Westphal G A, Bünger J, Schulz T G, Müller M M, Hallier E
Department of Occupational Health, Georg-August University Göttingen, Germany.
Arch Toxicol. 2000 Dec;74(10):638-41. doi: 10.1007/s002040000175.
N-Nitrosodiethylamine (NDEA) is carcinogenic in all investigated animal species at relatively low dosages. No threshold has been detected for these carcinogenic effects. The substance has been extensively investigated in various in vitro systems, revealing only weak mutagenicity at relatively high dosages. We reinvestigated NDEA in the Ames test with Salmonella typhimurium TA1535 to establish appropriate modifications of the standard Ames test protocol, to achieve a dose-dependent mutagenic response at a reasonably low dose range. Two main modifications were evaluated. Since the metabolism of dialkylnitrosamines is postulated to be mainly dependent on cytochrome P4502E1, a pyrazole-induced rat liver S9 was applied. The second modification involved a gastight preincubation, since metabolites of NDEA might evaporate from the incubation mixture. Cytochrome P4502E1 induction in Wistar rats was achieved by pyrazole treatment. For comparison, a rat liver S9-fraction produced by beta-naphtoflavone/phenobarbital induction was used. N-Nitrosopyrrolidine served as positive control for pyrazole-induced S9-mix with TA1535. NDEA showed no mutagenic response under all test conditions in the presence of pyrazole-induced S9-mix. A strong mutagenic response, exceeding the base rate up to 15-fold at a dose range of 25-1000 microg/plate, was observed using beta-naphtoflavone/phenobarbital-induced S9-mix, gastight preincubation and TA1535. In conclusion the Ames test with gastight preincubation can be useful for the testing of volatile compounds or substances leading to gaseous metabolites. The weak response of NDEA in the Ames test observed previously seems mainly to be due to the volatile character of its mutagenic metabolites. Our results do not support the hypothesis that cytochrome P4502E1 is a major toxifying enzyme for the formation of Ames-test-positive metabolites from NDEA.
N-亚硝基二乙胺（NDEA）在所有被研究的动物物种中，即使剂量相对较低也具有致癌性。尚未检测到这些致癌作用的阈值。该物质已在各种体外系统中进行了广泛研究，结果显示在相对高剂量下仅具有微弱的致突变性。我们用鼠伤寒沙门氏菌TA1535在艾姆斯试验中重新研究了NDEA，以确定对标准艾姆斯试验方案的适当修改，从而在合理的低剂量范围内实现剂量依赖性的致突变反应。评估了两项主要修改。由于假定二烷基亚硝胺的代谢主要依赖于细胞色素P4502E1，因此使用了吡唑诱导的大鼠肝脏S9。第二项修改涉及气密预孵育，因为NDEA的代谢产物可能会从孵育混合物中蒸发。通过吡唑处理实现了Wistar大鼠中细胞色素P4502E1的诱导。为了进行比较，使用了由β-萘黄酮/苯巴比妥诱导产生的大鼠肝脏S9组分。N-亚硝基吡咯烷用作吡唑诱导的S-9混合物与TA1535的阳性对照。在存在吡唑诱导的S9混合物的所有测试条件下，NDEA均未显示出致突变反应。使用β-萘黄酮/苯巴比妥诱导的S9混合物、气密预孵育和TA1535，在25-1000微克/平板的剂量范围内观察到了强烈的致突变反应，超过基础发生率达15倍。总之，气密预孵育的艾姆斯试验可用于检测挥发性化合物或导致气态代谢产物的物质。先前在艾姆斯试验中观察到的NDEA的微弱反应似乎主要是由于其致突变代谢产物的挥发性。我们的结果不支持细胞色素P4502E1是从NDEA形成艾姆斯试验阳性代谢产物的主要毒性酶这一假设。
