Lang D H, Rettie A E, Böcker R H
Department of Medicinal Chemistry, University of Washington, Seattle 98195, USA.
Chem Res Toxicol. 1997 Sep;10(9):1037-44. doi: 10.1021/tx970081l.
Compounds of the s-triazine family are among the most heavily used herbicides over the last 30 years. Some of these derivatives are suspected to be carcinogens. In this study the identity of specific phase-I enzymes involved in the metabolism of s-triazine derivatives (atrazine, terbuthylazine, ametryne, and terbutryne) by human liver microsomes was determined. Kinetic studies demonstrated biphasic kinetics for all pathways examined (S-oxidation, N-dealkylation, and side-chain C-oxidation). Low K(m) values were in a range of about 1-20 microM, whereas high K(m) values were up to 2 orders of magnitude higher. For a correlation study, 30 human liver microsomal preparations were screened for seven specific P450 activities, and these were compared to activities for the metabolites derived from these s-triazines. A highly significant correlation in the high-affinity concentration range was seen with cytochrome P450 1A2 activities. Chemical inhibition was most effective with alpha-naphthoflavone and furafylline at low s-triazine concentrations and additionally with ketoconazole and gestodene at high substrate concentrations. Studies with 10 heterologously expressed P450 forms demonstrated that several P450 enzymes are capable of oxidizing these s-triazines, with different affinities and regioselectivities. P450 1A2 was confirmed to be the low-K(m) P450 enzyme involved in the metabolism of these s-triazines. A potential participation of flavin-containing monooxygenases (FMOs) in sulfoxidation reactions of the thiomethyl derivatives ametryne and terbutryne in human liver was also evaluated. Sulfoxide formation in human liver microsomes as a function of pH, heat, and chemical inhibition indicated no significant involvement of FMOs. Finally, purified recombinant FMO3, the major FMO in human liver, exhibited no significant activity (< 0.1 nmol (nmol of FMO3)-1 min-1) in the formation of the parent sulfoxides of ametryne and terbutryne. Therefore, P450 1A2 alone is likely to be responsible for the hepatic oxidative phase-I metabolism of the s-triazine derivatives in exposed humans.
三嗪类化合物是过去30年中使用最为广泛的除草剂之一。其中一些衍生物被怀疑具有致癌性。在本研究中，确定了人肝微粒体参与三嗪衍生物（莠去津、特丁津、莠灭净和特丁净）代谢的特定I相酶的身份。动力学研究表明，所有检测途径（S-氧化、N-脱烷基化和侧链C-氧化）均呈现双相动力学。低Km值在约1-20μM范围内，而高Km值则高出2个数量级。为进行相关性研究，对30份人肝微粒体制剂进行了7种特定细胞色素P450活性的筛选，并将其与这些三嗪衍生物代谢产物的活性进行了比较。在高亲和力浓度范围内，观察到细胞色素P450 1A2活性存在高度显著的相关性。在低三嗪浓度下，化学抑制作用以α-萘黄酮和呋拉茶碱最为有效，在高底物浓度下，酮康唑和孕二烯酮也有额外的抑制作用。对10种异源表达的细胞色素P450形式的研究表明，几种细胞色素P450酶能够氧化这些三嗪，其亲和力和区域选择性各不相同。已证实细胞色素P450 1A2是参与这些三嗪代谢的低Km细胞色素P450酶。还评估了含黄素单加氧酶（FMO）在人肝中硫甲基衍生物莠灭净和特丁净的硫氧化反应中的潜在作用。人肝微粒体中硫氧化物的形成作为pH、温度和化学抑制的函数表明，FMO没有显著参与。最后，纯化的重组FMO3（人肝中的主要FMO）在莠灭净和特丁净母体硫氧化物的形成中没有显著活性（<0.1 nmol（FMO3的nmol）-¹ min-¹）。因此，单独的细胞色素P450 1A2可能是暴露人群中三嗪衍生物肝脏氧化I相代谢的原因。
