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. 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），其与暴露亲本的后代出现多种发育与行为不良结局密切相关。本研究以斑马鱼为模型，探究亲本孕前膳食暴露于BaP后，F1及F2代子代的性别依赖性不良结局，并与卵子、精子以及受精后10小时（hours post fertilization, hpf）胚胎的转录组学和表观遗传学变化进行对比分析。

本研究将成年野生型（5D品系）斑马鱼以1%体重的投喂量、每日两次的频率，喂食含708 μg BaP/g饲料（实测浓度）的食物，每日每尾鱼的BaP暴露剂量为14 μg BaP/g，连续投喂21天。采用交叉交配设计进行繁育，同时测定亲本（F0代）的行为与繁殖指数。

分别在受精后96小时（hpf）对F1及F2代幼体的行为进行检测，并在F1代个体性成熟后再次开展行为学评估。与对照组相比，BaP暴露对F0代成鱼的行为无显著影响，但两性F1代成鱼的运动行为均显著增强。亲本BaP暴露后，F1与F2代幼体的光运动响应实验（96 hpf）结果均发生显著改变。

为解析亲本性别依赖的分子机制，本研究分别通过RNA测序（RNA-seq）和简化基因组亚硫酸氢盐测序（Reduced Representation Bisulfite Sequencing, RRBS），检测F0代精子、卵子以及F1代4种交配组合的10 hpf胚胎中BaP介导的差异基因表达与DNA甲基化变化。其中，BaP暴露雄鱼与对照雌鱼交配获得的胚胎，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DMRs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。

亲本膳食BaP暴露可引发持续性的行为改变，其中雄性生殖系对多代不良结局的贡献最为显著。

另一组实验中，将性成熟（120天受精后，days post fertilization, dpf）的5D品系斑马鱼分为两组，分别投喂仅含丙酮的 TetraMin® 热带鱼饲料，以及含BaP的 TetraMin® 热带鱼饲料（每尾鱼BaP暴露剂量为25 μg，对应饲料中BaP浓度为1250 μg/g）。每个处理组设置5个重复养殖箱，每箱配对饲养2尾斑马鱼（每组总样本量N=5个重复箱，共20尾鱼），先驯化1周，期间水温维持在25.5~28℃，每日投喂未处理的饲料与Gemma 300微米粒径饲料各一次。暴露期间，每日以1%体重的量两次投喂对应剂量的BaP处理饲料，同时每日一次投喂Gemma 300微米粒径饲料，连续21天。

第21天结束后，采用交叉交配设计开展繁育，分为4个实验组：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（此时已停止暴露）收集卵子以评估繁殖成功率。第24天（雌鱼）与第25天（雄鱼）对斑马鱼实施安乐死，采集性腺、肝脏与脑组织并进行生物样本库储存，用于后续分析。此外，还收集了BaP暴露组与对照组斑马鱼的精子或卵子，以及4种交配组合的10 hpf胚胎，用于RNA-seq与DNA甲基化分析。