Assessment of parental benzo[a]pyrene exposure-induced cross-generational neurotoxicity and changes in offspring sperm DNA methylome in medaka fish

Abstract

Previous studies have revealed that DNA methylation changes could serve as potential genomic markers for environmental benzo[a]pyrene (BaP) exposure and intergenerational inheritance of various physiological impairments (e.g., obesity and reproductive pathologies). As a typical aromatic hydrocarbon pollutant, direct BaP exposure has been shown to induce neurotoxicity. To unravel the inheritance mechanisms of the BaP-induced bone phenotype in freshwater medaka, we conducted whole-genome bisulfite sequencing of F1 sperm and identified 776 differentially-methylated genes (DMGs). Ingenuity Pathway Analysis (IPA) revealed DMGs were significantly enriched in pathways associated with neuronal development and function. Therefore, it was hypothesized that parental BaP exposure (1 μg/L, 21 days) causes offspring neurotoxicity. Furthermore, the possibility for sperm methylation as an indicator for a neurotoxic phenotype was investigated. The F0 adult brains and F1 larvae were analyzed for BaP-induced direct and inherited toxicity. Acetylcholinesterase activity was significantly reduced in the larvae, together with decreased swimming velocity. Molecular analysis revealed that the marker genes associated with neuron development and growth (alpha1-tubulin, mbp, syn2a, shh, and gap43) as well as brain development (dlx2, otx2, and krox-20) were universally downregulated in the F1 larvae (3-days post-hatching). While parental BaP exposure at an environmentally relevant concentration could induce neurotoxicity in the developing larvae, the brain function of the exposed F0 adults was unaffected. This indicates that developmental neurotoxicity in larvae may result from impaired neuronal development and differentiation, causing delayed brain growth. The present study demonstrates that the possible adverse health effects of BaP in the environment are more extensive than currently understood. Thus, the possibility of multigenerational BaP toxicity should be included in environmental risk assessments.

Short-Term Exposure to Benzo(a)Pyrene Causes Disruption of GnRH Network in Zebrafish Embryos

“…To test whether BaP affects GnRH3 development, we investigated the expression of several markers of antioxidant response and apoptosis. Confirming those reported by others [ 31 ], BaP caused neurotoxicity as revealed by the increased expression of antioxidant and apoptotic genes. Coherently, embryos treated with 5 or 50 nM BaP exhibited increased cell death in several brain areas, as olphactory placode, retina, and hypothalamus.…”

Sperm handling and management in the teleost model fish Japanese medaka (Oryzias latipes)

“…Medaka is used for the evaluation of the spawning cycle as well as the transgenerational studies because the spawning is easily induced, and maturation time is relatively short 1 , 2 . These advantages have made it appropriate for using medaka as a model organism for functional genomics and epigenetics 3 , 4 , reproductive physiology 5 , 6 and toxicology 7 . As medaka has become a more and more popular vertebrate model system in various research fields, methods for sperm handling and management have become necessary for experimental approaches.…”

“…As medaka has become a more and more popular vertebrate model system in various research fields, methods for sperm handling and management have become necessary for experimental approaches. Recently, most researchers that used medaka sperm characteristics as endpoint extracted milt by crushing dissected testes 3 , 8 and to the best of our knowledge, the two available published articles about medaka sperm handling and storage are based on sperm collection after testes dissection 2 , 9 . Extracting the milt directly into the activating medium by abdominal massage has also been used in some previous studies 10 – 12 , however, there are some limitations with this method as visualizing the amount and color of extracted milt are difficult, recording milt volume is impossible and dilution variations are unavoidable.…”

The role of DNA methylation on gene expression in the vertebrae of ancestrally benzo[a]pyrene exposed F1 and F3 male medaka

“…However, methylation data for F1 and F2 generations were not investigated. Deregulation of DNA methylation was highlighted in the sperm of the F1 generation upon larval neurobehavioral and-developmental impacts in response to parental BaP exposure [ 16 ].…”