Ancestral Benzo[a]pyrene Exposure Induces Multigenerational Skeletal Deformities and Neurological Impairment in Fish: Role of DNA Methylation

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are widely present in aquatic environments and have been reported to cause different developmental impacts on mammals and teleosts. Benzo[a]pyrene (BaP), a typical representative of PAH, has been shown to induce skeletal deformities in both mice and fish by skewing the balance of bone formation and resorption. Additionally, skeletal deformities have been consistently observed across generations (F1, F2, and F3) in fish, although only the F0 generation has been directly exposed to BaP.

Epigenetic mechanisms (e.g., DNA methylation, histone modification, and non-coding miRNAs) have been regarded as the important regulators responsible for transgenerational effects, and BaP was previously reported to be an epigenetic modifier. Therefore, freshwater medaka fish were used to investigate whether ancestral BaP exposure-induced (F0) skeletal deformities (reduced bone thickness in adult males) could be explained by DNA methylation changes in fish vertebrae.

Experiments were conducted to explore the causes of skeletal deformities at the tissue, genetic, and epigenetic levels. Exposure experiments to BaP were performed by Mo (see details in Mo’s thesis, 2020). Briefly, adult medaka (6 months old) were exposed to BaP at 1 μg/L for 21 days and embryos were collected and raised to adulthood to obtain F1, F2, and F3.

The osteoblast (bone-formation cells) population was assessed by trichrome staining on bone tissue coupled with histometric analysis. The methylome of vertebrae and sperm were examined by Whole-genome bisulfite sequencing (WGBS). The transcriptome of vertebrae was profiled by RNA-seq and validated via real-time qPCR.

It was found that ancestral BaP exposure significantly reduced the osteoblast (bone formation cells) population in the F1 and F3 generations. Functional analysis determined that differentially methylated genes (DMGs) were associated with osteoblastogenesis (F1 and F3), chondrogenesis (F1 and F3), and osteoclastogenesis (F3). However, RNA-seq data did not support the role of DNA methylation in the regulation of genes involved in skeletogenesis because there was very little correlation between the level of differential methylation and expression of the genes related to skeletogenesis. Notably, RNA-seq and WGBS data indicated that genes related to nervous system development are more sensitive to ancestral BaP exposure. Sperm methylome was further examined to trace the origin of methylation marks in vertebrae. DMGs in both F1 and F2 sperm further supported that the predominant impacts of ancestral BaP exposure were on neurological activities. Additionally, cxcr3, sparc, and col12a1, involved in bone remodeling, were conserved among F1 and F2 sperm and F3 vertebrae, which were likely to partially explain the transgenerational skeletal deformities in the F3.

Because methylome data of sperm suggested potential impacts on neurological activity, the exposure was repeated to explore the neurotoxicity following parental BaP exposure. Parental exposure did not cause neurotoxicity to adult fish (F0) but induced detoxification responses (upregulated expression of cyp1a1 and ugt) in the liver of adult males. Interestingly, neurotoxicity was observed in the developing larva (F1), including decreased swimming speed and inhibited acetylcholinesterase activity. Moreover, genes involved in axon guidance (arhgef7), synaptogenesis (adcy8 and nlgn2), neuron development and growth (alpha1-tubulin, mbp, syn2a, shh, and gap43), and brain development (dlx2, otx2, and krox-20) were universally downregulated in the F1 larvae. Therefore, methylation marks in sperm might serve as good markers for multigenerational or even transgenerational neurotoxicity induced by BaP.

Date of Award	7 Jan 2022
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Wai Ting Doris AU (Supervisor) & Yuen Chong Richard KONG (Supervisor)