. We anticipate that successful development of these reactive tungsten alloys will allow the Navy to achieve superior inert warhead penetrator designs in the future, incorporating these materials as replacements for conventional components, and as lethality-enhancing projectiles and pellets. The commercial market, originally based in military munitions, could expand to include applications in mining, anti-terrorism, energy exploration, and other industries.
Hanns CEO/Chinatungten।com
History of Paperweights
History of Paperweights
http://www.museum.state.il.us/exhibits/barker/hist_pw.php
Nineteenth century revival of the glass industry
In early nineteenth-century Europe, a new creative potential developed in the decorative arts. An increasingly urban population and an expanding market of goods created by the Industrial Revolution stimulated the manufacture of many new decorative novelties. In the mid-1840s, glass paperweights appeared. They were a wholly modern, functional glass form that drew upon the ancient glassmaking techniques of millefiori and lampwork and the late-eighteenth century technique of cameo incrustation
The sudden emergence and popularity of paperweights can be attributed not only to their decorative appeal but also to a growing Victorian leisure-time interest in letter writing. This fashionable upper and middle class pastime assured their profitable manufacture along with many other glass accessories related to letter writing, all of which were purchased inexpensively at stationery and novelty shops.
Appearance of French paperweights
The exact year and origin of the manufacture of the first glass paperweight is problematical, but the first documented appearance can be traced to the Exhibition of Austrian Industry held in Vienna in 1845. The paperweights of Pietro Bigaglia of Venice were displayed at this exhibition. Knowledge of their existence was reportedly soon brought to the attention of the Saint-Louis glass factory in France, which immediately began to manufacture its own weights. A paperweight from Saint Louis dated 1845 is known, as well as one from Murano, Italy.
A second major French glasshouse, the Clichy factory, is also thought to have been manufacturing weights as early as 1845. A close concentric millefiori pedestal weight in the Barker collection is the earliest-dated known weight produced by the Clichy factory. The glided mount on this weight bears the inscription ";ESCALIER DE CRISTAL 1845." It is highly speculative, however, that the engraved date actually refers to the year of the weight's manufacture. The Escalier de Cristal was a novelty shop in Paris; consequently, the mount could have been added at any time.
The entry of a third leading French glasshouse, the Baccarat factory, into paperweightmaking is marked by existing weights enclosing the date 1846. Factories in Bohemia and England followed suit with the earliest-dated known weights from each locale inscribed "1848." In the decade or so following 1845, the three great French glasshouses of Saint Louis, Clichy, and Baccarat competed with one another in the manufacture of the most beautiful and the best executed weights. The results were a myriad of artistically conceived millefiori designs and lampworked motifs, near technical perfection of the glassmaker's skill, and great quantities of weights produced.
The Classic Period
This period of competitive manufacture, which captures paperweightmaking at its best, had come to be termed the Classic Period of French paperweights. It ranged in date from circa 1845 to 1855, although the time span is arbitrary and may extend slightly earlier or later (possibly through 1860) than the given decade. Perhaps the most highly praised paperweights of the French Classic Period are those produced by the Clichy factory. Clichy was the only French glasshouse whose weights were displayed at the Great Exposition at the Crystal Palace in London in 1851, and again, at the New York Crystal Palace in 1853. These public celebrations of the union of science and art in technology brought paperweights to the attention of the world. They were viewed by thousands of visitors, including a large American audience, and served to usher in the American Classic Period of paperweightmaking, which extended from 1852 through the 1870s, long after the popularity of paperweights had declined in Europe.
Modern paperweights
Paperweights continued to be produced in the twentieth century. Baccarat and Saint Louis continue to produce elegant weights reminiscent of the Classic Period, as well as modern designs. American glass companies and glass artists also continue creating paperweights in the traditional styles and create new traditions of their own.
Antique paperweights were made primarily in three French factories, between 1845 and 1860, in Baccarat, St. Louis, and Clichy. Weights (mainly of lesser quality) were also made in the United States, Great Britain, and elsewhere, though Bacchus (UK) and New England Glass Company (USA) produced some that equaled the best of the French. Modern weights have been made from about 1950 to the present.
The Morton D. Barker Paperweight Collection contains objects that were manufactured in Europe, the United States, and Asia.
The three manufacturers most renowned for the production of paperweights in the ninetheenth century were the French firms of Baccarat, Saint Louis, and Clichy. They produced the highest quality glass in Europe at that time. A fourth French paperweight maker was Pantin. Paperweights were being manufactured from about 1845 in France, and production continued until about 1860, when they went out of fashion.
American glass companies represented in the Barker Collection are the New England Glass Company, Boston & Sandwich Glass Company, Morgantown Glassware Guild, and Whitall & Tatum Company. Individual glass artists represented are Ronald Hansen, and Charles Kaziun, and Emil Larson, who also worked for several glass companies.
British glass companies are also represented in the collection by a millefiori weight by George Bacchus and Sons of Birmingham, and two objects attributed to Apsley Pellatt of London.
Glass paperweights or other objects from Sweden, Bohemia, and China are also found in the collection. A few paperweights cannot be positively attributed to any specific manufacturer or country.
Hanns CEO/Chinatungten।com
http://www.museum.state.il.us/exhibits/barker/hist_pw.php
Nineteenth century revival of the glass industry
In early nineteenth-century Europe, a new creative potential developed in the decorative arts. An increasingly urban population and an expanding market of goods created by the Industrial Revolution stimulated the manufacture of many new decorative novelties. In the mid-1840s, glass paperweights appeared. They were a wholly modern, functional glass form that drew upon the ancient glassmaking techniques of millefiori and lampwork and the late-eighteenth century technique of cameo incrustation
The sudden emergence and popularity of paperweights can be attributed not only to their decorative appeal but also to a growing Victorian leisure-time interest in letter writing. This fashionable upper and middle class pastime assured their profitable manufacture along with many other glass accessories related to letter writing, all of which were purchased inexpensively at stationery and novelty shops.
Appearance of French paperweights
The exact year and origin of the manufacture of the first glass paperweight is problematical, but the first documented appearance can be traced to the Exhibition of Austrian Industry held in Vienna in 1845. The paperweights of Pietro Bigaglia of Venice were displayed at this exhibition. Knowledge of their existence was reportedly soon brought to the attention of the Saint-Louis glass factory in France, which immediately began to manufacture its own weights. A paperweight from Saint Louis dated 1845 is known, as well as one from Murano, Italy.
A second major French glasshouse, the Clichy factory, is also thought to have been manufacturing weights as early as 1845. A close concentric millefiori pedestal weight in the Barker collection is the earliest-dated known weight produced by the Clichy factory. The glided mount on this weight bears the inscription ";ESCALIER DE CRISTAL 1845." It is highly speculative, however, that the engraved date actually refers to the year of the weight's manufacture. The Escalier de Cristal was a novelty shop in Paris; consequently, the mount could have been added at any time.
The entry of a third leading French glasshouse, the Baccarat factory, into paperweightmaking is marked by existing weights enclosing the date 1846. Factories in Bohemia and England followed suit with the earliest-dated known weights from each locale inscribed "1848." In the decade or so following 1845, the three great French glasshouses of Saint Louis, Clichy, and Baccarat competed with one another in the manufacture of the most beautiful and the best executed weights. The results were a myriad of artistically conceived millefiori designs and lampworked motifs, near technical perfection of the glassmaker's skill, and great quantities of weights produced.
The Classic Period
This period of competitive manufacture, which captures paperweightmaking at its best, had come to be termed the Classic Period of French paperweights. It ranged in date from circa 1845 to 1855, although the time span is arbitrary and may extend slightly earlier or later (possibly through 1860) than the given decade. Perhaps the most highly praised paperweights of the French Classic Period are those produced by the Clichy factory. Clichy was the only French glasshouse whose weights were displayed at the Great Exposition at the Crystal Palace in London in 1851, and again, at the New York Crystal Palace in 1853. These public celebrations of the union of science and art in technology brought paperweights to the attention of the world. They were viewed by thousands of visitors, including a large American audience, and served to usher in the American Classic Period of paperweightmaking, which extended from 1852 through the 1870s, long after the popularity of paperweights had declined in Europe.
Modern paperweights
Paperweights continued to be produced in the twentieth century. Baccarat and Saint Louis continue to produce elegant weights reminiscent of the Classic Period, as well as modern designs. American glass companies and glass artists also continue creating paperweights in the traditional styles and create new traditions of their own.
Antique paperweights were made primarily in three French factories, between 1845 and 1860, in Baccarat, St. Louis, and Clichy. Weights (mainly of lesser quality) were also made in the United States, Great Britain, and elsewhere, though Bacchus (UK) and New England Glass Company (USA) produced some that equaled the best of the French. Modern weights have been made from about 1950 to the present.
The Morton D. Barker Paperweight Collection contains objects that were manufactured in Europe, the United States, and Asia.
The three manufacturers most renowned for the production of paperweights in the ninetheenth century were the French firms of Baccarat, Saint Louis, and Clichy. They produced the highest quality glass in Europe at that time. A fourth French paperweight maker was Pantin. Paperweights were being manufactured from about 1845 in France, and production continued until about 1860, when they went out of fashion.
American glass companies represented in the Barker Collection are the New England Glass Company, Boston & Sandwich Glass Company, Morgantown Glassware Guild, and Whitall & Tatum Company. Individual glass artists represented are Ronald Hansen, and Charles Kaziun, and Emil Larson, who also worked for several glass companies.
British glass companies are also represented in the collection by a millefiori weight by George Bacchus and Sons of Birmingham, and two objects attributed to Apsley Pellatt of London.
Glass paperweights or other objects from Sweden, Bohemia, and China are also found in the collection. A few paperweights cannot be positively attributed to any specific manufacturer or country.
Hanns CEO/Chinatungten।com
Tungsten Heavy Aalloy Paperweights
Tungsten Alloy Paperweights
What is paperweight?
Paperweight are decorative objects, designed to hold sheets of paper on a surface to prevent wind from blowing them away. Paperweight has a long history in China, even can be traced back to the birth date of paper.
Type of paperweight
Antique paperweights
In ancient China, paperweight is one of the necessary equipment in sanctum. Antique paperweights are commonly made of jade, copper, china and high wood, using modeling rabbit, horse, sheep, deer, toads and other animals of the three-dimensional shape. They are often collected as examples of fine workmanship, and appreciated for their aesthetic as opposed to their rather than utilitarian aspect.
Copper paperweight
Jade paperweight
China paperweight
Modern paperweights
With the development of modern society, there are more and more types of peperweights. They retain the classical peaceful atmosphere, and at the same time, add a variety of new ideas and new elements have been integrating. WhetherWhatever material or specification, modern paperweight are moving heading towards diversification direction.
*Glass paperweight
*Crystal paperweight
*Stone Paperweight
*Ceramic Paperweight
*Wood paperweight
Tungsten Heavy Ally (WHA) Paperweight
——Never worn, never rust, high-density, high-performance paperweight
Tungsten heavy alloy bears the high density ranging 15.4-18.5g/cc (80-97W), with the components of W-Ni-Cu, W-Ni-Fe Or W-Ni-Cu-Fe and etc. It has a range of excellent features, to achieve full compliance with the requirements as paperweight material:
①High density More than twice as dense as steel, can press any book firmly;
②High strength The tensile strength is about 700-1400Mpa and the thermal expansion coefficient just only 1/2-1/3 of iron or steel, wear-resistant, never rust, can be preserved permanently;
③ Good weldability and machinability It is also possible to have "custom" bars made to your specifications
Chinatungsten Online (Xiamen) Manu. & Sales Corp., is one of most famous and professional manufacturers and exporters to supply various high-quality tungsten worldwide for over 20 years. We always provide different tungsten products base on specific requirement with dimensions, drawings and so on.
Any of your enquiries will be welcome to us. And we are sure to offer our excellent service to all of our esteemed clients.
Contact us by sales@chinatungsten.com now and get a good price from us!
Hanns CEO/Chinatungten।com
What is paperweight?
Paperweight are decorative objects, designed to hold sheets of paper on a surface to prevent wind from blowing them away. Paperweight has a long history in China, even can be traced back to the birth date of paper.
Type of paperweight
Antique paperweights
In ancient China, paperweight is one of the necessary equipment in sanctum. Antique paperweights are commonly made of jade, copper, china and high wood, using modeling rabbit, horse, sheep, deer, toads and other animals of the three-dimensional shape. They are often collected as examples of fine workmanship, and appreciated for their aesthetic as opposed to their rather than utilitarian aspect.
Copper paperweight
Jade paperweight
China paperweight
Modern paperweights
With the development of modern society, there are more and more types of peperweights. They retain the classical peaceful atmosphere, and at the same time, add a variety of new ideas and new elements have been integrating. WhetherWhatever material or specification, modern paperweight are moving heading towards diversification direction.
*Glass paperweight
*Crystal paperweight
*Stone Paperweight
*Ceramic Paperweight
*Wood paperweight
Tungsten Heavy Ally (WHA) Paperweight
——Never worn, never rust, high-density, high-performance paperweight
Tungsten heavy alloy bears the high density ranging 15.4-18.5g/cc (80-97W), with the components of W-Ni-Cu, W-Ni-Fe Or W-Ni-Cu-Fe and etc. It has a range of excellent features, to achieve full compliance with the requirements as paperweight material:
①High density More than twice as dense as steel, can press any book firmly;
②High strength The tensile strength is about 700-1400Mpa and the thermal expansion coefficient just only 1/2-1/3 of iron or steel, wear-resistant, never rust, can be preserved permanently;
③ Good weldability and machinability It is also possible to have "custom" bars made to your specifications
Chinatungsten Online (Xiamen) Manu. & Sales Corp., is one of most famous and professional manufacturers and exporters to supply various high-quality tungsten worldwide for over 20 years. We always provide different tungsten products base on specific requirement with dimensions, drawings and so on.
Any of your enquiries will be welcome to us. And we are sure to offer our excellent service to all of our esteemed clients.
Contact us by sales@chinatungsten.com now and get a good price from us!
Hanns CEO/Chinatungten।com
AN ADVANTAGE RESEARCH OF DEPLETED URANIUM & TUNGSTEN HEAVY ALLOY
Neurotoxic Potential of Depleted Uranium—Effects in Primary Cortical Neuron Cultures and in Caenorhabdit is elegans
ToxSci Advance Access originally published online on July 16, 2007
http://toxsci.oxfordjournals.org/cgi/content/full/99/2/553
Toxicological Sciences 2007 99(2):553-565; doi:10.1093/toxsci/kfm171
George C.-T. Jiang*, Kristen Tidwell , Beth Ann McLaughlin , Jiyang Cai , Ramesh C. Gupta , Dejan Milatovic¶, Richard Nass¶ and Michael Aschner¶,1
* Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1083 Department of Neurology Vanderbilt Eye Institute, Vanderbilt University, Nashville, Tennessee 37232 Toxicology Department, Murray State University, Hopkinsville, Kentucky 42240 ¶ Department of Pediatrics, Vanderbilt University, Nashville, Tennessee 37232
1 To whom correspondence should be addressed at Departments of Pediatrics and Pharmacology, and the Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University, 6110 MRBIII, 465 21st Ave. S, Nashville, TN 37232-2495. Fax: (615) 322-6541. E-mail: michael.aschner@vanderbilt.edu
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Received April 17, 2007; accepted June 13, 2007
ABSTRACTDepleted uranium (DU) is an extremely dense metal that is used in radiation shielding, counterbalances, armor, and ammunition. In light of the public concerns about exposure to DU and its potential role in Gulf War Syndrome (GWS), this study evaluated the neurotoxic potential of DU using focused studies on primary rat cortical neurons and the nematode Caenorhabditis elegans. We examined cell viability, cellular energy metabolism, thiol metabolite oxidation, and lipid peroxidation following exposure of cultured neurons to DU, in the form of uranyl acetate. We concurrently evaluated the neurotoxicity of uranyl acetate in C. elegans using various neuronal–green fluourescent protein reporter strains to visualize neurodegeneration. Our studies indicate that uranyl acetate has low cytotoxic potential, and uranium exposure does not result in significant changes in cellular energy metabolism, thiol metabolite oxidation, or lipid peroxidation. Furthermore, our C. elegans studies do not show any significant neurodegeneration following uranyl acetate exposure. Together, these studies suggest that DU, in the form of uranyl acetate, has low neurotoxic potential. These findings should alleviate the some of public concerns regarding DU as an etiologic agent of neurodegenerative conditions associated with GWS.
Key Words: depleted uranium; primary neurons; neurotoxicity; Gulf War Syndrome; C. elegans.
INTRODUCTIONDepleted uranium (DU) is a by-product of the enrichment of naturally occurring uranium for its most radioactive isotope, 235U. The extremely dense and pyrophoric properties of DU make it an excellent metallic substrate for radiation shielding, counterbalances, and in armor and ammunition (Jiang and Aschner, 2006 ). As a heavy metal, internalized DU is cleared by the kidneys, and numerous studies have demonstrated nephrotoxicity after exposure to high levels of DU (Andrews and Bates, 1987 ; Carriere et al., 2005 ; Goldman et al., 2006 ; Kobayashi et al., 1984 ; Taulan et al., 2004 ). Other than the effects on the kidneys, DU exposure is thought to result in neurologic sequelae. Indeed, it has been hypothesized that DU may contribute to the etiology of Gulf War Syndrome (GWS) (Abu-Qare and Abou-Donia, 2002 ; Bem and Bou-Rabee, 2004 ; Doucet, 1994 ; Durakovic, 2003 ; Gronseth, 2005 ; Jamal et al., 1996 ; Jiang and Aschner, 2006 ). Follow-up studies on Gulf War veterans exposed to DU demonstrated decreased cognitive performance compared to unexposed veterans, which provided evidence for such a theory (McDiarmid et al., 2000 ). The increased usage and health concerns have led researchers to scrutinize the effects of DU exposure on the central nervous system (CNS).
The recent interest in the effects of DU exposure on the CNS has led to a number of studies with small animals. Such studies have shown that uranium (U) indeed crosses the blood–brain barrier (Abou-Donia et al., 2002 ; Barber et al., 2005 ; Briner and Murray, 2005 ; Fitsanakis et al., 2006 ; Houpert et al., 2004 ; Leggett and Pellmar, 2003 ; Lestaevel et al., 2005 ; Paquet et al., 2006 ; Pellmar et al., 1999a ,b), accumulates in a dose-dependent manner in specific brain structures (Fitsanakis et al., 2006 ; Pellmar et al., 1999a ), and results in increased lipid oxidation (Briner and Murray, 2005 ), nitric oxide generation (Abou-Donia et al., 2002 ), and sensorimotor deficits (Abou-Donia et al., 2002 ). These studies have attempted to correlate the observed neurobiological changes with potential functional changes in cognitive behavior (Abou-Donia et al., 2002 ; Belles et al., 2005 ; Briner and Murray, 2005 ; Houpert et al., 2005 ). To date, however, there remains a significant gap in understanding the specific effects of uranium on cells of the CNS, and the potential molecular changes involved upon DU exposure.
The cellular effects of DU have only been evaluated in a limited number of cell culture models. Studies in Chinese hamster ovary cells have demonstrated cytogenetic toxicity of uranium (Lin et al., 1993 ), and induction of hypoxanthine (guanine) phosphoribosyltransferase (hprt) mutations and DNA adducts (Albertini et al., 2003 ; Stearns et al., 2005 ). Studies with immortalized human osteoblast cells to evaluate the effects of DU have corroborated this finding, further demonstrating that DU results in genotoxicity, and that it can be neoplastic (Miller et al., 1998a , 2001, 2002, 2003). Uranium has also been shown to induce activation of stress gene expression in human liver carcinoma cells (HepG2) (Miller et al., 2004 ). In the mouse macrophage cell line, J774, uranium treatment resulted in time- and concentration-dependent uptake of uranium, cytotoxicity, and induction of apoptosis (Kalinich et al., 2002 ). Concentration-dependent cytotoxicity was also observed in NRK-52E cells, another immortalized cell culture model representative of rat kidney proximal epithelium cells (Carriere et al., 2004 ). Researchers have also evaluated the transcriptomic and proteomic responses of HEK293 kidney cells, and renal tissue from rats exposed to DU, and found that there were several oxidative-response–related transcripts that were upregulated, and significantly increased peroxide levels that support the implication of oxidative stress (Prat et al., 2005 ; Taulan et al., 2004, 2006 ). In rat brain endothelial cells, the closest in vitro model to cells of CNS origin, researchers demonstrated that uranium did not result in significant cytotoxicity (Dobson et al., 2006 ).
To date, researchers have not undertaken focused studies to determine the effects of DU on cells of CNS origin. Numerous CNS cell models are available for study, including primary cultures and immortalized cell lines. Although primary cultures have a finite life span compared to immortalized cell lines, the former offer many advantages as cell lines will often show numerous changes in cell cycle and proliferation, morphology, and chromosomal variations. Furthermore, primary are cultured in the context of their naturally occurring neighboring cell types. In these studies, we have attempted to fill the gap in the knowledge of DU neurotoxicity by performing focused studies using primary rat cortical neurons to examine the acute neurotoxic potential of DU and the specific cellular effects in neurons. We are testing the hypothesis that DU results in significant concentration-dependent cytotoxicity, and oxidative stress, as has been previously seen in other cell culture models.
The nematode, Caenorhabditis elegans, is an excellent model organism that has been used in a number of toxicological studies (Anderson and Wild, 1994 ; Dhawan et al., 1999 ; Reichert and Menzel, 2005 ; Swain et al., 2004 ). The worms are easily grown and maintained, and have a rapid replication cycle, allowing for thousands of worms to be evaluated within a number of days (Brenner, 1974 ). The nematode is a model organism, with its complete genome determined, numerous genetic mutants freely available, and multicolor reporter constructs, e.g., green fluorescent protein (GFP), can be easily introduced into the system (Hobert and Loria, 2006 ; Link and Johnson, 2002 ; Miller, et al., 1999 ). Furthermore, there are only 302 neurons in the nematode, in which all the projection pathways have been determined (Gally and Bessereau, 2003 ; Wadsworth and Hedgecock, 1992 ). All of these C. elegans characteristics make it a powerful organism to evaluate the toxicological potential of a wide array of compounds. For our studies, C. elegans is an organism in which we can evaluate the in vivo effects of uranium on CNS cells. We tested the hypothesis that uranium exposure results in significant concentration-dependent neurotoxicity as can be visualized by neurodegeneration.
In light of the public concerns regarding DU, this study sought to evaluate the neurotoxicity of DU, in the form of uranyl acetate, using focused studies of a relatively homogeneous cell population of CNS origin. Here, we investigate the cytotoxic effects of U in primary rat neuronal cultures, subsequent changes in cellular metabolism, and concurrently evaluate the neurotoxicity of U in C. elegans using neuronal-GFP reporter strains.
MATERIALS AND METHODS
Materials
Uranyl acetate (UO2(CH3COO)2·2H2O) was purchased from Ted Pella, Inc. (Redding, CA). All other chemicals were purchased from Sigma (St Louis, MO) unless otherwise stated. Coverslips for cell culture were purchased from Carolina Biological Supply (Burlington, NC). All tissue culture media and supplements were purchased from Invitrogen (Carlsbad, CA), except for Hyclone Fetal Bovine Serum and Hyclone F12, which were purchased from VWR (Suwanee, GA). Nematode growth reagents and plasticware were purchased from VWR.
Cell culture conditions and uranyl acetate treatments.All experiments were approved by the Institutional Animal Care and Use Committee of Vanderbilt University and were performed according to Guidelines for Animal Experimentation as set forth by Vanderbilt University. Rat cortical neuron cultures were prepared from E17 rat pups, as previously described (McLaughlin et al., 1998 ). Briefly, E17 Harlan Sprague–Dawley rat embryos were decapitated, and the brains rapidly removed and placed in a 35-mm petri-dishes with cold Hank's balanced salt solution (HBSS). The cortices were dissected under a dissection microscope and then were placed in another dish containing HBSS to further remove blood vessels and meninges from cortical tissues. The isolated cortices were then transferred to a petri-dish containing 0.6% (wt/vol) trypsin in HBSS for 30 min. After two washes in HBSS, the cortical tissues were mechanically dissociated with a glass Pasteur pipette. Dissociated cortical cells were plated on poly-L-ornithine-treated glass coverslips in six-well plates, using a plating medium of glutamine-free Dulbecco's modified Eagle's medium–Eagle's salts (Invitrogen), supplemented with Ham's F12 (Hyclone, Logan, UT), heat-inactivated fetal bovine serum (Hyclone), and penicillin/streptomycin (Sigma), at a density of 700,000 cells per well. After 2 days in vitro, nonneuronal cell division was halted by a 1-day exposure to 10µM cytosine arabinoside (Sigma), and cultures were shifted to Neurobasal media (Invitrogen), supplemented with B27 (Invitrogen) and penicillin/streptomycin. Cells were maintained by changing the media every 2–3 days and grown at 37°C in a humidified atmosphere of 5% CO2 in air.
Cells were treated 3 weeks after isolation with DU (uranyl acetate), prepared as sterile solutions in treatment buffer, for 24 h, at 37°C in a humidified atmosphere of 5% CO2 in air. Treatment buffer consisted of minimal essential media (Invitrogen) supplemented with 25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 10 ml N2 media supplement (Invitrogen), 0.001% BSA (Sigma). N-methyl-D-aspartate (NMDA, Sigma) was used as a positive control for cytotoxicity at a final concentration of 100µM in conjunction with 10µM glycine.
Cell viability determinations.Primary rat cortical neuron viability was determined by fluorescence activated cell sorting (FACS) using the LIVE/DEAD viability/cytotoxicity kit (Molecular Probes, Eugene, OR). Both floating and attached cells were collected and stained with 2 µl of calcein and 8 µl of ethidium homodimer in phosphate buffered saline (PBS) as previously described (Chen et al., 2002 ). The percentage of viable cells was analyzed by flow cytometry (BD Immunocytometry Systems, San Jose, CA). For each sample, at least 10,000 cells were counted on a BD FACScan (Becton Dickinson, San Jose, CA). Data analyses were performed with WinMDI (Windows Multiple Document Interafce for Flow Cytometry) (http://facs.scripps.edu).
Cell viability and proliferation were evaluated by lactate dehydrogenase (LDH) (Sigma) and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) (Sigma) assays. LDH release was measured with an in vitro toxicology assay kit (Sigma) by assaying 40 µl sample medium spectrophotometrically (490:630 nm) according to the manufacturer's protocol, to obtain a measure of cytoplasmic LDH released from dead and dying neurons (Legrand et al., 1992 ). MTT is yellow until reduced to purple formazan in the mitochondria of living cells. The reduction of MTT to formazan occurs only when mitochondrial reductase enzymes are active, and thus conversion is a measurement of mitochondrial inhibition, and can be correlated to the number of viable (living) cells. LDH release and MTT analyses were determined according to manufacturer's instructions. LDH release results were confirmed qualitatively by visual inspection of the cells and, in several instances, quantitatively by cell counts by the method of Rosenberg and Aizenman (1989) .
Thiol metabolite determination.Quantification of levels of glutathione (GSH) and its related products were performed by high-performance liquid chromatography (HPLC) as previously described (Jones, 2002 ; Jones et al., 1998 ; Nelson et al., 1999 ). Briefly, treated cells were washed with PBS, and resuspended in 0.5% perchloric acid with 0.2M boric acid and 10µM -Glu–Glu (internal standard), and sonicated with a Sonics Vibra-Cell, two times for 20 s at 25% power. Extracts were derivatized with iodoacetic acid and dansyl chloride. The acid soluble cysteine (Cys), cystine (CySS), GSH, and oxidized glutathione (GSSG) were analyzed by HPLC using fluorescence detection on a Waters 2695 Alliance HPLC system (Waters, Milford, MA). Samples were loaded onto an YMC Pack NH2 (amino) column (Waters) and were eluted with a gradient of sodium acetate. The solvent used for mobile phase was 80% methanol. The peaks were quantified by integration relative to the internal standard. Using this method, samples were analyzed for Cys, CySS, GSH, and GSSG content. Redox status for the GSH/GSSG redox couple (Eh GSH), and the Cys/CySS redox couple (Eh Cys) were calculated using the Nernst equation.
Total adenosine nucleotides determination.Changes in adenosine nucleotides were measured by isocratic reversed-phase HPLC as previously described (Yang et al., 2004 ). For HPLC analysis, treatment media was removed from the cell samples before adding 950 µl of chilled 0.3M perchloric acid with 1mM disodium ethylenediaminetetraacetate to each well to harvest cell extracts into microcentrifuge tubes. An aliquot of 2M potassium hydroxide (170 µl) was then added to each sample, followed by centrifugation at 9000 x g to remove precipitates of KClO4. The supernatant was then stored at – 80°C until HPLC analysis on a Waters HPLC system (Waters), coupled with a dual -absorbance UV detector (Model 2487) equipped to a computer system with Waters Millennium software program (Workstation v. 4.0) for data processing. The mobile phase used was 0.1M ammonium dihydrogen phosphate (pH 6.0) with 1% methanol. Using the Symmetry Shield C-18 column and a flow rate of 0.6 ml/min, the peaks of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were eluted at retention times of 3.462, 3.868, and 5.694 min, respectively, with a variation window of 0.2 min in both standard and sample extracts. The peak height responses for all three nucleotides were recorded at 206 nm. The concentration of each nucleotide was determined in a 15-µl sample extract injected to HPLC and finally expressed in terms of nmol nucleotide per ml extract. The total adenosine nucleotides (TAN) content was calculated by TAN = ATP + ADP + AMP, while the energy charge potential (ECP) was calculated by the equation ECP = [ATP + 0.5 (ADP)]/TAN, as previously described (Yang et al., 2004 ).
F2-IsoP quantitation.Quantification of F2-isoprostanes (F2-IsoP) levels was determined using a stable isotope dilution method with detection by gas chromatography/mass spectrometry and selective ion monitoring as previously described (Milatovic et al., 2005 ; Morrow and Roberts, 1991 ; Roberts and Morrow, 1994 ). Briefly, samples were extracted and saponified, a stable isotope internal standard added, and then prepared for gas chromatography through a series of purifications by C-18 and Silica Sep-Pak cartridges and thin layer chromatography (TLC). Gas chromatography was performed using a 15 m long, 0.25 mm diameter, 0.25-µm film thickness, DB1701 fused silica capillary column (Fisons, Folsom, CA). The injector temperature was 265°C and oven (column) temperature was programmed from 200°C to 300°C at 15°C/min. Helium was used as the carrier gas at a flow rate of 1 ml/min. Ion source temperature was 250°C, electron energy was 70 eV, and filament current was 0.25 mA. For analysis, compounds were dissolved in undecane that was dried over a bed of calcium hydride. Negative ion chemical ionization mass spectrometry was performed using an Agilent Technologies G1789A GC/MSD instrument with a Hewlett–Packard computer system with ChemStation-NT. Total protein content was determined by BCA assay (Pierce, Rockford, IL) with bovine serum albumin as the standard (Smith et al., 1985 ).
Strains and maintenance.Caenorhabditis elegans strains were cultured on bacterial lawns of either NA-22 or OP-50, seeded on 8P or nematode growth medium (NGM) plates respectively, at 20°C according to standard methods (Brenner, 1974 ). Caenorhabditis elegans strain N2 (var. Bristol) is the wild-type strain, and was a gift of Dr Richard Nass (Vanderbilt University, Nashville, TN). The BY250 strain was developed and obtained from Dr Richard Nass (Vanderbilt University, Nashville, TN). Strain NW1229 (dpy-20(e1362) IV; evIs111) was obtained from the C. elegans Genetics Center (CGC, University of Minnesota, Minneapolis, MN).
Exposure of C. elegans to uranyl acetate.Embryos were obtained by hypochlorite treatment of gravid adults (Lewis and Fleming, 1995 ). After 17–24 h incubation in M9 buffer to obtain synchronized L1s, such that all nematodes are at the same point in their life cycle, the worms were washed once in 10 ml of dH2O, and then diluted to 50 worms per µl. L1 worms were treated with DU (uranyl acetate), prepared from a 1M stock solution in water. Five thousand worms were used in each siliconized microcentrifuge tube (Denver Scientific Inc., Metuchen, NJ) per treatment assay, and incubated with gentle shaking at 800 rpm for 30 min on a VWR Digital Mini Vortex Mixer (VWR Scientific, Suwanee, GA). Worms were then spread on NGM/OP-50 plates and incubated for 24 h at 20°C before further evaluation. For quantitative analyses of uranyl acetate-induced changes in worm viability, total number of live worms was determined for each concentration by counting each plate under a Stemi-2000 dissecting microscope (Zeiss, Thornwood, NY).
Photomicroscopy.Cell morphology was visually inspected on a Zeiss Axiovert 40 inverted microscope (Zeiss, Thornwood, NY). Cortical culture images were captured on an inverted Nomarski microscope (Zeiss Axiovert 200M) with AxioCam and AxioVision 4.4 software (Zeiss), using fixed exposures for all image captures between different treatments. Nematode images were captured on a Zeiss upright LSM510 confocal microscope (Zeiss), using laser scanning fluorescence and DIC (Nomarski) imaging. Worms were photographed under oil immersion with a 40x/1.30 Plan-Neofluar objective using fixed exposure settings for all image captures between different treatments. Images were exported using the Zeiss LSM Image Browser. Images were quantified for their fluorescence using Adobe Photoshop 6.0 (Adobe, San Jose, CA) and NIH ImageJ software. The fluorescent intensities were subsequently used to test if the levels of fluorescence were decreased upon treatment with U. With BY250 worms, cell bodies and dendrites were also manually scored as present if fluorescence could be seen. Dendrites were scored as abnormal if they had breaks or were barely visible. The ratio of abnormal:normal dendrites was used to calculate ratios for the different treatments, which were then compared for significance as previously described (Nass and Blakely, 2003 ).
Data analysis।All results are given as mean ± standard error of the mean. Differences between groups were analyzed statistically with one-way ANOVA followed by post hoc tests for multiple comparisons with p <>
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ToxSci Advance Access originally published online on July 16, 2007
http://toxsci.oxfordjournals.org/cgi/content/full/99/2/553
Toxicological Sciences 2007 99(2):553-565; doi:10.1093/toxsci/kfm171
George C.-T. Jiang*, Kristen Tidwell , Beth Ann McLaughlin , Jiyang Cai , Ramesh C. Gupta , Dejan Milatovic¶, Richard Nass¶ and Michael Aschner¶,1
* Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1083 Department of Neurology Vanderbilt Eye Institute, Vanderbilt University, Nashville, Tennessee 37232 Toxicology Department, Murray State University, Hopkinsville, Kentucky 42240 ¶ Department of Pediatrics, Vanderbilt University, Nashville, Tennessee 37232
1 To whom correspondence should be addressed at Departments of Pediatrics and Pharmacology, and the Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University, 6110 MRBIII, 465 21st Ave. S, Nashville, TN 37232-2495. Fax: (615) 322-6541. E-mail: michael.aschner@vanderbilt.edu
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Received April 17, 2007; accepted June 13, 2007
ABSTRACTDepleted uranium (DU) is an extremely dense metal that is used in radiation shielding, counterbalances, armor, and ammunition. In light of the public concerns about exposure to DU and its potential role in Gulf War Syndrome (GWS), this study evaluated the neurotoxic potential of DU using focused studies on primary rat cortical neurons and the nematode Caenorhabditis elegans. We examined cell viability, cellular energy metabolism, thiol metabolite oxidation, and lipid peroxidation following exposure of cultured neurons to DU, in the form of uranyl acetate. We concurrently evaluated the neurotoxicity of uranyl acetate in C. elegans using various neuronal–green fluourescent protein reporter strains to visualize neurodegeneration. Our studies indicate that uranyl acetate has low cytotoxic potential, and uranium exposure does not result in significant changes in cellular energy metabolism, thiol metabolite oxidation, or lipid peroxidation. Furthermore, our C. elegans studies do not show any significant neurodegeneration following uranyl acetate exposure. Together, these studies suggest that DU, in the form of uranyl acetate, has low neurotoxic potential. These findings should alleviate the some of public concerns regarding DU as an etiologic agent of neurodegenerative conditions associated with GWS.
Key Words: depleted uranium; primary neurons; neurotoxicity; Gulf War Syndrome; C. elegans.
INTRODUCTIONDepleted uranium (DU) is a by-product of the enrichment of naturally occurring uranium for its most radioactive isotope, 235U. The extremely dense and pyrophoric properties of DU make it an excellent metallic substrate for radiation shielding, counterbalances, and in armor and ammunition (Jiang and Aschner, 2006 ). As a heavy metal, internalized DU is cleared by the kidneys, and numerous studies have demonstrated nephrotoxicity after exposure to high levels of DU (Andrews and Bates, 1987 ; Carriere et al., 2005 ; Goldman et al., 2006 ; Kobayashi et al., 1984 ; Taulan et al., 2004 ). Other than the effects on the kidneys, DU exposure is thought to result in neurologic sequelae. Indeed, it has been hypothesized that DU may contribute to the etiology of Gulf War Syndrome (GWS) (Abu-Qare and Abou-Donia, 2002 ; Bem and Bou-Rabee, 2004 ; Doucet, 1994 ; Durakovic, 2003 ; Gronseth, 2005 ; Jamal et al., 1996 ; Jiang and Aschner, 2006 ). Follow-up studies on Gulf War veterans exposed to DU demonstrated decreased cognitive performance compared to unexposed veterans, which provided evidence for such a theory (McDiarmid et al., 2000 ). The increased usage and health concerns have led researchers to scrutinize the effects of DU exposure on the central nervous system (CNS).
The recent interest in the effects of DU exposure on the CNS has led to a number of studies with small animals. Such studies have shown that uranium (U) indeed crosses the blood–brain barrier (Abou-Donia et al., 2002 ; Barber et al., 2005 ; Briner and Murray, 2005 ; Fitsanakis et al., 2006 ; Houpert et al., 2004 ; Leggett and Pellmar, 2003 ; Lestaevel et al., 2005 ; Paquet et al., 2006 ; Pellmar et al., 1999a ,b), accumulates in a dose-dependent manner in specific brain structures (Fitsanakis et al., 2006 ; Pellmar et al., 1999a ), and results in increased lipid oxidation (Briner and Murray, 2005 ), nitric oxide generation (Abou-Donia et al., 2002 ), and sensorimotor deficits (Abou-Donia et al., 2002 ). These studies have attempted to correlate the observed neurobiological changes with potential functional changes in cognitive behavior (Abou-Donia et al., 2002 ; Belles et al., 2005 ; Briner and Murray, 2005 ; Houpert et al., 2005 ). To date, however, there remains a significant gap in understanding the specific effects of uranium on cells of the CNS, and the potential molecular changes involved upon DU exposure.
The cellular effects of DU have only been evaluated in a limited number of cell culture models. Studies in Chinese hamster ovary cells have demonstrated cytogenetic toxicity of uranium (Lin et al., 1993 ), and induction of hypoxanthine (guanine) phosphoribosyltransferase (hprt) mutations and DNA adducts (Albertini et al., 2003 ; Stearns et al., 2005 ). Studies with immortalized human osteoblast cells to evaluate the effects of DU have corroborated this finding, further demonstrating that DU results in genotoxicity, and that it can be neoplastic (Miller et al., 1998a , 2001, 2002, 2003). Uranium has also been shown to induce activation of stress gene expression in human liver carcinoma cells (HepG2) (Miller et al., 2004 ). In the mouse macrophage cell line, J774, uranium treatment resulted in time- and concentration-dependent uptake of uranium, cytotoxicity, and induction of apoptosis (Kalinich et al., 2002 ). Concentration-dependent cytotoxicity was also observed in NRK-52E cells, another immortalized cell culture model representative of rat kidney proximal epithelium cells (Carriere et al., 2004 ). Researchers have also evaluated the transcriptomic and proteomic responses of HEK293 kidney cells, and renal tissue from rats exposed to DU, and found that there were several oxidative-response–related transcripts that were upregulated, and significantly increased peroxide levels that support the implication of oxidative stress (Prat et al., 2005 ; Taulan et al., 2004, 2006 ). In rat brain endothelial cells, the closest in vitro model to cells of CNS origin, researchers demonstrated that uranium did not result in significant cytotoxicity (Dobson et al., 2006 ).
To date, researchers have not undertaken focused studies to determine the effects of DU on cells of CNS origin. Numerous CNS cell models are available for study, including primary cultures and immortalized cell lines. Although primary cultures have a finite life span compared to immortalized cell lines, the former offer many advantages as cell lines will often show numerous changes in cell cycle and proliferation, morphology, and chromosomal variations. Furthermore, primary are cultured in the context of their naturally occurring neighboring cell types. In these studies, we have attempted to fill the gap in the knowledge of DU neurotoxicity by performing focused studies using primary rat cortical neurons to examine the acute neurotoxic potential of DU and the specific cellular effects in neurons. We are testing the hypothesis that DU results in significant concentration-dependent cytotoxicity, and oxidative stress, as has been previously seen in other cell culture models.
The nematode, Caenorhabditis elegans, is an excellent model organism that has been used in a number of toxicological studies (Anderson and Wild, 1994 ; Dhawan et al., 1999 ; Reichert and Menzel, 2005 ; Swain et al., 2004 ). The worms are easily grown and maintained, and have a rapid replication cycle, allowing for thousands of worms to be evaluated within a number of days (Brenner, 1974 ). The nematode is a model organism, with its complete genome determined, numerous genetic mutants freely available, and multicolor reporter constructs, e.g., green fluorescent protein (GFP), can be easily introduced into the system (Hobert and Loria, 2006 ; Link and Johnson, 2002 ; Miller, et al., 1999 ). Furthermore, there are only 302 neurons in the nematode, in which all the projection pathways have been determined (Gally and Bessereau, 2003 ; Wadsworth and Hedgecock, 1992 ). All of these C. elegans characteristics make it a powerful organism to evaluate the toxicological potential of a wide array of compounds. For our studies, C. elegans is an organism in which we can evaluate the in vivo effects of uranium on CNS cells. We tested the hypothesis that uranium exposure results in significant concentration-dependent neurotoxicity as can be visualized by neurodegeneration.
In light of the public concerns regarding DU, this study sought to evaluate the neurotoxicity of DU, in the form of uranyl acetate, using focused studies of a relatively homogeneous cell population of CNS origin. Here, we investigate the cytotoxic effects of U in primary rat neuronal cultures, subsequent changes in cellular metabolism, and concurrently evaluate the neurotoxicity of U in C. elegans using neuronal-GFP reporter strains.
MATERIALS AND METHODS
Materials
Uranyl acetate (UO2(CH3COO)2·2H2O) was purchased from Ted Pella, Inc. (Redding, CA). All other chemicals were purchased from Sigma (St Louis, MO) unless otherwise stated. Coverslips for cell culture were purchased from Carolina Biological Supply (Burlington, NC). All tissue culture media and supplements were purchased from Invitrogen (Carlsbad, CA), except for Hyclone Fetal Bovine Serum and Hyclone F12, which were purchased from VWR (Suwanee, GA). Nematode growth reagents and plasticware were purchased from VWR.
Cell culture conditions and uranyl acetate treatments.All experiments were approved by the Institutional Animal Care and Use Committee of Vanderbilt University and were performed according to Guidelines for Animal Experimentation as set forth by Vanderbilt University. Rat cortical neuron cultures were prepared from E17 rat pups, as previously described (McLaughlin et al., 1998 ). Briefly, E17 Harlan Sprague–Dawley rat embryos were decapitated, and the brains rapidly removed and placed in a 35-mm petri-dishes with cold Hank's balanced salt solution (HBSS). The cortices were dissected under a dissection microscope and then were placed in another dish containing HBSS to further remove blood vessels and meninges from cortical tissues. The isolated cortices were then transferred to a petri-dish containing 0.6% (wt/vol) trypsin in HBSS for 30 min. After two washes in HBSS, the cortical tissues were mechanically dissociated with a glass Pasteur pipette. Dissociated cortical cells were plated on poly-L-ornithine-treated glass coverslips in six-well plates, using a plating medium of glutamine-free Dulbecco's modified Eagle's medium–Eagle's salts (Invitrogen), supplemented with Ham's F12 (Hyclone, Logan, UT), heat-inactivated fetal bovine serum (Hyclone), and penicillin/streptomycin (Sigma), at a density of 700,000 cells per well. After 2 days in vitro, nonneuronal cell division was halted by a 1-day exposure to 10µM cytosine arabinoside (Sigma), and cultures were shifted to Neurobasal media (Invitrogen), supplemented with B27 (Invitrogen) and penicillin/streptomycin. Cells were maintained by changing the media every 2–3 days and grown at 37°C in a humidified atmosphere of 5% CO2 in air.
Cells were treated 3 weeks after isolation with DU (uranyl acetate), prepared as sterile solutions in treatment buffer, for 24 h, at 37°C in a humidified atmosphere of 5% CO2 in air. Treatment buffer consisted of minimal essential media (Invitrogen) supplemented with 25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 10 ml N2 media supplement (Invitrogen), 0.001% BSA (Sigma). N-methyl-D-aspartate (NMDA, Sigma) was used as a positive control for cytotoxicity at a final concentration of 100µM in conjunction with 10µM glycine.
Cell viability determinations.Primary rat cortical neuron viability was determined by fluorescence activated cell sorting (FACS) using the LIVE/DEAD viability/cytotoxicity kit (Molecular Probes, Eugene, OR). Both floating and attached cells were collected and stained with 2 µl of calcein and 8 µl of ethidium homodimer in phosphate buffered saline (PBS) as previously described (Chen et al., 2002 ). The percentage of viable cells was analyzed by flow cytometry (BD Immunocytometry Systems, San Jose, CA). For each sample, at least 10,000 cells were counted on a BD FACScan (Becton Dickinson, San Jose, CA). Data analyses were performed with WinMDI (Windows Multiple Document Interafce for Flow Cytometry) (http://facs.scripps.edu).
Cell viability and proliferation were evaluated by lactate dehydrogenase (LDH) (Sigma) and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) (Sigma) assays. LDH release was measured with an in vitro toxicology assay kit (Sigma) by assaying 40 µl sample medium spectrophotometrically (490:630 nm) according to the manufacturer's protocol, to obtain a measure of cytoplasmic LDH released from dead and dying neurons (Legrand et al., 1992 ). MTT is yellow until reduced to purple formazan in the mitochondria of living cells. The reduction of MTT to formazan occurs only when mitochondrial reductase enzymes are active, and thus conversion is a measurement of mitochondrial inhibition, and can be correlated to the number of viable (living) cells. LDH release and MTT analyses were determined according to manufacturer's instructions. LDH release results were confirmed qualitatively by visual inspection of the cells and, in several instances, quantitatively by cell counts by the method of Rosenberg and Aizenman (1989) .
Thiol metabolite determination.Quantification of levels of glutathione (GSH) and its related products were performed by high-performance liquid chromatography (HPLC) as previously described (Jones, 2002 ; Jones et al., 1998 ; Nelson et al., 1999 ). Briefly, treated cells were washed with PBS, and resuspended in 0.5% perchloric acid with 0.2M boric acid and 10µM -Glu–Glu (internal standard), and sonicated with a Sonics Vibra-Cell, two times for 20 s at 25% power. Extracts were derivatized with iodoacetic acid and dansyl chloride. The acid soluble cysteine (Cys), cystine (CySS), GSH, and oxidized glutathione (GSSG) were analyzed by HPLC using fluorescence detection on a Waters 2695 Alliance HPLC system (Waters, Milford, MA). Samples were loaded onto an YMC Pack NH2 (amino) column (Waters) and were eluted with a gradient of sodium acetate. The solvent used for mobile phase was 80% methanol. The peaks were quantified by integration relative to the internal standard. Using this method, samples were analyzed for Cys, CySS, GSH, and GSSG content. Redox status for the GSH/GSSG redox couple (Eh GSH), and the Cys/CySS redox couple (Eh Cys) were calculated using the Nernst equation.
Total adenosine nucleotides determination.Changes in adenosine nucleotides were measured by isocratic reversed-phase HPLC as previously described (Yang et al., 2004 ). For HPLC analysis, treatment media was removed from the cell samples before adding 950 µl of chilled 0.3M perchloric acid with 1mM disodium ethylenediaminetetraacetate to each well to harvest cell extracts into microcentrifuge tubes. An aliquot of 2M potassium hydroxide (170 µl) was then added to each sample, followed by centrifugation at 9000 x g to remove precipitates of KClO4. The supernatant was then stored at – 80°C until HPLC analysis on a Waters HPLC system (Waters), coupled with a dual -absorbance UV detector (Model 2487) equipped to a computer system with Waters Millennium software program (Workstation v. 4.0) for data processing. The mobile phase used was 0.1M ammonium dihydrogen phosphate (pH 6.0) with 1% methanol. Using the Symmetry Shield C-18 column and a flow rate of 0.6 ml/min, the peaks of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were eluted at retention times of 3.462, 3.868, and 5.694 min, respectively, with a variation window of 0.2 min in both standard and sample extracts. The peak height responses for all three nucleotides were recorded at 206 nm. The concentration of each nucleotide was determined in a 15-µl sample extract injected to HPLC and finally expressed in terms of nmol nucleotide per ml extract. The total adenosine nucleotides (TAN) content was calculated by TAN = ATP + ADP + AMP, while the energy charge potential (ECP) was calculated by the equation ECP = [ATP + 0.5 (ADP)]/TAN, as previously described (Yang et al., 2004 ).
F2-IsoP quantitation.Quantification of F2-isoprostanes (F2-IsoP) levels was determined using a stable isotope dilution method with detection by gas chromatography/mass spectrometry and selective ion monitoring as previously described (Milatovic et al., 2005 ; Morrow and Roberts, 1991 ; Roberts and Morrow, 1994 ). Briefly, samples were extracted and saponified, a stable isotope internal standard added, and then prepared for gas chromatography through a series of purifications by C-18 and Silica Sep-Pak cartridges and thin layer chromatography (TLC). Gas chromatography was performed using a 15 m long, 0.25 mm diameter, 0.25-µm film thickness, DB1701 fused silica capillary column (Fisons, Folsom, CA). The injector temperature was 265°C and oven (column) temperature was programmed from 200°C to 300°C at 15°C/min. Helium was used as the carrier gas at a flow rate of 1 ml/min. Ion source temperature was 250°C, electron energy was 70 eV, and filament current was 0.25 mA. For analysis, compounds were dissolved in undecane that was dried over a bed of calcium hydride. Negative ion chemical ionization mass spectrometry was performed using an Agilent Technologies G1789A GC/MSD instrument with a Hewlett–Packard computer system with ChemStation-NT. Total protein content was determined by BCA assay (Pierce, Rockford, IL) with bovine serum albumin as the standard (Smith et al., 1985 ).
Strains and maintenance.Caenorhabditis elegans strains were cultured on bacterial lawns of either NA-22 or OP-50, seeded on 8P or nematode growth medium (NGM) plates respectively, at 20°C according to standard methods (Brenner, 1974 ). Caenorhabditis elegans strain N2 (var. Bristol) is the wild-type strain, and was a gift of Dr Richard Nass (Vanderbilt University, Nashville, TN). The BY250 strain was developed and obtained from Dr Richard Nass (Vanderbilt University, Nashville, TN). Strain NW1229 (dpy-20(e1362) IV; evIs111) was obtained from the C. elegans Genetics Center (CGC, University of Minnesota, Minneapolis, MN).
Exposure of C. elegans to uranyl acetate.Embryos were obtained by hypochlorite treatment of gravid adults (Lewis and Fleming, 1995 ). After 17–24 h incubation in M9 buffer to obtain synchronized L1s, such that all nematodes are at the same point in their life cycle, the worms were washed once in 10 ml of dH2O, and then diluted to 50 worms per µl. L1 worms were treated with DU (uranyl acetate), prepared from a 1M stock solution in water. Five thousand worms were used in each siliconized microcentrifuge tube (Denver Scientific Inc., Metuchen, NJ) per treatment assay, and incubated with gentle shaking at 800 rpm for 30 min on a VWR Digital Mini Vortex Mixer (VWR Scientific, Suwanee, GA). Worms were then spread on NGM/OP-50 plates and incubated for 24 h at 20°C before further evaluation. For quantitative analyses of uranyl acetate-induced changes in worm viability, total number of live worms was determined for each concentration by counting each plate under a Stemi-2000 dissecting microscope (Zeiss, Thornwood, NY).
Photomicroscopy.Cell morphology was visually inspected on a Zeiss Axiovert 40 inverted microscope (Zeiss, Thornwood, NY). Cortical culture images were captured on an inverted Nomarski microscope (Zeiss Axiovert 200M) with AxioCam and AxioVision 4.4 software (Zeiss), using fixed exposures for all image captures between different treatments. Nematode images were captured on a Zeiss upright LSM510 confocal microscope (Zeiss), using laser scanning fluorescence and DIC (Nomarski) imaging. Worms were photographed under oil immersion with a 40x/1.30 Plan-Neofluar objective using fixed exposure settings for all image captures between different treatments. Images were exported using the Zeiss LSM Image Browser. Images were quantified for their fluorescence using Adobe Photoshop 6.0 (Adobe, San Jose, CA) and NIH ImageJ software. The fluorescent intensities were subsequently used to test if the levels of fluorescence were decreased upon treatment with U. With BY250 worms, cell bodies and dendrites were also manually scored as present if fluorescence could be seen. Dendrites were scored as abnormal if they had breaks or were barely visible. The ratio of abnormal:normal dendrites was used to calculate ratios for the different treatments, which were then compared for significance as previously described (Nass and Blakely, 2003 ).
Data analysis।All results are given as mean ± standard error of the mean. Differences between groups were analyzed statistically with one-way ANOVA followed by post hoc tests for multiple comparisons with p <>
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AN ADVANTAGE RESEARCH OF TUNGSTEN ALLOY & CANCER BIOLOGY
AN ADVANTAGE RESEARCH OF TUNGSTEN ALLOY & CANCER BIOLOGY
Neoplastic transformation of human osteoblast cells to the tumorigenic phenotype by heavy metal–tungsten alloy particles: induction of genotoxic effects
Alexandra C. Miller3, Steve Mog1, LuAnn McKinney1, Lei Luo2, Jennifer Allen, Jiaquan Xu and Natalie Page2
Applied Cellular Radiobiology Department and 1 Veterinary Sciences Department, Armed Forces Radiobiology Research Institute, Bethesda, MD 20889-5603 and 2 Molecular Oncology Branch, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
Abstract
Heavy metal–tungsten alloys (HMTAs) are dense heavy metal composite materials used primarily in military applications. HMTAs are composed of a mixture of tungsten (91–93%), nickel (3–5%) and either cobalt (2–4%) or iron (2–4%) particles. Like the heavy metal depleted uranium (DU), the use of HMTAs in military munitions could result in their internalization in humans. Limited data exist, however, regarding the long-term health effects of internalized HMTAs in humans. We used an immortalized, non-tumorigenic, human osteoblast-like cell line (HOS) to study the tumorigenic transforming potential of reconstituted mixtures of tungsten, nickel and cobalt (rWNiCo) and tungsten, nickel and iron (rWNiFe). We report the ability of rWNiCo and rWNiFe to transform immortalized HOS cells to the tumorigenic phenotype. These HMTA transformants are characterized by anchorage-independent growth, tumor formation in nude mice and high level expression of the K-ras oncogene. Cellular exposure to rWNiCo and rWNiFe resulted in 8.90 ± 0.93- and 9.50 ± 0.91-fold increases in transformation frequency, respectively, compared with the frequency in untreated cells. In comparison, an equivalent dose of crystalline NiS resulted in a 7.7 ± 0.73-fold increase in transformation frequency. The inert metal tantalum oxide did not enhance HOS transformation frequency above untreated levels. The mechanism by which rWNiCo and rWNiFe induce cell transformation in vitro appears to involve, at least partially, direct damage to the genetic material, manifested as increased DNA breakage or chromosomal aberrations (i.e. micronuclei). This is the first report showing that HMTA mixtures of W, Ni and Co or Fe cause human cell transformation to the neoplastic phenotype. While additional studies are needed to determine if protracted HMTA exposure produces tumors in vivo, the implication from these in vitro results is that the risk of cancer induction from internalized HMTAs exposure may be comparable with the risk from other biologically reactive and insoluble carcinogenic heavy metal compounds (e.g. nickel subsulfide and nickel oxide).
Abbreviations: ALP, alkaline phosphatase; DU, depleted uranium; HMTA, heavy metal–tungsten alloys; HOS, human osteosarcoma; MN, micronuclei; MN-CB, micronucleated cytokinesis-blocked; ROS, reactive oxygen species.
Introduction
Heavy metals such as depleted uranium (DU) and tungsten are used as kinetic energy penetrators in military applications. While the use of DU in these applications has been limited to the USA, heavy metal–tungsten alloy (HMTA) penetrators (tungsten/nickel/iron and tungsten/nickel/cobalt) are manufactured and have been tested in numerous countries and are deployed world wide. A friendly fire accident that occurred during the Gulf War, resulting in US soldiers with retained large DU fragments (~2–20 mm), has focused attention on the potential health effects of internalized heavy metals like tungsten and DU used in military applications.
Several recent studies have investigated the potential health effects of militarily relevant heavy metals (1–5). These investigations have not only demonstrated the transforming ability (1) and mutagenicity (2) of DU, but also its neurotoxicity (5). In contrast, there is no information regarding the health effects of imbedded HMTAs. Studies have shown that occupational exposure to hard metal dust, a mixture of cobalt- and tungsten carbide-containing particles, is associated with development of different pulmonary diseases, including fibrosing alveolitis and lung cancer (6,7). The toxic properties of hard metal particles are not only attributed to an interaction between cobalt metal and carbide particles (8), but also to the production of hydroxyl radicals, which have been implicated in their genotoxic effects (9). The HMTAs used in military applications, however, are somewhat different from conventional hard metal. HMTA penetrators consist of a combination of tungsten, nickel and either cobalt or iron (tungsten >90%, nickel 1–6%, iron 1–6% or cobalt ~1–6%), in contrast to hard metal dust, which is a mixture of cobalt metal (5–10%) and tungsten carbide particles (>80%) (10). The differences in metal composition and percentages of hard metal particles and tungsten alloys used in military applications preclude the assumption that the biological effects of hard metal particles and tungsten alloy particles would be the same.
There are no studies that address the potential health effects of internalized tungsten or HMTAs in terms of genotoxicity, mutagenicity or carcinogenicity. The long-term health risks associated with internal chronic exposure to HMTA particles are not defined but are crucial to developing carcinogenesis risk standards for personnel who could be injured by HMTA shrapnel. Therefore, in view of carcinogenesis risk estimates and medical management questions relevant to possible future incidents of tungsten internalization, an examination of molecular and cellular effects, including the potential transforming ability of tungsten and tungsten alloys, are necessary to understanding the potential carcinogenic effects of this material. The use of cell culture models to investigate potential or known carcinogens can provide important insights into the cellular and molecular mechanisms of carcinogenesis.
The in vitro transformation assay has not previously been used to study the transforming ability of HMTAs. This assay has been widely used in conjunction with metal salts to assess the potential carcinogenicity of metal compounds (e.g. DU, nickel, chromium and lead) (11–15). We have therefore chosen to use this assay to initiate an assessment of the potential carcinogenicity of HMTAs. The HOS TE85 cell line, an immortalized, non-tumorigenic, osteoblast-like cell line, has been successfully used to demonstrate the transformation of non-tumorigenic human cells to the tumorigenic phenotype by metals (1,13,14) and chemical carcinogens (161,17). Several metal powders were chosen for this study. A fine tungsten powder, pure crystalline nickel, iron and cobalt were used since they are the components of one of several possible tungsten alloys used in military applications. Crystalline NiS has previously been shown to transform cells in vitro (13) and was tested as a positive control. Tantalum oxide (Ta2O5) was also used for comparison since tantalum, which is widely used in prosthetic devices, is considered an inert metal with few reported toxic effects (5).
To understand the potential health effects of internalized HMTAs used in military applications, we compared the effects of various metals that compose these HMTAs on human cell survival, transformation and induction of DNA damage. Our data demonstrate, for the first time, that a mixture of the metals used in tungsten alloys can transform human cells to the tumorigenic phenotype, similar to results observed with some DU (1) and Ni compounds (1,12–16). HMTA transformants form anchorage-independent colonies, show aberrant growth rates and produce tumors in athymic mice.
Materials and methods
Cell lines and cultureHuman osteosarcoma (HOS) cells (TE85, clone F-5) were obtained from the American Type Culture Collection (Rockville, MD). Cell cultures were propagated in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 10% heat-inactivated fetal calf serum (Gibco Laboratories, Grand Island, NY), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma, St Louis, MO). Cells were tested for Mycoplasma with a MycoTect Kit (Sigma) and only cells negative for Mycoplasma were used.
Metal powdersThe aim of the study was to examine the transforming capability of two HMTAs (tungsten/nickel/cobalt or tungsten/nickel/iron) currently used in military munitions. The weight percentage compositions of these alloys when used for military applications is approximately 91–93% tungsten, 5–3% nickel and either 2–4% iron or 2–4% cobalt (10). Since the HMTAs used by the military are not commercially available, we used a mixture of these metals, in the same percentages used by the military, to model the particles of the alloys. In this study we therefore tested the effects of a pure mixture of these materials. The powders were obtained from Alfa Aesar (Ward Hill, MA). The following powders were used: (i) extrafine cobalt metal (Alfa Aesar 10455, 99.5% purity), median particles size (d50) 1–4 µm, called hereafter Co; (ii) extrafine nickel metal (Alfa Aesar 10256, 99% purity), d50 3–5 µm, called hereafter Ni; (iii) extrafine iron metal (Alfa Aesar 40337, 98% purity), d501–3 µm, called hereafter Fe; (iv) extrafine tungsten metal (Alfa Aesar 10400, 99.9% purity), d50 1–3 µm, called hereafter W; (v) a pure mixture of W (92%), Ni (5%) and Co (3%) particles made in the laboratory without extensive milling, called hereafter rWNiCo; (vi) a pure mixture of W (92%), Ni (5%) and Fe (3%) particles made in the laboratory without extensive milling, called hereafter rWNiFe.
Prior to each experiment the insoluble metal particles were washed once in sterile H2O and again in acetone. They were then suspended in acetone, agitated with a magnetic stirring bar and dispensed into cell cultures. The suspensions were carefully mixed and dispersed before being added to the cells. Dose–response experiments were conducted by altering the amounts of each metal powder, based upon its percentage of 100% of the total amount of powders. For example, 100 µg rWNiCo powder/ml consists of 92 µg W, 5 µg Ni and 3 µg Co, while 50 µg rWNiCo powder/ml consists of 46 µg W, 2.5 µg Ni and 1.5 µg Co. In this manner the total amount (weight) of metal powder mixture was varied while the ratios of the component metals were held constant.
Cellular survival assayCytotoxicity was assayed by measuring a reduction in plating efficiency. Exponentially growing cells were seeded at 104 cells/100 mm dish with three dishes per treatment group. Cultures were then treated 24 h later with 100 µl volumes of metal particles for 24 h. Cells were then rinsed with Dulbecco's phosphate-buffered saline. Cultures were detached with trypsin/EDTA and counted with a Coulter counter (Hialeah, FL). Appropriate numbers of cells (100 or 500) were then plated onto 60 mm diameter Petri dishes and cultures were returned to the incubator for 10 days to allow for colony formation. Cultures were then fixed with methanol and stained with 1% crystal violet. Plates with >15 colonies and colonies with >50 cells were counted as survivors.
Transformation and cell growth studiesFor transformation assays 104 cells/100 mm dish were seeded and exposed to metal particles 24 h later, with three flasks per experiment. Prior to each experiment the insoluble metal particles were washed once in sterile H2O and again in acetone. They were then suspended in acetone, agitated with a magnetic stirring bar and dispensed into cell cultures. Immediately after the 24 h metal particle exposure cells were rinsed (three times with sterile serum-free medium), trypsinized, counted and seeded in 100 mm tissue culture dishes at a density calculated to yield ~95–200 surviving cells per dish. The cultures were incubated for 5 weeks with weekly changes of nutrient medium. At the end of the incubation period cells were fixed, stained and examined for the appearance of transformed foci (18). Transformed foci were assayed using the criteria developed by Reznikoff et al. (18) and the modified scoring protocols were derived from Landolph (19) and the International Agency on Cancer Research/National Cancer Institute/Environmental Protection Agency (IARC/NCI/EPA) Working Group (20). The Working Group report indicated that: (i) because of the continuum of focus morphology, some foci can be intermediate (I/II or II/III) in character; (ii) these foci should be scored conservatively and assigned to the category of lower aggressive behavior. With HOS cells it was easy to distinguish between a type I and a type II focus, but somewhat difficult to differentiate between type II and type III foci. Therefore, in our experiments only type II and type II/type III foci were scored as transformants. In Table I the number of dishes with foci equals the number of foci of type II + type III. To determine the transformation frequency the Poisson formula is used, employing the first term of the Poisson distribution: Po = exp(–mN), m = –lnPo. Details of the transformation frequency calculations and the standard error have been described elsewhere (1,21–1,22) but are detailed below. Transformed foci are not contact inhibited and may be dislodged during refeeding, forming satellite colonies. To avoid this source of errors, the average number of transformed foci per dish ( ) was computed from the proportion of dishes free of transformed colonies, f (i.e. = –lnf). To determine the transformation frequency, the following formula was used:
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Table I. Neoplastic transformation of immortalized human osteoblast cells by reconstituted tungsten heavy alloy: dose dependence
transformation frequency = /no. surviving cells per dish.
This calculation has been widely used in radiation-induced neoplastic transformation studies to quantitatively compare transformation frequencies. The transformation data are presented from three independent experiments unless otherwise noted.
Cell survival fraction was determined using the conventional clonogenic assay (1,16). Cytotoxicity and survival assays were conducted in parallel with each transformation assay as described (1,16). Several transformed foci were picked with cloning cylinders and expanded by mass culture to establish transformed clonal lines. The transformation dose–response curve was determined as indicated above except that increasing concentrations of pure metal powders were used. The optimum dose level for transformation was selected following a preliminary toxicity test based upon colony-forming efficiency. The high dose selected resulted in >60% toxicity and the low dose selected resulted in minimal toxicity.
To determine saturation densities, cells were plated at 1x105 cells per 100 mm diameter plate in complete growth medium and monitored for growth as previously described (1,16). For the soft agar clonability assay cells were plated at a density of 2x103 cells per well in a 6-well plate sandwiched by 1 ml bottom agar (0.6%) and 1 ml top agar (0.3%). Cells were fed weekly by adding a new layer of top agar. Colonies >0.5 mm diameter were scored using a microscope after 2 weeks. The plating efficiency value for each clone represents the mean number of colonies scored from three wells. For both saturation density determination and plating efficiency in soft agar data are representative of three independent experiments. To determine DNA synthesis, incorporation of tritiated thymidine into DNA was measured as previously described (1).
Invasion through MatrigelThe ability of transformed cells to degrade and cross tissue barriers was assessed in an in vitro invasion assay that utilizes Matrigel, a reconstituted basement membrane (Collaborative Research, Waltham, MA). For qualitative evaluation of cell behavior 5x104 cells were plated onto 16 mm dishes (Costar, Cambridge, MA) which had been previously coated with 250 µl of Matrigel (10 mg/ml). The net-like formation characteristic of invasive cells occurred within 12 h; invasion into the Matrigel was evident after 4 days.
Alkaline phosphatase (ALP) activityThe percentage of cells exhibiting ALP activity on their cell surface was evaluated on cytospins obtained 4 days after cell seeding using a cytochemical method (Kit 86R; Sigma) (23). The percentage of cells was calculated on at least 350 cells. Cellular ALP activity was also analyzed 4 days after seeding. Approximately 106 cells were resuspended in homogenization buffer (1 mmol/l MgCl2, 1 mmol/l CaCl2, 20 mol/l ZnCl2, 0.1 mol/l NaCl, 0.1% Triton X-100, 0.05 mol/l Tris–HCl, pH 7.4) and disrupted by gentle vortexing. The homogenate was used for the ALP assay, which was performed with p-nitrophenol phosphate as substrate as per the Kit 86R instructions. L/B/K ALP activity was normalized for the content of protein in the sample. Protein was measured using the Bradford method (1,2). One unit of L/B/K ALP activity is defined as the amount of enzyme capable of transforming 1 µmol substrate in 1 min at 25°C.
Tumorigenicity assayExperiments were performed with 4–5 week old female athymic mice (Division of Cancer Treatment, NCI Animal Program, Frederick Cancer Research Facility, Frederick, MD). For this assay 5x106 cells in a 0.2 ml sterile suspension were injected s.c. in the right scapula. Animals were then observed for tumor growth at the sites of injection for 180 days. Tumor area was measured using a caliper measurement of two perpendicular diameters. When tumors were >100 mm2 the animal was killed.
Northern blot analysis and DNA probesCytoplasmic RNA was extracted from exponentially growing cells and separated by electrophoresis in 1% agarose–formaldehyde gels. RNA preparation and blotting onto nytran filters, hybridization with radiolabeled DNA probes and autoradiography were previously described (1,16). The ras probe, a SacI 2.9 kb fragment of the human ras gene, and a 1.8 kb fragment of the human ß-actin gene were obtained from Oncor (Gaithersburg, MD). 32P-labeled DNA probes were prepared using a random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN).
Micronuclei (MN) analysisThe induction of MN in control- and metal powder(s)-exposed cells was assessed using the conventional fluorescence plus Giemsa harlequin staining protocol (24). Following a 1 h exposure, the medium containing the metal powder was removed and cells were rinsed (three times with sterile serum-free medium) and incubated again at 37°C in complete medium. Mitomycin C was used as a positive control (24 h exposure). Cytochalasin B (6 µg/ml) was added after 24 h to block cytokinesis. At 48 h post-treatment cells were dropped onto slides using a cytospin (Shandon, St Louis, MO) for 5 min at 600 r.p.m. Slides were fixed with 5% Giemsa.
Alkaline elution testThe DNA breakage potencies of the metal powders were examined using the rapid alkaline elution test based on the method of Kohn et al. (25) and using the Millipore Multiscreen Assay System as described by Anard and co-workers (26). A 96-well filtration plate with a hydrophilic polyvinylidene fluoride microporous membrane (pore size 0.65 µm) sealed to its bottom was used along with a vacuum manifold to remove solutions from the filters. Cells were labeled for 18 h with 1 µCi/ml [3H]TTP (ICN, Costa Mesa, CA) prior to analysis of DNA damage by alkaline elution. Approximately 7x104 labeled cells were dispersed into each well of the filtration plate and exposed to the different powders suspended in complete medium. After 1 h exposure 1 mM sodium formate was added and the cells were lysed in a solution containing 0.04 M Na4EDTA, 2 M NaCl, 0.2% N-lauroylsarcosine, 0.5 mg/ml proteinase K and 1 M sodium formate, pH 9.0. The DNA remaining on the filters was washed with a solution containing 0.02 mM Na4EDTA. Elution buffer (0.1 M tetrapropylammonium hydroxide, 0.02 M EDTA, pH 12.1) was added in a volume of 300 µl and the DNA was eluted by vacuum (~16 kPa) in a single fraction. The DNA breakage potency of the different powders was assessed by quantifying the radioactivity recovered with the eluted fraction.
Histopathology of tumorsFor routine staining, tumor tissues were fixed in buffered 10% formalin, embedded in paraffin, sectioned and stained by routine hematoxylin and eosin methods (1,16).
StatisticsStatistics for the MN and alkaline elution assays were performed with the 2 test and the Tukey–Kramer multiple comparisons test, respectively.
ResultsCharacterization of the model system: heavy metal powder cytotoxicityAn assessment of metal powder exposure on cell growth and survival was done to initially characterize our HOS model system and to determine suitable amounts of metal powders to be used in the transformation studies. Cytotoxicity data were computed relative to control (untreated) cultures. Figure 1 compares the effect of increasing concentrations (0.75–200 µg powder/ml) of the various powders (W, Ni, Co, Fe or the reconstituted mixtures rWNiCo and rWNiFe) on HOS cell survival. Colony formation data demonstrated that cellular exposure to either the pure powders (W, Ni, Co or Fe) or the reconstituted powders (rWNiCo or rWNiFe) induced a dose-dependent decrease in cell survival (Figure 1 ). At concentrations up to 2.5 µg powder/ml Co, Ni and Fe were non-toxic, while W and the reconstituted tungsten powders were non-toxic at concentrations up to 46–50 µg powder/ml. The LD50, determined from a series of plots (survival percentage versus powder amount/ml; data not shown) was ~7–10 µg powder/ml for Co, Ni and Fe and 185–200 µg powder/ml for W, rWNiCo and rWNiFe.
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Fig. 1. Effect of various metals (tungsten, nickel, cobalt, iron, tungsten + nickel + cobalt mixture and tungsten + nickel + iron mixture) on HOS cell survival following a 24 h exposure. Data are means ± SD from three experiments.
The effect of rWNiCo and rWNiFe on cell proliferation, measured as tritiated thymidine incorporation into DNA, is shown in Figure 2 . Both rWNiCo and rWNiFe inhibited thymidine incorporation in HOS cells in the range 50–200 µg powder/ml in a dose-dependent manner. Experiments were conducted under optimal growth conditions, i.e. in the presence of 10% fetal calf serum. Growth rate analysis by cell enumeration (data not shown) demonstrated similar results.
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Fig. 2. Effect of the tungsten alloy mixtures (rWNiCo and rWNiFe) on DNA synthesis. HOS cells were incubated for 24 h with rWNiCo or rWNiFe (0–200 µg powder/ml). During the last 4 h of incubation [3H]thymidine was added to the medium. Each point corresponds to the mean of three estimations of acid-precipitable radioactivity at each amount of metal mixture powder, expressed as a percentage of the untreated control. Error bars denote SD. P < 0.001 relative to untreated control in Student's t-test.
To preclude excessive cell killing or proliferation effects due to heavy metal toxicity, the non-toxic, non-cytostatic concentration of both rWNiCo and rWNiFe chosen for the transformation experiment was 50 µg powder/ml (24 h exposure, surviving fraction 95.5 ± 5%). To establish transformation dose–response curves for these powders, two additional powder concentrations that manifest ~75 (100 µg powder/ml) and 35% (200 µg powder/ml) cell survival were also chosen.
Transformation of HOS cells by rWNiCo and rWNiFe: comparison with pure W, Ni, Co and FeTo assess morphological cell transformation, the standard focus formation assay previously described by others for both C3H/10T1/2 and HOS cells was used (1,11–19); we also previously used this assay to examine the effects of DU on HOS cells (1). Based on our toxicity and growth data we selected a non-toxic and non-cytostatic exposure of the two metal mixtures to examine their transforming potentials. The morphology of untreated and rWNiCo-treated HOS cells is shown in Figure 3 . HOS cells exhibit a flat epithelial-like morphology and appear to grow in a monolayer (Figure 3A ). In contrast, exposure to rWNiCo caused a morphological change in HOS cells (Figure 3B ). Following treatment with rWNiCo and weekly changes of nutrient medium for 5 weeks, diffused type II foci appeared (Figure 3B ). The morphology of the foci is distinctly different from the surrounding cells (Figure 3b ). The foci exhibit a slight multi-layered pattern, which is somewhat different from the `piled up' appearance seen in transformed C3H10T1/2 cells (1,16–18). Similar morphological changes were observed for cells exposed to rWNiFe (data not shown). The transforming potential of each pure metal was also tested. Table I shows measured values for the transformation frequencies (normalized per surviving cell) for HOS cells treated with W (46 µg/ml), Ni (2.5 µg/ml), Co (1.5 µg/ml), Fe (1.5 µg/ml), rWNiCo (50–200 µg powder/ml) and rWNiFe (50–200 µg powder/ml). The data demonstrate that treatment with rWNiCo and rWNiFe resulted in a transformation frequency of 37.6 ± 3.90x10–4 and 40.1 ± 3.85x10–4, respectively. These increased transformation frequencies represent 8.90 ± 0.93- and 9.50 ± 0.91-fold increases in transformation frequency, respectively, compared with the frequency in untreated HOS cells. In comparison with rWNICo and rWNiFe, cellular exposure to the pure powders W, Co and Fe (1.5–184 µg/ml) did not significantly increase the transformation frequency above untreated control levels (Table I ). Data are not shown for Co and Fe (3 and 6 µg/ml) and W (92 and 184 µg/ml). Incubation with pure nickel (2.5, 5, 10 µg/ml), however, did result in a small but statistically significant increase in transformation frequency (control, 4.21 ± 0.41x10–4; 2.5 µg/ml, 7.55 ± 0.75x10–4; 5 µg/ml, 9.11 ± 0.95 x10–4; 10 µg/ml, 10.55 ± 1.85x10–4). Data in Table I are only shown for the lowest amount of pure nickel. In comparison, cellular exposure to a higher dose of the known transforming agent crystalline NiS (50 µg/ml) increased the transformation frequency to 32.5 ± 3.10x10–4. Not only has Ni exposure been epidemiologically linked to cancer but the ability of Ni to transform cells in vitro was previously demonstrated by this laboratory and others and is shown here for comparison (1,12–14). In contrast, the biologically inert Ta2O5 did not induce an increase in HOS transformation frequency above untreated levels (Table I ). These data also demonstrated a metal dose-dependent increase in transformation frequency. Several rWNiCo- and rWNiFe-transformed foci were picked with cloning cylinders and expanded by mass culture to establish transformed clonal lines. A spontaneous focus arising from untreated HOS cells was also selected and expanded. These transformed clones were selected for growth analysis (invasiveness in Matrigel; saturation density; growth in agar), ALP activity and tumorigenicity.
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Fig. 3. Morphology of HOS cells and focus formation following rWNiCo exposure and growth of cells in matrigel. Phase contrast micrograph, magnification x40. (A) Morphology of control HOS cells. (B) Focus formation in HOS cells. The edge of a focus of transformed cells is seen against a background of parental HOS cells with normal morphology. (C) Growth of control HOS cells in matrigel. (D) Growth of rWNiCo transformants in matrigel.
The cellular potential for invasion through reconstituted basement membrane Matrigel was assessed using untreated HOS cells and metal transformants. A comparison of these cells is shown in Figure 3 for untransformed parental HOS cells (Figure 3C ) and a single rWNiCo transformant (Figure 3D ). The rWNiCo transformant grew in Matrigel developing characteristic net-like structures, eventually degrading the extracellular matrix components. In marked contrast, untransformed parental HOS cells formed small non-invasive colonies on top of the Matrigel. The spontaneously transformed HOS cells were not used in this assay.
Biological characterization of the transformed phenotypeAlteration in growth control is critical to neoplastic transformation and therefore the rWNiCo- and rWNiFe-transformed clones were further characterized by quantitative differences in growth properties associated with the neoplastic phenotype, e.g. saturation density and soft agar colony-forming efficiency. Additionally, ALP activity and tumorigenicity were assessed in untreated and transformed HOS cells.
The saturation densities of rWNiCo- and rWNIFe-transformed cells were approximately three times higher than that of the parental HOS cells (Table II ). Saturation density data obtained for cells treated with W, Ni, Co and Fe powder were similar to that for parental HOS cells. An assessment of anchorage-independent growth ability was also done. A comparison of the transformants ability to grow in soft agar revealed that the rWNiCo and rWNiFe transformants generated colonies within 1 week with colony-forming efficiencies of 34 and 47%, respectively. Parental HOS cells and cells treated with the pure metals did not form a significant number of colonies in soft agar (Table II ). We have previously shown that DU, N-methyl-N'-nitro-N-nitrosoguanidine and EJ-ras transformants formed soft agar colonies whose size and frequency were comparable with those observed with the rWNiCo and rWNiFe transformants (1,15).
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Table II. Biological and biochemical properties of HOS cells transformed by rWNiCo and rWNiFe: saturation density, plating efficiency in soft agar and alkaline phosphatase activity
Since ALP activity was recently shown to correlate with the malignant phenotype in transformed HOS cells (23), it was also evaluated in control, metal-treated and transformed HOS cells. Parental HOS cells and HOS cells treated with each of the pure metals showed positive staining for ALP in all of the cells and high levels of cellular ALP activity (Table II ). In contrast, rWNiCo and rWNiFe transformants exhibited a low percentage of cells positive for ALP activity and low cellular ALP activity.
Inoculation of athymic nude mice with rWNiCo and rWNiFe transformants resulted in the development of animal tumors at the site of injection within 4 weeks (Table III ). In contrast, parental HOS cells and cells treated with W, Ni, Co or Fe (46, 2.5, 1.5 and 1.5 µg powder/ml, respectively) injected into nude mice did not result in tumor formation during a period of 6 months after cell inoculation. Tumor tissue was excised and used for histochemical and immunohistochemical analyses. Some tumor tissue was also used to establish cell lines for further study.
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Table III. Tumorigenicity of HOS cells transformed by tungsten/nickel/cobalt
Histological examination of the tumors formed by rWNiCo revealed an infiltrative neoplasm consisting of cuboidal cells forming acini, papillary fronds and tubules (Figure 4 ). No neoplastic spindled cells were present. This diagnosis suggests adenocarcinoma, a malignant epithelial neoplasm. Immunohistochemistry for cytokeratin, an epithelial marker, and for vimentin, a mesenchymal marker, were `positive' and `negative', respectively (Figure 4 ). Microscopic examination of sections of these tumors revealed tumor tissue similar to that observed with tumors induced by neoplastic transformation of HOS cells by DU (1) and NiS (14). Tumors were re-established in tissue culture and confirmed as human; their resemblance to the cells of origin was determined by karyological analysis (not shown) (1,16). These re-established cells were also assayed for the formation of mineralized matrix. Transformed cells grown in matrix development medium showed weak diffused staining for alkaline phosphatase and Ca3(PO4)2. A similar pattern of growth and staining was observed for DU-transformed cells (1; Table II ). This diagnosis is similar to that previously observed with metal- and chemical-transformed HOS cells (1,3).
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Fig. 4. Histopathology of tumors growing in nude mice following injection of rWNiCo-transformed cells. H&E, original magnification x212.
ras analysis in HMTA transformantsNeoplastic cell transformation is hypothesized to result from a multi-step process involving activation of oncogenes and inactivation of tumor suppressor genes (27). Both metal- and radiation-induced neoplastic transformation has been shown to be associated with genetic alterations in specific oncogenes and tumor suppressor proteins, e.g. ras (1,12,16). Therefore, possible molecular changes in ras associated with HMTA-induced transformation were studied using rWNiCo- and rWNiFe-transformed cell lines. Northern blot analysis, shown in Figure 5 , revealed high levels of K-ras mRNA in each of the HMTA clones tested. In contrast, K-ras mRNA levels were undetectable in parental HOS cells. The level of EJ-ras mRNA in EJ-ras-transformed HOS cells is also shown for comparison. The amount of actin mRNA was similar in all clones tested (bottom).
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Fig. 5. Analysis of ras oncogene expression in parental HOS, rWNiCo-, rWNiFe- and EJ-ras-transformed clones. Data are representative of three separate experiments. Northern blot analysis of cytoplasmic RNA (20 µg): lane 1, HOS; lane 2, rWNiCo-transformed cells; lane 3, rWNiFe-transformed cells; lane 4, EJ-ras-transformed cells. (Upper) Hybridization with a 32P-labeled K-ras probe (ras transcripts were undetectable in HOS cells). A SacI 2.9 kb fragment of the human ras gene and a 1.8 kb fragment of the human ß-actin gene were used. (Lower) Hybridization with 32P-labeled actin was used to indicate that the relative amounts of RNA loaded into each lane were the same.
Micronucleus induction in HOS cells exposed to rWNiCo, rWNiFe, W, Ni, Co and FeThe MN test was used to assess the ability of the metal powders to induce chromosomal aberrations (chromosome/genome mutations) and hence their genotoxic potential. MN induction in HOS cells after treatment with the pure or reconstituted powders was analyzed at each concentration (Table IV ). Mitomycin C, a known inducer of MN, is shown for comparison. The frequencies of micronucleated cytokinesis-blocked (MN-CB) HOS cells treated for 1 h with metal particles and harvested after 48 h are presented in Table IV for each powder and reconstituted mixture. There was a statistically significant increase in MN-CB observed with each metal tested in comparison with untreated cells. For the reconstituted mixtures, rWNiCo and rWNiFe, a dose-dependent, statistically significant increase in MN-CB was observed.
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Table IV. Micronucleus induction in HOS cells exposed to Co, Fe, Ni, W or mixture rWNiCo or rWNiFe: comparison with mitomycin C
Induction of DNA breaks in HOS cells exposed to rWNiCo, rWNiFe, W, Ni, Co and FeThe induction of DNA breaks in HOS cells after exposure to the pure metals or the reconstituted mixtures was measured at each concentration using the alkaline elution assay (Figure 6 ). Pure Co, Ni and Fe particles induced a small but statistically significant dose-dependent increase in the production of DNA breaks. In contrast, W alone did not induce any significant DNA breakage. The DNA breakage potency of the individual metals was, however, significantly less than that of either rWNiCo or rWNiFe. At the highest dose tested cellular exposure to either rWNiCo or rWNiFe yielded an increase in DNA breaks of 850–900% above untreated levels. The induction of DNA breakage by the metal mixtures appeared to be synergistic since the sum of the breakage increase for all the pure metals was less than that for the reconstituted mixture.
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Fig. 6. DNA single-strand break analysis using alkaline elution of HOS cells exposed to various metals (tungsten, nickel, cobalt, iron, tungsten + nickel + cobalt mixture and tungsten + nickel + iron mixture) for 1 h. Results are the means ± SD from three determinations. The control (unexposed HOS cells) and the positive control (100 mM ethyl methyl sulfonate) are represented by three horizontal lines (mean ± SD). Error bars denote SD. P < 0.001 relative to untreated control in Student's t-test.
DiscussionThis study was undertaken to assess the transforming and genotoxic potential of two heavy metal–tungsten alloy metals used in military munitions. Since these tungsten alloys are not commercially available, we used a mixture of the pure metals that compose each heavy metal–tungsten alloy. These metal mixtures, which mimic the ones used in military applications, are composed of tungsten, nickel and cobalt or tungsten, nickel and iron.
Our studies demonstrate for the first time that the malignant transformation of immortalized human cells can be achieved by exposure to a mixture of W, Ni and Co or a mixture of W, Ni and Fe. Transformants with both metal mixtures showed morphological changes, anchorage-independent growth in soft agar, induced tumors when transplanted into nude mice and exhibited alterations in ras oncogene expression. Cellular exposure to these mixtures also induced DNA breakage and chromosomal aberrations, i.e. MN induction, indicating that these mixtures are genotoxic. This is the first report to demonstrate not only that a tungsten alloy mixture can transform human cells, but also that, based upon equivalent concentrations and metal toxicities, the magnitude of this transformation is ~1.3-fold greater than that observed for NiS, a well-known transforming agent and carcinogen (13). Furthermore, both rWNiCo and rWNiFe caused a dose-dependent increase in the transformation frequency of HOS cells. Since there are little data regarding the potential tumorigenic and genotoxic effects of internalized tungsten and tungsten alloys, these results are important to the understanding of the mechanism of the potential late health effects, i.e. carcinogenicity, of tungsten alloys used in military applications.
These studies also confirm previous results demonstrating that HOS cells can be used to assess quantitative transformation in addition to morphological and neoplastic transformation (1,13,14,16). Morphological transformation studies have primarily involved rodent cell lines such as C3H and 10T1/2 (11,15). As with our previous DU studies (1), our current results again demonstrate that human cell models are available not only for morphological but also for quantitative transformation studies. The current studies with tungsten alloy mixtures demonstrate another step in understanding the transformation process of HOS cells.
The precise mechanism(s) by which rWNiCo and rWNiFe induce transformation in HOS cells is not fully known, although the data suggest several possibilities. Alterations in specific oncogenes (e.g. ras) and/or inactivation of tumor suppressor genes (e.g. p53) involved in the conversion of these cells to the malignant phenotype have been considered. HOS cells contain a mutation at codon 156 of the p53 gene, resulting in a mutated form of p53 protein that is believed to be partially responsible for their immortalization (28). Since neoplastic conversion is postulated to result from a multi-step process involving cell immortalization and gene alterations (27,29–31), transformation of immortalized HOS cells by rWNiCo and rWNiFe to the malignant phenotype may involve other cellular oncogenes in this process. Our data demonstrate that the ras oncogene was activated in the transformation process induced by both rWNiCo and rWNiFe exposure. The ras oncogenes have been implicated in both chemical- and radiation-induced animal tumors (1,29–32) and in spontaneous human tumors (33). Studies also show that the c-myc protooncogene plays an important role in the nickel- and lead-induced transformation of mammalian cells, possibly by stabilizing protooncogene RNAs (34,35). Additionally, effects on tumor suppressor proteins have also been observed in metal-transformed clones. DU- (1) and Ni-induced (1,12) transformations of HOS cells were both shown to affect the encoded protein of the Rb tumor suppressor gene by altering phosphorylation of Rb protein in the transformed clones. Similarly, long-term exposure of human epithelial cells to nickel resulted in p53 gene point mutations (35). Considering these findings, we speculate that the transformation of human cells by tungsten alloy mixtures may result from a conversion process that definitely involves activation of tumor-promoting gene(s) and that may involve alterations in tumor suppressor proteins. This hypothesis awaits further testing.
Histological and histochemical analyses of tumors formed by both the rWNiCo- and rWNiFe-transformed cells indicated a gland-like pattern with random calcium deposition rather than an osteogenic sarcoma pattern of growth. Specifically, this histological tumor examination revealed an infiltrative neoplasm consisting of cuboidal cells forming acini, papillary fronds and tubules with no neoplastic spindled cells present, which is consistent with a diagnosis of adenocarcinoma. The finding of a malignant epithelial neoplasm rather than a sarcoma, a malignant mesenchymal neoplasm, was not expected. However, transformation of a spindled cell line, like HOS, with subsequent transplantation into nude mice may result in dedifferentiation at some point, resulting in growth of an epithelial neoplasm. A previous study of NiS-induced transformation of HOS cells reported the same unexpected histomorphology of neoplasms in recipient nude mice; that study also included supportive immunohistochemistry with positive cytokeratin and positive carcinoembryonic antigen staining, both epithelial markers (14). Furthermore, in our study untreated HOS cells formed ALP-positive foci which calcified on extended culture, while the HMTA-transformed cells lacked the ability to form multilayered cellular structures and ALP-positive foci. Changes in ALP activity have been shown to be associated with expression of the malignant phenotype in human osteosarcoma cells (23). Additionally, both rWNiCo- and rWNiFe-transformed cells deposited calcium randomly in areas showing clumped growth of cells. Taken together, these observations suggest that HMTA-transformed cells had dedifferentiated and lost some osteoblastic characteristics. The similarity of our results to previous studies of HOS transformation and tumorigenesis strongly suggests that HOS tumorigenic cells dedifferentiate in vivo (1,14).
As indicated previously, the tungsten alloy mixtures and the pure metals (except Ni) have not been tested for transforming potential. However, studies addressing hard metal lung disease examined the cytotoxic and cytogenetic effects of Co, WC and a WC + Co mixture (36,37). The results demonstrated that WC + Co had a greater cellular toxicity than pure Co metal particles (8,9) and that cellular uptake of Co was enhanced when it was present in the form of WC + Co. The increased toxicity did not result from the increased bioavailability of Co, however, and it appears that WC + Co behaved as a specific toxic entity (9,35–37). Similarly, our data also show that the tungsten alloy metal mixtures W/Ni/Co and W/Ni/Fe exhibit a greater toxicity than that of any of the pure metals. We have not yet determined whether there is any enhanced bioavailabilty of any of the pure metals in the mixture or whether this potentially increased bioavailability would affect the toxicity or transforming ability of the tungsten alloy mixtures. Ongoing studies on HMTA uptake and bioavailability may help to answer these questions.
The mechanism by which rWNiCo and rWNiFe induce cell transformation in vitro appears to involve, at least partially, direct damage to the genetic material, manifested as increased DNA breakage and chromosomal aberrations (i.e. MN). Our data clearly show that direct DNA damage and induction of chromosomal aberrations result from cellular exposure to the tungsten alloy mixtures. In contrast, only the highest doses of pure W, Ni and Co induced any significant increase in DNA breakage above background. Ni and Co were previously shown to be genotoxic at higher, toxic concentrations (38), so our data with Ni and Co at low, non-toxic doses are not too surprising. The HMTA mixtures demonstrate a synergistic increase in DNA breakage when the pure metals are mixed together to compose the tungsten alloy mixture. In contrast, MN induction by the HMTA mixtures, while significantly greater than the level of MN induction by each pure metal, did not exhibit a synergistic relationship. The MN test may be somewhat less sensitive in assessing the synergistic DNA-damaging potential of the mixture demonstrated by the DNA breakage assay. The MN assay does, however, detect chromosomal aberrations and not just repairable DNA breakage. Therefore, the combination of the DNA single-strand break and MN assays provides a better understanding of the mechanisms responsible for the genotoxic nature of the HMTA mixtures.
Other mechanisms may be involved by which rWNiCo and rWNiFe induce cell transformation in vitro. Studies have demonstrated that non-genotoxic, carcinogenic metals like lead may induce transformation via an indirect mechanism such as changes in DNA conformation or enzymatic disturbances (39). Other studies have also demonstrated that inhibition of DNA replication, leading to an elevation in sister chromatid exchanges, may be involved in metal-induced transformation (40,41). All of these mechanisms could potentially be involved in the HMTA transformation process, since transformation induced by metals like Ni appears to involve multiple mechanisms essential to the neoplastic process. For example, the involvement of epigenetic mechanisms of action has also been postulated for Ni. Chromatin condensation, de novo methylation and the resultant aberrant genetic activity may be partially responsible for the carcinogenic action of Ni (34,42). Similarly, recent unpublished data from our laboratory demonstrate that cellular exposure to rWNiCo resulted in hypermethylation of DNA (unpublished data). Although we do not have any direct evidence that neoplastic transformation by the HMTA mixtures involves epigenetic mechanisms, we cannot rule out their potential involvement in the transformation process.
Another possible mechanism in HMTA mixture transformation may involve reactive oxygen species (ROS). The formation of oxygen radicals in metal transformation is well documented, however, for some metals the contribution of these free radicals to the carcinogenic process is somewhat unclear (38). For Ni it is clear that ROS are involved. On a cellular level, Ni has been shown to increase protein oxidation and induce formation of cell oxidants (42) and the binding of Ni to peptides increases its accessibility to critical DNA sites. Animal studies have confirmed the role of Ni and Ni-mediated ROS in the mechanism of Ni carcinogenesis (43). Similarly, Fe potentiates oxygen toxicity via the Fenton reaction, producing hydroxyl radicals and inducing oxidative stress (44). Animal carcinogenesis studies provide evidence that high doses of Fe are also carcinogenic (45). In contrast, Co, which also generates substantial amounts of oxygen radicals, is not mutagenic or carcinogenic (46). When Co and WC particles are mixed together, however, hydroxyl radicals are generated that contribute to the mixture's induction of DNA damage (9,37). Epidemiological studies demonstrate that occupational exposure to hard metals may cause different types of lung diseases (6,7), including cancer (47). Based on these findings with other metals, it is interesting to speculate that another mechanism by which the HMTA mixtures induce transformation, chromosomal aberrations and DNA breakage may involve oxidative damage. This hypothesis awaits further testing.
In summary, we used a model system of in vitro human osteoblast cells exposed for 24 h to rWNiCo (50 µg powder/ml) and rWNiFe (50 µg powder/ml) to assess the relative transforming potential of HMTAs in an effort to better understand the potential health risks from long-term exposure to internalized HMTAs. Cellular exposure to HMTAs induces neoplastic transformation. These HMTA transformants are characterized by anchorage-independent growth, tumor formation in nude mice and expression of high levels of the K-ras oncogene. The mechanism by which rWNiCo and rWNiFe induce cell transformation in vitro appears to involve, at least partially, direct damage to the genetic material manifested as increased DNA breakage or chromosomal aberrations (i.e. MN). These HMTAs appear to have transforming ability somewhat greater than that of many other trace heavy metals which also induce neoplastic cell transformation in vitro as well as cause tumor formation in animals (42). While additional animal studies are needed to address the effect of protracted exposure and tumor induction in vivo, the implication from our model system study is that the risk of neoplastic induction from internalized HMTA exposure may be similar to other biologically reactive and carcinogenic heavy metal compounds (e.g. Ni).
NotesTo whom correspondence should be addressed at: AFRRI, Applied Cellular Radiobiology Department, 8901 Wisconsin Avenue, Bethesda, MD 20889-5603, USA
Acknowledgments
The authors are grateful for the helpful guidance and assistance of Drs David McClain and John Kalinich. We also thank Dr David Ledney for his suggestions. This research was supported in part by the Armed Forces Radiobiology Research Institute under work unit no. AFRRI-99-4. The views presented are those of the authors and do not necessarily reflect the official views of the Department of Defense or the US government.
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Neoplastic transformation of human osteoblast cells to the tumorigenic phenotype by heavy metal–tungsten alloy particles: induction of genotoxic effects
Alexandra C. Miller3, Steve Mog1, LuAnn McKinney1, Lei Luo2, Jennifer Allen, Jiaquan Xu and Natalie Page2
Applied Cellular Radiobiology Department and 1 Veterinary Sciences Department, Armed Forces Radiobiology Research Institute, Bethesda, MD 20889-5603 and 2 Molecular Oncology Branch, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
Abstract
Heavy metal–tungsten alloys (HMTAs) are dense heavy metal composite materials used primarily in military applications. HMTAs are composed of a mixture of tungsten (91–93%), nickel (3–5%) and either cobalt (2–4%) or iron (2–4%) particles. Like the heavy metal depleted uranium (DU), the use of HMTAs in military munitions could result in their internalization in humans. Limited data exist, however, regarding the long-term health effects of internalized HMTAs in humans. We used an immortalized, non-tumorigenic, human osteoblast-like cell line (HOS) to study the tumorigenic transforming potential of reconstituted mixtures of tungsten, nickel and cobalt (rWNiCo) and tungsten, nickel and iron (rWNiFe). We report the ability of rWNiCo and rWNiFe to transform immortalized HOS cells to the tumorigenic phenotype. These HMTA transformants are characterized by anchorage-independent growth, tumor formation in nude mice and high level expression of the K-ras oncogene. Cellular exposure to rWNiCo and rWNiFe resulted in 8.90 ± 0.93- and 9.50 ± 0.91-fold increases in transformation frequency, respectively, compared with the frequency in untreated cells. In comparison, an equivalent dose of crystalline NiS resulted in a 7.7 ± 0.73-fold increase in transformation frequency. The inert metal tantalum oxide did not enhance HOS transformation frequency above untreated levels. The mechanism by which rWNiCo and rWNiFe induce cell transformation in vitro appears to involve, at least partially, direct damage to the genetic material, manifested as increased DNA breakage or chromosomal aberrations (i.e. micronuclei). This is the first report showing that HMTA mixtures of W, Ni and Co or Fe cause human cell transformation to the neoplastic phenotype. While additional studies are needed to determine if protracted HMTA exposure produces tumors in vivo, the implication from these in vitro results is that the risk of cancer induction from internalized HMTAs exposure may be comparable with the risk from other biologically reactive and insoluble carcinogenic heavy metal compounds (e.g. nickel subsulfide and nickel oxide).
Abbreviations: ALP, alkaline phosphatase; DU, depleted uranium; HMTA, heavy metal–tungsten alloys; HOS, human osteosarcoma; MN, micronuclei; MN-CB, micronucleated cytokinesis-blocked; ROS, reactive oxygen species.
Introduction
Heavy metals such as depleted uranium (DU) and tungsten are used as kinetic energy penetrators in military applications. While the use of DU in these applications has been limited to the USA, heavy metal–tungsten alloy (HMTA) penetrators (tungsten/nickel/iron and tungsten/nickel/cobalt) are manufactured and have been tested in numerous countries and are deployed world wide. A friendly fire accident that occurred during the Gulf War, resulting in US soldiers with retained large DU fragments (~2–20 mm), has focused attention on the potential health effects of internalized heavy metals like tungsten and DU used in military applications.
Several recent studies have investigated the potential health effects of militarily relevant heavy metals (1–5). These investigations have not only demonstrated the transforming ability (1) and mutagenicity (2) of DU, but also its neurotoxicity (5). In contrast, there is no information regarding the health effects of imbedded HMTAs. Studies have shown that occupational exposure to hard metal dust, a mixture of cobalt- and tungsten carbide-containing particles, is associated with development of different pulmonary diseases, including fibrosing alveolitis and lung cancer (6,7). The toxic properties of hard metal particles are not only attributed to an interaction between cobalt metal and carbide particles (8), but also to the production of hydroxyl radicals, which have been implicated in their genotoxic effects (9). The HMTAs used in military applications, however, are somewhat different from conventional hard metal. HMTA penetrators consist of a combination of tungsten, nickel and either cobalt or iron (tungsten >90%, nickel 1–6%, iron 1–6% or cobalt ~1–6%), in contrast to hard metal dust, which is a mixture of cobalt metal (5–10%) and tungsten carbide particles (>80%) (10). The differences in metal composition and percentages of hard metal particles and tungsten alloys used in military applications preclude the assumption that the biological effects of hard metal particles and tungsten alloy particles would be the same.
There are no studies that address the potential health effects of internalized tungsten or HMTAs in terms of genotoxicity, mutagenicity or carcinogenicity. The long-term health risks associated with internal chronic exposure to HMTA particles are not defined but are crucial to developing carcinogenesis risk standards for personnel who could be injured by HMTA shrapnel. Therefore, in view of carcinogenesis risk estimates and medical management questions relevant to possible future incidents of tungsten internalization, an examination of molecular and cellular effects, including the potential transforming ability of tungsten and tungsten alloys, are necessary to understanding the potential carcinogenic effects of this material. The use of cell culture models to investigate potential or known carcinogens can provide important insights into the cellular and molecular mechanisms of carcinogenesis.
The in vitro transformation assay has not previously been used to study the transforming ability of HMTAs. This assay has been widely used in conjunction with metal salts to assess the potential carcinogenicity of metal compounds (e.g. DU, nickel, chromium and lead) (11–15). We have therefore chosen to use this assay to initiate an assessment of the potential carcinogenicity of HMTAs. The HOS TE85 cell line, an immortalized, non-tumorigenic, osteoblast-like cell line, has been successfully used to demonstrate the transformation of non-tumorigenic human cells to the tumorigenic phenotype by metals (1,13,14) and chemical carcinogens (161,17). Several metal powders were chosen for this study. A fine tungsten powder, pure crystalline nickel, iron and cobalt were used since they are the components of one of several possible tungsten alloys used in military applications. Crystalline NiS has previously been shown to transform cells in vitro (13) and was tested as a positive control. Tantalum oxide (Ta2O5) was also used for comparison since tantalum, which is widely used in prosthetic devices, is considered an inert metal with few reported toxic effects (5).
To understand the potential health effects of internalized HMTAs used in military applications, we compared the effects of various metals that compose these HMTAs on human cell survival, transformation and induction of DNA damage. Our data demonstrate, for the first time, that a mixture of the metals used in tungsten alloys can transform human cells to the tumorigenic phenotype, similar to results observed with some DU (1) and Ni compounds (1,12–16). HMTA transformants form anchorage-independent colonies, show aberrant growth rates and produce tumors in athymic mice.
Materials and methods
Cell lines and cultureHuman osteosarcoma (HOS) cells (TE85, clone F-5) were obtained from the American Type Culture Collection (Rockville, MD). Cell cultures were propagated in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 10% heat-inactivated fetal calf serum (Gibco Laboratories, Grand Island, NY), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma, St Louis, MO). Cells were tested for Mycoplasma with a MycoTect Kit (Sigma) and only cells negative for Mycoplasma were used.
Metal powdersThe aim of the study was to examine the transforming capability of two HMTAs (tungsten/nickel/cobalt or tungsten/nickel/iron) currently used in military munitions. The weight percentage compositions of these alloys when used for military applications is approximately 91–93% tungsten, 5–3% nickel and either 2–4% iron or 2–4% cobalt (10). Since the HMTAs used by the military are not commercially available, we used a mixture of these metals, in the same percentages used by the military, to model the particles of the alloys. In this study we therefore tested the effects of a pure mixture of these materials. The powders were obtained from Alfa Aesar (Ward Hill, MA). The following powders were used: (i) extrafine cobalt metal (Alfa Aesar 10455, 99.5% purity), median particles size (d50) 1–4 µm, called hereafter Co; (ii) extrafine nickel metal (Alfa Aesar 10256, 99% purity), d50 3–5 µm, called hereafter Ni; (iii) extrafine iron metal (Alfa Aesar 40337, 98% purity), d501–3 µm, called hereafter Fe; (iv) extrafine tungsten metal (Alfa Aesar 10400, 99.9% purity), d50 1–3 µm, called hereafter W; (v) a pure mixture of W (92%), Ni (5%) and Co (3%) particles made in the laboratory without extensive milling, called hereafter rWNiCo; (vi) a pure mixture of W (92%), Ni (5%) and Fe (3%) particles made in the laboratory without extensive milling, called hereafter rWNiFe.
Prior to each experiment the insoluble metal particles were washed once in sterile H2O and again in acetone. They were then suspended in acetone, agitated with a magnetic stirring bar and dispensed into cell cultures. The suspensions were carefully mixed and dispersed before being added to the cells. Dose–response experiments were conducted by altering the amounts of each metal powder, based upon its percentage of 100% of the total amount of powders. For example, 100 µg rWNiCo powder/ml consists of 92 µg W, 5 µg Ni and 3 µg Co, while 50 µg rWNiCo powder/ml consists of 46 µg W, 2.5 µg Ni and 1.5 µg Co. In this manner the total amount (weight) of metal powder mixture was varied while the ratios of the component metals were held constant.
Cellular survival assayCytotoxicity was assayed by measuring a reduction in plating efficiency. Exponentially growing cells were seeded at 104 cells/100 mm dish with three dishes per treatment group. Cultures were then treated 24 h later with 100 µl volumes of metal particles for 24 h. Cells were then rinsed with Dulbecco's phosphate-buffered saline. Cultures were detached with trypsin/EDTA and counted with a Coulter counter (Hialeah, FL). Appropriate numbers of cells (100 or 500) were then plated onto 60 mm diameter Petri dishes and cultures were returned to the incubator for 10 days to allow for colony formation. Cultures were then fixed with methanol and stained with 1% crystal violet. Plates with >15 colonies and colonies with >50 cells were counted as survivors.
Transformation and cell growth studiesFor transformation assays 104 cells/100 mm dish were seeded and exposed to metal particles 24 h later, with three flasks per experiment. Prior to each experiment the insoluble metal particles were washed once in sterile H2O and again in acetone. They were then suspended in acetone, agitated with a magnetic stirring bar and dispensed into cell cultures. Immediately after the 24 h metal particle exposure cells were rinsed (three times with sterile serum-free medium), trypsinized, counted and seeded in 100 mm tissue culture dishes at a density calculated to yield ~95–200 surviving cells per dish. The cultures were incubated for 5 weeks with weekly changes of nutrient medium. At the end of the incubation period cells were fixed, stained and examined for the appearance of transformed foci (18). Transformed foci were assayed using the criteria developed by Reznikoff et al. (18) and the modified scoring protocols were derived from Landolph (19) and the International Agency on Cancer Research/National Cancer Institute/Environmental Protection Agency (IARC/NCI/EPA) Working Group (20). The Working Group report indicated that: (i) because of the continuum of focus morphology, some foci can be intermediate (I/II or II/III) in character; (ii) these foci should be scored conservatively and assigned to the category of lower aggressive behavior. With HOS cells it was easy to distinguish between a type I and a type II focus, but somewhat difficult to differentiate between type II and type III foci. Therefore, in our experiments only type II and type II/type III foci were scored as transformants. In Table I the number of dishes with foci equals the number of foci of type II + type III. To determine the transformation frequency the Poisson formula is used, employing the first term of the Poisson distribution: Po = exp(–mN), m = –lnPo. Details of the transformation frequency calculations and the standard error have been described elsewhere (1,21–1,22) but are detailed below. Transformed foci are not contact inhibited and may be dislodged during refeeding, forming satellite colonies. To avoid this source of errors, the average number of transformed foci per dish ( ) was computed from the proportion of dishes free of transformed colonies, f (i.e. = –lnf). To determine the transformation frequency, the following formula was used:
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Table I. Neoplastic transformation of immortalized human osteoblast cells by reconstituted tungsten heavy alloy: dose dependence
transformation frequency = /no. surviving cells per dish.
This calculation has been widely used in radiation-induced neoplastic transformation studies to quantitatively compare transformation frequencies. The transformation data are presented from three independent experiments unless otherwise noted.
Cell survival fraction was determined using the conventional clonogenic assay (1,16). Cytotoxicity and survival assays were conducted in parallel with each transformation assay as described (1,16). Several transformed foci were picked with cloning cylinders and expanded by mass culture to establish transformed clonal lines. The transformation dose–response curve was determined as indicated above except that increasing concentrations of pure metal powders were used. The optimum dose level for transformation was selected following a preliminary toxicity test based upon colony-forming efficiency. The high dose selected resulted in >60% toxicity and the low dose selected resulted in minimal toxicity.
To determine saturation densities, cells were plated at 1x105 cells per 100 mm diameter plate in complete growth medium and monitored for growth as previously described (1,16). For the soft agar clonability assay cells were plated at a density of 2x103 cells per well in a 6-well plate sandwiched by 1 ml bottom agar (0.6%) and 1 ml top agar (0.3%). Cells were fed weekly by adding a new layer of top agar. Colonies >0.5 mm diameter were scored using a microscope after 2 weeks. The plating efficiency value for each clone represents the mean number of colonies scored from three wells. For both saturation density determination and plating efficiency in soft agar data are representative of three independent experiments. To determine DNA synthesis, incorporation of tritiated thymidine into DNA was measured as previously described (1).
Invasion through MatrigelThe ability of transformed cells to degrade and cross tissue barriers was assessed in an in vitro invasion assay that utilizes Matrigel, a reconstituted basement membrane (Collaborative Research, Waltham, MA). For qualitative evaluation of cell behavior 5x104 cells were plated onto 16 mm dishes (Costar, Cambridge, MA) which had been previously coated with 250 µl of Matrigel (10 mg/ml). The net-like formation characteristic of invasive cells occurred within 12 h; invasion into the Matrigel was evident after 4 days.
Alkaline phosphatase (ALP) activityThe percentage of cells exhibiting ALP activity on their cell surface was evaluated on cytospins obtained 4 days after cell seeding using a cytochemical method (Kit 86R; Sigma) (23). The percentage of cells was calculated on at least 350 cells. Cellular ALP activity was also analyzed 4 days after seeding. Approximately 106 cells were resuspended in homogenization buffer (1 mmol/l MgCl2, 1 mmol/l CaCl2, 20 mol/l ZnCl2, 0.1 mol/l NaCl, 0.1% Triton X-100, 0.05 mol/l Tris–HCl, pH 7.4) and disrupted by gentle vortexing. The homogenate was used for the ALP assay, which was performed with p-nitrophenol phosphate as substrate as per the Kit 86R instructions. L/B/K ALP activity was normalized for the content of protein in the sample. Protein was measured using the Bradford method (1,2). One unit of L/B/K ALP activity is defined as the amount of enzyme capable of transforming 1 µmol substrate in 1 min at 25°C.
Tumorigenicity assayExperiments were performed with 4–5 week old female athymic mice (Division of Cancer Treatment, NCI Animal Program, Frederick Cancer Research Facility, Frederick, MD). For this assay 5x106 cells in a 0.2 ml sterile suspension were injected s.c. in the right scapula. Animals were then observed for tumor growth at the sites of injection for 180 days. Tumor area was measured using a caliper measurement of two perpendicular diameters. When tumors were >100 mm2 the animal was killed.
Northern blot analysis and DNA probesCytoplasmic RNA was extracted from exponentially growing cells and separated by electrophoresis in 1% agarose–formaldehyde gels. RNA preparation and blotting onto nytran filters, hybridization with radiolabeled DNA probes and autoradiography were previously described (1,16). The ras probe, a SacI 2.9 kb fragment of the human ras gene, and a 1.8 kb fragment of the human ß-actin gene were obtained from Oncor (Gaithersburg, MD). 32P-labeled DNA probes were prepared using a random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN).
Micronuclei (MN) analysisThe induction of MN in control- and metal powder(s)-exposed cells was assessed using the conventional fluorescence plus Giemsa harlequin staining protocol (24). Following a 1 h exposure, the medium containing the metal powder was removed and cells were rinsed (three times with sterile serum-free medium) and incubated again at 37°C in complete medium. Mitomycin C was used as a positive control (24 h exposure). Cytochalasin B (6 µg/ml) was added after 24 h to block cytokinesis. At 48 h post-treatment cells were dropped onto slides using a cytospin (Shandon, St Louis, MO) for 5 min at 600 r.p.m. Slides were fixed with 5% Giemsa.
Alkaline elution testThe DNA breakage potencies of the metal powders were examined using the rapid alkaline elution test based on the method of Kohn et al. (25) and using the Millipore Multiscreen Assay System as described by Anard and co-workers (26). A 96-well filtration plate with a hydrophilic polyvinylidene fluoride microporous membrane (pore size 0.65 µm) sealed to its bottom was used along with a vacuum manifold to remove solutions from the filters. Cells were labeled for 18 h with 1 µCi/ml [3H]TTP (ICN, Costa Mesa, CA) prior to analysis of DNA damage by alkaline elution. Approximately 7x104 labeled cells were dispersed into each well of the filtration plate and exposed to the different powders suspended in complete medium. After 1 h exposure 1 mM sodium formate was added and the cells were lysed in a solution containing 0.04 M Na4EDTA, 2 M NaCl, 0.2% N-lauroylsarcosine, 0.5 mg/ml proteinase K and 1 M sodium formate, pH 9.0. The DNA remaining on the filters was washed with a solution containing 0.02 mM Na4EDTA. Elution buffer (0.1 M tetrapropylammonium hydroxide, 0.02 M EDTA, pH 12.1) was added in a volume of 300 µl and the DNA was eluted by vacuum (~16 kPa) in a single fraction. The DNA breakage potency of the different powders was assessed by quantifying the radioactivity recovered with the eluted fraction.
Histopathology of tumorsFor routine staining, tumor tissues were fixed in buffered 10% formalin, embedded in paraffin, sectioned and stained by routine hematoxylin and eosin methods (1,16).
StatisticsStatistics for the MN and alkaline elution assays were performed with the 2 test and the Tukey–Kramer multiple comparisons test, respectively.
ResultsCharacterization of the model system: heavy metal powder cytotoxicityAn assessment of metal powder exposure on cell growth and survival was done to initially characterize our HOS model system and to determine suitable amounts of metal powders to be used in the transformation studies. Cytotoxicity data were computed relative to control (untreated) cultures. Figure 1 compares the effect of increasing concentrations (0.75–200 µg powder/ml) of the various powders (W, Ni, Co, Fe or the reconstituted mixtures rWNiCo and rWNiFe) on HOS cell survival. Colony formation data demonstrated that cellular exposure to either the pure powders (W, Ni, Co or Fe) or the reconstituted powders (rWNiCo or rWNiFe) induced a dose-dependent decrease in cell survival (Figure 1 ). At concentrations up to 2.5 µg powder/ml Co, Ni and Fe were non-toxic, while W and the reconstituted tungsten powders were non-toxic at concentrations up to 46–50 µg powder/ml. The LD50, determined from a series of plots (survival percentage versus powder amount/ml; data not shown) was ~7–10 µg powder/ml for Co, Ni and Fe and 185–200 µg powder/ml for W, rWNiCo and rWNiFe.
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Fig. 1. Effect of various metals (tungsten, nickel, cobalt, iron, tungsten + nickel + cobalt mixture and tungsten + nickel + iron mixture) on HOS cell survival following a 24 h exposure. Data are means ± SD from three experiments.
The effect of rWNiCo and rWNiFe on cell proliferation, measured as tritiated thymidine incorporation into DNA, is shown in Figure 2 . Both rWNiCo and rWNiFe inhibited thymidine incorporation in HOS cells in the range 50–200 µg powder/ml in a dose-dependent manner. Experiments were conducted under optimal growth conditions, i.e. in the presence of 10% fetal calf serum. Growth rate analysis by cell enumeration (data not shown) demonstrated similar results.
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Fig. 2. Effect of the tungsten alloy mixtures (rWNiCo and rWNiFe) on DNA synthesis. HOS cells were incubated for 24 h with rWNiCo or rWNiFe (0–200 µg powder/ml). During the last 4 h of incubation [3H]thymidine was added to the medium. Each point corresponds to the mean of three estimations of acid-precipitable radioactivity at each amount of metal mixture powder, expressed as a percentage of the untreated control. Error bars denote SD. P < 0.001 relative to untreated control in Student's t-test.
To preclude excessive cell killing or proliferation effects due to heavy metal toxicity, the non-toxic, non-cytostatic concentration of both rWNiCo and rWNiFe chosen for the transformation experiment was 50 µg powder/ml (24 h exposure, surviving fraction 95.5 ± 5%). To establish transformation dose–response curves for these powders, two additional powder concentrations that manifest ~75 (100 µg powder/ml) and 35% (200 µg powder/ml) cell survival were also chosen.
Transformation of HOS cells by rWNiCo and rWNiFe: comparison with pure W, Ni, Co and FeTo assess morphological cell transformation, the standard focus formation assay previously described by others for both C3H/10T1/2 and HOS cells was used (1,11–19); we also previously used this assay to examine the effects of DU on HOS cells (1). Based on our toxicity and growth data we selected a non-toxic and non-cytostatic exposure of the two metal mixtures to examine their transforming potentials. The morphology of untreated and rWNiCo-treated HOS cells is shown in Figure 3 . HOS cells exhibit a flat epithelial-like morphology and appear to grow in a monolayer (Figure 3A ). In contrast, exposure to rWNiCo caused a morphological change in HOS cells (Figure 3B ). Following treatment with rWNiCo and weekly changes of nutrient medium for 5 weeks, diffused type II foci appeared (Figure 3B ). The morphology of the foci is distinctly different from the surrounding cells (Figure 3b ). The foci exhibit a slight multi-layered pattern, which is somewhat different from the `piled up' appearance seen in transformed C3H10T1/2 cells (1,16–18). Similar morphological changes were observed for cells exposed to rWNiFe (data not shown). The transforming potential of each pure metal was also tested. Table I shows measured values for the transformation frequencies (normalized per surviving cell) for HOS cells treated with W (46 µg/ml), Ni (2.5 µg/ml), Co (1.5 µg/ml), Fe (1.5 µg/ml), rWNiCo (50–200 µg powder/ml) and rWNiFe (50–200 µg powder/ml). The data demonstrate that treatment with rWNiCo and rWNiFe resulted in a transformation frequency of 37.6 ± 3.90x10–4 and 40.1 ± 3.85x10–4, respectively. These increased transformation frequencies represent 8.90 ± 0.93- and 9.50 ± 0.91-fold increases in transformation frequency, respectively, compared with the frequency in untreated HOS cells. In comparison with rWNICo and rWNiFe, cellular exposure to the pure powders W, Co and Fe (1.5–184 µg/ml) did not significantly increase the transformation frequency above untreated control levels (Table I ). Data are not shown for Co and Fe (3 and 6 µg/ml) and W (92 and 184 µg/ml). Incubation with pure nickel (2.5, 5, 10 µg/ml), however, did result in a small but statistically significant increase in transformation frequency (control, 4.21 ± 0.41x10–4; 2.5 µg/ml, 7.55 ± 0.75x10–4; 5 µg/ml, 9.11 ± 0.95 x10–4; 10 µg/ml, 10.55 ± 1.85x10–4). Data in Table I are only shown for the lowest amount of pure nickel. In comparison, cellular exposure to a higher dose of the known transforming agent crystalline NiS (50 µg/ml) increased the transformation frequency to 32.5 ± 3.10x10–4. Not only has Ni exposure been epidemiologically linked to cancer but the ability of Ni to transform cells in vitro was previously demonstrated by this laboratory and others and is shown here for comparison (1,12–14). In contrast, the biologically inert Ta2O5 did not induce an increase in HOS transformation frequency above untreated levels (Table I ). These data also demonstrated a metal dose-dependent increase in transformation frequency. Several rWNiCo- and rWNiFe-transformed foci were picked with cloning cylinders and expanded by mass culture to establish transformed clonal lines. A spontaneous focus arising from untreated HOS cells was also selected and expanded. These transformed clones were selected for growth analysis (invasiveness in Matrigel; saturation density; growth in agar), ALP activity and tumorigenicity.
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Fig. 3. Morphology of HOS cells and focus formation following rWNiCo exposure and growth of cells in matrigel. Phase contrast micrograph, magnification x40. (A) Morphology of control HOS cells. (B) Focus formation in HOS cells. The edge of a focus of transformed cells is seen against a background of parental HOS cells with normal morphology. (C) Growth of control HOS cells in matrigel. (D) Growth of rWNiCo transformants in matrigel.
The cellular potential for invasion through reconstituted basement membrane Matrigel was assessed using untreated HOS cells and metal transformants. A comparison of these cells is shown in Figure 3 for untransformed parental HOS cells (Figure 3C ) and a single rWNiCo transformant (Figure 3D ). The rWNiCo transformant grew in Matrigel developing characteristic net-like structures, eventually degrading the extracellular matrix components. In marked contrast, untransformed parental HOS cells formed small non-invasive colonies on top of the Matrigel. The spontaneously transformed HOS cells were not used in this assay.
Biological characterization of the transformed phenotypeAlteration in growth control is critical to neoplastic transformation and therefore the rWNiCo- and rWNiFe-transformed clones were further characterized by quantitative differences in growth properties associated with the neoplastic phenotype, e.g. saturation density and soft agar colony-forming efficiency. Additionally, ALP activity and tumorigenicity were assessed in untreated and transformed HOS cells.
The saturation densities of rWNiCo- and rWNIFe-transformed cells were approximately three times higher than that of the parental HOS cells (Table II ). Saturation density data obtained for cells treated with W, Ni, Co and Fe powder were similar to that for parental HOS cells. An assessment of anchorage-independent growth ability was also done. A comparison of the transformants ability to grow in soft agar revealed that the rWNiCo and rWNiFe transformants generated colonies within 1 week with colony-forming efficiencies of 34 and 47%, respectively. Parental HOS cells and cells treated with the pure metals did not form a significant number of colonies in soft agar (Table II ). We have previously shown that DU, N-methyl-N'-nitro-N-nitrosoguanidine and EJ-ras transformants formed soft agar colonies whose size and frequency were comparable with those observed with the rWNiCo and rWNiFe transformants (1,15).
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Table II. Biological and biochemical properties of HOS cells transformed by rWNiCo and rWNiFe: saturation density, plating efficiency in soft agar and alkaline phosphatase activity
Since ALP activity was recently shown to correlate with the malignant phenotype in transformed HOS cells (23), it was also evaluated in control, metal-treated and transformed HOS cells. Parental HOS cells and HOS cells treated with each of the pure metals showed positive staining for ALP in all of the cells and high levels of cellular ALP activity (Table II ). In contrast, rWNiCo and rWNiFe transformants exhibited a low percentage of cells positive for ALP activity and low cellular ALP activity.
Inoculation of athymic nude mice with rWNiCo and rWNiFe transformants resulted in the development of animal tumors at the site of injection within 4 weeks (Table III ). In contrast, parental HOS cells and cells treated with W, Ni, Co or Fe (46, 2.5, 1.5 and 1.5 µg powder/ml, respectively) injected into nude mice did not result in tumor formation during a period of 6 months after cell inoculation. Tumor tissue was excised and used for histochemical and immunohistochemical analyses. Some tumor tissue was also used to establish cell lines for further study.
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Table III. Tumorigenicity of HOS cells transformed by tungsten/nickel/cobalt
Histological examination of the tumors formed by rWNiCo revealed an infiltrative neoplasm consisting of cuboidal cells forming acini, papillary fronds and tubules (Figure 4 ). No neoplastic spindled cells were present. This diagnosis suggests adenocarcinoma, a malignant epithelial neoplasm. Immunohistochemistry for cytokeratin, an epithelial marker, and for vimentin, a mesenchymal marker, were `positive' and `negative', respectively (Figure 4 ). Microscopic examination of sections of these tumors revealed tumor tissue similar to that observed with tumors induced by neoplastic transformation of HOS cells by DU (1) and NiS (14). Tumors were re-established in tissue culture and confirmed as human; their resemblance to the cells of origin was determined by karyological analysis (not shown) (1,16). These re-established cells were also assayed for the formation of mineralized matrix. Transformed cells grown in matrix development medium showed weak diffused staining for alkaline phosphatase and Ca3(PO4)2. A similar pattern of growth and staining was observed for DU-transformed cells (1; Table II ). This diagnosis is similar to that previously observed with metal- and chemical-transformed HOS cells (1,3).
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Fig. 4. Histopathology of tumors growing in nude mice following injection of rWNiCo-transformed cells. H&E, original magnification x212.
ras analysis in HMTA transformantsNeoplastic cell transformation is hypothesized to result from a multi-step process involving activation of oncogenes and inactivation of tumor suppressor genes (27). Both metal- and radiation-induced neoplastic transformation has been shown to be associated with genetic alterations in specific oncogenes and tumor suppressor proteins, e.g. ras (1,12,16). Therefore, possible molecular changes in ras associated with HMTA-induced transformation were studied using rWNiCo- and rWNiFe-transformed cell lines. Northern blot analysis, shown in Figure 5 , revealed high levels of K-ras mRNA in each of the HMTA clones tested. In contrast, K-ras mRNA levels were undetectable in parental HOS cells. The level of EJ-ras mRNA in EJ-ras-transformed HOS cells is also shown for comparison. The amount of actin mRNA was similar in all clones tested (bottom).
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Fig. 5. Analysis of ras oncogene expression in parental HOS, rWNiCo-, rWNiFe- and EJ-ras-transformed clones. Data are representative of three separate experiments. Northern blot analysis of cytoplasmic RNA (20 µg): lane 1, HOS; lane 2, rWNiCo-transformed cells; lane 3, rWNiFe-transformed cells; lane 4, EJ-ras-transformed cells. (Upper) Hybridization with a 32P-labeled K-ras probe (ras transcripts were undetectable in HOS cells). A SacI 2.9 kb fragment of the human ras gene and a 1.8 kb fragment of the human ß-actin gene were used. (Lower) Hybridization with 32P-labeled actin was used to indicate that the relative amounts of RNA loaded into each lane were the same.
Micronucleus induction in HOS cells exposed to rWNiCo, rWNiFe, W, Ni, Co and FeThe MN test was used to assess the ability of the metal powders to induce chromosomal aberrations (chromosome/genome mutations) and hence their genotoxic potential. MN induction in HOS cells after treatment with the pure or reconstituted powders was analyzed at each concentration (Table IV ). Mitomycin C, a known inducer of MN, is shown for comparison. The frequencies of micronucleated cytokinesis-blocked (MN-CB) HOS cells treated for 1 h with metal particles and harvested after 48 h are presented in Table IV for each powder and reconstituted mixture. There was a statistically significant increase in MN-CB observed with each metal tested in comparison with untreated cells. For the reconstituted mixtures, rWNiCo and rWNiFe, a dose-dependent, statistically significant increase in MN-CB was observed.
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Table IV. Micronucleus induction in HOS cells exposed to Co, Fe, Ni, W or mixture rWNiCo or rWNiFe: comparison with mitomycin C
Induction of DNA breaks in HOS cells exposed to rWNiCo, rWNiFe, W, Ni, Co and FeThe induction of DNA breaks in HOS cells after exposure to the pure metals or the reconstituted mixtures was measured at each concentration using the alkaline elution assay (Figure 6 ). Pure Co, Ni and Fe particles induced a small but statistically significant dose-dependent increase in the production of DNA breaks. In contrast, W alone did not induce any significant DNA breakage. The DNA breakage potency of the individual metals was, however, significantly less than that of either rWNiCo or rWNiFe. At the highest dose tested cellular exposure to either rWNiCo or rWNiFe yielded an increase in DNA breaks of 850–900% above untreated levels. The induction of DNA breakage by the metal mixtures appeared to be synergistic since the sum of the breakage increase for all the pure metals was less than that for the reconstituted mixture.
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Fig. 6. DNA single-strand break analysis using alkaline elution of HOS cells exposed to various metals (tungsten, nickel, cobalt, iron, tungsten + nickel + cobalt mixture and tungsten + nickel + iron mixture) for 1 h. Results are the means ± SD from three determinations. The control (unexposed HOS cells) and the positive control (100 mM ethyl methyl sulfonate) are represented by three horizontal lines (mean ± SD). Error bars denote SD. P < 0.001 relative to untreated control in Student's t-test.
DiscussionThis study was undertaken to assess the transforming and genotoxic potential of two heavy metal–tungsten alloy metals used in military munitions. Since these tungsten alloys are not commercially available, we used a mixture of the pure metals that compose each heavy metal–tungsten alloy. These metal mixtures, which mimic the ones used in military applications, are composed of tungsten, nickel and cobalt or tungsten, nickel and iron.
Our studies demonstrate for the first time that the malignant transformation of immortalized human cells can be achieved by exposure to a mixture of W, Ni and Co or a mixture of W, Ni and Fe. Transformants with both metal mixtures showed morphological changes, anchorage-independent growth in soft agar, induced tumors when transplanted into nude mice and exhibited alterations in ras oncogene expression. Cellular exposure to these mixtures also induced DNA breakage and chromosomal aberrations, i.e. MN induction, indicating that these mixtures are genotoxic. This is the first report to demonstrate not only that a tungsten alloy mixture can transform human cells, but also that, based upon equivalent concentrations and metal toxicities, the magnitude of this transformation is ~1.3-fold greater than that observed for NiS, a well-known transforming agent and carcinogen (13). Furthermore, both rWNiCo and rWNiFe caused a dose-dependent increase in the transformation frequency of HOS cells. Since there are little data regarding the potential tumorigenic and genotoxic effects of internalized tungsten and tungsten alloys, these results are important to the understanding of the mechanism of the potential late health effects, i.e. carcinogenicity, of tungsten alloys used in military applications.
These studies also confirm previous results demonstrating that HOS cells can be used to assess quantitative transformation in addition to morphological and neoplastic transformation (1,13,14,16). Morphological transformation studies have primarily involved rodent cell lines such as C3H and 10T1/2 (11,15). As with our previous DU studies (1), our current results again demonstrate that human cell models are available not only for morphological but also for quantitative transformation studies. The current studies with tungsten alloy mixtures demonstrate another step in understanding the transformation process of HOS cells.
The precise mechanism(s) by which rWNiCo and rWNiFe induce transformation in HOS cells is not fully known, although the data suggest several possibilities. Alterations in specific oncogenes (e.g. ras) and/or inactivation of tumor suppressor genes (e.g. p53) involved in the conversion of these cells to the malignant phenotype have been considered. HOS cells contain a mutation at codon 156 of the p53 gene, resulting in a mutated form of p53 protein that is believed to be partially responsible for their immortalization (28). Since neoplastic conversion is postulated to result from a multi-step process involving cell immortalization and gene alterations (27,29–31), transformation of immortalized HOS cells by rWNiCo and rWNiFe to the malignant phenotype may involve other cellular oncogenes in this process. Our data demonstrate that the ras oncogene was activated in the transformation process induced by both rWNiCo and rWNiFe exposure. The ras oncogenes have been implicated in both chemical- and radiation-induced animal tumors (1,29–32) and in spontaneous human tumors (33). Studies also show that the c-myc protooncogene plays an important role in the nickel- and lead-induced transformation of mammalian cells, possibly by stabilizing protooncogene RNAs (34,35). Additionally, effects on tumor suppressor proteins have also been observed in metal-transformed clones. DU- (1) and Ni-induced (1,12) transformations of HOS cells were both shown to affect the encoded protein of the Rb tumor suppressor gene by altering phosphorylation of Rb protein in the transformed clones. Similarly, long-term exposure of human epithelial cells to nickel resulted in p53 gene point mutations (35). Considering these findings, we speculate that the transformation of human cells by tungsten alloy mixtures may result from a conversion process that definitely involves activation of tumor-promoting gene(s) and that may involve alterations in tumor suppressor proteins. This hypothesis awaits further testing.
Histological and histochemical analyses of tumors formed by both the rWNiCo- and rWNiFe-transformed cells indicated a gland-like pattern with random calcium deposition rather than an osteogenic sarcoma pattern of growth. Specifically, this histological tumor examination revealed an infiltrative neoplasm consisting of cuboidal cells forming acini, papillary fronds and tubules with no neoplastic spindled cells present, which is consistent with a diagnosis of adenocarcinoma. The finding of a malignant epithelial neoplasm rather than a sarcoma, a malignant mesenchymal neoplasm, was not expected. However, transformation of a spindled cell line, like HOS, with subsequent transplantation into nude mice may result in dedifferentiation at some point, resulting in growth of an epithelial neoplasm. A previous study of NiS-induced transformation of HOS cells reported the same unexpected histomorphology of neoplasms in recipient nude mice; that study also included supportive immunohistochemistry with positive cytokeratin and positive carcinoembryonic antigen staining, both epithelial markers (14). Furthermore, in our study untreated HOS cells formed ALP-positive foci which calcified on extended culture, while the HMTA-transformed cells lacked the ability to form multilayered cellular structures and ALP-positive foci. Changes in ALP activity have been shown to be associated with expression of the malignant phenotype in human osteosarcoma cells (23). Additionally, both rWNiCo- and rWNiFe-transformed cells deposited calcium randomly in areas showing clumped growth of cells. Taken together, these observations suggest that HMTA-transformed cells had dedifferentiated and lost some osteoblastic characteristics. The similarity of our results to previous studies of HOS transformation and tumorigenesis strongly suggests that HOS tumorigenic cells dedifferentiate in vivo (1,14).
As indicated previously, the tungsten alloy mixtures and the pure metals (except Ni) have not been tested for transforming potential. However, studies addressing hard metal lung disease examined the cytotoxic and cytogenetic effects of Co, WC and a WC + Co mixture (36,37). The results demonstrated that WC + Co had a greater cellular toxicity than pure Co metal particles (8,9) and that cellular uptake of Co was enhanced when it was present in the form of WC + Co. The increased toxicity did not result from the increased bioavailability of Co, however, and it appears that WC + Co behaved as a specific toxic entity (9,35–37). Similarly, our data also show that the tungsten alloy metal mixtures W/Ni/Co and W/Ni/Fe exhibit a greater toxicity than that of any of the pure metals. We have not yet determined whether there is any enhanced bioavailabilty of any of the pure metals in the mixture or whether this potentially increased bioavailability would affect the toxicity or transforming ability of the tungsten alloy mixtures. Ongoing studies on HMTA uptake and bioavailability may help to answer these questions.
The mechanism by which rWNiCo and rWNiFe induce cell transformation in vitro appears to involve, at least partially, direct damage to the genetic material, manifested as increased DNA breakage and chromosomal aberrations (i.e. MN). Our data clearly show that direct DNA damage and induction of chromosomal aberrations result from cellular exposure to the tungsten alloy mixtures. In contrast, only the highest doses of pure W, Ni and Co induced any significant increase in DNA breakage above background. Ni and Co were previously shown to be genotoxic at higher, toxic concentrations (38), so our data with Ni and Co at low, non-toxic doses are not too surprising. The HMTA mixtures demonstrate a synergistic increase in DNA breakage when the pure metals are mixed together to compose the tungsten alloy mixture. In contrast, MN induction by the HMTA mixtures, while significantly greater than the level of MN induction by each pure metal, did not exhibit a synergistic relationship. The MN test may be somewhat less sensitive in assessing the synergistic DNA-damaging potential of the mixture demonstrated by the DNA breakage assay. The MN assay does, however, detect chromosomal aberrations and not just repairable DNA breakage. Therefore, the combination of the DNA single-strand break and MN assays provides a better understanding of the mechanisms responsible for the genotoxic nature of the HMTA mixtures.
Other mechanisms may be involved by which rWNiCo and rWNiFe induce cell transformation in vitro. Studies have demonstrated that non-genotoxic, carcinogenic metals like lead may induce transformation via an indirect mechanism such as changes in DNA conformation or enzymatic disturbances (39). Other studies have also demonstrated that inhibition of DNA replication, leading to an elevation in sister chromatid exchanges, may be involved in metal-induced transformation (40,41). All of these mechanisms could potentially be involved in the HMTA transformation process, since transformation induced by metals like Ni appears to involve multiple mechanisms essential to the neoplastic process. For example, the involvement of epigenetic mechanisms of action has also been postulated for Ni. Chromatin condensation, de novo methylation and the resultant aberrant genetic activity may be partially responsible for the carcinogenic action of Ni (34,42). Similarly, recent unpublished data from our laboratory demonstrate that cellular exposure to rWNiCo resulted in hypermethylation of DNA (unpublished data). Although we do not have any direct evidence that neoplastic transformation by the HMTA mixtures involves epigenetic mechanisms, we cannot rule out their potential involvement in the transformation process.
Another possible mechanism in HMTA mixture transformation may involve reactive oxygen species (ROS). The formation of oxygen radicals in metal transformation is well documented, however, for some metals the contribution of these free radicals to the carcinogenic process is somewhat unclear (38). For Ni it is clear that ROS are involved. On a cellular level, Ni has been shown to increase protein oxidation and induce formation of cell oxidants (42) and the binding of Ni to peptides increases its accessibility to critical DNA sites. Animal studies have confirmed the role of Ni and Ni-mediated ROS in the mechanism of Ni carcinogenesis (43). Similarly, Fe potentiates oxygen toxicity via the Fenton reaction, producing hydroxyl radicals and inducing oxidative stress (44). Animal carcinogenesis studies provide evidence that high doses of Fe are also carcinogenic (45). In contrast, Co, which also generates substantial amounts of oxygen radicals, is not mutagenic or carcinogenic (46). When Co and WC particles are mixed together, however, hydroxyl radicals are generated that contribute to the mixture's induction of DNA damage (9,37). Epidemiological studies demonstrate that occupational exposure to hard metals may cause different types of lung diseases (6,7), including cancer (47). Based on these findings with other metals, it is interesting to speculate that another mechanism by which the HMTA mixtures induce transformation, chromosomal aberrations and DNA breakage may involve oxidative damage. This hypothesis awaits further testing.
In summary, we used a model system of in vitro human osteoblast cells exposed for 24 h to rWNiCo (50 µg powder/ml) and rWNiFe (50 µg powder/ml) to assess the relative transforming potential of HMTAs in an effort to better understand the potential health risks from long-term exposure to internalized HMTAs. Cellular exposure to HMTAs induces neoplastic transformation. These HMTA transformants are characterized by anchorage-independent growth, tumor formation in nude mice and expression of high levels of the K-ras oncogene. The mechanism by which rWNiCo and rWNiFe induce cell transformation in vitro appears to involve, at least partially, direct damage to the genetic material manifested as increased DNA breakage or chromosomal aberrations (i.e. MN). These HMTAs appear to have transforming ability somewhat greater than that of many other trace heavy metals which also induce neoplastic cell transformation in vitro as well as cause tumor formation in animals (42). While additional animal studies are needed to address the effect of protracted exposure and tumor induction in vivo, the implication from our model system study is that the risk of neoplastic induction from internalized HMTA exposure may be similar to other biologically reactive and carcinogenic heavy metal compounds (e.g. Ni).
NotesTo whom correspondence should be addressed at: AFRRI, Applied Cellular Radiobiology Department, 8901 Wisconsin Avenue, Bethesda, MD 20889-5603, USA
Acknowledgments
The authors are grateful for the helpful guidance and assistance of Drs David McClain and John Kalinich. We also thank Dr David Ledney for his suggestions. This research was supported in part by the Armed Forces Radiobiology Research Institute under work unit no. AFRRI-99-4. The views presented are those of the authors and do not necessarily reflect the official views of the Department of Defense or the US government.
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Received April 14, 2000; revised September 6, 2000; accepted September 25, 2000.
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