Polycyclic aromatic hydrocarbons (PAHs), including the Group 1 human carcinogen benzo[a]pyrene (BaP), are produced by the incomplete combustion of organic matter and thus are present in tobacco smoke, charbroiled food and diesel exhaust. The nematode Caenorhabditis elegans is an established model organism, however it lacks the genetic components of the classical mammalian cytochrome P450 (CYP)-mediated BaP-diol-epoxide metabolism pathway. We therefore introduced human CYP1A1 or CYP1A2 together with human epoxide hydrolase (EPHX) into the worm genome by Mos1-mediated Single Copy Insertion (MosSCI) and evaluated their response to BaP exposure via toxicological endpoints. Compared to wild-type control, CYP-humanised worms were characterised by an increase in pharyngeal pumping rate and a decrease in volumetric surface area. Furthermore, BaP exposure reduced reproductive performance, as reflected in smaller brood size, which coincided with the downregulation of the nematode-specific major sperm protein as determined by transcriptomics (RNAseq). BaP-mediated reproductive toxicity was exacerbated in CYP-humanised worms at higher exposure levels. Collagen-related genes were downregulated in BaP-exposed animals, which correlate with the reduction in volumetric size. Whole genome DNA sequencing revealed a higher frequency of T > G (A > C) base substitution mutations in worms expressing human CYP1A1;EPHX which aligned with an increase in DNA adducts identified via an ELISA method (but not classical 32P-postlabelling). Overall, the CYP-humanised worms provided new insights into the value of genome-optimised invertebrate models by identifying the benefits and limitations within the context of the (3Rs) concept which aims to replace, reduce and refine the use of animals in research.

中文翻译：

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表达人细胞色素 P450 （CYP1A1 和 CYP1A2） 通路的基因组修饰秀丽隐杆线虫：环境致癌和药理学研究的实验模型


多环芳烃 （PAH），包括第 1 类人类致癌物苯并[a]芘 （BaP），是由有机物不完全燃烧产生的，因此存在于烟草烟雾、炭烤食品和柴油机尾气中。线虫秀丽隐杆线虫是一种已建立的模式生物，但它缺乏经典哺乳动物细胞色素 P450 （CYP） 介导的 BaP-二醇-环氧化物代谢途径的遗传成分。因此，我们通过 Mos1 介导的单拷贝插入 （MosSCI） 将人 CYP1A1 或 CYP1A2 与人环氧化物水解酶 （EPHX） 一起引入蠕虫基因组，并通过毒理学终点评估它们对 BaP 暴露的反应。与野生型对照相比，CYP 人源化蠕虫的特征是咽泵速度增加和体积表面积减少。此外，BaP 暴露降低了繁殖性能，这反映在较小的育雏大小上，这与转录组学 （RNAseq） 确定的线虫特异性主要精子蛋白的下调相吻合。BaP 介导的生殖毒性在 CYP 人化蠕虫中在较高暴露水平下加剧。胶原蛋白相关基因在 BaP 暴露的动物中下调，这与体积大小的减小相关。全基因组 DNA 测序显示，表达人 CYP1A1 的蠕虫中 T > G （A > C） 碱基取代突变的频率较高;EPHX 与通过 ELISA 方法鉴定的 DNA 加合物的增加一致（但不是经典的 32P 后标记）。 总体而言，CYP 人源化蠕虫通过在 （3R） 概念的背景下确定其益处和局限性，为基因组优化的无脊椎动物模型的价值提供了新的见解，该概念旨在取代、减少和改进动物在研究中的使用。