MUTATIONAL SPECTRUM INDUCED BY CHROMIUM(III) IN SHUTTLE VECTORS REPLICATED IN HUMAN-CELLS - RELATIONSHIP TO CR(III)-DNA INTERACTIONS

Trivalent chromium (Cr(III)), the ultimate species of chromium(VI) int racellular reduction, can associate with DNA forming Cr(III) monoadduc ts and DNA-DNA cross-links. However, the mutational specificity of Cr( LII) has not been determined partly because Cr(III) has difficulty ent ering cells. In this study, we have characterized the types of Cr(III) -induced DNA lesions in two buffer systems and the mutational spectrum of Cr(III)-treated shuttle vectors replicated in human 293 cells. Pla smids were treated with Cr(III) in buffers consisting of either 10 mM potassium phosphate, pH 7.5 (designated as KP), or 0.2 mM Tris-HCl and 20 mu M EDTA, pH 7.4 (designated as TE/50). The amounts of Cr(III) bo und to DNA increased as Cr(III) concentration increased in both buffer s; these Cr(III)-DNA associations were stable in both buffers during a 24-h dialysis. The electrophoretic mobility of supercoiled DNA was ma rkedly retarded in samples treated with Cr(III) in TE/50 but not KP bu ffer, suggesting that Cr(III)-mediated DNA-DNA cross-links were genera ted in TE/50 but did not form in KP. Polymerase-stop assay showed that DNA polymerases were mostly blocked at the 3' adjacent bases of guani nes on templates treated with Cr(III) in TE/50 but were not observed o n those treated in KP. The signals of Cr(III)-mediated cross-links gen erated in TE/50 buffers were reduced when they were dialyzed against K P buffers. Similarly, Cr(III)-DNA monoadducts formed in KP were conver ted to primer-template cross-links by dialysis against TE/50. The muta tion frequency of Cr(III) in the supF gene of pSP189 or pZ189 shuttle vectors replicated in human cells increased as Cr(III) concentration i ncreased in both buffers. DNA sequencing analysis showed that single-b ase substitutions (61-68%), two-base substitutions (3-5%), and deletio ns (21-34%) were induced in similar frequencies in plasmids treated wi th Cr(III) in either TE/50 or KP. The Cr(III)-induced base-substitutio n hot spots are different from those occurring spontaneously. Cr(III) enhances G . C base substitutions, particularly G . C --> C . G transv ersions, at 5'(G) under bar A, 5'C (G) under bar, and 5'A (G) under ba r sites. Base-substitution hot spots did not correlate with strong pol ymerase-stop sites, suggesting that base substitutions are derived fro m Cr(III) monoadducts, not from DNA-DNA cross-links.