Unraveling the Genetic and Epigenetic Pathways Underlying Transgenerational Vertebral Deformity Induced by Benzo[a]pyrene Using the Unique Double Transgenic Medaka Bone Model

以獨特的雙轉基因青鱂魚骨頭研究模型闡明苯並[a]芘誘導跨代骨骼畸形的遺傳及表觀遺傳通路

Student thesis: Doctoral Thesis

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Award date24 Jul 2020

Abstract

Benzo[a]pyrene (BaP), a model polycyclic aromatic hydrocarbon (PAH), is released into the environment from both natural and anthropogenic sources, such as forest fires, oil spills, volcanic eruptions, and incomplete combustion of fossil fuels, tobacco etc. As an organic persistent pollutant, it presents ubiquitously in the ecosystem. BaP is genotoxic and carcinogenic in vertebrates, and studies have showed that BaP exposure impairs development of bone cells, promotes bone loss and fracture, induces vertebral deformities, and delays bone healing in mammals. New evidences in mutilgenerational studies using fish indicate that F0 ancestral exposure to BaP (1 µg/L, 21 days) is able to induce bone deformity in medaka (Oryzias latipes) larvae in the F1-F3 generation, associated with the dysregulated expression of col10a1 and osx in whole larvae. However, when and how the impairment of vertebrae occurs during bone development remain to be elucidated.

Moreover, paternal preconception exposure to BaP alters the expression profiles of miRNA and mRNA in early F1 mouse embryos, including miRNAs related to bone metabolism and genes encoding epigenetic enzymes (epigenetic genes). The previous studies discovered BaP is a transgenerational skeletal toxicant, and the transgenerational bone deformity in F3 larvae is persistent to adulthood. Specifically, a bone thinning phenotype was identified in F3 medaka adult male ancestrally (F0) exposed to BaP, associated with the aberrant expression of selected bone miRNAs/target genes in the vertebrae. However, the underlying epigenetic and genetic mechanisms remain virtually unknown.

In this study a multigenerational approach was undertaken to assess the impacts of environmental BaP on bone metabolism of F1-F3 medaka offspring and decipher the genetic and epigenetic mechanisms underlying the transgenerational bone deformities induced by F0 ancestral BaP exposure. The double transgenic col10a1:nlGFP/osx:mCherry medaka fish, a unique bone research model, was exposed to an environmentally realistic concentration of BaP at 1 μg/L for 21 days. The bone development of fish was evaluated by an array of endpoints including: temporal (in vivo) and spatial (in situ) expression of col10a1:nlGFP and osx:mCherry, abundance and distribution of osteoblasts (osteoblast progenitors and premature osteoblasts) and bone mineralization at tissue level during early bone development (9 days post fertilization (dpf) - 17 days post harching (dph)) in F1-F3 offspring. Ancestral BaP-exposure delayed the onset of col10a1:nlGFP and osx:mCherry expression and reduced the abundance of col10a1:nlGFP-positive osteochondral progenitors cells and col10a1:nlGFP/osx:mCherry double-positive premature osteoblasts during critical windows of early vertebral bone formation, resulting in reduced bone mineralization in embryos (0 dph) and larvae (17 dph), compressed vertebral segments in larvae (17 dph), and reduced bone thickness in adult male fish (6 months) of the F1-F3 generations with ancestral exposure. Regulatory regions of col10a1 and osx were identified as potential targets of epigenetic modifications underlying the transgenerational inheritance of BaP bone toxicity. The current study provides novel knowledge of the underlying mechanisms at the cellular and molecular levels of the transgenerational toxicity of BaP.

To connect the transgenerational bone thinning phenotype at tissue level in F3 male fish with the molecular level (expression of miRNA and genes), deciphering the genetic and epigenetic pathways underlying transgenerational vertebral deformity induced by ancestral BaP exposure, the transcriptome and small RNA transcriptome were analyzed using RNA-seq and small RNA-seq, respectively. Bioinformatics analysis on the differentially expressed genes (DEGs) using DAVID identified the altered biological processes, molecular factors, cellular components and KEGG pathways, while the upstream regulators, canonical pathways, regulator effects and molecular networks were revealed using the Ingenuity Pathways Analysis (IPA). Prediction of conserved and novel miRNAs was performed using the miRDeep2 algorithm, followed by identification of differentially expressed miRNAs (DEMs) with small RNA analysis pipelines. Prediction of DEMs targeting on DEGs was performed using the miRanda algorithm. RT-qPCR was used to validate the selected DEMs and DEGs to confirm the miRNA-mRNA pairing involved in the transgenerational BaP bone toxicity.

Using a threshold cutoff of adj-p value < 0.05, a total of 1094 DEGs (706 up-DEGs and 361 down-DEGs) were identified in the transcriptomic analysis results, while 10 DEMs (1 up-DEM and 9 down-DEMs) were found with the threshold settings (p value < 0.05, log2-foldchange < -1 or >1) in the small RNA transcriptome data. Gene enrichment, ontology and pathway analysis uncovered two major canonical pathway clusters: bone metabolism pathways and typical BaP-responsive canonical pathways.

The bone metabolism pathways include role of osteoblasts, osteoclasts and chondrocytes in rheumatoid arthritis, WNT pathway, BMP signaling pathway, Rank signaling in osteoclast, calcium signaling, protein ubiquitination pathway, RhoA signaling, regulation of actin-based motility by Rho and signaling by Rho family GTPases, which highlights the major roles of the impaired bone formation and minor actions of the augmented bone resorption underlying the transgenerational BaP bone toxicity. Pairs of gene/miRNA cluster (BMP3/ola-miR-96-5p/ola-miR-181b-5p/ola-miR-199a-5p/ola-miR-205-5p/ola-miR-455-5p, FRZB/ola-miR-1-3p/ola-miR-183-5p/ola-miR-206, SFRP5/ola-miR-96-5p/ola-455-5p) were identified to be involved in the inhibition of bone formation. By contrast, gene/miRNA clusters (C-FOS/ola-miR-183-5p/ola-miR-205-5p) contributed to the promotion of bone resorption. The reduced bone formation is likely the decisive factor in the increased overall bone turnover, associated with the transgenerational BaP bone thinning in F3 adult males. The typical BaP-responsive canonical pathways, including AHR signaling, xenobiotic metabolism signaling and NRF2-mediated oxidative stress response, were also involved in the regulation of osteoblastogenesis and osteoclastogenesis underlying the transgenerational BaP bone toxicity.

In addition of being regulated by miRNAs, these dysregulated genes of bone metabolism were subjected to the transcription and/or post-transcription regulation by epigenetic enzymes (e.g. HDAC6, HDAC7, KDM5B) in the transgenerational BaP bone toxicity, and these dysregulated epigenetic genes were also subjected to the regulation of miRNAs (KDM5B/ola-miR-8071, KAT6B/ola-miR-8071).

This study has deciphered the miRNA-mRNA regulatory networks underlying the transgenerational BaP bone toxicity in the F3 adult male fish. The involvement of histone modification enzyme genes in the transgenerational bone toxicity indicates that epigenetic modifications on these identified candidate genes (e.g. SFRP5, FRZB, BMP3 and C-FOS), warrants for further studies. The dysregulated miRNAs identified in this study may serve as potential biomarkers for diagnosis of osteoporosis (OP).

Overall, these novel results highlight the miRNA-mRNA regulatory roles in the transgenerational BaP bone toxicity. This comprehensive study provides a toxicological basis for multigenerational risk assessment associated with BaP in the aquatic environments. The findings of BaP induced transgenerational bone impairments in fish also shed light on the potential multigenerational impacts of BaP exposure in other vertebrates including human.

    Research areas

  • Benzo[a]pyrene, Molecular and cellular mechanisms, Transgenerational inheritance, Bone development and metabolism, microRNAmRNA networks, Transgenic col10a1:nlGFP/osterix:mCherry medaka