1 Epigenetic Modulation of Nrf2 and Ogg1 Gene Expression in Testicular

Epigenetic modulation of Nrf2 and Ogg1 gene expression in testicular germ cells by methyl parathion exposure

Item Type Article

Authors Hernandez-Cortes, D.; Alvarado-Cruz, I.; Solís-Heredia, M.J.; Quintanilla-Vega, B.

Citation D. Hernandez-Cortes, I. Alvarado-Cruz, M.J. Solís-Heredia, B. Quintanilla-Vega, Epigenetic modulation of Nrf2 and Ogg1 gene expression in testicular germ cells by methyl parathion exposure, Toxicology and Applied Pharmacology, 346, pp 19-27, https:// doi.org/10.1016/j.taap.2018.03.010

DOI 10.1016/j.taap.2018.03.010

Publisher ACADEMIC PRESS INC ELSEVIER SCIENCE

Journal TOXICOLOGY AND APPLIED PHARMACOLOGY

Download date 03/10/2021 00:38:50

Item License http://rightsstatements.org/vocab/InC/1.0/

Version Final accepted manuscript

Link to Item http://hdl.handle.net/10150/627974 Epigenetic modulation of Nrf2 and Ogg1 gene expression in testicular germ cells by methyl parathion exposure

Hernandez-Cortes D1#, Alvarado-Cruz I1, Solís-Heredia MJ1, Quintanilla-Vega B1*

1Department of Toxicology, Cinvestav, Mexico City, Mexico, 07360.

*Corresponding Author: Betzabet Quintanilla-Vega, Department of Toxicology, Cinvestav, Ave.

IPN 2508, Col Zacatenco, Mexico City, Mexico, 07360. Phone (52) +5557473310. E-mail: [email protected]

#Current affiliation address: Department of Pharmacology and Toxicology, College of

Pharmacy, The University of Arizona. 1703 E. Mabel St., Tucson, Arizona, USA. 85721. E- mail: [email protected]

Running title: Epigenetic modulation as a novel mechanism of methyl parathion toxicity

List of abbreviations

Acetylcholinesterase AChE Antioxidant response element ARE Base excision repair BER Bovine serum albumin BSA Cytochrome P450 CYP DNA methyltransferase DNMT Fetal bovine serum FBS Methyl parathion Me-Pa Nuclear factor erythroid 2-related factor 2 Nrf2/NRF2 Organophosphate OP Reactive oxygen species ROS Room temperature RT 5-hydroxymethylcytosine 5-hmC 5-methylcytosine 5-mC 5,5’-dithiobis-2-nitrobenzoic acid DTNB 8-hydroxy-2’-deoxyguanosine 8-OHdG 8-oxo-2’-deoxyguanosine 8-oxo-dG 8-oxoguanine DNA glycosidase Ogg1/OGG1

1 Abstract

2 Methyl parathion (Me-Pa) is an oxidizing organophosphate (OP) pesticide that generates

3 reactive oxygen species (ROS) through its biotransformation. Some studies have also

4 suggested that OP pesticides have the capacity to alkylate biomolecules, including DNA. In

5 general, DNA methylation in gene promoters represses transcription. NRF2 is a key

6 transcription factor that regulates the expression of antioxidant, metabolic and detoxifying

7 genes through the antioxidant response element (ARE) situated in promoters of regulated

8 genes. Furthermore, DNA repair genes, including 8-oxoguanine DNA glycosidase (OGG1),

9 have been proposed as NRF2 target genes. Me-Pa exposure produces poor semen quality,

10 genetic and oxidative damage in sperm cells, and reduced fertility. However, the Me-Pa effects

11 on the methylation status and the expression of antioxidant (NRF2) or DNA repair (OGG1)

12 genes in male germ cells have not been investigated. Therefore, mice were exposed to Me-Pa

13 to evaluate the global (%5-mC) and specific methylation of Nrf2 and Ogg1 genes using

14 pyrosequencing, gene expression, and total protein carbonylation in male germ cells. The

15 results showed that Me-Pa significantly decreased the global DNA methylation pattern and

16 significantly increased the methylation of two CpG sites within Ogg1 promoter and one CpG

17 site within Nrf2 promoter. In addition, Ogg1 or Nrf2 expression did not change after Me-Pa

18 exposure despite the oxidative damage produced. Altogether, our data suggest that Me-Pa

19 toxicity alters Ogg1 and Nrf2 promoter methylation in male germ cells that may be modulating

20 their gene expression.

22 Keywords: methyl parathion, DNA methylation, sperm cells, epigenetics, gene expression,

23 oxidation.

24 Introduction

25 Exposure to OP pesticides is characterized by inhibition of the acetylcholinesterase

26 (AChE) enzyme in the nervous system (Edwards and Tchounwou, 2005), but other studies

27 have demonstrated the ability of OP pesticides to produce detrimental effects on the male

28 reproductive system including poor semen quality, hormone imbalance, decreased fertilization

29 rate, and impairment of embryo development (Joshi et al., 2003; Narayana et al., 2006).

30 Methyl parathion (Me-Pa; O,O-dimethyl O-4-p-nitrophenyl phosphorothioate) is an OP

31 compound still utilized in many countries, even though it is classified as highly or extremely

32 toxic by international authorities (EPA, 1986; WHO, 1993). Me-Pa has been associated with

33 genotoxic and mutagenic effects in cells, producing chromosome aberrations and generating

34 micronuclei (Malhi and Grover, 1987; Vijayaraghavan and Nagarajan, 1994). In particular, our

35 group has demonstrated that Me-Pa causes genetic damage in sperm cells at different stages

36 of spermatogenesis (Piña-Guzmán et al., 2006). Furthermore, we have previously reported

37 that oxidative stress is the main mechanism of Me-Pa toxicity due to the generation of reactive

38 oxygen species (ROS) during its biotransformation, which target mature and immature male

39 germ cells (Piña-Guzmán et al., 2009). In addition, epidemiological studies have reported poor

40 semen quality, DNA fragmentation, and sperm aneuploidies after exposure to OPs, such as

41 Me-Pa (Padungtod et al., 1999; Recio et al., 2001; Sánchez-Peña et al., 2004).

42 NRF2 is a transcription factor that promotes the antioxidant response element (ARE)-mediated

43 expression of protective and detoxifying enzymes by binding to the promoter region (Ma, 2013).

44 Mutational assays revealed that GTGACA***GC is the ARE consensus sequence (Rushmore

45 et al., 1991; Xie et al., 1995). Furthermore, nearby sequences and other elements influence

46 the ARE-mediated expression and induction of detoxifying genes (Prestera et al., 1993;

47 Wasserman and Fahl, 1997; Xie et al., 1995). 8-oxoguanine glycosylase (OGG1) is an enzyme

48 that belongs to the base excision repair (BER) pathway responsible for repairing oxidative

49 modifications in nitrogenous bases damaged by ROS, including 8-oxo-deoxyguanosine (8-oxo-

50 dG), one of the most abundant and well characterized pre-mutagenic lesions in DNA

51 (Klunglanda and Bjellandb, 2007).

52 In addition to oxidative stress, OP compounds and particularly Me-Pa have shown the capacity

53 to generate methylated adducts in guanine, adenine and cytosine in different organs, such as

54 the testes of mice exposed to mixtures or single doses of OP compounds (Dedek et al., 1984;

55 Segerback, 1981; Wiaderkiewicz et al., 1986), which suggests that OP pesticides have the

56 capacity to modify the genome methylation status.

57 DNA methylation is an epigenetic mechanism present in the genome as 5-methylcytosine (5-

58 mC) in critical sections close to regulatory elements in the promoter called CpG islands. In

59 general, gene promoter hypermethylation is associated with gene silencing, while promoter

60 hypomethylation is related to gene expression. Global genomic hypomethylation has also been

61 associated with chromosomal instability, aging, and chronic diseases, including cancer (Millar

62 et al., 2005). Interestingly, several studies have identified that OP compounds as well as other

63 environmental toxicants modify the epigenetic state through DNA methylation alterations in

64 different genes (Collotta et al., 2013).

65 Due to the epigenome undergoes extensive reprogramming throughout fetal development in

66 gametogenesis and early embryo preimplantation, these stages are susceptible to

67 environmental exposures. During gametogenesis, a genome-wide demethylation occurs in the

68 primordial germ cells to erase previous paternal imprinting, followed by a re-methylation to

69 reestablish sex-specific imprinting by a meiotic cellular division (Dolinoy et al. 2007).

70 Regarding DNA methylation, this epigenetic mechanism plays an important role in male fertility

71 due to its contribution to many processes during the early and late stages of spermatogenesis.

72 Thus, aberrant epigenetic reprogramming of male germ cells could result in alterations,

73 ranging from poor semen quality to infertility (Dada et al., 2012). Limited information regarding

74 the methylation capacity of OP compounds is available; specifically, alterations in methylation

75 at global and promoter-specific levels of antioxidant and DNA repair genes, and their influence

76 on gene expression caused by in vivo exposure to OP pesticides have not been determined.

77 Therefore, we evaluated the effect of Me-Pa on DNA global and promoter-specific methylation

78 and the gene expression of the DNA repair and antioxidant genes Ogg1 and Nrf2, respectively,

79 in male germ cells from mice exposed to the pesticide.

81 Material and methods

82 Chemicals

83 Chemical grade (99 % purity) Me-Pa was obtained from Chem Service (West Chester, PA).

84 Trypsin-EDTA (0.25 %), and fetal bovine serum (FBS) was obtained from Gibco

85 (ThermoFisher Scientific; Carlsbad, CA). Bovine serum albumin (BSA), collagenase II,

86 hyaluronidase IS, DNase I, Nile red, and Triton X-100 were purchased from Sigma-Aldrich (St.

87 Louis, MO). Methylated DNA Quantification Kit was acquired from Epigentek (Farmingdale,

88 NY), and EZ-96 DNA Methylation Kit for non-methylated cytosine conversion was purchased

89 from Zymo Research (Irvine, CA). GoTaq Hot Start Polymerase and Green Master Mix were

90 purchased from Promega (Madison, WI). Reagents and primers for pyrosequencing were

91 acquired from Qiagen Inc. (Hilden, Germany). TaqMan 20X probes, Master Mix, and reagents

92 for gene amplifications were obtained from Applied Biosystems (Foster City, CA). TRIzol

93 reagent was purchased from Invitrogen (Carlsbad, CA). RIPA and extraction buffers, halt

94 protease and phosphatase inhibitor cocktail, and the Pierce BCA Protein Assay Kit were

95 purchased from ThermoFisher Scientific (Carlsbad, CA). All other reagents, including those for

96 protein blotting, were molecular grade.

97 Animals

98 Adult male ICR-CD1 mice (13-15 weeks old) were obtained from our institutional animal

99 facilities. They were housed in filtered cages and maintained under a 12-h dark-light cycle with

100 food and water available ad libitum. Me-Pa was dissolved in α-tocopherol-free corn oil and

101 administered (ip) at repeated weight-adjusted doses of 6 mg/kg for 5 days, while control

102 rodents only received the vehicle during the same period (n= 6 control and 10 treated mice).

103 This scheme of treatment represents an occupational exposure in agricultural fields in

104 developing countries. All animal procedures were approved by the Institutional Animal Care

105 and Use Committee (CICUAL, for its initials in Spanish) in compliance with international

106 guidelines for the use and care of laboratory animals.

107

108 Germ cell isolation

109 Mice were euthanized 24 h after the last Me-Pa administration. Testes were dissected, and the

110 capsule was removed to obtain the seminiferous tubules that were subsequently mechanically

111 dissected in ice-cold serum-free RPMI-1640 medium (Gibco, ThermoFisher Scientific;

112 Carlsbad, CA). Then, seminiferous tubules were treated with a mixture of enzymes,

113 collagenase II (2 mg/ml), hyaluronidase IS (10 mg/ml) and DNase I (1 mg/ml) (Sigma-Aldrich,

114 St. Louis, MO), to remove the interstitial, myoid and Leydig cells. Later, 0.25 % trypsin-EDTA

115 was added to dissociate seminiferous tubules, releasing Sertoli and germ cells. Finally, DNase

116 I (1 mg/ml) was added again to reduce viscosity and cell aggregation of the final suspension

117 (Boucheron and Baxendale, 2012). After the cellular suspension was obtained (containing

118 Sertoli and germ cells mostly), it was incubated with 0.25 µg/ml Nile red for 15 min at room

119 temperature (RT). This compound has a high affinity for lipid droplets primarily found in the

120 phagosomes of Sertoli cells. Then, purification of germ cells was performed using a cell sorter

121 (MoFlo XPD High-Speed Cell Sorter; Beckman Coulter): positive Sertoli cells for Nile red were

122 discarded and germ cells were isolated through sorting. Finally, germ cells were recovered and

123 counted, and their characterization was indirectly performed through the absence of GATA-4, a

124 specific marker for Sertoli cells (Riboldi et al., 2012).

125

126 Immunocytochemistry

127 Isolated germ cells were fixed with methanol for 40 min and 1 % BSA to block unspecific sites

128 was added, and then they were incubated for 45 min at RT with 0.1 % Triton X-100 and 10 %

129 FBS for permeabilization. Later, cells were incubated with primary anti-GATA-4 antibody

130 (1:1200) (Rabbit monoclonal; Abcam Inc., Cambridge, MA) overnight at 4 ºC. The next day, the

131 cells were washed and incubated with the secondary antibody (1:2000) (Goat Anti-Rabbit IgG-

132 Alexa Fluor 488; Abcam Inc., Cambridge, MA) for 1 h at RT. Finally, the data from

133 approximately 5,000 events were collected using an LSR Fortessa Flow Cytometer (Becton

134 Dickinson, Mountain View, CA). HepG2 cells were used as a positive control for the assay

135 under the same conditions.

136

137 RNA extraction, reverse transcription and real-time RT-PCR for Ogg1 and Nrf2

138 Total RNA was isolated from 7x106 male germ cells using the TRIzol reagent. RNA

139 quantification and purity were assessed, and integrity was determined on a 1 % agarose gel.

140 Aliquots of 30 µg of RNA were converted to cDNA using 1 µl of M-MLV reverse transcriptase

141 following the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Real-time

142 PCR was conducted using TaqMan probes for Ogg1 (Mm00501781_m1), Nrf2

143 (Mm00477784_m1), and β-actin (Mm00607939_s1). PCR reactions were performed using

144 0.625 µl Taq Man probe, 250 ng cDNA template, 6.25 µl TaqMan Gene Expression Master Mix,

145 and 3.625 µl nuclease-free molecular grade water. Reactions were run in two independent

146 experiments in triplicate. Real-time PCR was conducted using an Applied Biosystems 7500

147 Real-Time PCR System (Applied Biosystems, Foster City, CA).

148

149 DNA extraction and ELISA for global DNA methylation patterns

150 For DNA isolation, 8x106 testicular germ cells were incubated with lysis buffer (0.32 M Sucrose,

151 5 mM MgCl2 6H2O, 10 mM Tris HCl, 1 % Triton X-100), and then the samples were washed

152 with suspension buffer (0.152 M NaCl, 60 mM EDTA, 34.7 mM SDS, and 0.4 M Tris-HCl) and

153 5 M sodium perchlorate to denature proteins. Finally, DNA was extracted with chloroform,

154 precipitated with absolute ethanol, and washed with 70 % ethanol. Once isolated, the quantity

155 and purity of DNA were assessed, and the integrity was determined on a 1 % agarose gel.

156 DNA was aliquoted in nuclease-free molecular grade water at a concentration of 13.3 ng/µl for

157 the ELISA analysis and 50 ng/µl for pyrosequencing. The relative quantification of 5-mC global

158 content was measured using the fluorometric assay in the MethylFlash Methylated DNA

159 Quantification Kit (Epigentek Group Inc., Farmingdale, NY) according to the manufacturer’s

160 protocol. Aliquots of 100 ng of DNA per well were placed in microplates, and colorimetric

161 quantification was performed at 450 nm. Two independent experiments were run and analyzed

162 in duplicate. Positive (containing 50 % 5-mC) and negative (containing 50 % cytosine) control

163 primers were included in the kit. A standard curve was generated by plotting different

164 concentrations of the positive control supplied in the kit, showing a linear response in the range

165 of 0.5 – 5.1 % 5-mC with an R2= 0.9974.

166

167 Pyrosequencing for DNA methylation patterns of the Ogg1 and Nrf2 promoters

168 DNA methylation analysis was performed using the highly quantitative bisulfite-PCR

169 pyrosequencing assay. To select the CpG sites on Ogg1 and Nrf2 genes, an in silico analysis

170 of Ogg1 and Nrf2 genes was performed. We used two web-based tools to predict NRF2-

171 binding sites: ConSite (http://consite.genereg.net) and Alggen (http://www.lsi.upc.es/~alggen).

172 Specific criteria for regions selection were: sequences containing an ARE (NRF2-binding site)

173 that modulates the transcription of these genes, located inside the promoter of each gene,

174 within a CpG island, close to regions where transcriptional factors and repressors bind, and

175 histone posttranslational modifications (H3K27Ac) have been found; avoiding polymorphisms

176 and repetitive sequences to improve the specificity. Specifically, we chose sections right before

177 the transcription start site of Nrf2 and Ogg1 genes. According to these requirements, two short

178 target sequences were selected on Ogg1 containing three and five CpGs, respectively, and

179 one target sequence was chosen on Nrf2 containing three CpGs (Fig. 1). To determine

180 whether these sequences were optimal for the identification of methylation using

181 pyrosequencing, the custom PyroMark® CpG assay was used. Then, specific primers for PCR

182 and pyrosequencing were designed using the PyroMark® Assay Design software version 2.0.1

183 (Qiagen, Hilden, Germany) to target multiple CpG sites for each selected sequence.

184 For pyrosequencing, 1 µg of genomic DNA was bisulfite-treated using the EZ DNA Methylation

185 Gold Kit and eluted in a final volume of 40 µl according to the manufacturer’s instructions.

186 Then, 1 µl of bisulfite-converted DNA was amplified by PCR using the GoTaq Hot Start

187 Polymerase, and specific primers for each region were designed for bisulfite-converted DNA

188 sequences. PCR was carried out using 15 µl of GoTaq Green Master Mix, 10 pmol forward

189 and 10 pmol reverse primers, 50 ng of bisulfite-treated DNA, and water to reach 30 µl final

190 volume. PCR products were purified, and CpG methylation was evaluated using 0.3 µM of

191 sequencing primers. Pyrosequencing was performed using the PyroMark® Q24

192 pyrosequencing system according to the manufacturer’s instructions (Qiagen, Hilden,

193 Germany). Samples were run in duplicate; non-CpG cytosines present in the analyzed

194 sequences as well as positive (containing 50 % 5-mC) and negative (containing 50 % cytosine)

195 primers were used as pyrosequencing controls. In addition, thymines were added intentionally

196 along the run to determine the efficiency of bisulfite conversion. The global methylation was

197 expressed as the percent of 5-mC (%5-mC) using the PyroMark® Analysis software version

198 2.0.7 (Qiagen, Hilde, Germany). Samples were run in two independent experiments. Table 1

199 shows the target sequences and their location, the locations of PCR and biotinylated-

200 sequencing primers, and the gene amplicon lengths.

201

202 Protein carbonylation

203 To determine protein oxidation, 10x106 sperm cells were isolated by flow cytometry and then

204 lysed with RIPA buffer plus halt protease and phosphatase inhibitor cocktail previously mixed.

205 Briefly, protein samples were quantified using the Pierce BCA Protein Assay Kit and treated for

206 derivatization of the carbonyl groups using hydrazine. Derivatized samples were separated in a

207 12 % SDS-PAGE gel at 100 V for 120 min, and then proteins were transferred to a 0.45-µm

208 nitrocellulose membrane in a semi-dry chamber for 90 min at 18 V. Later, the membrane was

209 incubated with blocking solution overnight at 4 ºC, and the next day, it was treated with the

210 primary antibody anti-2,4-dinitrophenylhydrazone (DNPHz) (1:5000) for 2 h. The membrane

211 was rinsed with TBS buffer and then incubated with the secondary antibody goat anti-rabbit

212 IgG H&L (1:8000) for 2 h. Finally, the membrane was washed again and developed with the

213 ECL Prime Western Blotting Detection Kit (GE Healthcare Life Sciences, Pittsburgh, PA)

214 following the manufacturer’s instructions.

215

216 Erythrocyte acetyl cholinesterase (AChE) activity

217 Peripheral blood samples were obtained by laceration of the tail artery 24 h after the last Me-

218 Pa administration and collected in heparinized vials. AChE activity was determined by the

219 hydrolysis of acetylthiocholine iodide following Ellman’s method (Ellman et al., 1961) using

220 thiocholine as a substrate, quinidine sulfate as a plasma cholinesterase inhibitor, and 5,5’-

221 dithiobis-2-nitrobenzoic acid (DTNB). Samples were detected at 412 nm each minute for 6 min

222 at RT. The AChE activity was expressed as µmol thiocholine min-1 ml-1.

223

224 Statistical analyses

225 The data from the control and exposed groups were compared by the two-sided Wilcoxon rank

226 sum test using STATA/IC software version 15.0 for Mac (StataCorp, College Station, TX).

227 Statistical significance was assigned at a p value≤ 0.05.

228

229 Results

230 General toxicity

231 The common neurological signs associated with OP poisoning were observed in mice during

232 the administration of Me-Pa (6 mg/kg/day/5 days), and the animals recovered after 3 h of the

233 daily exposure. In addition, Me-Pa exposure was evaluated by the AChE activity 24 h after the

234 last administration, which was significantly inhibited to 66 % of its activity (p< 0.01), with values

235 of 1.204 ± 0.090 µmol thiocholine min-1 ml-1 in exposed mice compared to 1.816 ± 0.047 µmol

236 thiocholine min-1 ml-1 in the control group.

237

238 Germ cell isolation and immunocytochemistry

239 The extraction of male germ cells from mice was performed by cell sorting via flow cytometry

240 using the fluorochrome Nile red as a tracker. The cell population was first distributed by size

241 (FSC-Height) and granularity (SSC-Height) to discard cellular debris, selecting the R1 region

242 (Fig. 2A). Then, cells within this region were redistributed by size (FSC-Height) and

243 fluorescence intensity (FL2-Log-Height) to isolate germ cells through sorting of the R2 region

244 (Fig. 2B). Purity of isolated germ cells was analyzed by the presence of Sertoli cells using the

245 GATA-4 marker. The immunodetection of Sertoli cells was assessed before (2.355 ± 0.240

246 fluorescence units) and after (0.020 ± 0.012 fluorescence units) sorting, obtaining a final purity

247 of 99 % germ cells. HepG2 cells were used as a positive control for the immunodetection of

248 GATA-4 (Fig. 2C).

249

250 mRNA expression levels of Ogg1 and Nrf2 in germ cells

251 The mRNA levels of the Nrf2 and Ogg1 genes were evaluated to determine whether Me-Pa

252 treatment was capable of changing their expression. No difference (p> 0.05) in Ogg1 gene

253 expression between the control (1.543 ± 0.076) and treated (1.551 ± 0.175) groups was found

254 (Fig. 3A), suggesting a lack of induction. Likewise, although reaching borderline significance, a

255 non-significant difference (p= 0.0509) in Nrf2 gene expression between the control (1.001 ±

256 0.104) and treated (1.289 ± 0.092) groups was observed (Fig. 3B).

257

258 Global and specific methylation patterns in germ cells

259 An ELISA kit containing the primary antibody against 5-mC was used to determine the global

260 methylation status of the genome. Levels of 5-mC were significantly decreased (p< 0.05) in

261 mice exposed to the pesticide (1.411 ± 0.132) compared to control mice (2.059 ± 0.242), and

262 an approximately 32 % reduction in global DNA methylation was observed (Fig. 4).

263 In addition to global DNA methylation, the specific methylation within the promoters of Ogg1

264 and Nrf2 after Me-Pa exposure was determined. Sequenced sections were selected for

265 convenience (see Methods Section) assuming that these regions are critical sites that regulate

266 the expression of the two evaluated genes. For the Ogg1 promoter, two regions were selected,

267 while only one region of the Nrf2 promoter was chosen. From the three CpG sites that were

268 located in the first region of Ogg1, a significant difference in the first (p< 0.01) and second (p<

269 0.05) CpG sites was observed in Me-Pa-treated animals compared to control mice (Figs. 5A-B).

270 However, no difference (p> 0.05) was observed in the third CpG site between groups (Fig. 5C).

271 In addition to the comparison between individual CpG sites, the mean value of %5-mC of all

272 CpG sites per target region of each gene was also compared between groups. A significant

273 difference (p< 0.01) in the mean value of the three CpG sites of the first region of Ogg1

274 between control and exposed mice was observed (Fig. 5D). Regarding the second region

275 sequenced in the Ogg1 promoter, five CpG sites were evaluated. However, none of these sites

276 was significantly different (p> 0.05) between the treated and control groups (Figs. 6A-E).

277 Similarly, there was no difference (p> 0.05) in the mean values of all five CpG sites of the

278 second region of Ogg1 between the treated and control animals (Fig. 6F).

279 The methylation pattern of the Nrf2 promoter was determined in one region containing three

280 CpG sites. No differences (p> 0.05) in the first CpG site (Fig. 7A) or in the second CpG site

281 (Fig. 7B) between the exposed and control groups were observed. However, we observed a

282 significant difference (p< 0.05) in the third CpG site (Fig. 7C) between the treated and control

283 mice. Finally, there was no difference (p> 0.05) in the mean values of the three CpG sites

284 between the treated and control groups (Fig. 7D).

285

286 Protein carbonylation

287 We evaluated the carbonylation of total protein of male germ cells by immunodetection as a

288 broad indicator of protein oxidation in our model. Mice exposed to Me-Pa showed a significant

289 increase of 44 % in protein oxidation compared to control mice (p< 0.05) (Fig. 8).

290

291 Discussion

292 We identified for the first time alterations in the epigenetic status of male germ cells at both the

293 global and promoter-specific levels caused by in vivo exposure to an OP pesticide as a novel

294 mechanism of toxicity that might modulate Ogg1 and Nrf2 gene expression. OPs are highly

295 toxic pesticides of restricted use or banned by the European Union (Pesticide Action Network,

296 2008). In the United States, OP pesticides accounted for 33 % (20 millions of pounds) of all

297 insecticide products used nationwide in 2012 (EPA, 2012), and their use in developing

298 countries, such as Mexico, is still a health public concern (Secretaría de Salud, 2017).

299 In this study, repeated doses of Me-Pa decreased the global DNA methylation profile of

300 testicular germ cells. Tunc and Tremellen (2009) reported a negative correlation between DNA

301 methylation and DNA fragmentation as well as ROS production in sperm from infertile men,

302 suggesting that oxidative damage to sperm DNA impairs the methylation of cells. We did not

303 measure DNA damage or oxidative stress generation after Me-Pa exposure in this study.

304 However, our group has reported that the oxidative capacity of Me-Pa is involved in sperm

305 DNA damage, compromising the fertilization rate of germ cells (Piña-Guzmán et al., 2006;

306 2009; Salazar-Arredondo et al., 2008). Therefore, the significant decrease in global DNA

307 methylation observed in this study could be related to the oxidative stress generated by Me-Pa

308 exposure; nevertheless, more studies are needed to determine whether the decrease in the

309 fertilization capacity is a consequence of the global hypomethylation promoted by Me-Pa

310 oxidative stress in male germ cells. Biotransformation of OP pesticides through cytochrome

311 P450 (CYP) enzymes has been reported in the testis (Moustafa et al., 2008), and CYP-

312 mediated reactions generate ROS through redox-cycling activity (Bondy and Naderi, 1994),

313 supporting that OP-induced male reproductive toxicity arises from in situ metabolism in the

314 testis.

315 Two central cellular defense mechanisms against oxidative damage are the antioxidant

316 systems and the DNA repair enzymes. In this study, a slight but non-significant increase in the

317 expression of Nrf2 and no change in Ogg1 expression were observed. Apparently, Me-Pa

318 exposure was not sufficient to induce either Ogg1 or Nrf2 despite the presence of oxidative

319 damage detected as protein carbonylation in male germ cells from mice.

320 Literature regarding the effect of OP pesticides on the expression of genes involved in

321 antioxidant or DNA repair mechanisms is scarce; nonetheless, a reduction in the expression of

322 genes and proteins related to DNA repair, antioxidant response, and cellular metabolism after

323 in vitro (Mankame et al., 2006) or in vivo (Chen et al., 2015) exposure to OP pesticides has

324 been reported. In addition, even though OGG1 has not been reported as a downstream gene

325 of NRF2, silencing of NRF2 downregulates OGG1 protein levels and reduces OGG1 gene

326 activity because an ARE is located within the OGG1 promoter (Habib et al., 2016; Singh et al.,

327 2013), which could explain the lack of OGG1 gene induction.

328 We evaluated gene-specific DNA methylation because there is vast literature suggesting that

329 environmental exposures can produce changes on different epigenetic mechanisms, altering

330 gene expression as consequence. In addition, CpG islands have been associated to

331 housekeeping gene promoters and this characteristic is shared in both species; mouse and

332 humans (Antequera & Bird, 1993). An autonomous CpG site methylation level has been

333 proposed (Pfeifer et al., 1990), but the specific mechanisms that regulate this process are not

334 well understood.

335 Regarding specific methylation, we found that repeated doses of Me-Pa increased the

336 methylation of specific CpG sites within the Nrf2 and Ogg1 promoters. Even though germ cells

337 from all stages of spermatogenesis are found in the seminiferous tubules, we most likely

338 collected a homogeneous population of postmeiotic germ cells. However, since methylation

339 changes occur less in these cells, particularly during spermiogenesis (Oakes et al., 2012), we

340 suggest that the hypermethylation observed by repeated exposure to the pesticide (control vs

341 treated) may be due the ability of Me-Pa to methylate per se the DNA (Wiaderkiewicz et al.,

342 1986).

343 Information regarding promoter-specific methylation changes promoted by exposure to OP

344 pesticides is very limited. However, some OPs, including fonofos, parathion, and terbufos,

345 induced methylation in the promoters of genes related to DNA damage and repair, among

346 others, in human hematopoietic K562 cells (Zhang et al., 2012a). Furthermore, the OP

347 pesticide diazinon was capable to methylate promoters of tumor suppressors and other related

348 genes related to the hallmarks of cancer due to its alkylating ability in K562 cells, suggesting

349 the potential role of OP pesticides in cancer development via epigenetic modifications (Zhang

350 et al., 2012b).

351 We observed a global hypomethylation and promoter-specific hypermethylation in male germ

352 cells. It is not surprising to observe global DNA hypomethylation and hypermethylation in

353 specific genes, this is a phenomenon commonly described in cancer development (Fotouhi et

354 al., 2014), including prostate cancer (Cho et al., 2006), and it was recently reported in pesticide

355 applicators exposed to a mixture of pesticides (Rusiecki et al., 2017). Currently, there is no

356 clear mechanism that could explain the occurrence of both hypomethylation and

357 hypermethylation events at the same time, but these phenomena could be caused by

358 independent mechanisms (Roman-Gomez et al., 2006) that require being further investigated.

359 We observed that some CpG sites within the Ogg1 and Nrf2 promoters were significantly

360 methylated, while the expression levels of these genes were not significantly increased as

361 expected due to the presence of oxidative damage after Me-Pa treatment. In other words,

362 since we have previously shown the oxidative capacity of Me-Pa (Piña-Guzmán et al., 2006),

363 we hypothesized that Nrf2 and Ogg1 should have expressed to repair this damage. By not

364 detecting increases in their mRNA levels we suggest that the pesticide altered Nrf2 and Ogg1

365 expression. The relationship between promoter DNA methylation and downregulation of Nrf2

366 (Huang et al., 2012) and OGG1 (Singh et al., 2012) gene expression has been reported in

367 mice and human MCF-7 cells, respectively.

368 Likewise, this correlation has also been described in epidemiological studies performed in

369 breast cancer patients for OGG1 (Fleischer et al., 2014) and in prostate cancer patients for

370 NRF2 (Khor et al., 2014). Interestingly, despite the differences in length and number of CpG

371 sites between the human and mouse NRF2 gene promoters, these authors studied the

372 methylation of a region in NRF2 with the same characteristics as those selected in our study:

373 a section located within the promoter and right before the transcription start site and the first

374 exon of the gene. In human testis, NRF2 protects the disruption of spermatogenesis caused by

375 oxidative stress, low Nrf2 expression causes high ROS levels and defective spermatogenesis

376 that predispose individuals to infertility (Nakamura et al., 2010; Chen et al., 2012). Hence, we

377 suggest that this region within the Nrf2 gene could be an important target site for epigenetic

378 regulation. However, further in vitro studies regarding regulation of Nrf2 gene expression are

379 needed to evaluate the effect of methylation in the CpG sites evaluated in our study.

380 As a single phenomenon, only a few studies have simultaneously evaluated DNA methylation

381 and oxidation. However, they have suggested that the oxidation of either a single guanine into

382 8-oxoguanine (8-oxo-G) or 8-oxo-dG or 5-mC into 5-hydroxymethylcytosine (5-hmC), both

383 adjacent to CpG sites, is crucial for the binding of proteins involved in the condensation and

384 inactivation of chromatin (Valinluck et al., 2004) and for the binding of transcription factors to

385 DNA (Zawia et al., 2009). Moreover, the last group and Kasymov et al. (2013) reported that the

386 methylation of a cytosine adjacent to an oxidized guanine (8-hydroxy-2’-deoxyguanosine, 8-

387 OHdG) in CpG sites not only synergistically decreases Ogg1 gene expression but also impairs

388 the ability of OGG1 protein to repair the oxidative damage. Finally, ROS might induce site-

389 specific hypermethylation via the upregulation of DNMT expression or by increasing the

390 formation of DNA methyltransferase (DNMT) complexes (Wu and Ni, 2015). As an oxidative

391 compound, Me-Pa may increase the methylation of Ogg1 and Nrf2 promoters by these

392 mechanisms, which needs further investigation. In despite of the literature and our results, the

393 mechanism involved in the interaction between DNA methylation and oxidative stress is not

394 fully understood and needs to be elucidated.

395 In our study, we did not measure DNA oxidation caused by Me-Pa exposure. However,

396 evidence from our group has shown the oxidative capacity of repeated doses of Me-Pa in mice

397 through the presence of 8-OHdG in DNA from spermatozoa (Monroy-Pérez et al., 2012) and

398 carbonylation of total protein in testis homogenate (Tello-Mora, In preparation).

399 In summary, our study demonstrates that Me-Pa is capable of inducing methylation changes in

400 gene promoters of DNA repair and antioxidant pathways, which resulted in the absence of

401 gene induction. We suggest that gene silencing of Ogg1 and Nrf2 impaired the antioxidant

402 capacity as well as the DNA repair machinery in germ cells, which could contribute to the

403 toxicity of the pesticide. Further studies are needed to elucidate the mechanism by which Me-

404 Pa and potentially other OP pesticides change the epigenetic landscape of male germ cells,

405 whether OP compounds directly alkylate the DNA bases or disturb the methylation machinery

406 by increasing ROS levels. Furthermore, the role that methylation changes might play in the

407 development of detrimental effects on semen quality and fertility, as well as the biological

408 consequences of aberrant germ cell epigenetic marks after OP pesticide exposure, also

409 require additional investigation.

410 Acknowledgments. The authors want to thank Víctor Rosales-García for his assistance with

411 cell sorting, Gerardo Martínez-Aguilar for technical assistance, and Dr. Juliana Navarro-Yepes

412 for her valuable help with the protein carbonylation assay. DHC was a recipient of a

413 scholarship from CONACYT (National Council of Science and Technology)-Mexico.

414 Funding sources

415 The researchers did not receive any specific grant from funding agencies in the public,

416 commercial, or not-for-profit sectors.

417 Conflict of interest

418 The authors declare no conflicts of interest.

419

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Table 1. Selected regions of the Nrf2 and Ogg1 gene promoters for pyrosequencing.

PCR and Chromosome Number of Amplicon Gene Region sequencing Target sequence location CpGs size (bp) primer

chr6: F:GGTGGGAGTA CCCGGAAGAAC

113,326,873 – AATTGGG CATACTGGCGC First 3 176 113,326,908 GCCTTTCCAAA R:ACCACCAAAC CCT CCCTAAAC

Ogg1 chr6: F:GGTGGGAGTA CGGGGCCACGT

113,326,956 – AATTGGGATT CCGGAGGAAGA Second 5 79 113,326,993 GGCGGGGCGT R:CACACCACCA GCTGCA AACCCCTAA

chr2: F:TAAGGGGAG CCGAGGGCGG

75,704,799 – GGGGGGATA GGCATGGACCT Nrf2 First 3 165 75,704,832 GAGTGCTTCCG R:ACTTCAACCC GC ACCTAACT

Genomic locations of the evaluated regions according to UCSC genome browser version

GRCm38/mm10, Dec. 2011. The CpG sites in the target sequences are bold. Target sequences are given before bisulfite conversion.

Figure legends

Figure 1. Regions within the Ogg1 and Nrf2 promoters selected for sequencing. Location of the regions sequenced in the Ogg1 (A) and Nrf2 (B) genes according to their respective transcription start site (TSS) in the promoter. The CpG sites in the target sequences are bold.

Figure 2. Isolation of male germ cells from mice. (A) Flow cytometry of the cellular suspension from testis distributed by size and granularity. (B) Sorting of the germ cells taken from the cellular suspension and distributed by size and fluorescence. (C)

Immunocytochemistry of the Sertoli cell marker GATA-4 in germ cells before and after flow cytometry. Bars represent the mean ± SEM of 2 independent experiments. Significant difference (*p< 0.05) between groups according to the Wilcoxon rank-sum test. HepG2 cells were used as a control for GATA-4 expression.

Figure 3. mRNA expression levels of Ogg1 (A) and Nrf2 (B) in male germ cells after Me-

Pa exposure. Bars represent the mean ± SEM of 2 independent experiments performed in triplicate. The data were normalized to actin. There was no significant difference (p> 0.05) between groups according to the Wilcoxon rank-sum test, n = 6 controls and 10 treated.

Figure 4. Global DNA methylation levels in male germ cells after Me-Pa exposure. Bars represent the mean ± SEM of 2 independent experiments. Significant difference (**p< 0.01,

*p< 0.05) between groups according to the Wilcoxon rank-sum test, n = 6 controls and 10 treated.

Figure 5. DNA methylation levels of CpG sites within the first Ogg1 target region in male germ cells after Me-Pa exposure. Methylation of the first (A), second (B), and third (C) CpG sites and the mean value across the 3 CpG sites (D). Bars represent the mean ± SEM of 2 independent experiments performed in duplicate. Significant difference (**p< 0.01, *p< 0.05) between groups according to the Wilcoxon rank-sum test, n = 6 controls and 10 treated.

Figure 6. DNA methylation levels of CpG sites within the second Ogg1 target region in male germ cells after Me-Pa exposure. Methylation of the first (A), second (B), third (C), fourth (D), and fifth (E) CpG sites and the mean value across the 5 CpG sites (F). Bars represent the mean ± SEM of 2 independent experiments performed in duplicate. There was no significant difference (p> 0.05) between groups according to the Wilcoxon rank-sum test, n

= 6 controls and 10 treated.

Figure 7. DNA methylation levels of CpG sites within the Nrf2 target region in male germ cells after Me-Pa exposure. Methylation of the first (A), second (B), and third (C) CpG sites and the mean value across the 3 CpG sites (D). Bars represent the mean ± SEM from 2 independent experiments performed in duplicate. Significant difference (*p< 0.05) between groups according to Wilcoxon rank-sum test, n = 6 controls and 10 treated.

Figure 8. Densitometry analysis of total protein carbonylation in male germ cells of mice exposed to Me-Pa. Bars represent the mean ± SEM of one experiment. Significant difference

(*p< 0.05) between groups according to the Wilcoxon rank-sum test, n = 4 controls and 3 treated.