Grouping of chromiuminduced spectra with others

Recently, we began to investigate the mutagenic activity of chromate, Cr(VI), a known human carcinogen. Although chromate had been found to be mutagenic in a variety of test systems,72 73 the mechanism of mutagenesis was less understood. There was evidence that chromate was reduced in the cell by glutathione and other cellular reducing agents, generating reactive intermediates that cause oxidative damage to DNA.74 75 It had also been demonstrated that Cr(III), the stable end product of intracellular reduction of chromate, bound tightly to DNA.48 76 We reasoned that the mutational mechanism could be revealed through an analysis of mutation spectra. For these studies we used both protocols outlined in Figure 13.2. First, we treated

Figure 13.7 Results of cluster analysis of a collection of published spectra. The underlying spectra of seven groups are shown.

the vector DNA in vitro with chromate and glutathione, removed any bound chromium from the DNA and transfected it into mammalian cells (Cr-in vitro).71 For comparison, we introduced the undamaged vector into the mammalian cells and then treated the cells with chromate (Cr-in vivo).70 The results of these studies are reproduced here in Figure 13.8. We compared these results for the SupF gene (position 99-183) to similar studies with the oxidizing agent, H2O2 using the method of Adams and Skopek.26 This analysis revealed that the chromium spectra were not significantly different from each other (p = 0.11) and the Cr-in vitro spectrum did not differ significantly from the H2O2-in vitro spectrum (p = 0.20). However, the H2O2-in vivo spectrum differed significantly from the Cr-in vivo spectrum (p < 0.001), the Crin vitro spectrum (p = 0.043) and the H2O2-in vitro spectrum (p < 0.001).

In order to compare these Cr-induced spectra to a broader range of mutation spectra, we have employed the clustering methods and we have expanded the collection of mutation spectra to include published spectra in which pZ189 was treated with Cr(III) in the presence of amino acids to form DNA/Cr/amino acid complexes4748 (Table 13.4, bottom). This analysis for seven groups revealed that the two chromium spectra (Cr-in vivo and Cr-in vitro) group with each other and together with ionizing radiation spectra (group 7). Interestingly, most of the spectra from chromium-induced crosslinks grouped together (group 2) with several other agents that form DNA adducts and also with H2O2-in vitro. These results suggest that the mutational specificity of the chromium-induced crosslinks differs from that of the chromium-induced oxidative damage. Furthermore, they suggest that mutational specificity of chromium in vivo may be more like chromium-induced oxidative damage than like chromium-induced crosslinks. However, some of these differences could also be related to the cell type and vector used in the experiments.

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