Multigenerational Effects of Dietary Benzo[a]pyrene Exposure on Behavior, Gene Expression, and DNA Methylation. Multigenerational Effects of Dietary Benzo[a]pyrene Exposure on Behavior, Gene Expression, and DNA Methylation

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is implicated in many developmental and behavioral adverse outcomes in offspring of exposed parents. Following a dietary preconceptional exposure to BaP in zebrafish, the objective of this study was to compare parental sex-dependent adverse outcomes in F1 and F2 offspring with the transcriptomic and epigenetic changes in eggs, sperm, and 10-hour post fertilization (hpf) embryos. Adult wild-type (5D) zebrafish were fed 708 µg BaP/g diet (measured) at a rate of 1% body weight twice/day (14 µg BaP/g fish/day) for 21 days. Fish were spawned using a crossover design and parental (F0) behavior and reproductive indexes measured. In offspring behavioral effects were measured at 96 hours post fertilization (hpf) in F1 & F2 larvae, and again when F1s were adult. Compared to controls, there was no significant effect of BaP exposure on adult behavior in F0, but locomotor behavior was significantly increased in F1 adults of both sexes. Larval behavior (96 hpf, photomotor response assay) was significantly altered in both the F1 and F2 generations following parental BaP exposure. To assess parental sex-dependent molecular mechanisms, BaP-mediated differential gene expression and DNA methylation changes were measured using RNAseq and RRBS, respectively, on F0 sperm and eggs and the 10 hpf embryos from all four crosses in F1 generation. Embryos resulting from the BaP male and control female cross had the most differentially methylated regions (DMRs) and differentially expressed genes. Some DMRs were associated with genes encoding chromatin modifying enzymes suggesting regulation of chromatin conformation by DNA methylation. Parental dietary BaP exposure caused persistent behavioral changes wherein the male germline contributed most significantly to the multigenerational adverse outcomes. Overall design: Sexually mature (120 dpf) 5D strain of zebrafish were fed either acetone alone or BaP-treated (25 μg/g fish equivalent to 1250 μg/g food, respectively) TetraMin® Tropical Flakes. Paired (2x2) zebrafish in five replicate tanks per treatment group (N=5 replicate tanks for a total 20 fish/group) were allowed to acclimate for a week while maintained at 25.5-28°C and fed twice daily with untreated flakes and Gemma 300 micron food. During the exposure, fish were fed 1% body weight twice daily of the corresponding dose of BaP-treated flake food and once daily Gemma 300 micro food for 21 days. At the end of day 21, a cross-over breeding design was implemented to assess sex-specific contributions into the following groups: control males x control females, BaP females x control males, BaP males x control females, and BaP males x BaP females. On days 22 and 23 (no longer being exposed), eggs were collected to determine reproductive success. On day 24 (females) and day 25 (males), fish were euthanized, and gonad, liver, and brain were collected biobanked for future analysis. Furthermore, sperm or eggs from the BaP exposed and control fish, along with 10 hpf embryos from each cross (4 crosses) were collected for RNA-seq and DNA methylation analysis.

苯并[a]芘（Benzo[a]pyrene, BaP）是一种多环芳烃（polycyclic aromatic hydrocarbon, PAH），已被证实可引发暴露亲代的子代出现多种发育与行为不良结局。本研究以斑马鱼为模型，针对孕前饮食暴露于BaP的处理组，旨在比较亲代性别依赖的F1、F2子代不良结局，并分析卵、精子以及受精后10小时（hours post fertilization, hpf）胚胎的转录组与表观遗传变化。

将成年野生型（5D品系）斑马鱼以每日两次、每次按体重1%的投喂量，喂食经实测含708 μg BaP/g的饲料，即每克鱼每日暴露14 μg BaP，持续21天。采用交叉交配设计进行产卵，并检测亲代（F0）的行为与繁殖指标。

分别在受精后96小时（96 hpf）对F1、F2仔鱼开展行为检测，且在F1个体达到成年阶段时再次进行行为学评估。与对照组相比，BaP暴露未对F0成年个体的行为产生显著影响，但两性F1成年个体的运动行为均显著增强。亲代BaP暴露后，F1与F2代仔鱼的行为（96 hpf，光运动响应实验）均发生显著改变。

为探究亲代性别依赖的分子机制，本研究分别通过RNAseq与RRBS技术，对F0代的精子、卵以及F1代4种交配组合的10 hpf胚胎进行BaP介导的差异基因表达与DNA甲基化变化检测。结果显示，由BaP暴露雄鱼与对照雌鱼交配产生的胚胎，其差异甲基化区域（differentially methylated regions, DMRs）与差异表达基因数量最多。部分DMR与编码染色质修饰酶的基因相关，提示DNA甲基化可通过调控染色质构象发挥作用。

亲代饮食BaP暴露可引发持续性行为改变，其中雄性生殖系对多代不良结局的贡献最为显著。

实验设计概述：将性成熟（120 dpf）的5D品系斑马鱼分为两组，分别投喂仅含丙酮的对照饲料，或BaP处理的TetraMin®热带鱼饲料（剂量为每克鱼25 μg BaP，对应饲料中BaP含量为1250 μg/g）。每个处理组设置5个重复缸，每缸配对2条斑马鱼（每组共20条鱼，N=5个重复缸），先适应饲养1周，期间维持水温25.5~28℃，每日投喂两次未处理的饲料与Gemma 300微米粒径饲料。暴露期间，每日两次按体重1%的投喂量给予对应剂量的BaP处理饲料，同时每日一次投喂Gemma 300微米饲料，持续21天。第21天结束时，采用交叉交配设计构建以下4组以评估性别特异性贡献：对照雄鱼×对照雌鱼、BaP暴露雌鱼×对照雄鱼、BaP暴露雄鱼×对照雌鱼、BaP暴露雄鱼×BaP暴露雌鱼。第22、23天（不再进行暴露处理）收集鱼卵以评估繁殖成功率。第24天（雌鱼）与第25天（雄鱼）处死后，采集性腺、肝脏与脑组织并冻存保藏以备后续分析。此外，收集BaP暴露组与对照组鱼的精子或卵，以及4种交配组合的10 hpf胚胎，用于RNA-seq与DNA甲基化分析。