Chronic acrylamide exposure in male mice induces DNA damage to spermatozoa; Potential for amelioration by resveratrol

Reproductive Toxicology 63 (2016) 1–12

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Chronic acrylamide exposure in male mice induces DNA damage to spermatozoa; Potential for amelioration by resveratrol Aimee L. Katen a,b , Simone J. Stanger a,b , Amanda L. Anderson a,b , Brett Nixon a,b , Shaun D. Roman a,b,∗ a b

Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales 2308, Australia The Priority Research Centres for Reproductive Sciences and Chemical Biology, University of Newcastle, Callaghan, New South Wales 2308, Australia

a r t i c l e

i n f o

Article history: Received 26 November 2015 Received in revised form 3 May 2016 Accepted 7 May 2016 Available online 9 May 2016 Keywords: Acrylamide Glycidamide Resveratrol DNA damage CYP2E1 Spermatozoa

a b s t r a c t Humans are chronically exposed to acrylamide since carbohydrate rich foods contain the toxicant as a result of cooking at high temperatures. While acrylamide is unreactive with DNA, it is readily oxidised to glycidamide, which adducts with DNA. This metabolism occurs via the enzyme, cytochrome P450, family 2, subfamily E, polypeptide 1 (CYP2E1). Acrylamide was administered to male CD1 mice for three or six months at a dose of 0.18 mg/kg bodyweight/day. DNA damage was detected in germ cells and mature spermatozoa of exposed mice without compromising their overall fertility. The use of resveratrol, an antioxidant and known CYP2E1 inhibitor, was found to ameliorate the DNA damage in both germ cells and spermatozoa. However, extended resveratrol treatment (six months, 10.0 mg/kg bw/week) resulted in premature activation of these cells. Thus the DNA damage found in spermatozoa after chronic acrylamide administration can be alleviated but an alternative CYP2E1 inhibitor may be required. Crown Copyright © 2016 Published by Elsevier Inc. All rights reserved.

1. Introduction Acrylamide is mainly produced for use in its polymerised form, with many industrial uses for polyacrylamide ranging from water treatment, paper processing, mining and mineral processing [1]. Small amounts of residual acrylamide can contaminate these sources allowing for human exposure to the toxicant. Exposure also occurs from cigarette smoke. Alarmingly acrylamide is produced from asparagine during the high temperature cooking of many starch-rich foods including potato chips, French fries, breads, cereals and biscuits [2]. Human exposure to acrylamide therefore occurs in a chronic manner at dietary doses estimated by the World Health Organisation to be in the range of 1–4 ␮g/kg bodyweight (bw)/day [3]. Acrylamide is well established as a neurotoxin when acute high dose exposure occurs in humans. In rats, the no observed adverse effects level (NOAEL) for neurotoxicity is 0.2–0.5 mg/kg bw/day [4]. Reproductive toxicity occurs in male rodents with the NOAEL reported to be 5 mg/kg bw/day for reduced fertility and 2 mg/kg bw/day for premature embryonic death [5]. Most studies focus on the consequences of acute acrylamide exposure on reproductive capacity and loss of embryos; however, what is lacking is assess-

∗ Corresponding author at: Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, 2308, Australia. E-mail address: [email protected] (S.D. Roman). http://dx.doi.org/10.1016/j.reprotox.2016.05.004 0890-6238/Crown Copyright © 2016 Published by Elsevier Inc. All rights reserved.

ment of the integrity of the male germ line as a result of chronic acrylamide exposure. Acrylamide is cleared by conjugation to glutathione, however in mice 60% is first converted to glycidamide [6]. Acrylamide and glycidamide adduct with DNA in vitro, however only glycidamideDNA adducts are found in vivo, which supports the importance of glycidamide formation in acrylamide’s genotoxicity [7]. The formation of DNA adducts, if not repaired, can cause mispairing during DNA replication and thus give rise to deleterious mutations [8]. The epoxidation of acrylamide to glycidamide is catalysed by the enzyme CYP2E1, a member of the cytochrome P450 family. Ghanayem et al. [9] have demonstrated that CYP2E1 is the primary enzyme responsible for this conversion. CYP2E1null mice exposed to acrylamide have 52–66-fold lower levels of glycidamide-haemoglobin adducts than their wild-type counterparts. It has also been demonstrated that glycidamide is required for the reproductive toxicity generated by acrylamide exposure, and hence so is the presence of CYP2E1 [10,11]. We propose that the level of acrylamide exposure seen in humans, being chronic in nature, leads to DNA damage in the male germline. Nixon et al. [12] demonstrated a correlation between time and dose and DNA damage in the spermatocytes of mice treated with acrylamide using a range of time (1, 3, 6, 9 or 12 months) and doses (0.001, 0.01, 0.1, 1, and 10 ␮g/ml via the drinking water). These treatment regimens encompassed doses equating to human dietary intake estimates, with all doses too low to generate

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embryonic lethality or reduced fertility. Spermatocytes have been found to express CYP2E1 [13], and the damage to these cells with chronic exposure was attributed to glycidamide adduct formation using the formamidopyrimidine-DNA glycosylase (FPG) modified comet assay. As an extension of this work, the present study sought to examine the impact of acrylamide exposure on mature spermatozoa. For this purpose, we administered acrylamide to male mice at the lowest dose and time that generated glycidamide adducts in spermatocytes (1 ␮g/ml via the drinking water for six months) [12] as well as the same dose for a shorter time period of three months. In the present study we were able to assess DNA damage in the spermatozoa as single strand breaks using the alkaline comet assay. However, we were not able to modify the assay to utilise FPG to directly assess glycidamide adducts. Therefore, to determine the role of glycidamide, we utilised resveratrol, a noncompetitive inhibitor of CYP2E1 [14,15] to inhibit glycidamide formation. Resveratrol also acts as an antioxidant [16,17] and has been demonstrated to inhibit acrylamide induced glycidamide and oxidative adducts in vitro [13]. Resveratrol is a phytoalexin found in numerous plant species, which are part of the human diet; including mulberries, peanuts and grapes [18]. Mice were treated with acrylamide in the presence or absence of resveratrol (9.9 ± 0.31 mg/kg bw/week) to determine it’s potential for ameliorating acrylamide induced DNA damage. 2. Materials and methods 2.1. Chemicals and reagents Acrylamide (A3553) and resveratrol (R5010) were purchased from Sigma (St Louis, MO, USA). Primary antibodies, rabbit polyclonal anti-␥H2A.X phospho S139 (ab11174) was purchased from Abcam (Cambridge, UK), goat polyclonal anti-8Hydroxydeoxyguanosine (8OHdG) (Ab5830) was purchased from Merck Millipore, (Temecula, CA, USA), anti-phosphotyrosine monoclonal antibody, clone PT-66 (P3300) and mouse monoclonal anti-tubulin tyrosine antibody, clone TUB-1A2 (T9028) were purchased from Sigma. Alexa Fluor® 594 goat anti-rabbit IgG (A11012) was purchased from Life Technologies, (Eugene, OR, USA) and Alexa Fluor® 594 donkey anti-goat (A11058) was purchased from Invitrogen (Carlsbad, CA, USA). ApopTag® Fluorescein In Situ Apoptosis Detection Kit (S7110), Apoptag® TdT enzyme (S7107) and antiDigoxigenin-Fluorescein (90426) were purchased from Millipore (Temecula, CA, USA). Proteinase K (V3021) was purchased from Promega (Madison, WI, USA). CometAssay® LMAgarose (4250050-02) was purchased from Trevigen® (Gaithersburg, MD, USA). Agarose type VII low gelling temperature (A-4018) was purchased from Sigma. RQ1 RNase free DNase (M610A) was purchased from Promega. Dakin slides, fully frosted (G376) were purchased from ProSciTech (Kirwan, QLD, Australia). SYBR® Green 1 nucleic acid gel stain (50513) was purchased from Lonza (Rockland, ME, USA). Tris-Glycine NG 4–20% protein gels (NG11-420) were purchased from NuSep (Homebush, NSW, Australia). Mini complete protease inhibitor cocktail tablets (11836153001) were purchased from Roche (Mannheim, Germany). Goat anti-mouse IgG-HRP (SCZSC2005) and PageRulerTM Prestained Protein Ladder (26616) were purchased from Thermo Fisher (Waltham, MA, USA). 2.2. Experimental design Thirty-six 5–6 week old Swiss CD-1 male mice were randomly allocated to eight treatment groups (four different treatments at two time points). The four treatments were; control, resveratrol, acrylamide or resveratrol and acrylamide. The experimental overview is outlined in Fig. 1. There were three mice per treatment

Fig. 1. Experimental design. Mice were administered control (vehicle alone), acrylamide, resveratrol or acrylamide and resveratrol. Control mice were administered vehicle alone by ip injection once per week. Acrylamide was administered to male mice through the drinking water at a concentration of 1 ␮g/ml. Resveratrol was administered via an ip injection of 0.4 mg once/week. Mice were exposed for a period of three or six months (i.e., 8 treatment groups). Three mice were allocated per treatment for the 3 month time point and 6 mice per treatment were allocated for the 6 month time point, totalling 36 mice. Bodyweight and water consumption was monitored throughout the study. Testes were fixed and sectioned for histological analysis and immunohistochemistry. Spermatozoa were extracted from vas deferentia for comet analyses. Spermatozoa were extracted from one caput plus corpus region per mouse for sperm counts, motility and morphology assessment. Mice exposed for six months in the control and resveratrol treatment groups had caudal spermatozoa extracted for measurement of capacitation levels via assessment of tyrosine phosphorylation.

for the 3 month time point and 6 mice per treatment allocated for the 6 month time point. Due to circumstances outside of our control, two mice were lost during the 6 month study. Hence, at completion, there were 5 mice in the control and resveratrol groups. The results for mouse bodyweight, testis weight and water consumption analyses include five or six mice per treatment group. For all other analyses, mice were allocated into assessment groups with 3 mice allocated per group. Hence results for sperm morphology, testis H&E, apoptag, tyrosine tubulin, ␥H2A.X, 8OHdG and sperm alkaline and neutral comet were assessed for 3 mice per treatment. One of the mice in the 6 month resveratrol treated group had disrupted spermatogenesis and was not able to be assessed for Image J quantification of IHC. For tyrosine phosphorylation analysis, we failed to retrieve sufficient sperm from one of the control mice, therefore only two were used for analysis. Likewise, one mouse had disrupted spermatogenesis with no spermatozoa in the resveratrol treatment group at six months so only two mice were included in the analysis of morphology, motility and sperm counts. Experiments involving animals were conducted in accordance with the policies set out by the Animal Care and Ethics Committee of the University of Newcastle. Mice were housed under conditions of 16 h light, 8 h dark, with food and water provided ad libitum. Up to six individuals were housed in one cage, and all mice were monitored for adverse health effects. Mouse bodyweight and water consumption were recorded on a weekly basis. These records were used to calculate mean acrylamide daily intake (Supplementary Table 1). 2.3. Acrylamide and resveratrol treatment Acrylamide drinking water solutions were prepared weekly using research grade acrylamide (Sigma ≥ 99% purity, A3553) diluted in filtered, deionized water at a concentration of 1 ␮g/ml as described by Nixon et al. [12]. Acrylamide is stable in water for at least one week [19,20]. Based on the formula recommended by the Food and Drug Administration (FDA) for conversion of mouse NOAEL to human equivalent dose (HED) [21], we determined the HED of acrylamide for our study to be 15 ␮g/ kg bw/day. This dose translation formula is as follows: HED (mg/kg) = animal dose (mg/kg) × animal Km /human Km . The Km factor is calculated by dividing the body weight (kg) by body surface area (BSA) (m2 ). The mouse Km factor is 3 and the human Km factor is 37 (for a 60 kg person) [21,22]. Resveratrol was obtained from Sigma (R5010) and

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Fig. 2. Testis sections from mice treated for three months. A) Histology of testis sections stained with haematoxylin and eosin. Scale bars equal to 200 ␮m. B) Cell death was assessed by use of the Apoptag® Fluorescein In Situ Apoptosis Detection Kit before being counterstained with DAPI. Scale bars equal to 100 ␮m. C) Sections were probed with anti-tyrosine tubulin and the appropriate secondary antibody before being counterstained with DAPI. Scale bars equal to 100 ␮m. All images are representative of results obtained for three biological replicates per treatment.

administered via an intraperitoneal (ip) injection once per week at a concentration 4 mg/ml dissolved in 30% ethanol in PBS with each mouse receiving a 100 ␮l injection. The resveratrol dose was chosen so as to mimic a human dose of approximately 50 mg, a dose which is available to humans in the form of tablet supplementation. The conversion from mouse to human equivalent dose (HED) was determined based on the above calculation and resulted in a resveratrol HED of 48.2 mg for a 60 kg human. Control animals received filtered, deionised water and were administered once weekly ip injections of 100 ␮l 30% ethanol in Phosphate-buffered saline (PBS). Mice were exposed for

three months (12 weeks) or six months (24 weeks ± 1 week). Immediately after treatment, animals were euthanized by CO2 asphyxiation, and tissues were collected. 2.4. Immunohistochemistry After extraction, mouse testes were fixed in Bouins, embedded in paraffin wax and sectioned at 4 ␮m thickness. Sections were then deparaffinised and rehydrated before antigen retrieval was performed by microwaving for 3 × 3 min in 50 mM Tris (pH 10.6). Sections were then blocked with 3%BSA in PBS with

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Fig. 3. Testis sections from mice treated for six months. A) Histology of testis sections stained with haematoxylin and eosin. Scale bars equal to 200 ␮m. B) Cell death was assessed by use of the Apoptag® Fluorescein In Situ Apoptosis Detection Kit Scale bars equal to 100 ␮m. C) Sections were probed with anti-tyrosine tubulin and the appropriate secondary antibody before counterstaining with DAPI. Scale bars equal to 100 ␮m.

0.05% Tween-20 (PBST) at room temperature for 1 h. Sections were then incubated with anti-␥H2AX (1:500 with 1% BSA/PBST), anti-8OHdG (1:100 with 1% BSA/PBST) or anti-tyrosine tubulin (1:400) overnight at 4 ◦ C. Sections were washed and incubated with the appropriate fluorescent conjugated secondary antibody (1:200 with 1% BSA/PBST) for 1 h at room temperature. Sections were counterstained with 0.5 ␮g/ml 4 -6-diamidino-2phenylindole (DAPI) for 2 min. Slides were mounted in mowiol and observed under fluorescence on an Axio Imager A1 fluorescence microscope (Carl Zeiss MicroImaging Inc., Thornwood, NY). Images

were taken using an Olympus DP70 microscope camera (Olympus America, Center Valley, PA). 2.5. Fluorescence quantification Image J software was used to quantify the level of fluorescence for anti-␥H2AX and anti-8OHdG used for immunohistochemical analyses. A total of 100 tubules per animal were assessed. For ␥H2A.X analysis only the post meiotic cells were assessed. This was performed by circling around the later stage germ cells for each tubule excluding all spermatocytes and spermatogonia. The corrected total cell fluorescence (CTCF) for the ␥H2A.X image was

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Fig. 4. DNA damage after three months of treatment. A) Sections were probed with anti-␥H2A.X and the appropriate secondary antibody and counterstained with DAPI. Scale bars equal to 100 ␮m. B) Sections were probed with anti- 8OHdG and the appropriate secondary antibody and then counterstained with DAPI. Scale bars equal to 100 ␮m. C) The mean ␥H2A.X fluorescence of the post-meiotic germ cells of 100 tubules per mouse was quantified using Image J analysis and normalised to the corresponding DAPI fluorescence. The mean 8OHdG fluorescence was assessed using Image J analysis by normalising the fluorescence of 10 images per mouse and calculating as a percentage of the fluorescence of the corresponding DAPI images.

calculated, normalised to background fluorescence, and then calculated as a percentage of the CTCF for the corresponding circled area in the DAPI image within the individual tubule (which was also normalised to background fluorescence) (see Supplementary Fig. 1 for example image) as previously described by Camlin et al. [23] and McCloy et al. [24]. For 8OHdG quantification, ten individual images were assessed per mouse to that a total of approximately 100 tubules per mouse were assessed. The CTCF was calculated as described for ␥H2A.X quantification.

2.6. Apoptag® apoptosis detection The procedure was carried out as per manufacturer’s instructions. Briefly, testis sections were deparaffinised in xylene and ethanol and tissue samples were pre-treated with 20 ␮g/ml Proteinase K. A positive control was treated with DNase enzyme before all samples were equilibrated and overlayed with TdT enzyme. A fluorescein tagged anti-digoxigenin probe was added to the sec-

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Fig. 5. DNA damage after six months of treatment. A) Sections were probed with anti-␥H2A.X, as described in Fig. 4A. Scale bars equal to 100 ␮m. B) Sections were probed with anti-8OHdG as described in Fig. 4B. Scale bars equal to 100 ␮m. The ␥H2A.X (C) or 8OHdG (D) fluorescence was quantified as described for Fig. 4C and D. Two mice were assessed for the resveratrol treatment as the third mouse did not have any spermatozoa or late stage germ cells present and so was excluded from quantification.

tions for 30 min at 37 ◦ C. Slides were washed and counterstained with DAPI. 2.7. Alkaline and neutral comet assay on spermatozoa Spermatozoa were extracted from the vas deferens of mice immediately after euthanasia into PBS. DNA damage in isolated spermatozoa, measured as single strand breaks (SSBs) and double strand breaks (DSBs) was measured using the alkaline and neutral comet assays, respectively, as described by Ribas-Maynou et al. [25] with the following modifications. Spermatozoa were diluted

1 × 106 spermatozoa/ml in TKB, and 10 ␮l of the suspension was mixed with 70 ␮l of LMAgarsoe (4250-050-01, Trevigen). Then 80 ␮l of the spermatozoa agarose mixture was spread onto a fully frosted Dakin slides (ProSciTech, Australia). These slides had previously been pre-coated with 70 ␮l of 1% low melting point (LMP) agarose (A4018, Sigma). The spermatozoa and agarose mixture was then immediately covered with a coverslip and allowed to solidify at 4 ◦ C for 1 h. Coverslips were then removed and slides were incubated in lysis solution 1 (0.8 M Tris-HCl, 0.8 M DTT, 1% SDS, pH 7.5), and then lysis solution 2 (0.4 M Tris-HCl, 50 mM EDTA, 2 M NaCl,

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Fig. 6. DNA damage to spermatozoa of male mice treated for three months measured as SSBs using the alkaline comet assay (A) and DSBs as measured by the neutral comet assay (B). DNA damage was measured as mean tail DNA%.

Fig. 7. DNA damage to spermatozoa of male mice treated for six months measured as SSBs using the alkaline comet assay (A) and DSBs as measured by the neutral comet assay (B). DNA damage was measured as Mean Tail DNA%.

0.4 M DTT, pH 7.5) for 30 min each at room temperature, and then washed in TBE (0.445 M Tris-HCl, 0.445 M boric acid, 10 mM EDTA) for 10 min. The slides designated for the alkaline assay were then submerged in alkaline solution (0.03 M NaOH, 1 M NaCl) for 15 min at 4 ◦ C and then electrophoresed at 1 V/cm in alkaline buffer (0.03 M NaOH) for 4 min. The slides designated for the neutral comet assay were electrophoresed in TBE (pH 7.5) for 4 min at 1 V/cm, and then washed in 0.9% NaCl. All of the slides were then washed in neutralization solution (0.4 M Tris-HCl, pH 7.5) for 5 min. Slides were stained with SYBR® green, covered with a cover slip and visualised under fluorescence. The DNA integrity of 50 cells per slide was analysed using Comet Assay IV software (Perceptive Instruments, Suffolk, UK). The relative fluorescence intensity in the comet “tail” versus total intensity of the comet (Tail DNA%) was used as a measure of DNA damage.

2.8. Tyrosine phosphorylation The cauda epididymis and vas deferens were dissected from mice and deposited in HEPES-buffered Biggers, Whitten, and Whittingham media (BWW) (composed of 91.5 mM NaCl, 4.6 mM KCl, 1.7 mM CaCl2 ·2H2 O, 1.2 mM KH2 PO4 , 1.2 mM MgSO4 ·7H2 O, 25 mM NaHCO3 , 5.6 mM D-glucose, 0.27 mM sodium pyruvate, 44 mM sodium lactate, 5 U/ml penicillin, 5 ␮g/ml streptomycin, 20 mM HEPES buffer and 0.3% BSA) under water-saturated mineral oil at 37 ◦ C. Spermatozoa were collected into BWW medium from an isolated cauda epididymal tubule following retrograde perfusion via the vas deferens. Following dilution to 6 × 106 spermatozoa/ml, cells were incubated at 37 ◦ C under an atmosphere of 5% CO2 :95% air. Two controls were generated from additional mouse spermatozoa: a non-capacitated (NC) control and a control in which capacitation was actively driven (Cap). Non capacitated control spermatozoa were incubated in BWW prepared without NaHCO3 (BWW-HCO3 − ) with additional NaCl incorporated to maintain an

osmolarity of 300 mOsm/kg. The formation of bicarbonate in these samples was prevented by capping the tubes throughout the incubation. Capacitated control spermatozoa were incubated in BWW supplemented with 3 mM pentoxifylline (ptx) and 5 mM dibutyryl cyclic adenosine monophosphate (dbcAMP). All other samples used in this aspect of the study were from mice that had been treated for six months with either the vehicle or resveratrol and were incubated in BWW only. Samples were incubated for either 0 or 180 min as indicated (t = 0 and t = 180) with gentle mixing throughout incubation. 2.9. Immunolocalisation of fixed spermatozoa Tyrosine phosphorylation levels were assessed in fixed populations of spermatozoa with an anti-phosphotyrosine antibody as described by Redgrove et al. [26]. Fluorescence quantification was performed using Image J software to assess the level of fluorescence for each individual spermatozoon, which was normalised to background fluorescence and was reported as CTCF. 2.10. Western blotting Following incubation, spermatozoa were pelleted and resuspended in protein lysis buffer as described by Dun et al. [27]. Lysates were recovered and resolved by SDS-PAGE and transferred to nitrocellulose membrane. The membranes were probed for antiphosphotyrosine as described by Redgrove et al. [26]. Densitometry analysis was performed using Image J analysis and normalised to the loading control (hexokinase). 2.11. Statistics Statistical analyses were performed using JMP software Version 9 (SAS Institute, Cary, NC). All bodyweight, testis weight, water con-

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Fig. 8. Tyrosine phosphorylation assessed in caudal spermatozoa of mice treated with control or resveratrol for six months. A) Immunolocalisation of phosphotyrosine in spermatozoa was determined by probing fixed cells with anti-phosphotyrosine, followed by the appropriate secondary antibody. Phase images are also shown. Results are representative of fluorescent staining observed in all spermatozoa of n = 2 control treated mice and n = 3 resveratrol treated mice. B) Image J analysis was performed to determine corrected total cell fluorescence. Results are presented as increase compared to the control at time 0 min (Control t = 0) C) Tyrosine phosphorylation quantified by Western blotting. Non-capacitated (NC) sperm (incubated in BWW-HCO3 ) and sperm driven to capacitate (Cap) with ptx/dcAMP were used as controls. Band 1 (hexokinase) served as a loading control. Band 2 increases in intensity between NC and Cap spermatozoa. D) The densitometry results are presented as increase compared to NC treatment. Densitometry analysis for bands 3, 5 and 8 can be found in Supplementary Fig. 3. Results are representative of n = 2 mice for control and n = 3 mice for resveratrol treatment.

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sumption, DNA damage and tyrosine phosphorylation were tested for normality using the Shapiro-Wilk test. When data were not from the Gaussian distribution, then a post hoc Wilcoxon multiple comparisons test was used to examine specific significant differences between pairs of groups. When data was from the normal distribution, an analysis of variance (ANOVA) was performed followed by All Pairs Tukeys HSD to examine differences between pairs. A 5% rejection index of null hypothesis was applied to all tests performed. 3. Results 3.1. Mouse bodyweight, water consumption, and calculated acrylamide and resveratrol dose As with previous chronic administration of acrylamide via the drinking water [12] there were no adverse events exhibited by the mice in the current study. Acrylamide or resveratrol treatment had no impact on mouse bodyweight or water consumption, indicating no serious adverse effects on health of the mice. Mouse bodyweights were also used to calculate resveratrol dose. Mean mouse bodyweights at completion of treatment were 41.0 g (SD = 1.9), 42.2 g (SD = 0.5), 42.9 g (SD = 1.9), 42.9 g (SD = 1.8), for the three month treatment and 44.4 g (SD = 3.7), 46.1 g (SD = 2.4), 42.4 g (SD = 1.8), 43.4 g (SD = 3.7) for the six month treatment for control, acrylamide, resveratrol and acrylamide plus resveratrol treatment groups, respectively. The average daily acrylamide dose (0.16 and 0.19 mg/kg bw/day for three months in the presence or absence of resveratrol respectively, 0.18 and 0.21 mg/kg bw/day for six months in the presence or absence of resveratrol respectively) was determined based on water consumption and mouse bodyweight values (Supplementary Table 1). Average weekly resveratrol doses (9.8 and 10.2 mg/kg bw/week for three months and 10.0 and 9.5 mg/kg bw/week for six months of resveratrol alone and with acrylamide treatment respectively) were also calculated based on mouse bodyweight values and once/week ip injections. 3.2. Chronic acrylamide or resveratrol exposure does not lead to cell death in the testis No significant differences in testis to bodyweight ratios were observed between any of the treatments. Acrylamide treatment did not cause any gross morphological abnormalities within tubules (Figs. 2 and 3A). Resveratrol treatment did cause some abnormal testis morphology after six months of treatment, with some cells visually appearing to be undergoing apoptosis and fewer early germ cells apparent. However, there was no indication of any apoptotic cells in any treatments (Figs. 2 and 3B). Sertoli cell morphology was examined further by staining with an antibody raised against tyrosine tubulin, a marker of Sertoli cells. Acrylamide treatment did appear to have some effect on Sertoli cell morphology with a more diffuse pattern of tyrosine tubulin staining apparent. The staining pattern demonstrated by the controls was mostly reflected in the resveratrol plus acrylamide treatment (Figs. 2 and 3C). Resveratrol alone treatment also had no apparent negative effects on these cells. 3.3. Chronic acrylamide exposure induces DNA damage in germ cells While acrylamide did not lead to cell death within the testis, it did lead to DNA damage in the germ cells. DSBs were induced in germ cells with exposure of 1 ␮g/ml (0.18 ± 0.02 mg/kg bw/day) after three or six months of treatment (Figs. 4 and 5A). While spermatocytes had positive ␥H2A.X staining in all treatments, which is indicative of the DSBs which occur during normal meiosis, acrylamide treatment resulted in staining of the later stage germ cells

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indicating DNA damage was present in these cells. The ␥H2A.X staining was observed particularly in round and elongating spermatids and was found to be significantly increased in post meiotic germ cells following three or six months of acrylamide exposure (p < 0.05) (Figs. 4 and 5C). Co-treatment with resveratrol at 9.9 ± 0.31 mg/kg bw/week for three or six months respectively, caused a significant reduction in DSBs compared with acrylamide treatment alone. This demonstrated the capacity of resveratrol to reduce acrylamide induced DNA damage in male germ cells after chronic exposure. Lower levels of DSBs were observed in resveratrol treated mice than their control counterparts. After three or six months of acrylamide exposure, increased levels of oxidative adducts were present in the germ cells within the seminiferous tubules (Figs. 4 and 5B) and this was found to be significant (p < 0.01). It is clear that oxidative damage is playing a role in germ cell DNA damage caused by chronic acrylamide exposure. Again, resveratrol co-treatment reduced the levels of DNA damage caused by three or six months of chronic acrylamide treatment (Fig. 4D). However, resveratrol treatment alone, or with acrylamide treatment for six months resulted in increased oxidative adducts in the germ cells (Fig. 5D). 3.4. Chronic acrylamide exposure induces DNA damage in spermatozoa Three or six months of acrylamide exposure resulted in a significant increase in SSBs in the spermatozoa of male mice (p < 0.001) (Figs. 6 and 7A). Interestingly with three months of treatment, the level of SSBs was significantly lower with resveratrol treatment than in the control treatment group (p < 0.001). Most importantly, resveratrol treatment administered to mice also receiving acrylamide treatment resulted in the same levels of SSBs as resveratrol alone treatment, again lower than control levels (p < 0.001). Three or six months of acrylamide exposure did not lead to increased DSBs in the spermatozoa as measured by the neutral comet assay (Figs. 6 and 7B). The range of Tail DNA percentage intensity values for each individual spermatozoon assessed can be found in Supplementary Fig. 2, which shows all of the spermatozoa from acrylamide treated mice contain damaged DNA in terms of SSBs. 3.5. Six months of resveratrol exposure induces double strand breaks in spermatozoa Interestingly, resveratrol induced significant levels of DSBs in the spermatozoa of mice treated for six months (Fig. 7B) as detected by the neutral comet assay. These DSBs were also detected in the alkaline comet assay that utilises alkaline conditions to denature DNA strands, in order to detect SSBs, but will also detect DSBs (Fig. 7A). The amount of damage was reduced to levels that were comparable to that of the control when resveratrol and acrylamide treatment were administered together. 3.6. Six months of resveratrol exposure results in capacitation like changes in the spermatozoa As six months of resveratrol treatment resulted in abnormal testis morphology, oxidative damage to germ cells and increased DSBs in spermatozoa, we sought to determine the underlying mechanism of action. Resveratrol is known to act as a direct inhibitor of phosphodiesterase enzymes (PDE) which leads to an increase in intracellular cyclic adenosine 3 ,5 -monophosphate (cAMP) [28], a second messenger that holds a pivotal role in promoting the capacitation cascade in mammalian spermatozoa [29]. A modest increase in tyrosine phosphorylation, a measure of capacitation status, was observed in the flagellum of control spermatozoa after 180 min incubation in BWW (Fig. 8A). In contrast, substan-

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tive levels of tyrosine phosphorylation were already detectable in the spermatozoa from resveratrol treated mice immediately after their isolation. This level of tyrosine phosphorylation also increased beyond that observed in control spermatozoa after 180 min incubation in BWW medium. The intensity of fluorescence observed in these spermatozoa was quantified using Image J analysis (Fig. 8B) and, after normalisation against the t = 0 control, it was determined that spermatozoa from resveratrol treated mice displayed comparable levels of labelling at t = 0 to those of control spermatozoa incubated for an extended period of 180 min (Fig. 8B). Image J analysis also confirmed a significant increase in the intensity of phosphotyrosine labelling during the incubation of spermatozoa from resveratrol treated mice, culminating in levels that were approximately double that of the control spermatozoa incubated for an equivalent period of time. Western blot analysis confirmed global increases in tyrosine phosphorylation across a myriad of substrates ranging in size from approximately 30–150 kDa (Fig. 8C). The predominant substrates were designated as bands 1–8 (Fig. 8C). Under this scheme, band 1 corresponds to the constitutively phosphorylated hexokinase [30] that was employed as a loading control for densitometry analysis (Fig. 8D, Supplementary Fig. 3; all values were calculated relative to the NC control, which was normalised to an intensity of 1). With the exception of band 2, which was clearly more intensely stained in populations of control spermatozoa that were actively driven to capacitate, the remaining three bands assessed were more prominent in the sperm recovered from resveratrol treated mice irrespective of the duration of incubation.

4. Discussion Acute acrylamide exposure causes obvious reproductive toxicity in male mice. A dose of 24 mg/kg bw/day for 4 weeks results in sperm abnormalities, decreased sperm counts, decreased fertility, reduced litter sizes and increased embryo resorptions [31]. A single dose of 125 mg/kg bw results in specific locus mutations and with exposure to late spermatids and early spermatozoa results in dominant lethality [32]. This type of exposure rarely occurs in humans, with exposure to acrylamide typically occurring in a chronic manner. Foods which contain asparagine and a reducing sugar will produce acrylamide when cooked at temperatures above 120 ◦ C, with increasing temperatures resulting in a concurrent increase in acrylamide formation. These foods include, but are not limited to, potato crisps (689–693 ␮g/kg), French fries (326–328 ␮g/kg), soft bread (27–37 ␮g/kg), roasted coffee (225–231 ␮g/kg) and breakfast cereals (132–142 ␮g/kg) [33]. This results in an estimated human dietary exposure level of 1–4 ␮g/kg bw/day equating to 70–280 ␮g/day for a 70 kg person [3,34]. We have demonstrated that the effects of chronic acrylamide exposure are quite different from the reproductive toxicity observed with acute exposure. A human relevant dose of acrylamide administered to male mice had no observable detrimental effects on general health. There was no effect on bodyweight or testis: bodyweight ratio with six months of exposure at a dose of 0.18 ± 0.02 mg/kg bw/day. There were no gross morphological abnormalities or cell death observed in the testes. These results are in agreement with an earlier study which observed no effect on any of these parameters with acrylamide doses ranging from 0.0001–2 mg/kg bw/day for 1, 3, 6, 9 or 12 months [12]. While spermatogenesis appeared to progress normally and there were no apparent effects on sperm number, motility or morphology (Supplementary Table), the DNA within the germ cells was damaged as a result of chronic acrylamide treatment. Spermatocytes have previously been found to express CYP2E1 [13], the enzyme responsible for the conversion of acrylamide to gly-

cidamide [9]. Glycidamide is the major contributor to the genetic damage caused by acrylamide, as it is able to adduct directly adenine and guanine of DNA [35]. The present study found that DNA damage was increased in the germ cells of male mice with three or six months of acrylamide treatment. This was measured as DSBs using an antibody raised against ␥H2A.X and is consistent with the dose and time dependent increase in DSBs observed with chronic acrylamide exposure by Nixon et al. [12]. In addition, we have for the first time demonstrated that chronic acrylamide treatment causes an increase in oxidative adducts in the germ cells. This oxidative damage is likely to occur as a result of the function of the CYP2E1 enzyme which relies on activation by oxygen [36]. Decay of the oxygenated P450 complex can lead to the release of small amounts of the superoxide anion radical (O2 − ). Dismutation of this radical can lead to the production of hydrogen peroxide (H2 O2 ), which can also form from decay of the peroxy P450 complex. These reactive oxygen species (ROS) can directly damage DNA leading to mutations [37]. These findings are consistent with in vitro experiments where isolated spermatocytes treated with acrylamide (1 ␮M, 18 h) have significantly increased levels of glycidamide and oxidative adducts, which were ameliorated by CYP2E1 inhibition via resveratrol treatment (0.1 ␮M) [13]. With chronic acrylamide treatment, we also found resveratrol to be an efficient inhibitor of DNA damage. Resveratrol abrogated the DSBs and oxidative adducts in germ cells induced by 3 months of acrylamide exposure, as well as the DSBs induced by 6 months of acrylamide exposure. DNA damage occurring in germ cells can only be repaired in the early stages of spermatogenesis, as late stage spermatids and spermatozoa are DNA repair deficient [38,39]. Previously it has been determined that chronic acrylamide exposure induces DNA damage in the germ cells (spermatocytes) as a result of glycidamide adduction [12], however because spermatozoa contain a compact genome they could not be assessed using the FPG modified comet assay. Rather, to assess the levels of DNA damage in these cells, the alkaline and neutral comet assays were performed to measure SSBs and DSBs respectively. As the data in Figs. 4 and 5 demonstrates, acrylamide caused DNA damage in late spermatids. Importantly, using the alkaline comet assay, we were able to establish that acrylamide treatment resulted in DNA damage as SSBs in the spermatozoa (Figs. 6 and 7A). Interestingly, we detected increased levels of DNA damage in the spermatozoa with three months of acrylamide treatment. In our previous study DNA damage as glycidamide adducts was not found to be increased with three months of exposure at the same dose (or higher doses) in the spermatocytes of mice [12]. This perhaps shows the increased sensitivity of spermatozoa to chronic acrylamide exposure. While the alkaline comet assay cannot determine the nature of the damage beyond SSBs, our experiments suggest glycidamide adducts are the major contributor with oxidative damage also occurring. The NOAEL for reduced reproductive capacity for mice (5 mg/kg bw/day) [5] far exceeds the levels administered in this study. Hence the spermatozoa carrying increased levels of DNA damage are likely to be fully capable of fertilising an oocyte. Indeed, spermatozoa with high levels of DNA damage have been demonstrated to still be capable of fertilisation [40]. It is important to note that all of the spermatozoa from mice treated with acrylamide carried DNA damage, (presented in Supplementary Fig. 2A and C) whereas control, and resveratrol (in the presence or absence of acrylamide) treatments resulted in spermatozoa carrying a range of different levels of DNA damage. Hence every spermatozoon from acrylamide treated mice will pass on compromised genetic material to offspring [40]. Experiments by Brevik et al. [41] indicate that paternal acrylamide exposure leads to altered gene expression in early embryogenesis. This highlights the need for an intervention to ameliorate the effects of chronic acrylamide treatment on the paternal germline.

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Acute resveratrol treatment (single dose of 30 mg/kg bw) has previously been found to ameliorate the oxidative damage caused to rat brain, lung, liver, kidney and testis tissues caused by acute acrylamide treatment (single dose of 40 mg/kg bw) [16]. Xie et al. [15] have demonstrated the ability of resveratrol to inhibit glycidamide adduct formation with liver DNA in mice caused by acrylamide treatment at human exposure relevant doses. Here we have demonstrated the capacity of resveratrol to ameliorate the DNA damage caused to the male germline by chronic acrylamide administration. This further emphasises the role of CYP2E1 metabolism of acrylamide to glycidamide in acrylamide induced DNA damage. Extended exposure to resveratrol (10.0 mg/kg bw/week for six months) however resulted in abnormal testis morphology, increased oxidative adducts in germ cells, and an increase in DSBs in the spermatozoa as measured by the neutral comet assay which is suggestive of cell death. These DSBs would have contributed to the levels of DNA damage detected by the alkaline comet assay. As resveratrol acts as an inhibitor of CYP2E1 this result could suggest that CYP2E1 plays a role in spermatogenesis. However CYP2E1 null mice have been produced and found to grow and develop normally [42] and have normal fertility [11,43,44]. Other properties of resveratrol are therefore proposed to cause the detrimental effects observed. Resveratrol has many targets, including its capacity to inhibit phosphodiesterase enzymes (PDE) (reviewed in Katen and Roman, [45]). This can lead to an increase in intracellular cyclic adenosine 3 ,5 -monophosphate (cAMP) [28]. Such a mechanism of action is of particular relevance to mammalian spermatozoa since the majority of the processes involved in their activation (capacitation) are modulated by a cAMP-dependent signalling cascade [46]. Capacitation encompasses a number of physiological changes in spermatozoa and culminates in the tyrosine phosphorylation of multiple proteins that underpin their functional activity [47]. This process is normally induced only after the cells enter the female reproductive tract, but can be stimulated in vitro by incubation in conditioned media that artificially promotes elevation of intracellular cAMP levels. Consequently, protein tyrosine phosphorylation is widely utilized as a hallmark of a spermatozoon’s capacitation status [48]. Consistent with its ability to act as a PDE inhibitor, resveratrol treatment for a period of six months led to increased basal levels of tyrosine phosphorylation immediately after these cells were released from the epididymal environment. This suggests that tyrosine phosphorylation, a hallmark of capacitation, may have been initiated in the spermatozoa from resveratrol treated animals before they have left the epididymal environment where they are normally stored in a quiescent, non-activated state. An interesting observation arising from this study was that resveratrol promoted tyrosine phosphorylation of a slightly different subset of proteins to those observed when sperm were driven to capacitate in the presence of pentoxifylline, dbcAMP and bicarbonate. For instance, band 4 was found to be more intensely labelled in the spermatozoa of mice treated with resveratrol compared to all controls. Hence spermatozoa from resveratrol treated mice had immediate and novel phosphotyrosine targets present, suggesting that an abnormal process of capacitation could be stimulated long before these spermatozoa have the opportunity to reach the female reproductive tract, where normal capacitation would take place. Such premature activation of capacitation-like changes are likely to be detrimental to the stored spermatozoa since this pathway leads to elevation of oxidative stress [46,49] and ultimately the induction of an apoptotic cascade leading to cell death [50]. We propose that these combined mechanisms could account for the elevated levels of DSBs observed in the spermatozoa of resveratrol treated mice. The trend of reduction in spermatozoa motility and the significant decrease in percentage of spermatozoa with normal morphology

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observed after resveratrol treatment (Supplementary Table 2) provide some support for this hypothesis. It is noteworthy that the effects of resveratrol treatment differ with different lengths of administration. Three months of treatment resulted in protection of spermatozoa, with these cells carrying lower levels of DNA damage than spermatozoa from their control counterparts. However, by six months of resveratrol treatment detrimental effects were observed, characterized by aberrant testis morphology and DSBs in the spermatozoa, the latter of which are possibly caused by premature induction of capacitation. The effects of resveratrol administration should not be of concern for humans consuming resveratrol via the diet, with average weekly dose of resveratrol estimated to be around 6.5 mg/week [51]. However, chronic dietary supplementation of resveratrol via oral tablets which contain 50 mg (or more), which are often taken daily, could be of concern and further investigation is warranted. 5. Conclusions In summary, this study has provided evidence implicating oxidative adducts in contributing to the DNA damage induced in germ cells of mice chronically exposed to the toxicant acrylamide. We have also demonstrated that sensitivity to acrylamide exposure extends to mature spermatozoa, which possessed significantly elevated levels of DNA damage. Indeed, these cells may prove to be more susceptible to this form of insult given that DNA lesions were detected as early as three months of treatment, as opposed to the six months required to elicit similar DNA damage in spermatocytes. Finally, we have identified an approach to alleviating DNA damage but demonstrated that it is not without consequences. Overall, these data clearly demonstrate that the genetic material that will be passed on to future generations is compromised by chronic acrylamide treatment in the male mouse model. Conflict of interest There are no conflict of interest. Funding This work was supported by funding from the Reproductive Science Group, Faculty of Science & IT, University of Newcastle and the Priority Research Centres for Reproductive Sciences and Chemical Biology. A.L.K. is the recipient of an Australian Postgraduate Award Scholarship from the Commonwealth of Australia. Transparency document The http://dx.doi.org/10.1016/j.reprotox.2016.05.004 associated with this article can be found in the online version. Acknowledgements We thank Dr. Belinda Nixon for her help with monitoring and treatment of animals, and in collection of tissue samples. We also thank Dr. Alexander Sobinoff for his assistance in assessment of spermatozoa samples. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.reprotox.2016. 05.004.

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