Nakagawa Yoshio, Nakajima Kazuo, Suzuki Toshinari
Divisions of Pharmacology, Tokyo Metropolitan Institute of Public Health, 3-24-1 Hyakunin-cho, Shinjuku-ku, 169-0073, Japan.
Toxicology. 2004 Aug 5;200(2-3):123-33. doi: 10.1016/j.tox.2004.03.012.
The metabolism and action of chlorpropham (isopropyl N-(3-chlorophenyl)carbamate; CIPC, a post-harvest agent) and its metabolites were studied in freshly isolated rat hepatocytes and isolated rat hepatic mitochondria, respectively. The exposure of hepatocytes to CIPC caused a concentration (0.25-1.0 mM)- and time (0-3h)-dependent cell death accompanied by loss of cellular ATP and adenine nucleotides. CIPC at a weakly toxic level (0.5 mM) was metabolized to isopropyl N-(3-chloro-4-hydroxyphenyl)carbamate (4OH-CIPC) and subsequently to its glucuronide and sulfate conjugates (major metabolites) or alternatively to a minor metabolite 3-chloroaniline (3CA). The addition of SKF-525A (50 microM), an inhibitor of microsomal monooxygenase, enhanced the CIPC (0.5 mM)-induced cytotoxicity accompanied by loss of ATP and 4OH-CIPC and inhibited the decrease in the concentration of the parent compound. CIPC led to a strong decrease in cellular ATP content compared to its metabolites, 4OH-CIPC and 3CA. On the other hand, the exposure of isolated hepatic mitochondria to CIPC reduced State 3 respiration with a FAD-linked substrate (succinate plus rotenone) and/or with a NAD+ -linked substrate (pyruvate plus malate), whereas State 3 respiration with ascorbate plus tetramethyl-p-phenylendiamine (cytochrome oxidase-linked respiration) was not affected markedly by CIPC. Further, the addition of CIPC caused an increase in the rate of State 4 oxygen consumption, indicating an uncoupling effect, and a decrease in the rate of State 3 oxygen consumption in a concentration-dependent manner, respectively. In contrast, the addition of neither 4OH-CIPC nor 3CA markedly affected the rate of states 3 and/or 4 oxygen consumption. These results indicate that CIPC-induced cytotoxicity is mediated by the parent compound rather than by its metabolites such as 4OH-CIPC and 3CA, and that the toxicity is associated with a rapid depletion of ATP via impairment of mitochondrial function related to oxidative phosphorylation.
分别在新鲜分离的大鼠肝细胞和分离的大鼠肝线粒体中研究了氯苯胺灵（异丙基N-（3-氯苯基）氨基甲酸酯；CIPC，一种采后试剂）及其代谢产物的代谢和作用。将肝细胞暴露于CIPC会导致浓度（0.25 - 1.0 mM）和时间（0 - 3小时）依赖性的细胞死亡，并伴有细胞ATP和腺嘌呤核苷酸的损失。低毒水平（0.5 mM）的CIPC代谢为异丙基N-（3-氯-4-羟基苯基）氨基甲酸酯（4OH-CIPC），随后代谢为其葡萄糖醛酸和硫酸盐结合物（主要代谢产物），或者代谢为次要代谢产物3-氯苯胺（3CA）。添加微粒体单加氧酶抑制剂SKF-525A（50 microM）可增强CIPC（0.5 mM）诱导的细胞毒性，伴有ATP和4OH-CIPC的损失，并抑制母体化合物浓度的降低。与它的代谢产物4OH-CIPC和3CA相比，CIPC导致细胞ATP含量大幅下降。另一方面，将分离的肝线粒体暴露于CIPC会降低以FAD连接底物（琥珀酸加鱼藤酮）和/或NAD +连接底物（丙酮酸加苹果酸）进行的状态3呼吸，而以抗坏血酸加四甲基对苯二胺（细胞色素氧化酶连接呼吸）进行状态3呼吸则不受CIPC的明显影响。此外，添加CIPC分别导致状态4耗氧率增加，表明存在解偶联效应，以及状态3耗氧率以浓度依赖性方式降低。相比之下，添加4OH-CIPC和3CA均未明显影响状态3和/或状态4的耗氧率。这些结果表明，CIPC诱导的细胞毒性是由母体化合物介导的，而不是由其代谢产物如4OH-CIPC和3CA介导的，并且毒性与通过与氧化磷酸化相关的线粒体功能受损导致的ATP快速消耗有关。
