DIFFERENTIAL SENSITIVITY OF CHROMIUM-MEDIATED DNA INTERSTRAND CROSS-LINKS AND DNA-PROTEIN CROSS-LINKS TO DISRUPTION BY ALKALI AND EDTA

Some compounds of hexavalent chromium are well-established carcinogens . Chromium enters mammalian cells in the hexavalent form and is reduce d to chromium(III). Treatment of purified DNA with chromium(III) produ ces DNA-DNA interstrand crosslinks (DDC) which obstruct the progressio n of DNA polymerases in vitro. DDC were also detected in chromate-trea ted cultured normal human lung cells using the renaturing agarose gel electrophoresis (RAGE) assay and correlated with base-specific inhibit ion of DNA replication. Curiously, DDC have gone undetected in studies of cultured cells using the alkaline elution (AE) technique, whereas chromium-mediated DNA-protein crosslinks (DPC) were readily detected b y AE. We tested the hypothesis that AE conditions [60 mM tetraethyl am monium hydroxide (TEA), 20 mM EDTA, pH 12.6, for 16 h at room temperat ure] dissociate DDC but not DPC using chromium(III)-treated plasmid DN A and the RAGE assay. Dose-dependent chromium-induced DDC were unaffec ted by TEA (pH 11.8) alone or by more rigorous alkaline denaturation c onditions (200 mM NaOH, pH 13.5, for 16 h). DDC were, however, complet ely disrupted by EDTA (pH 12.6) alone or the combination of TEA and ED TA (pH 12.6). In contrast, DPC remained largely intact under these con ditions, Therefore, past AE-based studies which have failed to detect chromium-induced DDC do not prove the absence of this lesion. AE may n ot be suitable for detecting DDC induced by EDTA-chelatable agents suc h as metals. (C) 1998 Society of Toxicology.