Cornu M C, Moore G A, Nakagawa Y, Moldéus P
Karolinska Institutet, Institutionen för Toxikologi, Stockholm, Sweden.
Biochem Pharmacol. 1993 Oct 19;46(8):1333-8. doi: 10.1016/0006-2952(93)90096-f.
The toxicity of redox cycling compounds which generate the formation of active oxygen species is commonly accepted to be associated with a decrease of cellular reductants involved in cellular defence. However, when hepatocytes were incubated with diquat, an established redox cycler, in the presence of ascorbic acid (AA) (1 mM), the intracellular level of AA was increased. The effects of diquat on AA uptake were investigated in isolated rat hepatocytes. Incubation of hepatocytes with diquat plus AA (1 mM) resulted in about a 2-fold increased accumulation which occurred in a time-dependent manner reaching a steady state after 15 min at 37 degrees. The initial AA uptake rate was dependent on the AA concentration added. This process is described by Michaelis-Menten kinetics (apparent Km = 953 +/- 59 microM and Vmax = 2.68 nmol/min/10(6) cells). Characterization of AA accumulation showed it to be inhibited: by incubation at 4 degrees; with carbonyl cyanide p-trifluoromethoxyphenylhydrazone, an inhibitor of intracellular ATP production; by decreasing the extracellular Na+ concentration or incubating with ouabain; with pfloretin, a glucose transport inhibitor; and with glucose, a competitive inhibitor of AA transport. Replacement of AA with its oxidized form, dehydroascorbic acid, in the absence of diquat enhanced AA accumulation by 2.5-fold and apparently prevented further accumulation by added diquat. In addition, maintaining AA reduced with dithiothreitol inhibited the diquat effect. Diquat-induced AA accumulation was inhibited (65%) by desferrioxamine, a free-iron chelator, but not by catalase and/or superoxide dismutase or different antioxidants. In contrast, incubation with other active oxygen species generating systems including bipyridilium structural analogues, paraquat and benzyl viologen, had no effect on AA accumulation in hepatocytes. These results suggest that diquat-induced AA accumulation by hepatocytes occurs by a specific mediated transport system rather than as a consequence of cytotoxicity and may involve the presence of free-iron.
氧化还原循环化合物产生活性氧物种的毒性通常被认为与参与细胞防御的细胞还原剂减少有关。然而，当肝细胞在抗坏血酸（AA）（1 mM）存在的情况下与百草枯（一种已确定的氧化还原循环剂）一起孵育时，细胞内AA水平升高。在分离的大鼠肝细胞中研究了百草枯对AA摄取的影响。用百草枯加AA（1 mM）孵育肝细胞导致积累增加约2倍，这种积累呈时间依赖性，在37℃下15分钟后达到稳态。初始AA摄取率取决于添加的AA浓度。该过程由米氏动力学描述（表观Km = 953±59 microM，Vmax = 2.68 nmol/min/10(6)细胞）。AA积累的特征表明它受到以下因素的抑制：在4℃下孵育；用羰基氰对三氟甲氧基苯基腙，一种细胞内ATP产生的抑制剂；通过降低细胞外Na+浓度或用哇巴因孵育；用根皮素，一种葡萄糖转运抑制剂；以及用葡萄糖，AA转运的竞争性抑制剂。在没有百草枯的情况下用其氧化形式脱氢抗坏血酸替代AA可使AA积累增加2.5倍，并明显阻止添加百草枯后的进一步积累。此外，用二硫苏糖醇维持AA还原状态可抑制百草枯的作用。百草枯诱导的AA积累被去铁胺（一种游离铁螯合剂）抑制（65%），但不受过氧化氢酶和/或超氧化物歧化酶或不同抗氧化剂的抑制。相反，与其他活性氧物种产生系统（包括联吡啶结构类似物、敌草快和苄基紫精）一起孵育对肝细胞中AA积累没有影响。这些结果表明，百草枯诱导肝细胞积累AA是通过特定的介导转运系统发生的，而不是细胞毒性的结果，并且可能涉及游离铁的存在。
