BASE-SPECIFIC ARREST OF IN-VITRO DNA-REPLICATION BY CARCINOGENIC CHROMIUM - RELATIONSHIP TO DNA INTERSTRAND CROSS-LINKING

We have previously shown that trivalent chromium can bind to purified DNA and form lesions capable of obstructing DNA replication in vitro. Trivalent chromium is not, however, carcinogenic to humans. Rather, it is the end product of the intracellular reduction of hexavalent chrom ium, which is carcinogenic. The process of chromium reduction yields s everal reactive intermediates which may also interact with DNA, perhap s producing different lesions than those generated when trivalent chro mium binds DNA. The present study was undertaken to determine whether the treatment of DNA with hexavalent chromium in the presence of ascor bate (the intracellular reductant responsible for most in vivo chromiu m reduction), would also generate DNA lesions capable of obstructing r eplication. Using increasing chromium concentrations and a constant as corbate:chromium ratio of 0.5:1 to generate biologically relevant addu ct levels, a DNA polymerase arrest assay revealed that polymerase arre sting lesions were formed and were indistinguishable from those genera ted by trivalent chromium, in that the most prominent arrests sites we re one base upstream of guanine residues on the template strand. Measu rement of the amount of chromium bound to template DNA in relation to the number of arrests demonstrated that only a subset (18.5%) of the c hromium adducts were capable of causing polymerase arrest. Arrest assa ys performed with increasing ratios of ascorbate to chromium showed th at high ratios (greater than or equal to 5:1) resulted in decreased po lymerase arrests. DNA interstrand crosslinks in the arrest assay templ ate were detected by renaturing agarose gel electrophoresis, and were shown to decrease markedly with increasing ascorbate to chromium ratio s, whereas chromium binding levels remained unchanged. These results s trongly implicate DNA interstrand crosslinks as the polymerase arresti ng lesion. The present study confirms and extends our previous study w ith trivalent chromium, and suggests that while the initial chemical n ature of the DNA lesions formed by either trivalent chromium or reduct ive intermediates of hexavalent chromium may differ, their effect on D NA replication is the same.