Mutational signatures of genome-modified Caenorhabditis elegans expressing the human cytochrome P450 (CYP1A1 and CYP1A2) pathway

Polycyclic aromatic hydrocarbons (PAHs), such as 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 lacks the genetic components of the classical mammalian cytochrome P450 (CYP)-mediated BaP-diol-epoxide metabolism pathway thus CYP1A1 or CYP1A2 together with human epoxide hydrolase (EPHX) was introduced into the worm genome, thereby allowing to study potential physiological, genomic and transcriptional changes after BaP exposure in these CYP-humanised C. elegans strains. Whole genome sequencing revealed a higher frequency of T>G base substitution mutations in worms expressing human CYP1A1;EPHX thereby demonstrating the amenity of introducing human genes into the C. elegans genome and their utility to serve as a model for environmental carcinogenesis and pharmacological research. Overall design: Whole genome sequencing was carried out via the Illumina NovaSeq platform to detect SNPs, InDels, SVs and CNVs in C.elegans exposed to no, a low (40 µM) or high (640 µM) concentrations of benzo[a]pyrene from synchronized L1 stage to L4 stage. Three strains were used , namely a background strain, or strains that were genome-modified to express either the human CYP1A1 and EPHX or CYP1A2 and EPHX .

多环芳烃（Polycyclic Aromatic Hydrocarbons, PAHs），例如苯并[a]芘（benzo[a]pyrene, BaP），是有机物不完全燃烧的副产物，广泛存在于烟草烟雾、炭烤食品及柴油尾气中。秀丽隐杆线虫（Caenorhabditis elegans）缺失经典哺乳动物细胞色素P450（cytochrome P450, CYP）介导的BaP-二醇-环氧化物代谢通路的遗传组分，因此研究人员将人类CYP1A1或CYP1A2与环氧化物水解酶（epoxide hydrolase, EPHX）共同导入该线虫的基因组，由此可在这些经人类基因修饰的秀丽隐杆线虫品系中，探究BaP暴露后潜在的生理、基因组及转录组变化。全基因组测序结果显示，表达人类CYP1A1与EPHX的线虫品系中，T>G碱基替换突变的频率显著更高，这证实了将人类外源基因导入秀丽隐杆线虫基因组的可行性，同时验证了该基因工程线虫模型在环境致癌作用研究与药理学实验中的应用价值。整体实验设计：本研究依托Illumina NovaSeq平台开展全基因组测序，以检测秀丽隐杆线虫在不同浓度BaP暴露后的单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs）、插入缺失突变（Insertions and Deletions, InDels）、结构变异（Structural Variations, SVs）与拷贝数变异（Copy Number Variations, CNVs）。处理流程为：将同步化的L1期幼虫培养至L4期，设置空白对照组、低浓度（40 μM）组与高浓度（640 μM）组。实验共使用3种线虫品系：野生型背景品系，以及经基因组修饰分别表达人类CYP1A1与EPHX、CYP1A2与EPHX的工程化品系。