| Compounds containing hexavalent chromium [Cr(VI)] are well-documented human carcinogens that cause cancers in the respiratory system, including the lungs. In this paper, we examined the ability of the insoluble Cr(VI) compound, lead chromate, PbCrO4, to induce cytotoxicity and morphological transformation of C3H 10T1/2 mouse embryonic fibroblastic cells. We seeded the cells at 2,000 cells per 60 mm dish, then treated the cells one day after seeding with PbCrO4 at three concentrations, 1.72&mgr;g/mL, 2.5&mgr;g/mL, and 4.21&mgr;g/mL. Next, we also followed the Nesnow protocol where we seeded 2,000 cells per 60 mm dish, and then treated the cells on the fifth day after they were seeded, for 48 hours with lead chromate. Interestingly, we found very low induction of morphologically transformed foci induced by PbCrO4 when compared with the high yields of foci induced by 1&mgr;g/mL of the strong mutagenic and clastogenic carcinogen, 3-methylcholanthrene (MCA). This is surprising, considering that Cr(VI) compounds are strong human carcinogens to the respiratory system and also to internal organs. However, we realized from reading the scientific literature, that ascorbate, a dominant biological reducing agent, is required for reductive activation of the carcinogenic activity of Cr(VI). In addition, we realized that Cr(VI) is usually present in the micromolar range when humans are exposed to it occupationally, whereas by contrast ascorbate is present at concentrations in the millimolar range under physiological conditions in humans. Therefore, we investigated the cytotoxic effects of ascorbate on 10T1/2 mouse embryo cells, and the effect of non-cytotoxic concentrations of ascorbate on Cr(VI)-induced cytotoxicity in 10T1/2 cells. Our cell survival data revealed that ascorbate can itself exert a cytotoxic effect on the 10T1/2 mouse embryo cells. Furthermore, when 10T1/2 cells were treated with both Cr(VI) and ascorbate, we found that ascorbate played dual roles. It serves as a pro-oxidant at lower concentrations, and as an anti-oxidant at higher concentrations. We also found that treating the cells with <0.07mM ascorbate, caused only minimal or no cytotoxicity, and also showed that ascorbate at this concentration had a pro-oxidative activity, i.e. ascorbate increased the cytotoxicity of Cr(VI) to 10T1/2 cells. This information is crucial for our future experiments, when we design a protocol to incorporate ascorbate into our transformation assays assessing the cytotoxicity and cell transforming of Cr(VI) compounds. |
