Abstract

Chemicals in commerce or under development must be assessed for genotoxicity; assessment is generally conducted using validated assays (e.g. Tk mouse lymphoma assay) as part of a regulatory process. Currently, the MutaMouse FE1 cell mutagenicity assay is undergoing validation for eventual use as a standard in vitro mammalian mutagenicity assay. FE1 cells have been shown to be metabolically competent with respect to some cytochrome P450 (CYP) isozymes; for instance, they can convert the human carcinogen benzo[a]pyrene into its proximate mutagenic metabolite. However, some contradictory results have been noted for other genotoxic carcinogens that require two-step metabolic activation (e.g. 2-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoxaline). Here, we examined three known or suspected human carcinogens, namely acrylamide, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (4-ABP), together with their proximate metabolites (i.e. glycidamide, N-OH-PhIP and N-OH-4-ABP), to aid in the validation of the FE1 cell mutagenicity assay. Assessments of the parent compounds were conducted both in the presence and absence of an exogenous metabolic activation mixture S9; assessments of the metabolites were in the absence of S9. The most potent compound was N-OH-PhIP -S9, which elicited a mutant frequency (MF) level 5.3-fold over background at 5 µM. There was a 4.3-fold increase for PhIP +S9 at 5 µM, a 1.7-fold increase for glycidamide −S9 at 3.5 mM and a 1.5-fold increase for acrylamide +S9 at 4 mM. Acrylamide −S9 elicited a marginal 1.4-fold MF increase at 8 mM. Treatment with PhIP −S9, 4-ABP ±S9 and N-OH-4-ABP −S9 failed to elicit significant increases in lacZ MF with any of the treatment conditions tested. Gene expression of key CYP isozymes was quantified by RT-qPCR. Cyp1a1, 1a2 and 1b1 are required to metabolise PhIP and 4-ABP. Results showed that treatment with both compounds induced expression of Cyp1a1 and Cyp1b1 but not Cyp1a2. Cyp2e1, which catalyses the bioactivation of acrylamide to glycidamide, was not induced after acrylamide treatment. Overall, our results confirm that the FE1 cell mutagenicity assay has the potential for use alongside other, more traditional in vitro mutagenicity assays.

中文翻译：

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选定环境致癌物及其代谢物对 MutaMouse FE1 肺上皮细胞的体外致突变性

 抽象的

商业或正在开发的化学品必须进行遗传毒性评估；作为监管过程的一部分，通常使用经过验证的检测（例如Tk小鼠淋巴瘤检测）进行评估。目前，MutaMouse FE1 细胞致突变性测定正在接受验证，以最终用作标准的体外哺乳动物致突变性测定。 FE1 细胞已被证明对某些细胞色素 P450 (CYP) 同工酶具有代谢能力；例如，他们可以将人类致癌物苯并[ a ]芘转化为其最接近的致突变代谢物。然而，对于需要两步代谢激活的其他基因毒性致癌物（例如2-乙酰氨基芴和2-氨基-3-甲基咪唑并[4,5- f ]喹喔啉），已经注意到一些矛盾的结果。在这里，我们检查了三种已知或可疑的人类致癌物，即丙烯酰胺、2-氨基-1-甲基-6-苯基咪唑并[4,5 -b ]吡啶（PhIP）和4-氨基联苯（4-ABP）及其近似物质。代谢物（即缩水甘油酰胺、 N -OH-PhIP 和N -OH-4-ABP），以帮助验证 FE1 细胞致突变性测定。在存在和不存在外源代谢活化混合物 S9 的情况下对母体化合物进行了评估；代谢物的评估是在没有 S9 的情况下进行的。最有效的化合物是N -OH-PhIP -S9，它在 5 µM 浓度下引发的突变频率 (MF) 水平是背景的 5.3 倍。 PhIP +S9 在 5 µM 时增加了 4.3 倍，缩水甘油酰胺 -S9 在 3.5 mM 时增加了 1.7 倍，丙烯酰胺 +S9 在 4 mM 时增加了 1.5 倍。丙烯酰胺-S9 在 8 mM 时引起 MF 略微增加 1.4 倍。 在任何测试的处理条件下，用 PhIP -S9、4-ABP ±S9 和N -OH-4-ABP -S9 处理均未能引起lacZ MF 的显着增加。通过 RT-qPCR 定量关键 CYP 同工酶的基因表达。 PhIP 和 4-ABP 的代谢需要 Cyp1a1、1a2 和 1b1。结果表明，两种化合物的处理均诱导Cyp1a1和Cyp1b1的表达，但不诱导Cyp1a2 的表达。 Cyp2e1催化丙烯酰胺生物活化为缩水甘油酰胺，在丙烯酰胺处理后没有被诱导。总体而言，我们的结果证实 FE1 细胞致突变性测定具有与其他更传统的体外致突变性测定一起使用的潜力。