Effects of glutathione on chromium-induced DNA crosslinking and DNA polymerase arrest

Hexavalent chromium (Cr (VI)) is reduced intracellularly to Cr (V), Cr (IV) and Cr (III) by ascorbate (Asc), cysteine and glutathione (GSH). These met abolites induce a spectrum of genomic DNA damage resulting in the inhibitio n of DNA replication. Our previous studies have shown that treatment of DNA with Cr (III) or Cr (VI) plus Asc results in the formation of DNA-Cr-DNA c rosslinks (Cr-DDC) and guanine-specific arrests of both prokaryotic and mam malian DNA polymerases. GSH not only acts as a reductant of Cr (VI) but als o becomes crosslinked to DNA by Cr, thus, the focus of the present study wa s to examine the role of GSH in Cr-induced DNA damage and polymerase arrest s. Co-incubation of Cr (III) with plasmid DNA in the presence of GSH led to the crosslinking of GSH to DNA. GSH co-treatment with Cr (III) also led to a decrease in the degree of Cr-induced DNA interstrand crosslinks relative to Cr (III) alone, without affecting total Cr DNA binding. DNA polymerase arrests were observed following treatment of DNA with Cr (III) alone, but w ere markedly reduced when GSH was added to the reaction mixture. Pre-formed polymerase-arresting lesions (Cr-DDC) were not removed by subsequent addit ion of GSH. Treatment of DNA with Cr (VI), in the presence of GSH, resulted in crosslinking of GSH to DNA, but failed to produce detectable DNA inters trand crosslinks or polymerase arrests. The inhibitory effect of GSH on Cr- induced polymerase arrest was further confirmed in human genomic DNA using quantitative PCR (QPCR) analysis. Treatment of genomic DNA with Cr (III) re sulted in a marked inhibition of the amplification of a 1.6 kb target fragm ent of the p53 gene by Taq polymerase. This was almost completely prevented by co-treatment with GSH and Cr (III). These results indicate that Cr-indu ced DNA interstrand crosslinks, and not DNA-Cr-GSH crosslinks, are the prin cipal lesions responsible for blocking DNA replication. Moreover, the forma tion of DNA-Cr-GSH crosslinks may actually preclude the formation of the po lymerase arresting lesions.