Causes of DNA single-strand breaks during reduction of chromate by glutathione in vitro and in cells

Abstract

Carcinogenic chromates induce DNA single-strand breaks (SSB) that are detectable by conventional alkali-based assays. However, the extent of direct breakage has been uncertain because excision repair and hydrolysis of Cr–DNA adducts at alkaline pH also generate SSB. We examined mechanisms of SSB production during chromate reduction by glutathione (GSH) and assessed the significance of these lesions in cells using genetic approaches. Cr(VI) reduction was biphasic and the formation of SSB occurred exclusively during the slow reaction phase. Catalase or iron chelators completely blocked DNA breakage, as did the use of GSH purified by a modified Chelex procedure. Thus, the direct intermediates of GSH–chromate reactions were unable to cause SSB unless activated by H 2O 2. SSB repair-deficient XRCC1 −/− and proficient XRCC1 + EM9 cells had identical survival at doses causing up to 60% clonogenic death and accumulation of 1 mM Cr(VI). However, XRCC1 −/− cells displayed higher lethality in the more toxic range and the depletion of GSH made them hypersensitive even to moderate doses. Elevation of cellular catalase or GSH levels eliminated survival differences between XRCC1 −/− and XRCC1 + cells. In summary, formation of toxic SSB in cells occurs at relatively high chromate doses, requires H 2O 2, and is suppressed by high GSH concentrations.