Goeptar A R, te Koppele J M, Glatt H R, Groot E J, Seidel A, Barrenscheen M, Wölfel C, Doehmer J, Vermeulen N P
Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology, Vrije Universiteit, Amsterdam, The Netherlands.
Chem Biol Interact. 1995 Jul 14;97(2):149-68. doi: 10.1016/0009-2797(95)03611-o.
The objective of the present study was to investigate the cytotoxicity of Adriamycin (ADR) and mitomycin C (MMC) in tumor and non-tumor cells with respect to the role of cytochrome P450 (P450). Therefore, genetically engineered V79 Chinese hamster fibroblasts expressing only single enzymes of P450 were used. SD1 and XEM2 cells expressed rat P450IIB1 and P450IA1, respectively, whereas the V79 parental cells contained no detectable P450 levels. The cytotoxicity of ADR and MMC in the V79 cell system was compared with that in freshly isolated hepatocytes from phenobarbital (PB-hepatocytes)- and beta-naphthoflavone (beta NF-hepatocytes)-induced rats. Following 24 h of exposure to ADR equal cytotoxicity was observed in V79, SD1 and XEM2 cells. Addition of metyrapone (MP, an inhibitor of P450IIB1) and alpha-naphthoflavone (alpha NF, an inhibitor of P450IA1) had no effect on the ADR-induced cytotoxicity in SD1 and XEM2 cells, respectively. Likewise, MMC was equitoxic in V79 and SD1 cells. Co-incubation of SD1 cells with MP did not alter MMC-induced cytotoxicity. MMC, however, showed a decreased cytotoxicity in XEM2 cells when compared to the parental V79 cells. Unexpectedly, the cytotoxicity of MMC in XEM2 cells was increased by alpha NF to the same level as observed in the parental V79 cells. In contrast to V79- and V79-derived cells, in freshly isolated hepatocytes from PB or beta NF-induced rats, MMC was cytotoxic (measured as lactate dehydrogenase leakage) within 3 h of incubation. ADR, however, was only cytotoxic to the hepatocytes when intracellular glutathione was first depleted by diethylmaleate. The MMC- and ADR-induced cytotoxicity was found to be more pronounced in PB-hepatocytes than in beta NF-hepatocytes. Contrary to the findings in the V79-derived cells, MP afforded complete protection against both MMC- and ADR-induced cytotoxicity in PB-hepatocytes, whereas alpha NF only partially inhibited the cytotoxicity of MMC in beta NF-hepatocytes. In conclusion, we have demonstrated that PB-inducible P450s play a role in the cytotoxicity of both MMC and ADR in freshly isolated PB-hepatocytes but that P450IIB1 does not in genetically reconstituted SD1 cells. P450IA1, however, decreased the cytotoxicity of MMC in the XEM2 cells. The ADR-induced cytotoxicity, which was observed in XEM2 cells, was not mediated by P450IA1. The present study underscores the complexity in the comparison of ADR- and MMC-induced cytotoxicities in normal and tumor cells.
本研究的目的是就细胞色素P450（P450）的作用，研究阿霉素（ADR）和丝裂霉素C（MMC）对肿瘤细胞和非肿瘤细胞的细胞毒性。因此，使用了仅表达单一P450酶的基因工程V79中国仓鼠成纤维细胞。SD1和XEM2细胞分别表达大鼠P450IIB1和P450IA1，而V79亲本细胞未检测到P450水平。将ADR和MMC在V79细胞系统中的细胞毒性与苯巴比妥（PB-肝细胞）和β-萘黄酮（βNF-肝细胞）诱导的大鼠新鲜分离的肝细胞中的细胞毒性进行了比较。在暴露于ADR 24小时后，在V79、SD1和XEM2细胞中观察到同等的细胞毒性。分别添加美替拉酮（MP，P450IIB1的抑制剂）和α-萘黄酮（αNF，P450IA1的抑制剂）对SD1和XEM2细胞中ADR诱导的细胞毒性没有影响。同样，MMC在V79和SD1细胞中具有同等毒性。SD1细胞与MP共同孵育不会改变MMC诱导的细胞毒性。然而，与亲本V79细胞相比，MMC在XEM2细胞中的细胞毒性降低。出乎意料的是，αNF使XEM2细胞中MMC的细胞毒性增加到与亲本V79细胞中观察到的相同水平。与V79及其衍生细胞相反，在PB或βNF诱导的大鼠新鲜分离的肝细胞中，MMC在孵育3小时内具有细胞毒性（以乳酸脱氢酶泄漏衡量）。然而，只有当细胞内谷胱甘肽首先被马来酸二乙酯耗尽时，ADR才对肝细胞具有细胞毒性。发现MMC和ADR诱导的细胞毒性在PB-肝细胞中比在βNF-肝细胞中更明显。与V79衍生细胞中的发现相反，MP对PB-肝细胞中MMC和ADR诱导的细胞毒性均提供了完全保护，而αNF仅部分抑制了βNF-肝细胞中MMC的细胞毒性。总之，我们已经证明，PB诱导的P450在新鲜分离的PB-肝细胞中MMC和ADR的细胞毒性中起作用，但在基因重组的SD1细胞中P450IIB1不起作用。然而，P450IA1降低了XEM2细胞中MMC的细胞毒性。在XEM2细胞中观察到的ADR诱导的细胞毒性不是由P450IA1介导的。本研究强调了在正常细胞和肿瘤细胞中比较ADR和MMC诱导的细胞毒性的复杂性。
