The genetic consequences of paternal acrylamide exposure and potential for amelioration

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

Mouse spermatocytes express CYP2E1 and respond to acrylamide exposure

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

Acrylamide exposure elicits an increase in CYP gene expression in the male germ line.

<p>Gene expression levels were analysed in isolated spermatocytes by Q-PCR following incubation with acrylamide. Cells were isolated and cultured with acrylamide (1 uM, 18 h), RNA was extracted, reverse transcription performed and QPCR conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094904#s2" target="_blank">Materials and Methods</a>. CYP2E1 and CYP1B1 gene expression was significantly increased in spermatocytes following acrylamide exposure (<i>F</i>_1,16 = 20.1, *<i>p</i><0.05). Data are depicted as transformed values (2e−ΔΔC(t)) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094904#s2" target="_blank">Materials and Methods</a>, and is representative of n = 3 experiments (Mean ±SEM).</p

Oxidative stress may play a role in the DNA damage induced by acrylamide in spermatocytes.

<p>(A) The comet assay was conducted on spermatocytes treated with acrylamide (1 µM, 18 h) or glycidamide (0.5 µM, 18 h) in the presence or absence of hOGG1. Significant levels of DNA damage were not detected in the acrylamide treated spermatocytes in either the presence or absence of hOGG1. A modest but significant increase in Tail DNA % was observed in spermatocytes following glycidamide treatment in the absence of hOGG1 (<i>F</i>_7,677 = 134.4, *<i>p</i><0.001); however, hOGG1 treatment resulted in greater detection of Tail DNA % following glycidamide exposure. Treatment with H₂O₂ (500 µM, 5 min) was used as a positive control and produced significant increases in Tail DNA % in the presence of either enzyme (***<i>p</i><0.001). (B) Glutathione (GSH) levels in spermatocytes were measured using a GSH assay following acrylamide (1 µM, 18 h) or glycidamide exposure (0.5 µM, 18 h). No significant differences in GSH levels in male germ cells were observed following acrylamide or glycidamide treatment. Additionally, relatively low GSH levels were found in spermatocytes (0.19 µM) compared to P19 embryonal carcinoma cells (2.14 µM, <i>F</i>_3,6 = 1676.2, ***<i>p</i><0.001). Data are representative of n = 3 experiments (Average ±SEM).</p

Gene expression of CYP2E1 in the male germ line.

<p>Q-PCR analysis of CYP2E1 mRNA expression in mouse testis at different developmental stages 2, 6, 11, 14, 18, 22, and 36 d after birth, and adult (older than 56 d). Expression was also examined in isolated male germ cells, spermatogonia, spermatocytes and spermatids. Data are representative of n = 3 experiments and depicted as transformed values, 2e−ΔC(t) (Mean ±SEM), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094904#s2" target="_blank">Materials and Methods</a>. CYP2E1 gene expression was found predominantly in spermatogonia. Statistically significant differences were found in spermatogonia, 11, 14 and 18 d compared to 2 d testis (<i>F</i>_10,79 = 4.0,*<i>p</i><0.05).</p

Protein expression of CYP2E1 in the male germ line.

<p>(A) Immunolocalisation of CYP2E1 (red staining) in testis sections at different developmental stages, 11 d after birth, 22 d after birth, 36 d after birth, and adult (older than 56 d). Sections were sequentially probed with anti-CYP2E1 and appropriate secondary antibody before being counter-stained with DAPI (blue staining). CYP2E1 protein expression was found in spermatocytes at 22 d after birth to adult testis, with weaker staining observed in spermatids. Diagram outlining where different male germ cell types reside in the seminiferous tubule is shown in supplementary data. (B) Immunolocalisation of CYP2E1 in isolated spermatogonia, spermatocytes and spermatids, showing CYP2E1 expression (green staining) in spermatocytes. Germ cells were probed with anti-CYP2E1 and anti-GCNA, which labels spermatogonia and spermatocytes, or anti-PKA[C], which labels spermatids (red staining). Both tissue sections and cells were probed with rabbit serum in the absence of primary antibody as a control, which did not produce a detectable signal (blank images not shown). Scale bars equal to 50 µm.</p

Acrylamide treatment (1 µM, 18 h) or glycidamide treatment (0.5 µM, 18 h) did not impact on spermatocyte morphology or viability.

<p>(A) Adult mouse spermatocytes were treated with acrylamide (1 µM, 18 h) or glycidamide (0.5 µM, 18 h) and dual stained with FITC-PNA, which labels the developing acrosome (green), and PI (red) to observe cell morphology. Scale bar is equal to 50 µm. (B) The viability of spermatocytes treated with acrylamide (1 µM, 18 h) or glycidamide (0.5 µM, 18 h) assessed by trypan blue exclusion. Data are representative of n = 3 experiments, measured in triplicate (Mean, ±SEM), and >200 cells were scored per replicate. At the doses used in the current study, no differences in cell morphology or viability were observed following treatment with acrylamide or glycidamide.</p

Acrylamide and glycidamide induces DNA damage in the male germ line.

<p>DNA damage was assessed in spermatocytes treated with acrylamide or glycidamide using the comet assay in the presence or absence of FPG. (A) Representative comet images from control, acrylamide (1 µM, 18 h), glycidamide (0.5 µM, 18 h) and H₂O₂ (500 µM, 5 min) treated spermatocytes. (B) The average Tail DNA % was assessed for each sample and in the absence of FPG, a modest increase in Tail DNA % was observed in spermatocytes treated with glycidamide (<i>F</i>_7,663 = 61.7, *<i>p</i><0.05). In the presence of FPG however, both acrylamide and glycidamide produced significant increases in Tail DNA % (***<i>p</i><0.001) with a greater response observed following glycidamide exposure. Treatment of spermatocytes with H₂O₂ (500 µM, 5 min) was used as a positive control for damage, and induced significant increases in Tail DNA % in both the presence and absence of FPG. (C) Spermatocytes were assessed for DNA damage using the FPG comet assay following acrylamide exposure, at doses between 10 nM to 10 µM for 18 h. Spermatocytes treated with H₂O₂ (500 µM, 5 min) were used as a positive control for DNA damage. Significant increases in Tail DNA % were observed in spermatocytes following 100 nM acrylamide treatment and above (<i>F</i>_5,507 = 83.6, ***<i>p</i><0.001). Significant increases in Tail DNA % were also observed in cells treated with H₂O₂ (***<i>p</i><0.001).(D) Spermatocytes were assessed for DNA damage using the FPG comet assay following glycidamide exposure, at doses between 5 nM to 5 µM for 18 h (<i>F</i>_5,491 =  **<i>p</i><0.01). Spermatocytes treated with H₂O₂ (500 µM, 5 min) were used as a positive control for DNA damage. Significant increases in Tail DNA % were observed in spermatocytes following 5 nM glycidamide treatment and above. All data are representative of n = 3 experiments (Mean ±SEM).</p

Acrylamide is metabolized in spermatocytes to glycidamide via CYP2E1.

<p>The Isolated spermatocytes were treated with acrylamide (1 µM, 18 h), glycidamide (0.5 µM, 18 h), resveratrol (0.1 µM, 18 h), or a combination of acrylamide (1 µM) and resveratrol (0.1 µM, 18 h) or glycidamide (0.5 µM) and resveratrol (0.1 µM, 18 h). DNA damage was assessed by the comet assay modified by the addition of the FPG or hOGG1 enzyme. Significant levels of DNA damage were detected in the acrylamide treated spermatocytes in the presence of FPG (<i>F</i>_13,877 = 92.5, ***<i>p</i><0.001) or hOGG1 (**<i>p</i><0.001). A significant increase in DNA damage was observed with glycidamide treatment in the presence of FPG (***<i>p</i><0.001). Resveratrol treatment on its own had no effect on the level of DNA damage in spermatocytes with FPG or hOGG1 treatment. DNA damage assessed in cells treated with the combination of acrylamide and resveratrol was not significantly different from control. Treatment with glycidamide and resveratrol caused a significant induction in DNA damage when treated with FPG (***<i>p</i><0.001), but not hOGG1. Spermatocytes treated with H₂O₂ (500 µM, 5 min) were used as a positive control for DNA damage. Significant increases in Tail DNA % were observed in cells treated with H₂O₂ in the presence of either FPG or hOGG1 (***<i>p</i><0.001). All data are representative of n = 3 experiments (Mean ±SEM).</p