Standeven A M, Wetterhahn K E
Department of Chemistry, Dartmouth College, Hanover, NH 03755.
Carcinogenesis. 1992 Aug;13(8):1319-24. doi: 10.1093/carcin/13.8.1319.
Chromium(VI) reductase activity was measured in ultrafiltrates of rat lung after various pretreatments in vitro at 37 degrees C and pH 7.0. Pretreatment of ultrafiltrates with L-ascorbate oxidase (EC 1.10.3.3), which specifically eliminated ascorbate, blocked approximately 95% of chromium(VI) reductase activity in ultrafiltrates. Preincubation of ultrafiltrates with heat-denatured ascorbate oxidase or the sulfhydryl-blocking agent N-ethylmaleimide (NEM) had no significant effect on Cr(VI) reductase activity. In rat lung cytosols, L-ascorbate oxidase blocked approximately 95% and NEM blocked approximately 15% of Cr(VI) reductase activity. The extent of inhibition of Cr(VI) reductase activity in cytosols by L-ascorbate oxidase was significantly decreased to approximately 75% after addition of 1.0 mM NADPH. When Cr(VI) was incubated with salmon sperm nuclei suspended in rat lung cytosol for 15 min, Cr became bound to nuclear DNA. This Cr-DNA binding was completely inhibited by preincubation of rat lung cytosols with L-ascorbate oxidase and inhibited approximately 60% by preincubation with NEM. Taken together these data suggest that ascorbate and/or ascorbate-dependent factors are the principal reductants of Cr(VI) in both ultrafiltrates and cytosols prepared from rat lung and ascorbate-dependent metabolism of Cr(VI) results in Cr binding to nuclear DNA in vitro. Although sulfhydryl-containing factors and NADPH-dependent factors only make a minor contribution to Cr(VI) reduction in rat lung cytosols, sulfhydryls may be significantly involved in the binding of Cr to nuclear DNA.
在37℃和pH 7.0条件下，对经过各种体外预处理的大鼠肺超滤液进行铬（VI）还原酶活性测定。用L -抗坏血酸氧化酶（EC 1.10.3.3）对超滤液进行预处理，该酶能特异性去除抗坏血酸，可使超滤液中约95%的铬（VI）还原酶活性受到抑制。将超滤液与热变性的抗坏血酸氧化酶或巯基阻断剂N -乙基马来酰亚胺（NEM）预孵育，对铬（VI）还原酶活性无显著影响。在大鼠肺胞质溶胶中，L -抗坏血酸氧化酶可阻断约95%的铬（VI）还原酶活性，NEM可阻断约15%的该活性。加入1.0 mM NADPH后，L -抗坏血酸氧化酶对胞质溶胶中铬（VI）还原酶活性的抑制程度显著降低至约75%。当铬（VI）与悬浮于大鼠肺胞质溶胶中的鲑鱼精细胞核孵育15分钟后，铬会与核DNA结合。大鼠肺胞质溶胶与L -抗坏血酸氧化酶预孵育可完全抑制这种铬 - DNA结合，与NEM预孵育可抑制约60%。综合这些数据表明，抗坏血酸和/或抗坏血酸依赖性因子是大鼠肺制备的超滤液和胞质溶胶中铬（VI）的主要还原剂，铬（VI）的抗坏血酸依赖性代谢导致铬在体外与核DNA结合。尽管含巯基因子和NADPH依赖性因子对大鼠肺胞质溶胶中铬（VI）还原的贡献较小，但巯基可能在铬与核DNA的结合中起重要作用。
