Atrazine induced transgenerational reproductive effects in medaka (Oryzias latipes)

Environmental Pollution 251 (2019) 639e650

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Atrazine induced transgenerational reproductive effects in medaka (Oryzias latipes)+ Jacob A. Cleary a, Donald E. Tillitt b, Frederick S. vom Saal c, Diane K. Nicks b, Rachel A. Claunch b, Ramji K. Bhandari a, * a b c

Department of Biology, University of North Carolina Greensboro, Greensboro, NC 27412, USA U.S. Geological Survey, Columbia Environmental Research Center, Columbia, MO 65201, USA Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 March 2019 Received in revised form 23 April 2019 Accepted 4 May 2019 Available online 10 May 2019

Atrazine is presently one of the most abundantly used herbicides in the United States, and a common contaminant of natural water bodies and drinking waters in high-use areas. Dysregulation of reproductive processes has been demonstrated in atrazine exposed fish, including alteration of key endocrine pathways on hypothalamic-pituitary-gonadal (HPG) axis. However, the potential for atrazine-induced transgenerational inheritance of reproductive effects in fish has not been investigated. The present study examined the effects of early developmental atrazine exposure on transgenerational reproductive dysregulation in Japanese medaka (Oryzias latipes). F0 medaka were exposed to atrazine (ATZ, 5 or 50 mg/ L), 17a-ethinylestradiol (EE2, 0.002 or 0.05 mg/L), or solvent control during the first twelve days of development with no subsequent exposure over three generations. This exposure overlapped with the critical developmental window for embryonic germ cell development, gonadogenesis, and sex determination. Exposed males and females of the F0 generation were bred to produce an F1 generation, and this was continued until the F2 generation. Sperm count and motility were not affected in F0 males; however, both parameters were significantly reduced in the males from F2 Low EE2 (0.002 mg/L), Low ATZ (5 mg/L), and High ATZ (50 mg/L) lineages. Fecundity was unaffected by atrazine or EE2 in F0 through F2 generations; however, fertilization rate was decreased in low atrazine and EE2 exposure lineages in the F2 generation. There were significant transgenerational differences in expression of the genes involved in steroidogenesis and DNA methylation. These results suggest that although early life exposure to atrazine did not cause significant phenotypes in the directly exposed F0 generation, subsequent generations of fish were at greater risk of reproductive dysfunction. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Transgenerational inheritance Atrazine Reproduction Medaka fish Epigenetics

1. Introduction The United States alone uses approximately 1 billion pounds of pesticides annually, half of which are herbicides (Gilliom et al., 2006). Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5triazine) is a pre-emergent herbicidal triazine used to control broadleaf weed growth on crops, such as corn. Atrazine is among the most frequently applied agricultural herbicides in the world, with annual use in the U.S. estimated up to approximately 82 million pounds (Baker, 2016; Gilliom et al., 2006; Thelin and Stone,

+ This paper has been recommended for acceptance by Christian Sonne. * Corresponding author. E-mail address: [email protected] (R.K. Bhandari).

https://doi.org/10.1016/j.envpol.2019.05.013 0269-7491/© 2019 Elsevier Ltd. All rights reserved.

2010). Surface and ground waters are contaminated by runoff and leaching, making both natural bodies of water and well water systems in agricultural areas vulnerable (Jayachandran et al., 1994; Lerch et al., 2011). Although atrazine does not bioaccumulate or biomagnify, it is moderately persistent in soil (Jablonowski et al., 2011), and there remain concerns that ephemeral, sub-lethal atrazine exposures pose a risk to aquatic and semi-aquatic species, especially during early developmental stages such as gonadogenesis and sex determination, due to the disruption of key endocrine signaling pathways. Adverse outcomes from atrazine exposures span across all vertebrate classes with a wide range of characterized endocrinerelated effects, including intersex gonads and feminization in some species (Hayes et al., 2011; Papoulias et al., 2014; Solomon et al., 2008; Tillitt et al., 2010; Wirbisky and Freeman, 2015).

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Atrazine induced reproductive toxicity or neurobehavioral abnormalities are thought to be caused by alterations of the
hypothalamic-pituitary-gonadal (HPG) axis (Alvarez et al., 2015; Corvi et al., 2012; Gely-Pernot et al., 2015; Hayes et al., 2011; Lin et al., 2013; Tillitt et al., 2010). Atrazine has been found to target functions of the primary organs of both the male and female reproductive axes: hypothalamus (Russart and Rhen, 2016), pituitary (Kucka et al., 2012), ovaries (Silveyra et al., 2017), and testes (Gely-Pernot et al., 2015; Gely-Pernot et al., 2017). Adult fish chronically exposed to environmentally relevant concentrations of atrazine had reduced reproductive function. Adult Japanese medaka fish exposed to 0.5, 5, and 50 mg/L atrazine exhibited reduced egg production after an exposure of 25 days (Papoulias et al., 2014); whereas the same concentration of atrazine reduced egg production in fathead minnows by reducing spawning and altering ovarian morphology (Tillitt et al., 2010). Female fathead minnows and medaka that were affected by atrazine exposure produced fewer eggs and demonstrated downregulation of multiple genes required for reproduction and egg production (Richter et al., 2016). Direct effects of atrazine induced reproductive effects in adult fish may, therefore, be associated with alteration in molecular pathways that control steroidogenesis via the hypothalamus-pituitary-gonad axis, as demonstrated by Suzawa and Ingraham (2008) in a mammalian in vitro toxicity testing study. The HPG axis is the known target for atrazine-induced reproductive dysfunction in mammals (Cooper et al., 2000). When organisms are exposed to certain endocrine disrupting chemicals during critical life history stages, such as gametogenesis or sex determination, changes can occur in the germline which can then be transmitted to the next generation (Bhandari, 2016; Skinner, 2011). Transgenerational inheritance due to environmental exposure during development has been observed in various organisms, for example in C. elegans that experienced temperature change (Greer et al., 2011; Klosin et al., 2017), in fish exposed to dioxin, bisphenol A, ethinylestradiol, and methylmercury (Baker et al., 2014c; Bhandari et al., 2015b; Carvan et al., 2017; Volkova et al., 2015), in birds that experienced social isolation (Goerlich et al., 2012), in mammals exposed to BPA, atrazine, vinclozolin, dioxin, DDT (Anway et al., 2005; Manikkam et al., 2012; McBirney et al., 2017; Wolstenholme et al., 2012), and in humans that faced food restriction during war times (Pembrey, 2010). With recent discoveries of endocrine disruptors affecting transgenerational inheritance of disease susceptibility and phenotypic abnormalities (Nilsson et al., 2018), it is imperative to explore whether the endocrine disrupting chemical atrazine possesses similar capabilities given its ubiquity in the environment. Ancestral environmental chemical contaminant exposure can cause epigenetic transgenerational inheritance of altered phenotypes. Developmental exposures to environmental contaminants can result in one set of effects in the exposed generations and a completely different phenotype in the unexposed offspring. For example, zebrafish exposed to environmentally relevant concentrations of atrazine, between 0.3 and 30 parts per billion, during embryogenesis exhibited decreased spawning at adulthood, while their offspring inherited altered head length-to-body ratios (Wirbisky et al., 2016a). Gravid mice fed atrazine at 100 mg/kg/day during the germline reprogramming period (F0, E 6.5-E15.5) produced great grand-offspring (F3) males with aberrant meiotic and spermatogenic activity (Hao et al., 2016); whereas in the rats, embryonic atrazine exposure (F0 generation, 25 mg/kg BW/day, E8E14) caused incidences of testicular and mammary diseases alongside lean body phenotypes in the F3 generation (McBirney et al., 2017). While mammalian research is integral for insight into human health outcomes from atrazine exposure and mechanisms associated with it, our study was designed to elucidate the

population level effects in piscine organisms, which as a group are more frequently exposed to atrazine and, thus, possess a greater risk for adverse effects. The primary goal of this study was to explore whether developmental atrazine exposure promotes epigenetic transgenerational inheritance of reproductive dysfunction in male and female medaka (Oryzias latipes). We selected exposure concentrations of 5 and 50 mg/L of atrazine as environmentally relevant for this moderately persistent and seasonal pulsatile herbicide commonly found in water bodies adjacent to agricultural use areas (Brodeur et al., 2013; USEPA, 2016). 17b-ethinylestradiol (EE2) was used as a positive control for transgenerational inheritance, as EE2 has demonstrated this capacity in previous studies (Bhandari et al., 2015b). The life history characteristics of many aquatic species having spawning seasons parallel to large seasonal applications of atrazine in agricultural areas make this transient developmental exposure an appropriate model system to evaluate the potential transgenerational effects of atrazine. 2. Materials and methods 2.1. Experimental design The study was designed to evaluate direct and transgenerational reproductive effects in medaka from developmental exposure to atrazine. Treatment concentrations were 5 mg/L and 50 mg/L atrazine, 0.002 mg/L and 0.05 mg/L EE2, and solvent control (water). Fish were exposed during early embryonic development (0 through 12 days post-fertilization, dpf) overlapping with the critical period of gonadogenesis and sex determination in medaka that occurs between 5 and 10 dpf (Iwamatsu, 2004; Nishimura and Tanaka, 2014). Prior studies have confirmed EE2-induced transgenerational reproductive phenotype in medaka (Bhandari et al., 2015b), so EE2 (0.05 mg/L) was used as a positive control, along with a lower environmentally relevant concentration (0.002 mg/L). No outbreeding was performed in this experiment. Rather, fish were bred within the treatment groups to maintain a situation as would be expected in small agricultural ponds and local streams in agricultural regions of the United States. Fish were then cultured in clean water to adulthood, after this early life exposure, and were designated as F0 adults (directly exposed during development). Their offspring were designated as F1 and the offspring of F1 as F2. The F2 generation was not directly exposed to test chemicals, rather any effects observed in the F2 generation were the result of ancestral exposure. At sexual maturity (approximately 90e120 day post-hatch) spawning trials were conducted with individuals from the F0 and F2 generations. Transgenerational phenotypes can only be observed at F2 generation which is not directly exposed to the stressor, so we did not focus on F1 generation as effects in this generation are referred to as multigenerational exposure effects. (See Supplemental Fig. S1). The spawning trials to determine fecundity consisted of 6 breeding groups (replicates) for each treatment lineage. Breeding groups were comprised of 2 males and 6 females per chamber and egg production was measured daily for a period of 2 weeks. Morphological end points (length, wet weight, liver weight and gonad weights) were measured, reproductive organs were isolated, and sperm end points were measured at the end of each generation take down. 2.2. Fish care and handling Fish culture and exposures were conducted at the USGS Columbia Environmental Research Center (CERC). All animal procedures were conducted in accordance with the procedures described by American Institute of Fishery Research Biologists

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(AIFRB), “Guidelines for Use of Fishes in Field Research”; and with all US Geological Survey CERC guidelines for the humane treatment of test organisms during culture and experimentation. Experimental protocols and the study plan were approved by USGS-CERC Institutional Animal Care and Use Committee and Institutional Animal Care and Use Committee (IACUC) of the University of Missouri. 2.3. Exposure and maintenance of fish Japanese medaka (Oryzias latipes) Hd-rR strain were used. Adult broodstock medaka were housed in four tanks, each contained a breeding ratio of four males to six females (ten total fish per tank). Fertilized eggs (F0) were collected, pooled, and randomly sorted and assigned to fifteen glass petri dishes (5 treatments x 3 replicates/treatment). Fifty fertilized eggs were transferred to each petri dish containing 50 mL of filtered well water; as such, lineages from each petri dish were considered as biological replicates within the treatments. Atrazine (
100%, CAS # 90935) and EE2 (98%, CAS# E4876) were purchased from Sigma. Treatments consisted of a carrier solvent control (water), 0.002 mg/L and 0.05 mg/L EE2 (EE2 Low and EE2 High, respectively), and 5 mg/L and 50 mg/L of atrazine (ATZ Low and ATZ High, respectively). Exposure began approximately 8 h post-fertilization and was terminated at 12 days post fertilization (dpf). The exposure window overlapped with the window of epigenetic reprogramming of germ cells during medaka sex determination and gonadogenesis (Iwamatsu, 2004; Kondo et al., 2003; Nishimura and Tanaka, 2014). Treatment solutions were renewed with 80% new solution each day (every 24 h). No further exposures were performed after 12 dpf or in any of the subsequent generations (F1 or F2). Fish from all treatment groups were reared separately with 3 replicate tanks per treatment per generation. All fish were reared in 20-L aquaria with flow-through water and aeration. A schematic of the experimental design is presented in Supplemental Fig. 1. 2.4. Exposure solution and uptake measurement Atrazine exposure solutions used were quantified by ELISA using the protocol previously described (Papoulias et al., 2014). To quantify atrazine uptake in the medaka embryos, radioactivelabeled 14C-atrazine was used in a parallel experiment under identical conditions, using embryos from the same breeding pairs that produced embryos for the gene expression and phenotype analysis. Atrazine concentrations were measured in the exposure medium and embryo/fry using liquid scintillation counting of 14C -atrazine as previously described (Papoulias et al., 2014). EE2 uptake by medaka embryos under these conditions has been reported previously (Bhandari et al., 2015a,b). In 24 h, EE2 uptake by medaka embryo was 1.2 pg/mg embryo in 24 h and 4 pg/mg embryo in 7 days). Final atrazine uptake (pg/mg embryo) was calculated at three time points: 1, 7, and 12 days after exposure.

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successfully completed development until hatching (10 days after fertilization). After collection of fertilized eggs for the next generation, the reproductively active adults were sacrificed for morphometric analysis and tissue collection. Body length, gonad weight and liver weight were recorded for each fish. Tissues samples (gonads) were collected and preserved in 500 mL RNA later solution (ThermoFisher Scientific, # AM7021) for nucleic acid isolation. Sperm parameters were analyzed at the laboratory of Dr. Yuksel Agca in the Department of Veterinary Pathobiology, University of Missouri. Sperm were activated just prior to the total sperm count, mobile sperm count, and percentage motility measurement (of total). Sperm analysis was completed within 2e4 h of testis isolation. 2.6. RNA/DNA isolation, cDNA synthesis, real-time quantitative PCR, and 5-mC ELISA Gonads were collected from the adult fish and stored in RNALater solution at 20  C until nucleic acid isolation. A total of five ovaries and five testes were collected from each biological replicate within a treatment group at the end of the spawning trials from generations F0 through F2. However, based on financial and logistical limitations only the whole gonad tissues from F0 and F2 generation fish were used for phenotype characterization and molecular analysis. Whole gonad tissues were transferred from RNAlater© to 300 ml lysis buffer and homogenized manually with a pestle prior to homogenization with the Benchmark D1000 handheld homogenizer with three 4 s pulses. All samples were briefly centrifuged after homogenization and subjected to RNA/DNA isolation with ZymoResearch Z-R Duet™ MiniPrep Kit (#D7003) according to manufacturer's protocol with DNase I treatment to avoid genomic DNA contamination. RNA concentration and quality were confirmed with NanoDrop (ND-2000) spectrophotometer and bleach gel electrophoresis (Aranda et al., 2012), respectively. Supplementary Fig. S5 shows 28S and 18S bands of randomly selected representative RNA samples (8 out of 150 samples). cDNA was synthesized from each RNA sample by using Applied Biosystems' High-Capacity cDNA Reverse Transcription kit following standard manufacturer protocol. For quantitative PCR, 4 ng of each sample was used with Power-Up™ SYBR® Green Master Mix reagents (Applied Biosciences # A25743) and primers specific to medaka genes of interest (See supplementary information for primer sequences). Efficiency of the qPCR reaction for each of the primer pairs was between 94% and 100%. Beta actin and Ef1a were used as internal controls and gene expression was calculated relative to control using comparative 2DDCT method (Schmittgen and Livak, 2008). Global DNA methylation was quantified by a commercial 5-mC ELISA kit (#D5326) developed by Zymo Research. Methylation levels were determined according to manufacturer's instructions and have been presented as percent methylation (%). This kit works for medaka fish DNA methylation (5 mC). We have verified the kit with whole genome bisulfite sequencing of medaka embryo epigenome (Wang and Bhandari, 2019).

2.5. Medaka lineage maintenance and phenotype characterization 2.7. Statistical analysis All fish were maintained on a 14L:10D photoperiod with water temperature at 25 ± 0.5  C. Each tank was equipped with an overflow outlet and air supply. Exposure lineages were maintained in each generation by breeding adults from the same replicate tank, so parent-of-origin effects were not examined. Fecundity, fertilization rate, and embryo survival were recorded daily for 7e14 days. Fecundity was calculated as total egg production within each breeding tank/day, and fertilization rate was calculated by percent of fertilized eggs within the total daily egg production. Embryo survival was determined as the number of fertilized eggs that

The study was designed to examine exposure-induced transgenerational phenotypic and molecular endpoints. Statistical analyses of atrazine uptake, gonadosomatic and hepatosomatic indices, fecundity, fertilization, sperm parameters, and gene expression were performed by comparison of control and treatment groups within the same generation and across generations. There were 5 technical replicates sampled per biological replicate. Altogether, three biological replicates (tanks) were used in the determination of significance testing (n ¼ 3). The effects of exposure to different

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concentrations of chemicals within one generation were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's posthoc test. Student's t-test for independent samples was used to compare F0 generation data with F2 generation data for each endpoint. Data were represented as mean ± SEM (standard error of the mean). Significant differences among treatments within one generation are indicated by lower-case letters, and significant transgenerational differences in F2 generation from F0 are indicated by symbols above respective bars. 3. Results 3.1. Exposure concentration and uptake Measured concentrations in the petri dishes ranged from 3.4 to 4.8 mg/L for Low ATZ group and from 48.5 to 58.0 mg/L for High ATZ group (supplemental information, Fig. S2A). These data indicated that 5 mg/L exposure concentrations of ATZ were 18e32% below nominal concentration, while 50 mg/L treatments were 5e16% greater than the nominal concentration. Embryo atrazine uptake during the 12-day experimental period was between 15.8 and 22.9 pg/mg embryo for Low ATZ group and between 208 and 222 pg/mg embryo for High ATZ group (supplemental information, Fig. S2B).

3.3. Fecundity and fertilization Fecundity did not change due to exposure in any generation relative to control (Fig. 1A). The number of fertilized eggs increased by 15.27% in the ATZ High treatment lineage (p < 0.05) in the F0 generation relative to the F0 control treatment, whereas the number of fertilized eggs decreased by 22.42% and 18.38% in the EE2 Low (p < 0.05) and ATZ Low lineages, respectively (Fig. 1B), in the F2 generation relative to the F2 control treatment. 3.4. Sperm parameters in males Total sperm count, motile sperm count, and percent motility were unaffected by any of the treatments in the F0 generation relative to the controls in the F0 generation (Fig. 2AeC), whereas all the three parameters were decreased (32.49% in sperm count, 55.15% in motile sperm, and 32.14% in motility) in the EE2 Low lineage at F2 generation relative to controls in the F2 generation. Total sperm count and total motile sperm were significantly decreased (p < 0.05) by 48.82% and 57.79%, respectively, in the ATZ Low treatment lineage at F2 generation, relative to F2 generation control treatment. In the ATZ high lineage, total motile sperm count and sperm motility was significantly decreased (p < 0.05) by 55.28% and 38.27%, respectively, at F2 generation relative to the control for that generation. 3.5. Gene expression changes

3.2. Body somatic indices Body length and body weight were not changed by exposure in any generations (supplemental information, Fig. S3 A-D). Liver to body ratios (hepatosomatic index, HSI) were significantly reduced (1.23 times) as compared to controls in females from the EE2 Low (p < 0.05), 1.24 times in ATZ Low (p < 0.05), and 1.32 times in ATZ High (p < 0.01) treatment lineages within the F2 generation (supplemental information, Fig. S4B), whereas no significant difference was found in males (supplemental information, Fig. S4A). In both males and females, gonad to body ratio (gonadosomatic index, GSI) was not altered by any exposures (supplemental information, Fig. 4 C&D).

Testis: Transgenerational gene expression profiles of star, fshr, and ara were determined in the testis (Fig. 3). The expression of star, a gateway of steroidogenesis, was significantly decreased (3.25 fold) in the ATZ High lineage at F0 generation and increased (3.17 and 6.21 folds) in the ATZ Low and ATZ High lineage at the F2 generation relative to their respective control treatments. Expression of fshr was significantly decreased 5.26 fold in the EE2 High (p < 0.05), 11.23 fold in ATZ Low and 9.09 fold in ATZ High (p < 0.001) lineages at the F0 generation, whereas the expression was increased 3.40 times in the EE2 high and 2.88 times in ATZ Low lineages (p < 0.05) at the F2 generation. The expression of ara was increased (2.02 fold) in the EE2 Low lineage and decreased (1.72

Fig. 1. Transgenerational differences in fecundity (A) and fertilization rate (B) caused by EE2 and atrazine exposure during early life at F0 generation. The F0 embryos were exposed to EE2 and atrazine from 8 h post fertilization (dpf) to 12 dpf. Fish were raised to adulthood without further exposure in any generations. Effects observed here are due to early life exposure of grandparents on reproduction of grandchildren. Data are presented as mean ± SEM. Letters above bars indicate significant differences from each other within the same generation (ANOVA, p < 0.05).

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Fig. 2. Transgenerational differences in sperm parameters [total sperm count (A), motile sperm count (B) and sperm motility (C)] of the F0 and F2 generation adult males caused by EE2 and atrazine exposure during early life at F0 generation. Data are presented as mean ± SEM. Letters above bars indicate significant differences from each other within the same generation (ANOVA, p < 0.05). Total sperm count indicates total number of sperm per milliliter of sperm collected. Total motile sperm indicates the number of sperm that showed a movement and has been expressed as number of motile sperm per milliliter of total sperm collected. Sperm motility indicates a percentage of the sperm that were actively swimming during the measurement and has been expressed as % motility against total number of sperm collected.

Fig. 3. Transgenerational differences in expression of reproductive genes in the testis of adult medaka exposed to 17a-ethinylestradiol and atrazine. The F0 generation was directly exposed during early life (0e12 days post fertilization), whereas the F2 generation was never exposed to test chemicals. Gene symbols are given just above the graph. Data are presented as relative quantification (RQ) and mean ± SEM. Letters indicate significant differences among treatments within the same generation. Letters above bars indicate significant differences from each other within the same generation (ANOVA, p < 0.05) and symbols indicate significant differences in F2 generation when compared with F0 generation within the same exposure group (y, p < 0.05; z, p < 0.01; yz, p < 0.001).

fold) in the ATZ High (p < 0.01) lineage within the F0 generation, whereas no change was found at the F2 generation. Ovary: Transgenerational gene expression profiles of Star, Cyp19a1a, lhr, and esr1 were determined in the ovary (Fig. 4). Expression of star and lhr was not altered in any of the chemical exposure treatment lineages. The cyp19a1a expression was increased in the EE2 High (p < 0.05), ATZ Low (p < 0.05) and ATZ High (p < 0.01) groups within the exposed F0 generation, while no transgenerational differences were observed in Cyp19a1a gene expression in any lineages at F2 generation when compared to control. F2 generation ovaries of the fish from ATZ lineages maintained significantly lower levels of cyp19a1a expression when compared with the F0 generation ovaries (p < 0.01 & p < 0.001, indicated by symbols). Expression of esr1 was decreased (2.38 fold) in the ATZ High (p < 0.05) treatment lineage at the F0 generation, but no transgenerational (F2 generation) alterations in esr1 expression were observed in any treatment lineages.

epigenetic changes in the testis (Fig. 5) and ovary (Fig. 6). Testis: Dnmt1 expression was not altered in any treatment lineages within the F0 generation, whereas the expression of dnmt1 was increased (2.98 fold) in the EE2 high lineage within the F2 generation (p < 0.05) with a tendency toward increase (1.92 fold) in the ATZ Low lineage. Dnmt3aa expression was decreased (2.94 fold) in the ATZ Low (p < 0.005) and in ATZ High (3.36 fold, p < 0.01) lineages within the F0 generation, whereas the expression was increased in the EE2 High (1.83 fold, p < 0.05) at the F2 generation. Ovary: Dnmt1 expression was decreased in the EE2 High (p < 0.01) and ATZ High (p < 0.05) treatment lineages at the F0 generation, whereas within the F2 generation the dnmt1 expression was decreased in the ATZ Low lineage (p < 0.05). Dnmt3aa expression was decreased in the ATZ Low (p < 0.01) and ATZ High (p < 0.01) lineages at the F0 generation, whereas no significant effects were found in expression of Dnmt3aa in any lineages within the F2 generation. 3.7. Global DNA methylation

3.6. Expression of DNA methyltransferase genes Dnmt3aa and Dnmt1 expression was quantified as a measure of

Global DNA methylation was measured on DNA isolated from whole testis and ovaries (Fig. 7 A&B). Within the F0 generation,

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Fig. 4. Transgenerational differences in expression of reproductive genes in the ovaries of adult medaka exposed to 17a-ethinylestradiol and atrazine during early life at F0 generation. Here, the F0 generation was directly exposed, whereas the F2 generation was never exposed to test chemicals. Gene symbols are given just above the graph. Data are presented as relative quantification (RQ) and mean ± SEM. Letters above bars indicate significant differences from each other within the same generation (ANOVA, p < 0.05) and symbols indicate significant differences in F2 generation when compared with F0 generation within the same exposure group (z, p < 0.01; yz, p < 0.001).

global DNA methylation levels were significantly decreased in the ATZ High lineage testis (1.27 fold, p < 0.01) and ovaries (1.85 fold, p < 0.05) relative to controls. Within the F2 generation, global DNA methylation was significantly decreased in the testis of EE2 high lineage males (1.24 fold) as compared to control, whereas no significant differences were observed in the ovary of F2 females. F2 generation males from High EE2 lineage had a significant decrease (z, p < 0.01) in DNA methylation levels in the testis (1.26 fold) as compared to that in the F0 generation males; whereas ATZ High lineage males had a significant increase (yz, p < 0.001) in DNA methylation at F2 generation (1.15 fold) as compared to F0. 4. Discussion The present findings demonstrate that embryonic atrazine exposure results in some phenotypic abnormalities in adults of the exposed F0 generation but also exerts significant transgenerational effects on phenotype in the F2 generation by altering reproductive capacity in medaka. Embryonic atrazine exposure of the F0 generation induced abnormal sperm count and motility in the F2 generation. Analysis of select testis and ovary-specific transcripts revealed transgenerationally altered expression patterns of genes

that play critical roles in gametogenesis. Embryonic atrazine or EE2 exposure suppressed genes involved in DNA methylation (dnmt1 and dnmt3aa) in adults of the F0 generation, suggesting hypomethylation in gonads was caused by direct exposure. Additionally, F2 generation testes and ovaries showed increased expression patterns in the Dnmt1 gene in the low atrazine exposure concentration, suggesting epigenetic associations with transgenerational phenotypes in medaka. The present results also support the possibility that environmental chemical exposures could have far reaching transgenerational health consequences in fish, although the underlying mechanisms are yet to be defined (Bhandari et al., 2015a). Atrazine exposure did not alter fecundity (egg production) in either the F0 or F2 generations, indicating that egg numbers are not affected by embryonic atrazine exposure. Although egg production is indicative of reproductive health in females, it does not always predict successful production of offspring or their health (Chavarro et al., 2016; Kjørsvik et al., 1990). Offspring health and survival also depend on the quality of eggs that are produced. Therefore, it is difficult to conclude based on the present findings that the female reproduction was not affected by embryonic atrazine exposure. Direct effects of early life exposure to atrazine on embryonic

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Fig. 5. Transgenerational differences in expression of DNA methyltransferase genes in the testis of adult medaka exposed to atrazine and 17a-ethinylestradiol during early life at F0 generation. Gene symbols are given just above the graph. Data are presented as relative quantification (RQ) and mean ± SEM. F0 generation was directly exposed, whereas F2 generation was never exposed to test chemicals, but effects were mediated by the F1 generation germ cells. Letters above bars indicate significant differences from each other within the same generation (ANOVA, p < 0.05) and symbols indicate significant differences in F2 generation when compared with F0 generation within the same exposure group (y, p < 0.05; z, p < 0.01).

Fig. 6. Transgenerational differences in expression of DNA methyltransferase genes in the ovaries of adult medaka exposed to atrazine and 17a-ethinylestradiol during early life at F0 generation. Gene symbols are given just above the graph. Data are presented as relative quantification (RQ) and mean ± SEM. F0 generation was directly exposed, whereas the F2 generation was never exposed to test chemicals. Letters above bars indicate significant differences from each other within the same generation (ANOVA, p < 0.05) and symbols indicate significant differences in F2 generation when compared with F0 generation within the same exposure group (y, p < 0.05; V, p < 0.06).

development has been evident in the literature; however, information on latent effects on adult health initiated from embryonic stages is limited. In our prior studies, a significant reduction in egg production was observed when adult female fathead minnows or medaka were exposed to atrazine during the reproductive cycle (Papoulias et al., 2014; Tillitt et al., 2010). In zebrafish, embryonic atrazine exposure (30 mg/L) of the F0 generation induced follicular atresia and caused reduced spawning in adult zebrafish from that same F0 generation (Wirbisky et al., 2016a). Direct reproductive effects of atrazine exposure appear to be species and life history stage specific. The present study demonstrated a significant reduction in fertilization success of the F2 generation fish from either low ATZ or low EE2 embryonic exposure of the F0. Mechanisms underlying

transgenerational inheritance of altered phenotypes are not clearly understood; however, a majority of studies focused on environmentally induced transgenerational inheritance report altered pathologies, behavior, and molecular signatures in males, suggesting inheritance of sex-biased transgenerational effects (Bhandari, 2016; Hanson and Skinner, 2016; Nilsson and Skinner, 2015). In medaka, BPA and EE2 exposure caused similar reductions in fertilization rate at the F2 and F3 generations (Bhandari et al., 2015b), while in zebrafish, dioxin exposure at the juvenile stage (3 weeks and 7 weeks post-fertilization) caused significant reductions in fertilization success at the F2 generation (Baker et al., 2014a; Baker et al., 2014b, c). The present study is the first report to demonstrate a transgenerational reproductive impairment caused by atrazine exposure in fish. Prior to this, only one study has investigated

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Fig. 7. Transgenerational alterations in global DNA methylation (5 mC) levels in the F2 testis (A) and ovaries (B) of adult medaka exposed to 17a-ethinylestradiol and atrazine during early life at F0 generation. Data are presented as mean fold change (±SEM) against controls. Letters above bars indicate significant differences from each other within the same generation (ANOVA, p < 0.05) and symbols indicate significant differences in F2 generation when compared with F0 generation within the same exposure group (z, p < 0.01; yz, p < 0.001).

developmental atrazine exposure effects on reproduction at adulthood and in immediate offspring of zebrafish (Wirbisky et al., 2016b). Embryonic atrazine exposure was found to induce fertilization impairment at adulthood of the F0 generation fish. Since the study by Wirbisky et al. did not examine reproductive defects in subsequent generations, it is currently unknown if these effects persist into the F2 generation. In the same study, embryos exposed to 30 parts per trillion (30 ng/L) concentration of atrazine during the first 72 h of development showed a significant decrease in spawning and a significant increase in follicular atresia in the adult F0 females as well as structural defects in the F1 offspring. No change in body or testes weight, gonadosomatic index, testes histology, or levels of 11ketotestosterone or testosterone were observed in the F0 males with the same treatment (Wirbisky et al., 2016b). Taken together, the present results together with others indicate that environmental concentrations of atrazine affect reproduction of fish in the current generation. Additionally, the present study adds to the existing literature that atrazine can also affect the reproduction of subsequent, unexposed generations transgenerationally through ancestral exposure. Atrazine induced transgenerational reproductive phenotypes have been found in higher vertebrates as well. In CD-1 mice, atrazine exposure (100 mg/kg/day) during gestational day 6.5 and 15.5 affected meiosis, spermiogenesis and reduced the spermatozoa number in the male mice at the third generation (Hao et al., 2016). In the outbred Hsd:Sprague Dawley®™SD®™ (Harlan) rat, atrazine exposure (25 mg/kg BW/day) increased the incidence of testis disease in the F2 and F3 generations (McBirney et al., 2017). These results, including the present findings, suggest that early developmental exposure to atrazine may or may not pose immediate significant reproductive risks into adulthood of exposed fish, but certainly induces molecular changes in the gametes which could lead to transgenerational phenotypes potentially affecting future generations. Both low concentration EE2 and atrazine exposures during embryo development led to reductions in total sperm numbers, motile sperm counts, and sperm motility in the F2 generation lineages, suggesting atrazine effects on male reproduction. EE2 induction of sperm impairment has been previously reported in rainbow trout, but only in F0 generation males due to direct

exposure (Brown et al., 2008). Previously we found that in medaka, embryonic EE2 exposure at a concentration of 0.05 mg/L resulted in reduced fertilization rate in F2 generation males, but whether these males had impaired sperm motility or reduced sperm count was not examined (Bhandari et al., 2015b). In zebrafish, 11-day exposure to nominal atrazine concentrations of 2, 10, and 100 mg/L caused a significant decrease in sperm motility, mitochondrial function, and membrane integrity (Bautista et al., 2018). In CD-1 mice, developmental atrazine exposure did not affect the morphology or cell types in testis of the F3 generation males, but caused a 30% reduction in mature spermatozoa in the epididymis of the F1 and F3 generation males (Hao et al., 2016). The same study also found a 2.6 times decrease in levels of protamine 2 in ATZ-derived testis, suggesting impairment of histone modification and chromatin function during spermiogenesis. Acetylation at histone H4 (H4K5ac and H4K8ac) is essential for bromodomain-containing protein binding, and that binding is, in turn, critical for histone-toprotamine transition (Goudarzi et al., 2014). Evidence for histone impairment was demonstrated by Western blot analysis of H4K5Ac in a purified histone fraction of the spermatozoa collected from F3 generation males. In that study, an approximate 1.3-fold decrease in the level of H4K5Ac was observed in F3 atrazine-lineage males, suggesting that the decreased protamine and histone H4 acetylation levels could reflect an inefficient histone-to-protamine replacement in the F3 atrazine-lineage males (Goudarzi et al., 2014). A recent study demonstrated increased rate of transgenerational histone retention in rat sperm caused by embryonic atrazine exposure, which conforms to the previous findings (Goudarzi et al., 2014) that atrazine may cause epigenetic impairment by interfering with histone-to-protamine replacement process. Although histone modifications were not quantified in the present study, a similar abnormal sperm phenotype to the study by Goudarzi et al. (2014) suggests the possibility for epigenetic impairment of sperm in the F2 medaka testis. It is notable that mechanisms controlling biological processes are complementary among eukaryotes, although results pertaining to physiological relevance in higher vertebrates are not directly comparable to lower vertebrates because of complexity of body plan and physiological processes.

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The present results demonstrate down-regulation of steroidogenic acute regulatory protein (STAR) gene expression by developmental exposure to high concentration of atrazine in the testis of F0 males. Conversely, F2 generation males showed significant increases in star expression in both ATZ Low and ATZ High treatment lineages, suggesting that the consequences of early developmental exposures pose a markedly dissimilar risk to the grand-offspring (F2) than it does to the exposed generation (F0). We found that a number of transgenerational (F2) effects were different from direct effects of atrazine exposure. Careful examination of this phenomenon is required as germline effects at F0 generation can be inherited by both germ cells and somatic cells in the subsequent exposed generation (F1). The F1 generation germ cells were exposed during the F0 exposure period of development which included differentiation of testes and ovaries; as such, these effects could then be transmitted to the F2 and possibly subsequent generations via epigenetic marks in the germ cells. These transgenerationally-transmitted effects would only occur through atrazine-induced reprogramming of germ cells, including gametes in adult males. Previous studies characterizing atrazine's impact on steroidogenesis have demonstrated significant alterations in steroid hormone synthesis, but few of those studies examined embryonic exposure effects on adult or subsequent generations' steroid hormone gene expression or enzymatic activity. STAR is a rate limiting protein in the synthetic steroidogenic pathway and is required to transport cholesterol in the mitochondria (Kiriakidou, 1996). Without the proper expression of this transport protein gene, secretion of steroid hormones and regulation of lipid accumulation are affected (LaVoie, 2017). Star expression has been found to increase (Suzawa and Ingraham, 2008) or decrease (Pogrmic et al., 2009) after atrazine exposures. ARa is a nuclear steroid hormone receptor responsible for mediating the transcriptional effects of the circulating androgenic hormones, testosterone, and in teleost fishes, 11-ketotestosterone (Sperry and Thomas, 1999). Androgens and their receptors are responsible for appropriate male development, spermatogenesis, and fertility. Alteration in ara gene expression in the testis of the F0 generation indicates a direct effect on developing testis that retained developmentally programmed effects until adulthood; however, this effect was not inherited by the F2 generation. Direct exposure effects on ara expression in the testes of F0 generation males showed an interesting pattern. Low EE2 exposure increased ara transcript levels, whereas ATZ exposure (both low and high concentrations) tended to suppress ara expression, suggesting that ATZ affects reproductive genes through mechanisms different from environmental estrogen, EE2. Atrazine may have some estrogenic properties (Albanito et al., 2015) as it elicits an estrogen action by up-regulating aromatase activity in certain cancer cells (Sanderson et al., 2000), but not by binding to or activating era (Connor et al., 1996; Roberge et al., 2004; Tennant et al., 1994). The expression of the fshr gene decreased in EE2 and ATZexposed F0 generation testis, while the F2 generation inherited an elevated state of fshr expression. Effects were observed in males, while ATZ and EE2 exposure did not alter star, lhr, and esr1 in the ovary at any of the generation examined, suggesting sex-specific effects of these two environmental compounds on the reproductive axis signaling pathways. It is not uncommon to have opposite effects in transgenerational lineages compared to F0 generation lineages, as modifications in the germline are distributed to both somatic cells and germ cells in the offspring, causing unpredicted somatic cell defects by as yet unknown mechanisms (Heard and Martienssen, 2014). FSH and its receptor, FSHR, play a significant role in spermatogenesis in the testis (Pogrmic-Majkic et al., 2018; Schulz et al., 2001). Our observation of atrazine-induced suppression of fshr in the testis of F0 generation males conforms with

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previous reports of direct exposure effects of atrazine (Fa et al., 2013; Pogrmic-Majkic et al., 2018); however, the consequences of elevated fshr in F2 generation is currently unknown. In reproductive tissues, mis-expression of fshr leads to abnormal gonadal morphology, hemorrhagic cysts, altered levels of sex steroids, and teratomas (Peltoketo et al., 2010). Transgenerational induction of elevated fshr in males may also result in precocious maturation. Subsequently, these males may not be competitive enough at the normal reproductive age, as premature activation of hypothalamusreproductive axis causes precocious maturation and puberty (Guiry et al., 2010; McGee and Narayan, 2013). Given that F2 generation fish had both reduced sperm count/motility and reduced fertilization success in the EE2-Low and ATZ-low lineages, the possibility for having precociously mature males in F2 generation cannot be ignored. Although the present study did not examine precocious maturation, a careful examination of male phenotype during the mid-stage of sexual maturation at the F2 generation might be sufficient to explain the phenotype. Gene-environment interaction can result in the emergence of phenotypes involving genome-wide epigenetic alterations (Crews and Gore, 2014; Skinner et al., 2010). Epigenetic reprogramming, especially erasure and reestablishment of new epigenetic marks, occurs first during the early blastula stage and then again during sex determination and gonad differentiation (Reik et al., 2001). DNA methylation is one of the extensively studied epigenetic mechanisms and is an enzymatic process involving DNA methyltransferases (DNMTs). Although we did not examine DNA methylation at the single nucleotide level, the results still provide insights into methylation pattern in reproductive tissues at the whole genome level and are supported by expression pattern of the genes encoding DNA methyltransferase enzymes. Expression of the dnmt1 gene, which is involved in maintenance of methylation marks during mitosis, and the dnmt3aa gene, which is involved in de novo DNA methylation, was decreased relative to controls from EE2 and ATZ groups at the F0 generation, similar to that observed in common carp (Xing et al., 2014) and zebrafish (WirbiskyHershberger et al., 2017). This decrease in dnmt3aa and dnmt1 expression as well as a decrease in global DNA methylation levels in the F0 ATZ High group suggests that hypomethylation in the testes and ovaries was associated with direct exposure to high concentration of atrazine. In contrast, transgenerational increase in dnmt1 mRNA levels and corresponding increase in global 5 mC levels in the F2 ATZ High lineage males suggest an increased level of hypermethylation in the testis in grand offspring. Previously, it has been observed that exposure to atrazine or EE2 caused decreased DNA methyltransferase gene expression and decreased global DNA methylation in the gonads (Wang et al., 2014; WirbiskyHershberger et al., 2017). These decreases were not due to chronic exposure to these stressor chemicals but were due to a programmed effect that was initiated during embryogenesis and maintained throughout the life of the organism. This is not uncommon as previous studies suggest that newly programmed differentially methylated regions of DNA can be established in germ cells due to ancestral atrazine exposure (Hao et al., 2016; McBirney et al., 2017). Environmental contaminant exposure during early developmental periods of sex determination and gonadogenesis may imprint novel patterns of DNA methylation marks on primordial germ cell epigenome (Jirtle and Skinner, 2007; Skinner, 2011). This imprint may escape germ cell epigenetic reprogramming events just like genetic imprints are maintained in both germ cells and somatic cells, causing phenotypic differences in F2 generation offspring. Despite the fact that an association has been shown between environmental exposure and transgenerational epigenetic mutations (epimutations) in the gametes (Hao et al., 2016;

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Manikkam et al., 2012; McBirney et al., 2017; McCarrey, 2014; McCarrey et al., 2016), it is still unclear whether these epimutations were established during the window of germ cell reprogramming or during the time of gametogenesis. Studies have, however, demonstrated the ability of environmental contaminants to induce differential epimutations in genomic DNA at both F0 and F2 generations of fish (Carvan et al., 2017; Gold et al., 2018; Hanson and Skinner, 2016; Hao et al., 2016; Haque et al., 2016; McBirney et al., 2017). It is also hypothesized that if a perturbation occurs on DNA methylation during the period of germ cell reprogramming, it is likely to escape the event of programmed erasure of epigenome and behave as imprinted loci in the germline (Jirtle and Skinner, 2007; Skinner, 2016; Xin et al., 2015). Global hypomethylation of genomic DNA in the testis and ovary of the F0 generation fish exposed to high dose atrazine indicates epigenetic effects of exposure on gonads, whereas no significant differences were observed in global DNA methylation profile within the F2 generation. Low atrazine exposure-induced hypermethylation in ovaries of the F0 generation females suggests dose-dependent and sexspecific epigenetic alterations caused by direct exposure during embryonic development. 5. Conclusions Developmental exposure to atrazine or EE2 resulted in alterations in sperm parameters and fertilization efficiency of fish in future generations. Additionally, effects of direct exposure (F0 generation) were different than transgenerational effects (F2 generation) observed in the same exposure lineages. It is difficult to extrapolate data from laboratory studies to predict transgenerational reproductive effects of atrazine in the natural populations of fish. However, the present results suggest that atrazine and EE2 can induce transgenerational health effects in fish and that emergence of transgenerational phenotypes in natural populations warrants further study. Competing interests The authors declare no competing interests as defined by Environmental Pollution. Disclaimer Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Author contributions Conceived experiments- RKB, DET, FVS; performed experimentRKB, JAC, DN, RC; Data Analysis- JAC, RKB, wrote manuscript and prepared figures- JAC, RKB; Figure Art work: RKB., JAC; Proofread and improved manuscript- RKB, DET, FVS. Acknowledgements Authors thank James Candrl, Vanessa Velez, John Carroll, Dr. Catherine Richter, and Dr. Jessica Leet for assistance during experimental period and the FSP reviewer for invaluable suggestions on earlier versions of this manuscript. Measurement of sperm parameters was made possible by generous support from the laboratory of Dr. Yuksel Agca, Veterinary Pathology, University of Missouri-Columbia. This study was supported by the funds from United States Geological Survey (USGS G15AS00092) and UNCG Department of Biology to RKB.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.05.013. References Albanito, L., Lappano, R., Madeo, A., Chimento, A., Prossnitz, E.R., Cappello, A.R., Dolce, V., Abonante, S., Pezzi, V., Maggiolini, M., 2015. Effects of atrazine on estrogen receptor alpha- and G protein-coupled receptor 30-mediated signaling and proliferation in cancer cells and cancer-associated fibroblasts. Environ. Health Perspect. 123, 493e499. https://doi.org/10.1289/ehp.1408586.
Alvarez, N.B., Avigliano, L., Loughlin, C.M., Rodríguez, E.M., 2015. The adverse effect of the herbicide atrazine on the reproduction in the intertidal varunid crab Neohelice granulata (Dana, 1851). Regional Studies in Marine Science 1, 1e6. https://doi.org/10.1016/j.rsma.2014.12.001. Aranda, P.S., LaJoie, D.M., Jorcyk, C.L., 2012. Bleach gel: a simple agarose gel for analyzing RNA quality. Electrophoresis 33, 366e369. https://doi.org/10.1002/ elps.201100335. Anway, M.D., Cupp, A.S., Uzumcu, M., Skinner, M.K., 2005. Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 308, 1466e1469. https://doi.org/10.1126/science.1108190. Baker, N.T., 2016. Estimated annual agricultural pesticide use by major crop or crop group for states of the conterminous United States, 1992-2016,. In: N.W.-Q.A.N. (Ed.), Project. US Geological Survey, Reston, VA. Baker, T.R., King-Heiden, T.C., Peterson, R.E., Heideman, W., 2014a. Dioxin induction of transgenerational inheritance of disease in zebrafish. Mol. Cell. Endocrinol. 398, 36e41. https://doi.org/10.1016/j.mce.2014.08.011. Baker, T.R., Peterson, R.E., Heideman, W., 2014b. Adverse effects in adulthood resulting from low-level dioxin exposure in juvenile zebrafish. Endocr. Disruptors 2. https://doi.org/10.4161/endo.28309. Baker, T.R., Peterson, R.E., Heideman, W., 2014c. Using zebrafish as a model system for studying the transgenerational effects of dioxin. Toxicol. Sci. 138, 403e411. https://doi.org/10.1093/toxsci/kfu006. Bautista, F.E.A., Varela Junior, A.S., Corcini, C.D., Acosta, I.B., Caldas, S.S., Primel, E.G., Zanette, J., 2018. The herbicide atrazine affects sperm quality and the expression of antioxidant and spermatogenesis genes in zebrafish testes. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 206e207, 17e22. https://doi.org/10.1016/ j.cbpc.2018.02.003. Bhandari, R.K., 2016. Medaka as a model for studying environmentally induced epigenetic transgenerational inheritance of phenotypes. Environ Epigenet 2, dvv010. https://doi.org/10.1093/eep/dvv010. Bhandari, R.K., Deem, S.L., Holliday, D.K., Jandegian, C.M., Kassotis, C.D., Nagel, S.C., Tillitt, D.E., Vom Saal, F.S., Rosenfeld, C.S., 2015a. Effects of the environmental estrogenic contaminants bisphenol A and 17alpha-ethinyl estradiol on sexual development and adult behaviors in aquatic wildlife species. Gen. Comp. Endocrinol. 214, 195e219. https://doi.org/10.1016/j.ygcen.2014.09.014. Bhandari, R.K., Vom Saal, F.S., Tillitt, D.E., 2015b. Transgenerational effects from early developmental exposures to bisphenol A or 17alpha-ethinylestradiol in medaka, Oryzias latipes. Sci. Rep. 5, 9303. https://doi.org/10.1038/srep09303. Brodeur, J.C., Sassone, A., Hermida, G.N., Codugnello, N., 2013. Environmentallyrelevant concentrations of atrazine induce non-monotonic acceleration of developmental rate and increased size at metamorphosis in Rhinella arenarum tadpoles. Ecotoxicol. Environ. Saf. 92, 10e17. https://doi.org/10.1016/ j.ecoenv.2013.01.019. Brown, K.H., Schultz, I.R., Cloud, J., Nagler, J.J., 2008. Aneuploid sperm formation in rainbow trout exposed to the environmental estrogen 17a-ethynylestradiol. Proc. Natl. Acad. Sci. Unit. States Am. 105, 19786e19791. https://doi.org/10.1073/ pnas.0808333105. Carvan 3rd, M.J., Kalluvila, T.A., Klingler, R.H., Larson, J.K., Pickens, M., MoraZamorano, F.X., Connaughton, V.P., Sadler-Riggleman, I., Beck, D., Skinner, M.K., 2017. Mercury-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in zebrafish. PLoS One 12, e0176155. https://doi.org/10.1371/journal.pone.0176155. Chavarro, J.E., Minguez-Alarcon, L., Chiu, Y.H., Gaskins, A.J., Souter, I., Williams, P.L., Calafat, A.M., Hauser, R., Team, E.S., 2016. Soy intake modifies the relation between urinary bisphenol a concentrations and pregnancy outcomes among women undergoing assisted reproduction. J. Clin. Endocrinol. Metab. 101, 1082e1090. https://doi.org/10.1210/jc.2015-3473. Connor, K., Howell, J., Chen, I., Liu, H., Berhane, K., Sciarretta, C., Safe, S., Zacharewski, T., 1996. Failure of chloro-S-triazine-derived compounds to induce estrogen receptor-mediated responses in vivo and in vitro. Toxicol. Sci. 30, 93e101. https://doi.org/10.1093/toxsci/30.1.93. Cooper, R.L., Stoker, T.E., Tyrey, L., Goldman, J.M., McElroy, W.K., 2000. Atrazine disrupts the hypothalamic control of pituitary-ovarian function. Toxicol. Sci. 53, 297e307. https://doi.org/10.1093/toxsci/53.2.297. Corvi, M.M., Stanley, K.A., Peterson, T.S., Kent, M.L., Feist, S.W., La Du, J.K., Volz, D.C., Hosmer, A.J., Tanguay, R.L., 2012. Investigating the impact of chronic atrazine exposure on sexual development in zebrafish. Birth Defects Res B Dev Reprod Toxicol 95, 276e288. https://doi.org/10.1002/bdrb.21016. Crews, D., Gore, A.C., 2014. Current controversies and debates. Transgenerational epigenetics. In: Trygve Tollefsbol. Elsevier, pp. 371e390, 1. https://doi.org/10. 1016/B978-0-12-405944-3.00026-X.

J.A. Cleary et al. / Environmental Pollution 251 (2019) 639e650 Fa, S., Pogrmic-Majkic, K., Samardzija, D., Glisic, B., Kaisarevic, S., Kovacevic, R., Andric, N., 2013. Involvement of ERK1/2 signaling pathway in atrazine action on FSH-stimulated LHR and CYP19A1 expression in rat granulosa cells. Toxicol. Appl. Pharmacol. 270, 1e8. https://doi.org/10.1016/j.taap.2013.03.031. Gely-Pernot, A., Hao, C., Becker, E., Stuparevic, I., Kervarrec, C., Chalmel, F.,
gou, B., Smagulova, F., 2015. The epigenetic processes of meiosis in Primig, M., Je male mice are broadly affected by the widely used herbicide atrazine. BMC Genomics 16. https://doi.org/10.1186/s12864-015-2095-y. Gely-Pernot, A., Saci, S., Kernanec, P.Y., Hao, C., Giton, F., Kervarrec, C., Tevosian, S., Mazaud-Guittot, S., Smagulova, F., 2017. Embryonic exposure to the widely-used herbicide atrazine disrupts meiosis and normal follicle formation in female mice. Sci. Rep. 7, 3526. https://doi.org/10.1038/s41598-017-03738-1. Gilliom, R.J., Barbash, J.E., Crawford, C.G., Hamilton, P.A., Martin, J.D., Nakagaki, N., Nowell, L.H., Scott, J.C., Stackelberg, P.E., Thelin, G.P., Wolock, D.M., 2006. The Quality of Our Nation's WatersdPesticides in the Nation's Streams and Ground Water, 1992e2001. U.S. Geological Survey Circular U.S. Geological Survey, Reston, Virginia, p. 172. Goerlich, V.C., N€ att, D., Elfwing, M., Macdonald, B., Jensen, P., 2012. Transgenerational effects of early experience on behavioral, hormonal and gene expression responses to acute stress in the precocial chicken. Horm. Behav. 61, 711e718. https://doi.org/10.1016/j.yhbeh.2012.03.006. Gold, H.B., Jung, Y.H., Corces, V.G., 2018. Not just heads and tails: the complexity of the sperm epigenome. J. Biol. Chem., 001561 jbc. R117. Goudarzi, A., Shiota, H., Rousseaux, S., Khochbin, S., 2014. Genome-scale acetylation-dependent histone eviction during spermatogenesis. J. Mol. Biol. 426, 3342e3349. https://doi.org/10.1016/j.jmb.2014.02.023. Greer, E.L., Maures, T.J., Ucar, D., Hauswirth, A.G., Mancini, E., Lim, J.P., Benayoun, B.A., Shi, Y., Brunet, A., 2011. Transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans. Nature 479, 365e371. https:// doi.org/10.1038/nature10572. Guiry, A., Flynn, D., Hubert, S., O'Keeffe, A.M., LeProvost, O., White, S.L., Forde, P.F., Davoren, P., Houeix, B., Smith, T.J., Cotter, D., Wilkins, N.P., Cairns, M.T., 2010. Testes and brain gene expression in precocious male and adult maturing Atlantic salmon (Salmo salar). BMC Genomics 11, 211. https://doi.org/10.1186/ 1471-2164-11-211. Hanson, M.A., Skinner, M.K., 2016. Developmental origins of epigenetic transgenerational inheritance. Environ Epigenet 2. https://doi.org/10.1093/eep/ dvw002. Hao, C., Gely-Pernot, A., Kervarrec, C., Boudjema, M., Becker, E., Khil, P., Tevosian, S.,
gou, B., Smagulova, F., 2016. Exposure to the widely used herbicide atrazine Je results in deregulation of global tissue-specific RNA transcription in the third generation and is associated with a global decrease of histone trimethylation in mice. Nucleic Acids Res. 44, 9784e9802. https://doi.org/10.1093/nar/gkw840. Haque, M.M., Nilsson, E.E., Holder, L.B., Skinner, M.K., 2016. Genomic Clustering of differential DNA methylated regions (epimutations) associated with the epigenetic transgenerational inheritance of disease and phenotypic variation. BMC Genomics 17, 418. https://doi.org/10.1186/s12864-016-2748-5. Hayes, T.B., Anderson, L.L., Beasley, V.R., de Solla, S.R., Iguchi, T., Ingraham, H., Kestemont, P., Kniewald, J., Kniewald, Z., Langlois, V.S., Luque, E.H., McCoy, K.A., Munoz-de-Toro, M., Oka, T., Oliveira, C.A., Orton, F., Ruby, S., Suzawa, M., TaveraMendoza, L.E., Trudeau, V.L., Victor-Costa, A.B., Willingham, E., 2011. Demasculinization and feminization of male gonads by atrazine: consistent effects across vertebrate classes. J. Steroid Biochem. Mol. Biol. 127, 64e73. https:// doi.org/10.1016/j.jsbmb.2011.03.015. Heard, E., Martienssen, R.A., 2014. Transgenerational epigenetic inheritance: myths and mechanisms. Cell 157, 95e109. https://doi.org/10.1016/j.cell.2014.02.045. Iwamatsu, T., 2004. Stages of normal development in the medaka Oryzias latipes. Mech. Dev. 121, 605e618. https://doi.org/10.1016/j.mod.2004.03.012. Jablonowski, N.D., Schaffer, A., Burauel, P., 2011. Still present after all these years: persistence plus potential toxicity raise questions about the use of atrazine. Environ. Sci. Pollut. Res. Int. 18, 328e331. https://doi.org/10.1007/s11356-0100431-y. Jayachandran, K., Steinheimer, T., Somasundaram, L., Moorman, T.B., Kanwar, R.S., Coats, J.R., 1994. Occurrence of atrazine and degradates as contaminants of subsurface drainage and shallow groundwater. J. Environ. Qual. 23, 311e319. Jirtle, R.L., Skinner, M.K., 2007. Environmental epigenomics and disease susceptibility. Nat. Rev. Genet. 8, 253e262. https://doi.org/10.1038/nrg2045. Kiriakidou, M., 1996. Expression of steroidogenic acute regulatory protein (StAR) in the human ovary. J. Clin. Endocrinol. Metab. 81, 4122e4128. https://doi.org/ 10.1210/jc.81.11.4122. Kjørsvik, E., Mangor-Jensen, A., Holmefjord, I., 1990. Egg quality in fishes. In: Blaxter, J.H.S., Southward, A.J. (Eds.), Advances in Marine Biology. Academic Press, pp. 71e113. Klosin, A., Casas, E., Hidalgo-Carcedo, C., Vavouri, T., Lehner, B., 2017. Transgenerational transmission of environmental information in C. elegans. Science 356, 320e323. https://doi.org/10.1126/science.aah6412. Kondo, M., Nanda, I., Hornung, U., Asakawa, S., Shimizu, N., Mitani, H., Schmid, M., Shima, A., Schartl, M., 2003. Absence of the candidate male sex-determining gene dmrt1b (Y) of medaka from other fish species. Curr. Biol. 13, 416e420. Kucka, M., Pogrmic-Majkic, K., Fa, S., Stojilkovic, S.S., Kovacevic, R., 2012. Atrazine acts as an endocrine disrupter by inhibiting cAMP-specific phosphodiesterase4. Toxicol. Appl. Pharmacol. 265, 19e26. https://doi.org/10.1016/ j.taap.2012.09.019. LaVoie, H.A., 2017. Transcriptional control of genes mediating ovarian follicular growth, differentiation, and steroidogenesis in pigs. Mol. Reprod. Dev. 84,

649

788e801. https://doi.org/10.1002/mrd.22827. Lerch, R., Sadler, E.J., Sudduth, K., Baffaut, C., Kitchen, N., 2011. Herbicide transport in goodwater creek ExperimentalWatershed: I. Long-Term research on atrazine 1. JAWRA Journal of the American Water Resources Association 47, 209e223. Lin, Z., Dodd, C.A., Filipov, N.M., 2013. Short-term atrazine exposure causes behavioral deficits and disrupts monoaminergic systems in male C57BL/6 mice. Neurotoxicol. Teratol. 39, 26e35. https://doi.org/10.1016/j.ntt.2013.06.002. Manikkam, M., Guerrero-Bosagna, C., Tracey, R., Haque, M.M., Skinner, M.K., 2012. Transgenerational actions of environmental compounds on reproductive disease and identification of epigenetic biomarkers of ancestral exposures. PLoS One 7, e31901. https://doi.org/10.1371/journal.pone.0031901. McBirney, M., King, S.E., Pappalardo, M., Houser, E., Unkefer, M., Nilsson, E., SadlerRiggleman, I., Beck, D., Winchester, P., Skinner, M.K., 2017. Atrazine induced epigenetic transgenerational inheritance of disease, lean phenotype and sperm epimutation pathology biomarkers. PLoS One 12, e0184306. McCarrey, J.R., 2014. Distinctions between transgenerational and nontransgenerational epimutations. Mol. Cell. Endocrinol. 398, 13e23. https:// doi.org/10.1016/j.mce.2014.07.016. McCarrey, J.R., Lehle, J.D., Raju, S.S., Wang, Y., Nilsson, E.E., Skinner, M.K., 2016. Tertiary epimutations - a novel aspect of epigenetic transgenerational inheritance promoting genome instability. PLoS One 11, e0168038. https://doi.org/ 10.1371/journal.pone.0168038. McGee, S.R., Narayan, P., 2013. Precocious puberty and Leydig cell hyperplasia in male mice with a gain of function mutation in the LH receptor gene. Endocrinology 154, 3900e3913. https://doi.org/10.1210/en.2012-2179. Nilsson, E.E., Sadler-Riggleman, I., Skinner, M.K., 2018. Environmentally induced epigenetic transgenerational inheritance of disease. Environ Epigenet 4, dvy016. https://doi.org/10.1093/eep/dvy016. Nilsson, E.E., Skinner, M.K., 2015. Environmentally induced epigenetic transgenerational inheritance of disease susceptibility. Transl. Res. 165, 12e17. Nishimura, T., Tanaka, M., 2014. Gonadal development in fish. Sexual Development 8, 252e261. https://doi.org/10.1159/000364924. Papoulias, D.M., Tillitt, D.E., Talykina, M.G., Whyte, J.J., Richter, C.A., 2014. Atrazine reduces reproduction in Japanese medaka (Oryzias latipes). Aquat. Toxicol. 154, 230e239. https://doi.org/10.1016/j.aquatox.2014.05.022. Peltoketo, H., Strauss, L., Karjalainen, R., Zhang, M., Stamp, G.W., Segaloff, D.L., Poutanen, M., Huhtaniemi, I.T., 2010. Female mice expressing constitutively active mutants of FSH receptor present with a phenotype of premature follicle depletion and estrogen excess. Endocrinology 151, 1872e1883. https://doi.org/ 10.1210/en.2009-0966. Pembrey, M.E., 2010. Male-line transgenerational responses in humans. Hum. Fertil. 13, 268e271. https://doi.org/10.3109/14647273.2010.524721. Pogrmic, K., Fa, S., Dakic, V., Kaisarevic, S., Kovacevic, R., 2009. Atrazine oral exposure of peripubertal male rats downregulates steroidogenesis gene expression in Leydig cells. Toxicol. Sci. 111, 189e197. https://doi.org/10.1093/toxsci/kfp135. Pogrmic-Majkic, K., Samardzija, D., Stojkov-Mimic, N., Vukosavljevic, J., TrninicPjevic, A., Kopitovic, V., Andric, N., 2018. Atrazine suppresses FSH-induced steroidogenesis and LH-dependent expression of ovulatory genes through PDE-cAMP signaling pathway in human cumulus granulosa cells. Mol. Cell. Endocrinol. 461, 79e88. https://doi.org/10.1016/j.mce.2017.08.015. Reik, W., Dean, W., Walter, J., 2001. Epigenetic reprogramming in mammalian development. Science 293, 1089e1093. https://doi.org/10.1126/ science.1063443. Richter, C.A., Tillitt, D.E., Papoulias, D., Whyte, J.J., Villeneuve, D.L., Ankley, G.T., Tillitt, D.E., 2016. Mechanisms of atrazine-induced reproductive impairment in fathead minnow (Pimephales promelas) and Japanese medaka (Oryzias latipes). Environ. Toxicol. Chem. 35, 8. Roberge, M., Hakk, H., Larsen, G., 2004. Atrazine is a competitive inhibitor of phosphodiesterase but does not affect the estrogen receptor. Toxicol. Lett. 154, 61e68. Russart, K.L.G., Rhen, T., 2016. Atrazine alters expression of reproductive and stress genes in the developing hypothalamus of the snapping turtle, Chelydra serpentina. Toxicology 366e367, 1e9. https://doi.org/10.1016/j.tox.2016.08.001. Sanderson, J.T., Seinen, W., Giesy, J.P., van den Berg, M., 2000. 2-Chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity? Toxicol. Sci. 54, 121e127. Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101e1108. Schulz, R.W., Vischer, H.F., Cavaco, J.E., Santos, E.M., Tyler, C.R., Goos, H.J., Bogerd, J., 2001. Gonadotropins, their receptors, and the regulation of testicular functions in fish. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 129, 407e417. Silveyra, G.R., Canosa, I.S., Rodríguez, E.M., Medesani, D.A., 2017. Effects of atrazine on ovarian growth, in the estuarine crab Neohelice granulata. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 192, 1e6. https://doi.org/10.1016/ j.cbpc.2016.10.011. Skinner, M.K., 2011. Role of epigenetics in developmental biology and transgenerational inheritance. Birth Defects Res C Embryo Today 93, 51e55. https:// doi.org/10.1002/bdrc.20199. Skinner, M.K., 2016. Endocrine disruptors in 2015: epigenetic transgenerational inheritance. Nat. Rev. Endocrinol. 12, 68e70. https://doi.org/10.1038/ nrendo.2015.206. Skinner, M.K., Manikkam, M., Guerrero-Bosagna, C., 2010. Epigenetic transgenerational actions of environmental factors in disease etiology. Trends Endocrinol. Metabol. 21, 214e222. https://doi.org/10.1016/j.tem.2009.12.007. Solomon, K.R., Carr, J.A., Du Preez, L.H., Giesy, J.P., Kendall, R.J., Smith, E.E., Van Der

650

J.A. Cleary et al. / Environmental Pollution 251 (2019) 639e650

Kraak, G.J., 2008. Effects of atrazine on fish, amphibians, and aquatic reptiles: a critical review. Crit. Rev. Toxicol. 38, 721e772. https://doi.org/10.1080/ 10408440802116496. Sperry, T.S., Thomas, P., 1999. Identification of two nuclear androgen receptors in kelp bass (Paralabrax clathratus) and their binding affinities for xenobiotics: comparison with Atlantic croaker (Micropogonias undulatus) androgen receptors. Biol. Reprod. 61, 1152e1161. Suzawa, M., Ingraham, H.A., 2008. The herbicide atrazine activates endocrine gene networks via non-steroidal NR5A nuclear receptors in fish and mammalian cells. PLoS One 3, e2117. https://doi.org/10.1371/journal.pone.0002117. Tennant, M.K., Hill, D.S., Eldridge, J.C., Wetzel, L.T., Breckenridge, C.B., Stevens, J.T., 1994. Chloro-s-triazine antagonism of estrogen action: limited interaction with estrogen receptor binding. J. Toxicol. Environ. Health, Part A Current Issues 43, 197e211. Thelin, G.P., Stone, W.W., 2010. Method for estimating annual atrazine use for counties in the conterminous United States, 1992e2007. U.S. Geological Survey Scientific Investigations Report 2010e 5034, 129. Tillitt, D.E., Papoulias, D.M., Whyte, J.J., Richter, C.A., 2010. Atrazine reduces reproduction in fathead minnow (Pimephales promelas). Aquat. Toxicol. 99, 149e159. https://doi.org/10.1016/j.aquatox.2010.04.011. USEPA, 2016. AEMP Data: Atrazine, Flow, Precipitation and TSS Plots for 2016 Monitoring Year. USEPA, Washington DC, p. 10. Volkova, K., Reyhanian Caspillo, N., Porseryd, T., Hallgren, S., Dinnetz, P., Olsen, H., Porsch Hallstrom, I., 2015. Transgenerational effects of 17alpha-ethinyl estradiol on anxiety behavior in the guppy, Poecilia reticulata. Gen. Comp. Endocrinol. 223, 66e72. https://doi.org/10.1016/j.ygcen.2015.09.027. Wang, C., Zhang, Z., Yao, H., Zhao, F., Wang, L., Wang, X., Xing, H., Xu, S., 2014. Effects of atrazine and chlorpyrifos on DNA methylation in the liver, kidney and gill of the common carp (Cyprinus carpio L.). Ecotoxicol. Environ. Saf. 108, 142e151. https://doi.org/10.1016/j.ecoenv.2014.06.011.

Wang, X., Bhandari, R.K., 2019. DNA methylation dynamics during epigenetic reprogramming of medaka embryo. Epigenetics 1e12. https://doi.org/10.1080/ 15592294.2019.1605816. Wirbisky, S.E., Freeman, J.L., 2015. Atrazine exposure and reproductive dysfunction through the hypothalamus-pituitary-gonadal (HPG) Axis. Toxics 3, 414e450. https://doi.org/10.3390/toxics3040414. Wirbisky, S.E., Sepulveda, M.S., Weber, G.J., Jannasch, A.S., Horzmann, K.A., Freeman, J.L., 2016a. Embryonic atrazine exposure elicits alterations in genes associated with neuroendocrine function in adult male zebrafish. Toxicol. Sci. 153, 149e164. https://doi.org/10.1093/toxsci/kfw115. Wirbisky, S.E., Weber, G.J., Sepulveda, M.S., Lin, T.L., Jannasch, A.S., Freeman, J.L., 2016b. An embryonic atrazine exposure results in reproductive dysfunction in adult zebrafish and morphological alterations in their offspring. Sci. Rep. 6, 21337. https://doi.org/10.1038/srep21337. Wirbisky-Hershberger, S.E., Sanchez, O.F., Horzmann, K.A., Thanki, D., Yuan, C., Freeman, J.L., 2017. Atrazine exposure decreases the activity of DNMTs, global DNA methylation levels, and dnmt expression. Food Chem. Toxicol. 109, 727e734. https://doi.org/10.1016/j.fct.2017.08.041. Wolstenholme, J.T., Edwards, M., Shetty, S.R., Gatewood, J.D., Taylor, J.A., Rissman, E.F., Connelly, J.J., 2012. Gestational exposure to bisphenol a produces transgenerational changes in behaviors and gene expression. Endocrinology 153, 3828e3838. https://doi.org/10.1210/en.2012-1195. Xin, F., Susiarjo, M., Bartolomei, M.S., 2015. Multigenerational and transgenerational effects of endocrine disrupting chemicals: a role for altered epigenetic regulation? Semin. Cell Dev. Biol. 43, 66e75. https://doi.org/10.1016/ j.semcdb.2015.05.008. Xing, H., Zhang, Z., Yao, H., Liu, T., Wang, L., Xu, S., Li, S., 2014. Effects of atrazine and chlorpyrifos on cytochrome P450 in common carp liver. Chemosphere 104, 244e250. https://doi.org/10.1016/j.chemosphere.2014.01.002.