Dr Shaun Roman

© 2015. Acrylamide is a toxin that humans are readily exposed to due to its formation in many carbohydrate rich foods cooked at high temperatures. Acrylamide is carcinogenic, neurotoxic and causes reproductive toxicity when high levels of exposure are reached in mice and rats. Acrylamide induced effects on fertility occur predominantly in males. Acrylamide exerts its reproductive toxicity via its metabolite glycidamide, a product which is only formed via the cytochrome P450 detoxifying enzyme CYP2E1. Glycidamide is highly reactive and forms adducts with DNA. Chronic low dose acrylamide exposure in mice relevant to human exposure levels results in significantly increased levels of DNA damage in terms of glycidamide adducts in spermatocytes, the specific germ cell stage where Cyp2e1 is expressed. Since cells in the later stages of spermatogenesis are unable to undergo DNA repair, and this level of acrylamide exposure causes no reduction in fertility, there is potential for this damage to persist until sperm maturation and fertilisation. Cyp2e1 is also present within epididymal cells, allowing for transiting spermatozoa to be exposed to glycidamide. This could have consequences for future generations in terms of predisposition to diseases such as cancer, with growing indications that paternal DNA damage can be propagated across multiple generations. Since glycidamide is the major contributor to DNA damage, a mechanism for preventing these effects is inhibiting the function of Cyp2e1. Resveratrol is an example of an inhibitor of Cyp2e1 which has shown success in reducing damage caused by acrylamide treatment in mice.

Metabolism of xenobiotics by cytochrome P450s (encoded by the CYP genes) often leads to bio-activation, producing reactive metabolites that interfere with cellular processes and cause DNA damage. In the testes, DNA damage induced by xenobiotics has been associated with impaired spermatogenesis and adverse effects on reproductive health. We previously reported that chronic exposure to the reproductive toxicant, acrylamide, produced high levels of DNA damage in spermatocytes of Swiss mice. CYP2E1 metabolises acrylamide to glycidamide, which, unlike acrylamide, readily forms adducts with DNA. Thus, to investigate the mechanisms of acrylamide toxicity in mouse male germ cells, we examined the expression of the CYP, CYP2E1, which metabolises acrylamide. Using Q-PCR and immunohistochemistry, we establish that CYP2E1 is expressed in germ cells, in particular in spermatocytes. Additionally, CYP2E1 gene expression was upregulated in these cells following in vitro acrylamide exposure (1 µM, 18 h). Spermatocytes were isolated and treated with 1 µM acrylamide or 0.5 µM glycidamide for 18 hours and the presence of DNA-adducts was investigated using the comet assay, modified to detect DNA-adducts. Both compounds produced significant levels of DNA damage in spermatocytes, with a greater response observed following glycidamide exposure. A modified comet assay indicated that direct adduction of DNA by glycidamide was a major source of DNA damage. Oxidative stress played a small role in eliciting this damage, as a relatively modest effect was found in a comet assay modified to detect oxidative adducts following glycidamide exposure, and glutathione levels remained unchanged following treatment with either compound. Our results indicate that the male germ line has the capacity to respond to xenobiotic exposure by inducing detoxifying enzymes, and the DNA damage elicited by acrylamide in male germ cells is likely due to the formation of glycidamide adducts. © 2014 Nixon et al.

Retinoids, vitamin A (retinol) and its metabolic derivatives, are required for normal vertebrate development. In murine embryonic stem (ES) cells, which remain undifferentiated when cultured in the presence of LIF (leukemia inhibitory factor), little metabolism of exogenously added retinol takes place. After LIF removal, ES cells metabolize exogenously added retinol to 4-hydroxyretinol and 4-oxoretinol and concomitantly differentiate. The conversion of retinol to 4-oxoretinol is a high-capacity reaction because most of the exogenous retinol is metabolized rapidly, even when cells are exposed to physiological (¿1 µM) concentrations of retinol in the medium. No retinoic acid or 4-oxoRA synthesis from retinol was detected in ES cells cultured with or without LIF. The cytochrome P450 enzyme CYP26 (retinoic acid hydroxylase) is responsible for the metabolism of retinol to 4-oxoretinol, and CYP26 mRNA is greatly induced ( > 15-fold) after LIF removal. Concomitant with the expression of CYP26, differentiating ES cells grown in the absence of LIF activate the expression of the differentiation marker gene FGF-5 whereas the expression of the stem cell marker gene FGF-4 decreases. The strong correlation between the production of polar metabolites of retinol and the differentiation of ES cells upon removal of LIF suggests that one important action of LIF in these cells is to prevent retinol metabolism to biologically active, polar metabolites such as 4-oxoretinol.