Jemnitz Katalin, Veres Zsuzsa, Torok Geza, Toth Eva, Vereczkey Laszlo
Department of Biochemical Pharmacology, Chemical Research Center of the Hungarian Academy of Sciences, H1525 Budapest, Hungary.
Mutagenesis. 2004 May;19(3):245-50. doi: 10.1093/mutage/geh026.
We studied the replacement of hepatic S9 with in vivo and in vitro induced hepatocytes as a metabolic activation system with the aim of broadening the possibilities of mutagenic assays. Rats were pretreated with beta-naphthoflavone (BNF), phenobarbital (PB), 3-methylcholanthrene (MC) and a combination of BNF and PB (BNF + PB). Mutagenic activation of benzo[a]pyrene (BP) and 2-aminoanthracene (2AA) by hepatic S9 and hepatocytes was determined in the Ames test. Primary rat hepatocytes were used for in vitro induction and were used as the activating system in the Ames test. In vivo BNF treatment greatly increased the metabolic activation capacity of hepatic S9 and hepatocytes towards BP. With regard to 2AA activation, S9 and hepatocytes showed different BNF induction profiles. PB treatment reduced the mutagenicity of both compounds. Although ethoxyresorufin O-dealkylase (EROD) activity of S9 from BNF + PB-treated animals was almost 30-fold greater than the control, its effectiveness in activation of 2AA was below the control level. A large part of the EROD activity of control cells was lost during culture, together with the ability to activate 2AA, however, 72 h of MC induction increased EROD activity to 200-fold of the control, which corresponds to 28% of that of in vivo induced hepatocytes. The mutagenic potential of BP activated by in vitro induced hepatocytes was 10-fold above the control, which is 47% of the mutagenicity detected following in vivo induction. In vitro induced hepatocytes increased 2AA mutagenicity to 14.6-fold over the control, which corresponds to 68% of in vivo induction. Our results suggest that primary culture of hepatocytes provides a useful model for the study of the role of metabolic activation processes concerning enzyme activity of cytochromes P450 and other metabolic enzymes and induction profiles of different inducers.
我们研究了用体内和体外诱导的肝细胞替代肝S9作为代谢活化系统，目的是拓宽诱变试验的可能性。用β-萘黄酮（BNF）、苯巴比妥（PB）、3-甲基胆蒽（MC）以及BNF和PB的组合（BNF+PB）对大鼠进行预处理。在Ames试验中测定肝S9和肝细胞对苯并[a]芘（BP）和2-氨基蒽（2AA）的诱变活化作用。原代大鼠肝细胞用于体外诱导，并用作Ames试验中的活化系统。体内BNF处理大大增加了肝S9和肝细胞对BP的代谢活化能力。关于2AA活化，S9和肝细胞表现出不同的BNF诱导谱。PB处理降低了两种化合物的诱变性。虽然来自BNF+PB处理动物的S9的乙氧基异吩恶唑酮O-脱烷基酶（EROD）活性几乎比对照高30倍，但其对2AA的活化效果低于对照水平。对照细胞的大部分EROD活性在培养过程中丧失，同时丧失了激活2AA的能力，然而，72小时的MC诱导使EROD活性增加到对照的200倍，这相当于体内诱导肝细胞的28%。体外诱导的肝细胞激活的BP的诱变潜力比对照高10倍，这是体内诱导后检测到的诱变性的47%。体外诱导的肝细胞使2AA的诱变性比对照增加到14.6倍，这相当于体内诱导的68%。我们的结果表明，肝细胞原代培养为研究细胞色素P450和其他代谢酶的酶活性以及不同诱导剂的诱导谱的代谢活化过程的作用提供了一个有用的模型。
