The impact of the environmental hazard, acrylamide, on the male reproductive tract and transgenerational phenotype

The impact of exposure to environmental hazards on personal health and the health of our offspring has become of great importance over recent decades. The effect of gestational exposures across generations have been widely investigated, but what is less well understood is the impact of paternal preconception exposure. The research presented utilised the environmental hazard, acrylamide as a model to facilitate a greater understanding of paternal preconception exposure, and the multi- and transgenerational consequences. Throughout this research, the effect of acrylamide on the male reproductive tract was investigated, following single and multigenerational exposures, and the importance of the enzyme CYP2E1, known to modulate acrylamide-toxicity. CYP2E1 is a P450 metabolising enzyme, localised to the endoplasmic reticulum and/or the mitochondria throughout various tissues of the body, including in the pachytene spermatocytes of the testis and epithelial cells of the epididymis within the male reproductive tract. It was hypothesised that acrylamide would alter CYP2E1 protein abundance and DNA fragmentation in the male reproductive tract, and multigenerational exposure of the male germline would result in altered phenotypes in progeny. An in vivo acute exposure model and in vitro cell culture were utilised to establish the effect of acrylamide on CYP2E1 in the male reproductive tract. Prior to the examination of acrylamide exposures, we performed immunoblotting analysis of CYP2E1 and optimisation of subcellular fractionation techniques to isolate and purify the components of the cell that harbour CYP2E1. Mitochondrial fractions from liver tissue were extracted and purified, while microsomal fractions from the endoplasmic reticulum require further optimisation, and thus this technique was not utilised for further analyses. From the in vivo and in vitro exposure regimes it was determined that acrylamide increased the abundance of CYP2E1 in the spermatocytes of the testis (150% of vehicle) and mECap18 cells (130% of vehicle) and elevated DNA fragmentation in both the mECap18 cells (120% of vehicle) and mature spermatozoa (≥120% of vehicle). To postulate potential mechanisms of this induction comparison to the well-characterised CYP2E1 substrate ethanol was performed. Ethanol exposure also elevated CYP2E1 abundance in spermatocytes (130% of vehicle) and the mECap18 cells (150% of vehicle), in addition to Cyp2e1 transcript expression in the mECap18 cells only (≤500% of vehicle). To model environmental multigenerational exposure, a chronic regime of acrylamide exposure at a human-relevant dose following the paternal germline was employed. Paternal and grand-paternal acrylamide exposure modified the response to acrylamide in male offspring with significantly altered DNA fragmentation in mature spermatozoa and CYP2E1 abundance in spermatocytes with or without acrylamide exposure of the progeny. Additionally, acrylamide exposure at the human-relevant dose of ≈0.2 mg/kg bw/day resulted in the transgenerational phenotype of decreased testis to body weight ratio in the male F2 progeny following ancestral exposure to acrylamide (75-80% of unexposed lineage). The experiments outlined herein demonstrate novel understanding of acrylamide and its effects on the male reproductive tract, and the impact of preconception exposure to the reproductive health of multiple generations. These data provide new insight into the transgenerational impact of an environmental hazard at under a human-relevant regime, following paternal preconception exposure, to expand our understanding of environmental health.