Reactive oxygen species and oxidative DNA damage mediate the cytotoxicity of tungsten-nickel-cobalt alloys in vitro

Tungsten alloys (WA) have been introduced in an attempt to find safer alternatives to depleted uranium and lead munitions. However, it is known that at least one alloy, 91% tungsten-6% nickel-3% cobalt (WNC-91-6-3), causes rhabdomyosarcomas when fragments are implanted in rat muscle. This raises concerns that shrapnel, if not surgically removable, may result in similar tumours in humans. There is therefore a clear need to develop rapid and robust in vitro methods to characterise the toxicity of different WAs in order to identify those that are most likely to be harmful to human health and to guide development of new materials in the future. In the current study we have developed a rapid visual in vitro assay to detect toxicity mediated by individual WA particles in cultured L6-C11 rat muscle cells. Using a variety of techniques (histology, comet assay, caspase-3 activity, oxidation of 2'7'-dichlorofluorescin to measure the production of reactive oxygen species and whole-genome microarrays) we show that, in agreement with the in vivo rat carcinogenicity studies, WNC-91-6-3 was the most toxic of the alloys tested. On dissolution, it produces large amounts of reactive oxygen species, causes significant amounts of DNA damage, inhibits caspase-3, triggers a severe hypoxic response and kills the cells in the immediate vicinity of the alloy particles within 24h. By combining these in vitro data we offer a mechanistic explanation of the effect of this alloy in vivo and show that in vitro tests are a viable alternative for assessing new alloys in the future.