RESUMO
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.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Doxorrubicina/toxicidade , Fígado/efeitos dos fármacos , Mitomicina/toxicidade , Animais , Benzoflavonas/farmacologia , Morte Celular/efeitos dos fármacos , Células Cultivadas , Cricetinae , Cricetulus , Ciclofosfamida/toxicidade , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Di-Hidroxi-Di-Hidrobenzopirenos/toxicidade , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fibroblastos , L-Lactato Desidrogenase/metabolismo , Fígado/citologia , Masculino , Maleatos/farmacologia , Metirapona/farmacologia , Fenobarbital/farmacologia , Ratos , Ratos Wistar , Transfecção , beta-NaftoflavonaRESUMO
A V79 Chinese hamster cell line was constructed for stable expression of human cytochrome P450 2E1 (CYP2E1) by integration of a SV40 Early promoter recombinant CYP2E1 cDNA into the chromosomal DNA. The cDNA encoded CYP2E1 was effectively expressed and enzymatically active, as shown by hydroxylation of chlorzoxazone and of p-nitrophenol, at rates of about 70 pmol x mg-1 total protein x min-1. CYP2E1 content and activity was increased upon cultivation in the presence of ethanol indicating a substrate mediated stabilization effect. A similar stabilizing effect was also observed for inhibitors of CYP2E1, e.g. imidazole, 4-methylpyrazole, and isoniazid. The feasibility of the newly established cell line V79MZh2E1 for toxicological studies was shown by CYP2E1-mediated activation of N-nitrosodimethylamine and p-nitrophenol and a dose-dependent cytotoxic and mutagenic effect.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Oxirredutases N-Desmetilantes/biossíntese , Animais , Northern Blotting , Southern Blotting , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Clorzoxazona/metabolismo , Cricetinae , Cricetulus , Citocromo P-450 CYP2E1 , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/genética , Vetores Genéticos , Humanos , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Mutagênicos/toxicidade , Nitrofenóis/metabolismo , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Oxirredutases N-Desmetilantes/genética , Proteínas Recombinantes/biossíntese , TransfecçãoRESUMO
V79 derived Chinese hamster cells lines with specific xenobiotica metabolising functions were constructed by gene technological means. The significance of these cell lines as a test system in toxicology and pharmacology has been demonstrated by the evaluation of metabolite profiles of pharmaceuticals and xenobiotica, and the mutagenic and cytotoxic potency of the metabolites under defined and reproducible conditions.
