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. 2016 Jul 13;153(1):149–164. doi: 10.1093/toxsci/kfw115

Embryonic Atrazine Exposure Elicits Alterations in Genes Associated with Neuroendocrine Function in Adult Male Zebrafish

Sara E Wirbisky *, Maria S Sepúlveda *,, Gregory J Weber *, Amber S Jannasch , Katharine A Horzmann *, Jennifer L Freeman *,1
PMCID: PMC5013880  PMID: 27413107

Abstract

The developmental origins of health and disease (DOHaD) hypothesis states that exposure to environmental stressors early in life can elicit genome and epigenome changes resulting in an increased susceptibility of a disease state during adulthood. Atrazine, a common agricultural herbicide used throughout the Midwestern United States, frequently contaminates potable water supplies and is a suspected endocrine disrupting chemical. In our previous studies, zebrafish was exposed to 0, 0.3, 3, or 30 parts per billion (μg/l) atrazine through embryogenesis, rinsed, and allowed to mature to adulthood. A decrease in spawning was observed with morphological alterations in offspring. In addition, adult females displayed an increase in ovarian progesterone and follicular atresia, alterations in levels of a serotonin metabolite and serotonin turnover in brain tissue, and transcriptome changes in brain and ovarian tissue supporting neuroendocrine alterations. As reproductive dysfunction is also influenced by males, this study assessed testes histology, hormone levels, and transcriptomic profiles of testes and brain tissue in the adult males. The embryonic atrazine exposure resulted in no alterations in body or testes weight, gonadosomatic index, testes histology, or levels of 11-ketotestosterone or testosterone. To further investigate potential alterations, transcriptomic profiles of adult male testes and brain tissue was completed. This analysis demonstrated alterations in genes associated with abnormal cell and neuronal growth and morphology; molecular transport, quantity, and production of steroid hormones; and neurotransmission with an emphasis on the hypothalamus–pituitary–adrenal and hypothalamus–pituitary–thyroid axes. Overall, this data indicate future studies should focus on additional neuroendocrine endpoints to determine potential functional impairments.

Keywords: atrazine, developmental origins of health and disease, hormones, neuroendocrine system, transcriptomics, zebrafish

INTRODUCTION

Research investigating the genetic alterations observed in adult diseases has begun to show support of a developmental origin. These alterations are thought to be due to the reprogramming of cellular processes specifically during development in response to the surrounding environment. This concept has been termed the ‘developmental origins of health and disease’ (DOHaD) hypothesis (Barker and Osmond, 1986; Barker et al., 1993; reviewed in Hanson and Gluckman, 2014). Numerous disease states are hypothesized to have a developmental origin including cardiovascular and metabolic disorders, obesity, diabetes, heart disease, and cancer (reviewed in Dolinoy and Jirtle, 2008; reviewed in Chen and Zhang, 2011); however, the understanding of the genetic mechanisms behind the development of these diseases is still under investigation.

Endocrine disrupting chemicals (EDCs) are exogenous agents that disrupt endogenous hormone signaling pathways (reviewed in Swedenborg et al., 2009). EDCs are diverse in structure and are found in numerous products such as plasticizers, pharmaceuticals, and pesticides, making human exposure to these chemicals a likely event (Birnbaum and Fenton, 2003; Ma et al., 2010; Prins et al., 2007). Two primary challenges in understanding the mechanisms of EDCs are their characteristic non-monotonic dose–response and various latency periods between exposure and observable effects (reviewed in Vandenberg, 2012). Studies show that EDCs can alter tissue formation, reproduction, and play a role in the onset of obesity and cancer (Cooper et al., 2000; Hatch et al., 2011; Roy et al., 2009; Swedenborg et al., 2009; Wetzel et al., 1994). Furthermore, studies implicate that the adverse effects of EDCs can affect not only the exposed organism, but future offspring as well (Anway et al., 2005, 2006; Baker et al., 2014a,b; Guerrero-Bosagna et al., 2010; King Heiden et al., 2009).

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) is an agricultural herbicide that is applied throughout the Midwestern United States and other parts of the globe on a variety of crops including corn, sorghum grass, sugar cane, and wheat (Barr et al., 2007; Eldridge et al., 2008; Solomon et al., 2008). Due to atrazine’s water solubility, mobility in soil, and long half-life, atrazine frequently contaminates potable water supplies and reaches levels above the maximum contaminant level (MCL) as set by the U. S. Environmental Protection Agency (EPA) of 3 parts per billion (ppb; μg/l) (Fraites et al., 2009; Rohr and McCoy, 2010; U.S. EPA, 2002). As such the European Union banned the use of atrazine in 2003 (European Commission, 2003; reviewed in Sass and Colangelo, 2006). Epidemiological studies report several potential adverse health effects associated with maternal atrazine exposure including an increased risk of babies born small for their gestational age (SGA), intrauterine growth retardation (IUGR), and birth defects (Mattix et al., 2007; Munger et al., 1997; Ochoa-Acuña et al., 2009; Winchester et al., 2009).

Several in vivo studies have investigated the adverse effects of atrazine on the neuroendocrine system. Adult female models show reproductive dysfunction by way of the hypothalamus–pituitary–gonadal (HPG) axis through the inhibition of gonadotropin releasing hormone (GnRH) and a reduction in the pre-ovulatory surge of luteinizing hormone (LH), follicle stimulating hormone (FSH), and prolactin (PRL) (Cooper et al., 2000; Foradori et al., 2009, 2013). In addition, male studies report delays in puberty along with alterations in behavior, hormone levels (testosterone [T] and estradiol [E2]), testes and prostate morphology, and cellular and genetic mechanisms throughout the steroidogenesis pathway (Belloni et al., 2011; Friedmann, 2002; Jin et al., 2013; Pogrmic et al., 2009; Pogrmic-Majkic et al., 2010; Rayner et al., 2007; Rosenberg et al., 2008; Victor-Costa et al., 2010).

These previously mentioned studies utilized various rodent models investigating the adverse effects of gestational, peripubertal, and adult atrazine exposure; however, the zebrafish provides a strong complementary vertebrate model when investigating the hypothesis of a developmental origin of the neuroendocrine effects caused by atrazine. Strengths of the zebrafish model system in the DOHaD exposure paradigm include ex utero fertilization and development, rapid embryogenesis, a relatively short life span, and conserved genetic homology (reviewed in de Esch et al., 2012; Howe et al., 2013). In combination with these biological strengths are the functional and structural homology of the zebrafish endocrine and central nervous system (CNS) (reviewed in de Esch et al., 2012; reviewed in Löhr and Hammerschmidt, 2011).

Previous studies have investigated the DOHaD exposure paradigm and transgenerational effects of various EDCs in zebrafish. Paternal bisphenol A (BPA) exposure during spermatogenesis elicited an increase in the rate of heart failures of offspring up to the F2 generation (Lombó et al., 2015). Developmental exposure to 17α-ethinylestradiol (EE2) found an increase in anxiety and increased shoaling in adults at ∼4 months of age. In addition, decreases in breeding and behavioral alterations in offspring were reported (Volkova et al., 2015). The later-in-life and transgenerational alterations following an exposure to 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) has also been reported with adverse effects encompassing a skewed sex ratio of the developmentally exposed zebrafish along with skeletal malformations and a reduction in egg release and fertilization in the unexposed F1 and F2 generations (Baker et al., 2014a,b).

We previously reported that an embryonic atrazine exposure of 0.3, 3, or 30 ppb (1–72 hpf) in zebrafish elicited immediate changes to the transcriptome including alterations in genes associated with reproductive system function and development, cell cycle regulation, and cancer, and morphological alterations (Weber et al., 2013). More recently, we reported that an embryonic atrazine exposure of 0.3, 3, or 30 ppb (1–72 hpf) alters serotonin turnover and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in adult female zebrafish 9-month post-fertilization (mpf) with brain transcriptomic profiles indicating alterations in genes associated with nervous system development and function, behavior, and tissue development at 6 mpf (Wirbisky et al., 2015). In addition, our results show that an embryonic atrazine exposure of 0.3, 3, or 30 ppb (1–72 hpf) resulted in a decrease in spawning in the exposed population with adult females exhibiting increased ovarian weight, an increase in atretic ovarian follicles, and an increase in progesterone at 5–8 mpf. Transcriptomic analysis of ovarian tissue revealed alterations in genes associated with neuroendocrine system development and function, tissue development, and behavior (Wirbisky et al., 2016a). Furthermore, alterations in head-to-body ratios were observed in the offspring of the exposed generation at 72 hpf (Wirbisky et al., 2016a).

As reproductive dysfunction is also influenced by males, the current study aimed to address the later-in-life consequences in response to an embryonic atrazine exposure of 0.3, 3, or 30 ppb by assessing morphology and histology of the reproductive system, hormone levels, and transcriptomic profiles of testes and brain tissue of adult male zebrafish. Results were compared with previous studies from our laboratory in order to identify potential sex-specific disruptive effects of atrazine on the neuroendocrine system.

METHODS

Zebrafish Husbandry and Experimental Design

Zebrafish (wild-type AB strain) were housed in a Z-Mod System (Aquatic Habitats, Apopka, FL) on a 14:10-h light:dark cycle and maintained at 28°C ± 1 °C with a pH of 7.0–7.2 and conductivity range of 470–520 µS. Adult zebrafish were bred in cages and embryos were collected, staged, and rinsed with system water as described previously for experimental use (Weber et al., 2013; Wirbisky et al., 2015). Embryos were dosed with 0, 0.3, 3, or 30 ppb atrazine (CAS #1912-24-9; Chem Service, 98% purity) from 1- to 72-h post-fertilization (hpf) (through the end of embryogenesis) as previously described (Weber et al., 2013; Wirbisky et al., 2015). Atrazine sample concentrations were verified using a U.S. EPA approved immunoassay kit for atrazine (Abraxis Atrazine ELISA Kit, Warminster, PA) as previously described (Freeman et al., 2005). After the exposure, larvae were rinsed with clean fish system water and allowed to mature under normal growing conditions. Treatments were repeated for additional clutches to attain multiple biological replicates. All animal protocols were approved and performed in accordance with Purdue University’s Institutional Animal Care and Use Committee guidelines.

Fish and Gonadal Tissue Collection and Histology

Adult male zebrafish (∼5–8 mpf) were euthanized in MS-222 (Ethyl 3-aminobenzoate methanesulfonate, Sigma, St. Louis, MO) (4 mg/ml) and subsequently weighed to obtain body weight. A complete set of fish from each treatment group was collected for each endpoint as it was analyzed so all sets were age matched. Testes were collected and weighed for weight and calculation of gonadosomatic index (GSI) determined as (gonad weight/total body weight)×100. A sample of tissue was collected from both testes and fixed in Davidson’s Fixative (Johnson et al., 2009) overnight at room temperature and then transferred to histology grade 70% EtOH (Sigma). Hematoxylin and eosin (H&E) sections were prepared following standard procedures and sections examined under a light microscope (10–40×) for staging and evaluation of any abnormalities as outlined in Johnson et al. (2009). Testicular histological sections from 10 males per treatment per replicate (n = 4; 40 total fish per treatment group were assessed) were used to quantify thickness of germinal epithelium and percent area occupied by different cell types (primary spermatocyte, secondary spermatocyte, spermatid, spermatozoa or spermatogonia). This was done by first scanning the histological sections into Aperio Digital Pathology (Leica Biosystems Inc., Buffalo Grove, IL) and then using these digital images for calculating area or length using ImageJ (https://imagej.nih.gov/ij/index.html). These parameters were measured from a total of eight seminiferous tubules per slide. The number of Leydig cells surrounding individual seminiferous tubules was quantified to evaluate for Leydig cell hyperplasia and the histological sections were evaluated by a board certified veterinary pathologist for the presence of Leydig cell or Sertoli cell hypertrophy blinded to treatment groups. About 34–50 seminiferous tubules per replicate were examined.

Hormone Analysis of Adult Male Testes Tissue

Adult male zebrafish were euthanized in MS-222 (Ethyl 3-aminobenzoate methanesulfonate, Sigma, St. Louis, MO) (4 mg/ml) and testes were collected. Testes from six to nine males were pooled per treatment in each replicate (n = 3–4). The testes tissue was stored at −80 °C prior to extraction and analysis. Each sample was weighed, typically 8–20 mg, and placed in a 1.7-ml centrifuge tube. To each, 50 ng of 13C3-testosterone was added as an internal standard. Glass homogenizer beads were added and 0.1 ml of water. The tubes were briefly vortexed and then 1 ml of hexane/ethyl acetate (60/40 v/v) was added to each. The samples were again vortexed for 15 min on maximum speed. They were centrifuged at 15 000 rpm for 5 min and the top organic layer transferred to a new tube. They were then dried in a rotary evaporation device and stored at −20 °C until derivatization. The extracts were derivatized with the AB Sciex Keto derivatization kit (AB Sciex, Framingham, MA) just prior to LC/MS/MS analysis. To each sample, 50 µl of reagent was added. The reaction time was 60 min at room temperature.

An Agilent 1200 Rapid Resolution liquid chromatography (LC) system coupled to an Agilent 6460 series QQQ mass spectrometer (MS) was used to analyze 11-KT and testosterone in each sample. A Waters Xbridge C18 2.1 mm×100 mm, 3 µm column was used for LC separation. The buffers were: (A) water + 0.1% formic acid and (B) acetonitrile + 0.1% formic acid. The linear LC gradient was as follows: time 0 min, 5% B; time 1 min, 5% B; time 5 min, 100% B; time 15 min, 100% B; time 16 min, 5% B; time 20 min, 5% B. The flow rate was 0.3 ml/min. Multiple reaction monitoring was used for MS analysis. The data were acquired in positive electrospray ionization (ESI) mode by monitoring the following transitions: Testosterone 403.1 → 344.1 (20V), 164 (40V); 13C3-Testosterone 406.1 → 347.1 (20V), 167 (40V); 11-Ketotestosterone 417.3 → 358.3 (25V). The jet stream ESI interface had a gas temperature of 325 °C, gas flow rate of 8 l/min, nebulizer pressure of 40 psi, sheath gas temperature of 250 °C, sheath gas flow rate of 7 l/min, capillary voltage of 4000 V, and nozzle voltage of 1000 V.

Transcriptome Microarray Analysis of Adult Male Testes and Brain Tissue

Testes and brain tissue from six adult males (∼6 mpf) each from a different biological replicate (n = 6) were collected from each group exposed to an atrazine treatment (0, 0.3, 3, or 30 ppb) during embryogenesis, homogenized in Trizol (Life Technologies, Carlsbad, CA), and flash frozen in liquid nitrogen. Total RNA was isolated by the RNeasy Mini Kit (Qiagen, Venlo, Limburg). Transcriptomic microarray analysis was conducted using the one-color hybridization strategy to compare gene expression profiles among the atrazine treatments with a zebrafish custom 4 ×180 K expression platform (Agilent Technologies, Santa Clara, CA). This microarray is a multiplex format of four arrays each consisting of 180 K probes interrogating 36 000 known and predicted targets with approximately three to five probes per target and is based on the Ensembl and UCSC Genome Databases. Following hybridization, arrays were washed and scanned on an Agilent Technologies SureScan Microarray Scanner (Agilent Technologies, Santa Clara, CA). Array image data were extracted using Agilent Feature Extraction Software 11.5 (Agilent Technologies, Santa Clara, CA). Array image data were then uploaded to GeneSpring 12.5 (Agilent Technologies, Santa Clara, CA) for statistical analysis. Microarray analysis was performed following MIAME guidelines (Brazma et al., 2001). Each gene list was imported into Ingenuity Pathway Analysis (IPA) for gene ontology and molecular pathway analysis. Genes referred to in the methods and results sections reflect zebrafish nomenclature. Genes in the discussion section are reported as the human homologs of the zebrafish genes identified to be altered by microarrays.

Quantitative Polymerase Chain Reaction (qPCR) Confirmation of Microarray

Quantitative polymerase chain reaction (qPCR) was performed on a subset of selected genes altered in all three atrazine treatments. qPCR was performed on the same samples as used in the microarray analysis (n = 6). cDNA synthesis was performed using SuperScript® First-strand synthesis system (Invitrogen, Carlsbad, CA). Seven target genes were selected for confirmation in the male testes (deiodinase iodothyronine type 2 [dio2], insulin like growth factor 2 [igf2], thyroid hormone receptor alpha a [thraa], alpha-2-macroglobulin [a2m], spermatogenesis associated serine rich 2 [spats2], fatty acid elongase 6 [elovl6], and apolipoprotein M [apom]). An additional seven target genes were selected for confirmation in the male brain tissue (dopa decarboxylase [ddc], steroid-5-alpha-reductase, alpha polypeptide 2b [srd5a2b], metallopeptidase domain 8 [adam8], neuropeptide y [npy], hydroxysteroid [11-beta] dehydrogenase [hsd11b2], melanocortin 2 receptor [mc2r], and solute carrier family 2 member 9-like 1 [slc2a9l1]). These genes were selected based on their associated pathways and biological functions. Primers specific to target genes were designed using the Primer3 website (Supplementary Table 1). BioRad SSO Advance SYBR Green Supermix kit was used according to the manufacturer’s recommendations. qPCR was performed following MIQE guidelines (Bustin et al., 2009) and as previously described (Weber et al., 2013; Wirbisky et al., 2014, 2015). Similar to previous studies in our laboratory (Weber et al., 2013; Wirbisky et al., 2014) several genes were assessed to determine the best reference gene to be used for this data set (data not shown). β-actin was found to be the most consistent and least variable for this analysis. Experimental samples were run in triplicate (technical replicates) and gene expression was normalized to β-actin. Efficiency and specificity were checked with melting and dilution curve analysis and no-template controls.

Statistical Analysis

Statistical analyses were conducted using SAS statistical software. Total body weight, testes weight, GSI, thickness of germinal epithelium, Leydig cell frequency, and hormone levels were analyzed using a one-way analysis of variance (ANOVA). The frequency of testicular abnormalities and percent area occupied by different cell types was analyzed using chi-squared tests. Microarray analysis was completed to determine differentially expressed genes for each atrazine treatment as well as in all three atrazine treatments combined. This was first determined using an ANOVA with a Tukey’s post hoc test when a significant ANOVA was observed (α < 0.05). In addition, a mean absolute log2 expression ratio of at least 0.585 (50% increase or decrease in expression) must be satisfied. Microarray confirmation by qPCR was assessed by a Pearson’s correlation (α < 0.05).

RESULTS

Testes Tissue Morphology and Histology

Adult male zebrafish did not show any significant difference between control and atrazine treatments in body weight (P = 0.7878), testes weight (P = 0.6589) or GSI (P = 0.314) (Supplementary Figure 1). No alterations were noted in the percentage of primary spermatocytes (P = 0.2156), secondary spermatocytes (P = 0.2522), spermatids (P = 0.0789), spermatozoa (P = 0.9991), or spermatogonia (P = 0.3174) (Supplementary Figure 2A). In addition, no significant differences were observed in germinal epithelium thickness in any of the atrazine treatments (P = 0.0967; Supplementary Figure 2B). Testicular abnormalities including hypoplasia, atrophy, and underdevelopment were observed. However, no differences in the frequency of these testicular abnormalities were observed between treatments (X2 =2.0, P = 0.5678; Supplementary Figure 2C). No differences were also observed in Leydig cell frequency between treatment groups (P = 0.5320; Supplementary Figs. 3 and 4) with no Leydig cell hypertrophy or Sertoli cell hypertrophy observed.

Hormone Analysis of Adult Male Testes Tissue

Adult male zebrafish did not show any significant difference between control and atrazine treatments in testes tissue levels of 11-ketotestosterone (P = 0.2488; Supplementary Figure 5A) or testosterone (P = 0.5206; Supplementary Figure 5B).

Transcriptome Analysis of Adult Male Testes Tissue

Results from the male gonad revealed expression alterations in 239 mapped genes in the 0.3-ppb treatment group, 162 genes in the 3-ppb treatment group, and 288 genes in the 30-ppb treatment group. Of these differentially expressed genes, 88 were common among all three atrazine treatments (GSE72242; Figure 1A). The aforementioned gene numbers are the known human orthologs of the zebrafish genes found to be altered by microarrays and these human gene designations were used in subsequent gene enrichment analyses. Significantly altered gene lists for each of the treatment groups as well as genes altered in all three atrazine treatments were uploaded into IPA for subsequent gene ontology analyses. The most enriched molecular and cellular functions for the 0.3-ppb treatment group were lipid metabolism, gene expression, small molecule biochemistry, and cellular development (Supplementary Table 2). Genes were specifically involved in synthesis and concentration of lipid and quantity of steroid hormone. The 3-ppb treatment group showed a similar response as pathways enriched showed changes of genes associated with lipid metabolism, cell death and survival, small molecule biochemistry, cellular assembly and organization, and cell morphology (Supplementary Table 3). Again, genes were specifically involved in the synthesis and concentration of lipid and quantity of steroid hormone. Pathways enriched in the 30-ppb treatment group included molecular transport, gene expression, cellular growth and proliferation, cellular assembly and organization, and cell morphology revealing similarities to the 0.3- and 3-ppb treatment groups (Supplementary Table 4). Gene ontology analysis of the 88 genes altered in all three treatment groups revealed effects in lipid metabolism, molecular transport, small molecule biochemistry, and cell morphology (Table 1). These genes are involved in quantity of steroid hormone, in concentration and oxidation of lipid, and in synthesis and concentration of acylglycerols (eg, acylglycerol, diacylglycerol, and triacylglycerol). Canonical pathway and network analysis revealed down-regulated genes were associated with steroid hormone production and with quantity and conversion of l-triiodothyronine and quantity of TSH in blood (Figure 2).

FIG. 1.

FIG. 1.

Transcriptomic analysis in testes and brain of adult male zebrafish exposed to atrazine during embryogenesis. Venn diagrams indicating the unique and common genes with expression alterations (after gene ontology analysis) in testes (A) and brain (B) of adult male zebrafish exposed to 0.3, 3, or 30 ppb atrazine during embryogenesis.

TABLE 1.

Gene Enrichment of Molecular and Cellular Functions in Adult Male Testes in All Three Atrazine Treatments

Molecular and Cellular Functions P valuea Number of Genesb
Lipid Metabolism 1.56E-02–4.14E-05 16
 Concentration of lipid 4.66E-03 11
 Synthesis of acylglycerol 9.01E-04 4
 Quantity of steroid 1.09E-02 7
 Quantity of steroid hormone 4.83E-03 4
 Absorption of cholesterol 8.45E-03 2
 Concentration of corticosterone 1.46E-02 3
 Secretion of aldosterone 7.95E-03 2
Molecular Transport 1.56E-02–4.14E-05 20
 Quantity of diacylglycerol 6.48E-05 4
 Quantity of steroid 1.09E-02 7
 Concentration of fatty acid 1.27E-02 5
 Absorption of cholesterol 8.45E-03 2
Small Molecule Biochemistry 1.56E-02–4.14E-05 26
 Oxidation of lipid 1.53E-02 11
 Quantity of steroid hormone 4.83E-03 4
 Concentration of fatty acid 1.27E-02 5
 Concentration of lipid 4.66E-03 5
Cell Morphology 5.55E-05–1.29E-02 31
 Morphology of cells 1.85E-04 24
 Size of cells 3.01E-03 7
 Formation of cellular protrusions 2.30E-03 11
 Abnormal morphology of cells 2.26E-03 16

aDerived from the likelihood of observing the degree of enrichment in a gene set of a given size by chance alone.

bClassified as being differentially expressed that relate to the specified function category; a gene may be present in more than one category.

FIG. 2.

FIG. 2.

Canonical pathway and network analysis associate steroid hormone production as a target of atrazine toxicity. Canonical pathway analyses of altered genes in the microarray analysis of male gonad indicate genes associated with steroid hormone production including GHR, IGF2, ADRB2, DIO2, THRA, LSR, and PNPLA2 (A). Genes specific to quantity and conversion of l-triiodothyronine and quantity of TSH in blood are identified (B). Green indicates a down regulation.

Quantitative Polymerase Chain Reaction (qPCR) Confirmation of Male Testes Microarray Data

A subset of genes found to be significantly altered in all three atrazine treatments by microarray analysis (Table 2) were independently confirmed by qPCR. Seven target genes were assessed (dio2, igf2, thraa, a2m, spats2, elovl6, and apom). The data showed a significant positive correlation between the microarray and qPCR in the 3- and 30-ppb atrazine treatments (3 ppb: R = 0.901, P = 0.0056; 30 ppb: R = 0.943, P = 0.0014, respectively), whereas the 0.3-ppb atrazine treatment also showed a positive correlation the P value was slightly >0.05 (R = 0.748, P = 0.0530, Figure 3).

TABLE 2.

Summary of Microarray Values (log2 ratios) of Genes Targeted by qPCR

0.3 ppb 3 ppb 30 ppb
Log2 Log2 Log2
Testes Tissue
 Gene Symbol
  dio2 −3.92333 −3.09288 −3.32264
  igf2 −1.54005 −1.39077 −3.32883
  thraa −1.05178 −0.80124 −0.73048
  a2m 0.65901 1.71589 1.55684
  spats2 0.69795 0.63289 0.76239
  elovl6 −3.55275 −2.94823 −2.36902
  apom −3.28333 −2.09963 −3.0844
Brain Tissue
 Gene Symbol
  ddc −1.82782 −2.09255 −2.31208
  srd5ab2 1.21537 1.1122 1.57693
  adam8 −3.07142 −3.21008 −3.36499
  npy 1.89466 1.83823 2.13258
  hsd11b2 −1.88906 −2.45393 −2.45782
  mc2r −3.38681 −2.76998 −3.43416
  slc2a9l1 −5.15445 −3.55225 −4.95778

FIG. 3.

FIG. 3.

qPCR comparative analysis of microarray data for adult male testes tissue. qPCR analysis was performed to compare gene expression changes detected on the microarray on seven gene targets (dio2, igf2, thraa, a2m, spats2, elovl6, and apom). All targeted genes were significantly altered on the microarray. A linear correlation was completed and statistical significance of the correlation was determined using a Pearson’s correlation. The 0.3-ppb (A) atrazine treatment showed a positive correlation, but the P value was slightly >0.05 (R = 0.748, P = 0.0530). A significant positive correlation between the microarray and qPCR in the 3 (B) and 30 ppb (C) atrazine treatments was observed (3 ppb: R = 0.901, P = 0.0056; 30 ppb: R = 0.943, P = 0.0014, respectively). The microarray data are represented as log2 expression values and qPCR values are the ratios of relative expression against β-actin represented as log2.

Transcriptome Analysis of Adult Male Brain Tissue

Results from the male brain revealed expression alterations in 2748 mapped genes in the 0.3-ppb treatment group, 3034 genes in the 3-ppb treatment group, and 2826 genes in the 30-ppb treatment group. Of these differentiated genes 2290 were common among all three atrazine treatments (GSE72243; Figure 1B). Using IPA, the most enriched molecular and cellular functions for the 0.3-ppb treatment group were cell morphology, cellular function and maintenance, cell to cell signaling and interaction, and cellular development (Supplementary Table 5). Specific functions genes were associated included development, differentiation, and morphology of neurons; axonogenesis; long term potentiation; and neurotransmission. The 3-ppb treatment group showed identical pathways as the 0.3-ppb treatment with the additional inclusion of alterations in molecular transport (Supplementary Table 6). There were slight differences in the specific functions of each pathway in which genes were involved, but still indicated alterations in neurotransmission, axonogenesis, neuritogenesis, and long term potentiation. Pathways enriched in the 30-ppb treatment group were almost identical to the 3-ppb treatment group with only minor differences in specific functions (Supplementary Table 7). As such, gene ontology analysis of the 2290 genes altered in all three treatment groups revealed genes associated with cell to cell signaling, cellular morphology, molecular transport, cellular assembly and organization, and cellular function and maintenance (Table 3) with genes involved in neuritogenesis, synaptogenesis, and neurotransmission. Canonical pathway and network analysis indicated altered genes were associated with neurotransmitter pathways with most of these genes up regulated (Figure 4).

TABLE 3.

Gene Enrichment of Molecular and Cellular Functions in Adult Male Brain in All Three Atrazine Treatments

Molecular and Cellular Functions P valuea Number of Genesb
Cell-To-Cell signaling and Interaction 1.92E-30–8.59E-05 315
 Secretion of neurotransmitter 1.61E-12 51
 Synaptic transmission 1.16E-25 119
 Neurotransmission 1.92E-30 145
 Secretion of catecholamine 2.67E-06 22
 Long term potentiation of synapse 3.13E-14 52
Cell Morphology 1.42E-27–5.59E-05 610
 Morphology of neurons 4.02E-14 115
 Branching of neurites 9.57E-11 68
 Plasticity of synapse 9.37E-08 34
 Formation of cellular protrusions 3.42E-18 192
 Morphogenesis of neurites 4.73E-11 88
Cellular Assembly and Organization 1.28E-22–7.73E-05 421
 Growth of neurites 5.15E-12 118
 Release of vesicles 1.10E-05 11
 Organization of cytoskeleton 1.28E-22 293
 Branching of neurites 9.57E-11 68
Cellular Function and Maintenance 1.28E-22–7.73E-05 605
 Organization of cytoskeleton 1.28E-22 293
 Formation of cellular protrusions 3.42E-18 192
 Neuritogenesis 2.90E-18 135
 Synaptogenesis 1.79E-12 55
 Development of gap junctions 2.93E-12 58
Molecular Transport 1.37E-21–8.59E-05 475
 Concentration of corticosterone 2.48E-05 29
 Quantity of steroid hormone 3.61E-05 35
 Quantity of catecholamines 2.18E-06 37
 Secretion of neurotransmitter 1.61E-12 51
 Transport of molecule 1.37E-21 346

aDerived from the likelihood of observing the degree of enrichment in a gene set of a given size by chance alone.

bClassified as being differentially expressed that relate to the specified function category; a gene may be present in more than one category.

FIG. 4.

FIG. 4.

Canonical pathway and network analysis associate neurotransmitter function as a target of atrazine toxicity. Canonical pathway analyses of altered genes in the microarray analysis of male brain indicate genes associated with various neurotransmitter pathways including release of neurotransmitters, secretion of GABA, quantity of catecholamines and monoamines, and release and concentration of 5-hydroxytryptamine. Green indicates a down regulation and red indicates an up regulation.

qPCR Confirmation of Male Brain Microarray Data

A subset of genes found to be significantly altered in all three atrazine treatments by microarray analysis (Table 2) were independently confirmed by qPCR. Seven gene targets were confirmed and included ddc, srd5a2b, adam8, npy, hsd11b2, mc2r, and slc2a9l1. The data were statistically significant for this subset of genes with a positive correlation between the microarray and qPCR in all three atrazine treatments (0.3 ppb: R = 0.853, P = 0.0147; 3 ppb: R = 0.873, P = 0.0103, 30 ppb: R = 0.874, P = 0.0101) (Figure 5).

FIG. 5.

FIG. 5.

qPCR comparative analysis of microarray data for adult male brain tissue. qPCR analysis was performed to compare gene expression changes detected on the microarray on seven gene targets (ddc, srd5a2b, adam8, npy, hsd11b2, mc2r, and slc2a9l1). All targeted genes were significantly altered on the microarray. A linear correlation was completed and statistical significance of the correlation was determined using a Pearson’s correlation. A statistically significant positive correlation between the microarray and qPCR was observed in all three atrazine treatments (A: 0.3 ppb: R = 0.853, P = 0.0147; B: 3 ppb: R = 0.873, P = 0.0103; C: 30 ppb: R = 0.874, P = 0.0101). The microarray data are represented as log2 expression values and qPCR values are the ratios of relative expression against β-actin represented as log2.

DISCUSSION

In order to assess how an embryonic atrazine exposure affects the adult male reproductive system, testicular morphology and histology, hormone levels, and transcriptomic profiles of testes and brain were assessed. Investigation of alterations on the reproductive system from an embryonic atrazine exposure of 0.3, 3, or 30 ppb showed no significant changes in body weight, testes weight, or GSI. Results from our study are in agreement with other aquatic studies that examined the effects of juvenile or adult atrazine exposure in goldfish or fathead minnows in which no alterations in body weight or GSI were reported (Bringolf et al., 2004; Nadzialek et al., 2008; Spanò et al., 2004; Table 4). Although the differences in treatment, time of exposure, and time of analysis are different, these studies still support that atrazine exposure does not alter morphological characteristics in males. Rodent studies have begun to address the later-in-life consequences of maternal and peripubertal atrazine exposure in adult male offspring. Previous studies have shown similar results reporting no significant alterations in body or testes weight later-in-life following maternal and/or peripubertal atrazine exposure of up to 100 mg/kg (Rosenberg et al., 2008; Stanko et al., 2010; Stoker et al., 1999; Table 5). A more complex study conducted by Rayner et al. reported a decrease in body weight at postnatal day (PND) 120 following only lactational atrazine exposure. In addition, a significant decrease in body weight was observed at PND220 in only gestational treated animals (100 mg/kg). No alterations in testes weights were observed at either time point or exposure regimen (Rayner et al., 2007). Although the study conducted by Rayner et al. (2007) provides conflicting data regarding alterations in body weight, it is thought that these changes are not solely dependent upon atrazine exposure because of the lack of a clear trend. With this exception, our study is in agreement with the previously mentioned studies in that embryonic and/or developmental atrazine exposure does not elicit lasting effects on body or testes weight.

TABLE 4.

Summary Table of Discussed Aquatic Studies

Referencea Species Exposure (ppb) Duration Outcomes
Bringolf et al. (2004) Fathead minnow (Pimephales promelas) 0, 5, or 50 21 days (adult) No significant alterations in male total body weight or testes weight; no significant alterations in fecundity, offspring survival or hatchability; no alterations in vtg
Spanó et al. (2004) Goldfish (Carassius auratus) 0, 100, or 1000 21 days (adult) No alterations in GSI; Decrease in plasma testosterone and 11-ketotestosterone and an increase in plasma estradiol in the 1000-μg/l treatment group; structural disruption in testis tissue (1000 μg/l); follicular atresia in ovary tissue in both treatments; no changes in vtg
Coady et al. (2005) African clawed frog (Xenopus laevis) 0, 0.1, 1, 10, or 25 72 hph -2–3 mpm Concentrations of 0.1 and 25 μg/l did not alter mortality, metamorphosis, gonad development, or aromatase activity; 1 μg/l elicited a decrease in estradiol
Freeman et al. (2005) African clawed frog (Xenopus laevis) 0, 100, 450, or 800 Stage 47 or 54—1, 2, 3, 4, or 5 weeks Delayed metamorphosis at 100, 450, and 800 μg/l
Du Preez et al. (2008) African clawed frog (Xenopus laevis) 0, 1, 10, or 25 96 hpf–2 ypm No effect of clutch size, hatching rate, or time to metamorphosis in F1 generation; no alterations in sex ratio of offspring (F2); no alterations in testicular morphology (F1 or F2)
Oka et al. (2008) African clawed frog (Xenopus laevis) 0, 0.1, 1, 10, or 100 Tadpole (stage 49)—metamorphosis (stage 66) No effect on metamorphosis, gonad development, or aromatase expression; no alterations in levels of vtg
Nadzialek et al. (2008) Goldfish (Carassius auratus) 0, 100, or 1000 56 days (adult) No alterations in GSI, plasma concentrations of estradiol, or alterations in cyp19a1; decrease in 11-ketotestosterone after 56-day exposure in the 1000-μg/l treatment
Spolyarich et al. (2010) Spotted Marsh Frog (Limnodynastes tasmaniensis) 0, 0.1, 1, 3, or 30 Gosner stage 28–42 No alterations in tadpole growth, development, or sex ratios; testicular oocytes observed in one fish at 3 μg/l atrazine, although not statistically significant
Tillitt et al. (2010) Fathead minnow (Pimephales promelas) 0, 0.5, 5, or 50 14 or 30 days (adult) Decrease in total egg production; Reduction in total number of spawning events; no alterations in steroid hormone levels; testicular oocytes found in the 5-μg/l treatment group; ovaries with lipid accumulation and atretic follicles observed
Corvi et al. (2012) Zebrafish (Danio rerio) 0, 0.1, 1, or 10 μM 17–130 dpf No alterations in sex ratio; no alterations in gonad development
Weber et al. (2013) Zebrafish (Danio rerio) 0, 0.3, 3, or 30 1–72 hpf Significant increase in head length at 72 hpf in all atrazine treatments; transcriptomic data included genes altered in neuroendocrine and reproductive system function, cell cycle, and carcinogenesis
Papoulais et al. (2014) Japanese Medaka (Oryzias latipes) 0, 0.5, 5, or 50 14 or 38 days (adult) Decrease in total egg production in all atrazine treatments after day 25 of exposure; no alterations in spawning events, GSI, aromatase protein, or whole body estradiol or testosterone
Wirbisky et al. (2015) Zebrafish (Danio rerio) 0, 0.3, 3, or 30 1–72 hpf Decrease in serotonin metabolite 5-hydroxyindoleacetic acid and serotonin turnover in females; transcriptomic analysis revealed alterations in genes throughout the serotonergic pathway
Wirbisky et al. (2016a) Zebrafish (Danio rerio) 0, 0.3, 3, or 30 1–72 hpf Decrease in spawning events at 30 μg/l treatment; swollen abdomen and increase in atretic follicles in females exposed to 30 μg/l; increase in ovarian progesterone; transcriptomic analysis revealed alterations in genes involved in steroidogenesis

Abbreviations: dpf, days post-fertilization; GSI, gonadosomatic index; hpf, hours post-fertilization; hph, hours post-hatching; mpm, months post-metamorphosis; ppb, parts per billion (µg/l); µM, micromolar; vtg, vitellogenin.

aStudies listed in the table are studies referenced within the text; this is not an all-encompassing literature review.

TABLE 5.

Summary Table of Discussed Male Mammalian Studies

Reference Species/Strain Exposure Duration Outcomes
Stoker et al. (1999) Wistar rat 0, 6.25, 12.5, 25, or 50 mg/kg PND1–4 No significant difference in body, testes, or prostate weight at PND120; Prostate inflammation at PND120 at 25 and 50 mg/kg; Complete inhibition of suckling induced prolactin release at 50 mg/kg
Friedmann (2002) Sprague–Dawley rat 0 or 50 mg/kg/day PND46–48 (acute) Decrease in serum and intratesticular testosterone
PND22–48 (chronic)
Stoker et al. (2002) Wistar rat 0, 6.25, 12.5, 25, 50, 100, or 200 mg/kg of DEA, DIA, and DACT (atrazine equimolar doses) PND23–53 Decrease in PPS; Reduction in ventral and lateral prostate weight; Decrease in epididymal and seminal vesicle weight; Decrease in testosterone; Increase in estrone; No alterations in estradiol; No alterations in thyroid hormones
Rayner et al. (2007) Long-Evans rat 0 or 100 mg/kg/day GD15–19 Delay in preputial separation; Increase in lateral prostate weight at PND120; No alterations in testes and seminal vesicle weight at PND120 and PND220; No alterations in serum testosterone and androstenedione, but a decrease in prolactin at PND220
Rosenberg et al. (2008) Sprague–Dawley rat 0, 10, 50, 75, or 100 mg/kg/day GD14-Parturition Increase in dead pups at 75 and 100 mg/kg; Increase in preputial separation at 100 mg/kg/day; Decrease in angiogenital distance at 100 mg/kg/day; No gross morphology alterations; Decrease in serum and intratesticular testosterone
Fraites et al. (2009) Long–Evans rat 0 or 75 mg/kg (single dose) Adult Exposure Increase in adrenocorticotropic hormone (ACTH), corticosterone, and progesterone
Laws et al. (2009) Wistar rat 0, 5, 50, 100, or 200 mg/kg/day (single dose) Adult Exposure Increase in plasma ACTH and serum corticosterone and progesterone.
Pogrmic-Majkic et al. (2010) Wistar rat In vitro: 1–50 μM 24 or 72 h (in vitro); 1, 3, or 6 days (in vivo) In vitro: Increase cAMP; Increase in androgen production; Increase in steroidogenic gene expression. In vivo: Stimulatory but transient action of atrazine on cAMP signaling and androgenesis
In vivo: 200 mg/kg
Pogrmic et al. (2009) Wistar rat 0, 50, or 200 mg/kg/day PND23–50 Suppression of luteinizing hormone receptor gene expression, inhibition of cAMP production; decrease in mRNA of steroidogenic genes
Stanko et al. (2010) Long Evans rat 0 or 100 mg/kg GD15–19 No body weight or testes weight alterations; Delay in preputial separation; Increases in testosterone and estrone at PND120; Decrease in estradiol and estrone at PND180
Victor-Costa et al. (2010) Wistar Rat 0, 50, 200, or 300 mg/kg/day 7 (300 mg/kg), 15 (50 or 200 mg/kg), or 40 (200 mg/kg) days Decreased body weight, increased testes and adrenal weight; Decreased testosterone; Increased estradiol; Dilation of seminiferous tubules; Testicular atrophy
Belloni et al. (2011) CD1 mice 0, 1, or 100 μg/kg/day GD14–PND21 Feminization of behavioral profile; Decrease in sperm count and concentration
Fraites et al. (2011) Sprague–Dawley rat 0, 1, 5, 20, or 100 mg/kg/day GD14–21 No alterations in testosterone production, timing of puberty, play behavior, AGD, or male sex organ weights at any atrazine treatment at PND59
Kucka et al. (2012) Sprague–Dawley anterior pituitary and Leydig cells 0, 10–50 μM Various time points Increase in cAMP and prolactin in pituitary cells; Inhibition of PDE4 isoenzymes
Jin et al. (2013) ICR mice 0, 50, 100, or 200 mg/kg/day 3 weeks (peripubertal exposure) Decrease in body and liver weight; Increase in relative testes weight; Decrease in testosterone; Increase in estradiol; Decrease in expression of steroidogenic genes
DeSesso et al. (2014) Wistar rat 0, 1, 5, 25, or 125 mg/kg/day GD6–21 or PND2–21 No alterations in spermatid counts in testes, spermatozoa counts in epididymides, or plasma testosterone levels at PND70 or PND170; Increase in percentage of abnormal sperm on PND70 at 125 mg/kg/day; PND exposure showed a reduction in absolute testes and epididymis weights in the 125-mg/kg/day treatment group on PND70; No effect on plasma testosterone or sperm morphology at PND70 and PND170

Abbreviations: AGD: angiogenital distance: GD: gestational day; PDE: phosphodiesterase; PND: post-natal day; µM: micromolar

*Studies listed in the table are studies referenced within the text; this is not an all-encompassing literature review.

Studies reporting histological analysis of adult testes tissue following an embryonic atrazine exposure are limited in anuran, fish, and rodent models (Tables 4 and 5). Our results report no significant histological alterations in thickness of germinal epithelium, percentage of different cell types (primary and secondary spermatocytes, spermatid, spermatozoa, and spermatogonia), and testicular abnormalities (hypoplastic, atrophied, and underdeveloped). To our knowledge, no other studies utilizing fish models have assessed testicular histology following an embryonic atrazine exposure. Two studies assessing the adverse effect of adult atrazine exposure report the occurrence of testicular oocytes in zebrafish (Corvi et al., 2012) and fathead minnows (Tillitt et al., 2010); however, the prevalence of this effect is rare and was not statistically significant. An additional adult study conducted by Spanò et al. (2004) reported no alterations in the percentage of any male sex cells following a 21-day atrazine exposure of 100 or 1000 ppb. Anuran studies utilizing Xenopus laevis have also reported no significant testicular abnormalities following developmental atrazine exposure (Coady et al., 2005; Oka et al., 2008). Rodent models examining later-in-life effects of atrazine exposure on testes histology have reported significant reduction in sperm concentration at 100 μg/kg combined with no alterations in germinal epithelium at PND60-65 (Belloni et al., 2011). DeSesso et al. reported that an atrazine exposure of 1, 5, 25, or 125 mg/kg from gestational day (GD) 6–21 elicited no significant alterations in spermatid or spermatozoa counts at PND70 or PND170. However, an increase in abnormal sperm was reported at PND70 at 125 mg/kg and at PND170 at 25 mg/kg. In addition, prenatal atrazine exposure from PND2–PND21 had no effect on spermatid or sperm counts at PND70 and PND170, but an increase in abnormal sperm was observed in the 125-mg/kg treatment group at PND170 (DeSesso et al., 2014). Results from these studies are consistent with our findings in which no significant changes in cell types and germinal epithelium were observed. The largest contrast is found with the increase in abnormal sperm observed in DeSesso et al. (2014), but this difference could be attributed to differences in treatment and exposure periods.

In addition, we observed no alterations in testes levels of 11-ketotestosterone (main form of testosterone in fish) or testosterone. To our knowledge, this is the first study in which a DOHaD exposure paradigm was assessed in fish species that investigated hormonal alterations, but previous studies identifying hormonal alterations following juvenile or adult atrazine exposure have been reported. A study conducted by Nadzialek et al. (2008) reported a decrease in plasma levels of 11-KT following a 56-day atrazine exposure of 1000 ppb in juvenile goldfish. Spanò et al. (2004) reported no alterations in 11-KT or testosterone in goldfish testes tissue; however, a significant decrease was reported in plasma levels of 11-KT and testosterone following an adult exposure of 1000 ppb for 21 days. Additional adult studies of atrazine exposure in Japanese medaka and goldfish reported no alterations in whole body levels of testosterone or estradiol following an exposure of 0.5, 5, or 50 ppb (Papoulais et al., 2014; Tillitt et al., 2010). Although our study did not examine plasma levels of 11-KT or testosterone, our results are in agreement with Spanò et al. regarding tissue hormone levels, keeping in mind the differences in experimental design. As previously stated, plasma levels of 11-KT and/or testosterone have been shown to be decreased in juvenile and adulthood exposures; however, at high doses (1000 ppb). Previous rodent studies assessing later-in-life hormonal effects of atrazine exposure are in agreement with our findings (DeSesso et al., 2014; Fraites et al., 2011; Rayner et al., 2007; Stanko et al., 2010). A contrasting study conducted by Rosenberg et al. (2008) reported a decrease in serum and intratesticular testosterone levels at PND60 following a gestational atrazine exposure of 50, 75, or 100 mg/kg. Although contrasting results are reported, this could be because of differences in the exposure or latency period compared with previously mentioned studies. Our results combined with the previously mentioned fish and rodent studies support the hypothesis that the later-in-life impacts of atrazine on 11-KT and testosterone levels diminish as the time period between exposure and analysis increases.

To further investigate potential adverse health outcomes of the embryonic atrazine exposure, transcriptomic analysis of testes tissue of adult male zebrafish exposed to 0, 0.3, 3, or 30 ppb atrazine through embryogenesis was also assessed. While differentiation and maturation of cells in the 0.3-ppb treatment group, cell death and survival in the 3-ppb treatment group, and cellular proliferation and molecular transport (specifically associated with the thyroid pathway) in the 30-ppb treatment group were more pronounced compared with the other treatment groups, great similarities among all the treatment groups were observed for the molecular pathways in which the genes with altered expression in the testes are associated. Our transcriptomic results of testes tissue indicated significant alterations in gene expression associated with molecular and cellular functions including lipid metabolism, molecular transport, small molecular biochemistry, and cell morphology. Genes of interest in these categories included GHR, IGF2, LSR, PNPLA2, ADRB2, DIO2, and THRA. These genes are involved in lipid concentration, quantity of steroid hormones, absorption of cholesterol, quantity of l-triiodothyronine (T3) and thyroid stimulating hormone (TSH). Thyroid hormones are vital for proper regulation of body metabolism, growth, development, and reproduction. Current studies support the hypothesis in which thyroid hormones (as part of the hypothalamus–pituitary–thyroid [HPT] axis) interact with hormones of the HPG axis (reviewed in Habibi et al., 2012; Morais et al., 2013). In addition, sex steroid signaling and Sertoli cell differentiation involves triiodothyronine (T3) mediated down regulation of aromatase (CYP19A1) gene transcription and an up regulation of androgen receptor gene expression (Morais et al., 2013). Results from our transcriptomic data highlight the HPT pathway in all atrazine treatments, but especially in the 30-ppb treatment group, through the decrease in expression of THRA, DIO2, and GH. Interestingly, these genes were not identified in adult male brain tissue; highlighting the tissue specific effects of atrazine exposure. The testes tissue may be more susceptible to alterations in genes associated within the HPT axis because of its association with reproductive function. This reported decrease in THRA is important as thyroid hormone receptors (THRα and THRβ) in Sertoli and Leydig cells of zebrafish have been identified (Morais et al., 2013), although no difference in Leydig cell frequency or hypertrophy of Leydig or Sertoli cells were observed in this study. In addition, the reported decrease in DIO2 is of interest as it is an important regulator of circulating and intracellular thyroid hormone levels in fish (Xie et al., 2015). Gene ontology analysis of male testes tissue also revealed T3 as a top upstream regulator along with TR/RXR activation as a top canonical pathway. Although our study did not investigate levels of thyroid hormone in the adult male zebrafish, transcriptomic data and previous rodent and X. laevis studies (Freeman et al., 2005; Ghinea et al., 1979; Kornilovskaya et al., 1996; Stoker et al., 2002) indicate the need for further investigation to support the DOHaD exposure paradigm for HPT dysfunction.

Gene ontology analysis also revealed growth hormone (GH) as a top upstream regulator and canonical pathway. Genes associated with this pathway included growth hormone receptor (GHR) and insulin like growth factor 2 (IGF2) in which significant decreases in expression were observed. Testes and reproductive accessory organs are sites of both growth hormone (GH) production and action. GH is responsible for promoting testicular growth and development, gametogenesis, and steroidogenesis in adult males (reviewed in Hull and Harvey, 2014). In addition, GH is a steroidogenic factor which can stimulate Leydig cells to produce estrogen and androgens in vitro. In vitro studies have also reported that GH can stimulate the production of steroidogenic acute regulatory protein (StAR) which could lead to an increase in hormone production (reviewed in Hull and Harvey, 2014). Furthermore, it is hypothesized that GH acts at the level of the pituitary and gonads to modify gonadotropin releasing hormone (GnRH) actions (reviewed in Hull and Harvey, 2014). As GnRH is the primary regulator of the reproductive axis, understanding parallel regulators is key in determining the mechanism of atrazine toxicity.

In addition, transcriptomic analysis of brain tissue of adult male zebrafish exposed to 0, 0.3, 3, or 30 ppb atrazine through embryogenesis was assessed. To our knowledge, no transcriptomic profiling has been completed on adult male brain tissue following developmental atrazine exposure. The embryonic atrazine exposure had the largest effect in terms of the number of genes with altered expression on the male brain transcriptome compared with any other tissue we have analyzed to date (including 72 hpf zebrafish larvae (Weber et al., 2013), adult female brain (Wirbisky et al., 2015), adult ovaries (Wirbisky et al., 2016a), and adult testes). The large commonality in the number of genes observed to have altered expression in each treatment group resulted in very similar molecular pathways identified to be influenced in the brain of adult male zebrafish exposed to atrazine during embryogenesis. Results indicated significant alterations in gene expression associated with molecular and cellular functions including cell-to-cell signaling and interaction, cell morphology, cellular assembly and organization, cellular function and maintenance, and molecular transport. Subcategories included secretion of catecholamines and neurotransmitters, outgrowth of neurites, quantity of steroid hormone, and synaptogenesis.

Previous studies have assessed how atrazine targets steroidogenesis throughout the HPG axis in various animal and in vitro models (Kucka et al., 2012; Pogrmic et al., 2009, Pogrmic-Majkic et al., 2010; Roberge et al., 2004; Tables 4 and 5). Our transcriptomic results of brain tissue showed alterations in three primary steroidogenic genes in all three atrazine treatments. The first gene of interest is steroidogenic acute regulatory protein (StAR). This gene is responsible for the mobilization of cholesterol from the outer to the inner mitochondrial membrane where it is metabolized to pregnenolone, the precursor to glucocorticoids, mineralocorticoids, and sex steroids (Pogrmic et al., 2009). Although the majority of research investigating the effects of atrazine on StAR have focused on Leydig cells (Pogrmic et al., 2009; Pogrmic-Majkic et al., 2010), StAR is also found to be expressed in brain tissue of rodents, zebrafish, and humans (Furukawa et al., 1998; Kim et al., 2003; Richter et al., 2011; Sierra et al., 2003). How this down regulation of StAR affects lipid metabolism and hormone synthesis is still unknown. In conjunction with the decrease in StAR gene expression, our results revealed a decrease in CYP11A1, a gene associated with the mobilization of cholesterol. This finding indicates that in addition to reducing the mobilization of cholesterol, the metabolism of cholesterol may also be reduced, which in turn could lead to decreases in steroid hormone concentration, but further research is needed to confirm this hypothesis. Furthermore, an increase in aromatase (CYP19A1) was also observed. It has been hypothesized that atrazine elicits an increase in CYP19A1 therefore causing a decrease in testosterone and an increase in estradiol (Hayes et al., 2002, 2010). This remains controversial as numerous other studies have not been able to support this hypothesis (Coady et al., 2005; Du Preez et al., 2008; Nadzialek et al., 2008; Spolyarich et al., 2010). While our data show an increase in CYP19A1 in adult male brain tissue; no decreases in testes 11-KT or testosterone were observed. CYP19A1 was also increased in female ovary tissue following an embryonic atrazine exposure with no significant increase in estradiol (Wirbisky et al., 2016a). These results are interesting in that alterations in steroidogenic hormones appear to be sex and tissue specific (ie, no alterations of STAR, CYP11A1, or CYP19A1 were identified in testes tissue). With specific regards to the males, alterations in these three steroidogenic genes requires further investigation as no additional functional endpoints were significantly altered in this study.

Numerous genes were also found to be involved in the release of neurotransmitters, secretion of GABA, concentration and release of 5-hydroxytryptamine, quantity and release of catecholamines, and quantity of monoamines. In a previous study conducted by our laboratory (Wirbisky et al., 2015) no alterations in neurotransmitter levels (DA, DOPAC, 5-HT, 5-HIAA, and GABA) in adult male brain tissue following an embryonic atrazine exposure were reported. While the previous study indicates no alterations in neurotransmitter levels, transcriptome analysis shows that neurotransmitter systems are potentially affected by an embryonic atrazine exposure at the mRNA level; primary genes of interest found to be altered include serotonergic receptors (HTR1A, HTR1B, HTR2A, HTR2C), DDC, GABA receptors (GABBR1 and GABRB3), and cholinergic receptors (CHRNA5 and CHRNB3). Of interesting note, is that the transcriptomic profile of adult female brain tissue also reported alterations in neurotransmitter function specifically regarding the serotonergic system with gene expression alterations in similar serotonergic receptors (Wirbisky et al., 2015). These findings support the need for further research focused on neurotransmission alterations from an embryonic atrazine exposure in both genders.

Functioning alongside the HPG axis is the hypothalamus–pituitary–adrenal (HPA) axis; the body’s key stress response pathway. The HPA axis is an additional site in which atrazine has been shown to target by increasing concentrations of adrenocorticotropic hormone (ACTH) and corticosterone in male and female rodents (Fraites et al., 2009; Laws et al., 2009). This is important as the HPA axis is thought to modify the function of the reproductive axis through the indirect interactions of corticotropin-releasing factor (CRF) and GnRH neurons. In addition, steroid hormone feedback from corticosteroids can alter secretion of GnRH and luteinizing hormone (LH) (Fraites et al., 2009). Our transcriptomic results indicate a significant increase in corticotropin-releasing hormone (CRH) and in corticotropin-releasing hormone receptor 2 (CRHR2), further supporting the effects of atrazine on the HPA axis through a developmental origin. Furthermore, a significant decrease in expression of melanocortin 2 receptor (MC2R) was observed. This gene is responsible for the proper binding of pro-opiomelancortin (POMC) derived hormones; primarily adrenocorticotropic hormone (ACTH). These results demonstrate that an embryonic atrazine exposure alters gene expression throughout the HPA axis and further supports the developmental origin of adverse neuroendocrine effects of atrazine.

Transcriptomic analysis of adult male and female brain and gonad tissue following an embryonic atrazine exposure has revealed sex differences in gene expression alterations. The current proposed expected mechanism behind the DOHaD paradigm is suggested to be from epigenetic alterations. Epigenetic alterations occur through different mechanisms including DNA methylation, histone modifications, and non-coding RNAs (Bouwmeester et al., 2016). Although these factors have not been addressed in this study, we did recently report that an embryonic atrazine exposure (the same exposure period as in the current study) did result in immediate alterations in microRNA (miRNA) expression in miRNAs associated with neurodevelopment, cancer, and angiogenesis (Wirbisky et al., 2016b). Targeting analysis revealed connections between altered miRNAs and genes previously identified to be immediately altered by this atrazine exposure (Weber et al., 2013). These findings support the need of future studies to define epigenetic mechanisms underlying the later in life alterations observed from an embryonic atrazine exposure.

The disruption of the developing neuroendocrine system was identified in our initial gene expression assessment of zebrafish larvae in which alterations in CYP17A1, LH, ADCY1, and PDE10A were reported (Weber et al., 2013). Additionally, PER1 and PER3 were significantly altered which play a role in regulating circadian rhythm; a key process in reproductive function. The analysis of adult female brain tissue also reported alterations in genes associated with proper neuroendocrine function including CYP17A1, LHb, and THRA (Wirbisky et al., 2015). In addition, an embryonic atrazine exposure has been shown to decrease 5-HIAA and serotonin turnover (Wirbisky et al., 2015). Although neurotransmitter levels were not addressed in this study, neurotransmitters and neuropeptides are thought to affect GnRH release. Transcriptomic profiling of adult female ovary tissue following an embryonic atrazine exposure revealed enrichment of genes associated with endocrine system development and function and steroidogenesis which included CYP19A1, CYP1B1, STAR, and VIP. These genetic alterations provide a genetic link to the observed increase in ovarian progesterone and follicular atresia (Wirbisky et al., 2016a). Transcriptomic analysis of adult male testes and brain tissue revealed some similarities between the genders, but also demonstrated a wider range of neuroendocrine dysfunction by way of the HPA, HPT, and neurotransmitter pathways (Figure 6). Additionally, in contrast to the female study, no morphological or histological alterations were observed following an embryonic atrazine exposure. Therefore, additional neuroendocrine endpoints are required in order to further assess any functional alterations indicated by the transcriptomic analysis in adult males.

FIG. 6.

FIG. 6.

Schematic diagram of the effects of an embryonic atrazine exposure in zebrafish. Diagram representing the effects of an embryonic atrazine exposure at 72 hpf as well as in brain and gonad tissue of adult females and males exposed during embryogenesis compiling data from this study (Weber et al., 2013; Wirbisky et al., 2015, 2016a). The genes in green are representative of changes observed at 72 hpf; genes in purple are altered in adult female brain tissue; genes listed in pink are those altered in adult female ovarian tissue; red signifies the morphological alterations observed in adult females; genes listed in blue are those altered in adult male brain tissue; genes in orange are altered in adult male testes tissue.

CONCLUSIONS

Our transcriptomic data suggest a developmental origin of adverse effects on the neuroendocrine system in adult male zebrafish caused by an embryonic atrazine exposure. Many adult diseases and disorders are thought to originate from an in utero or developmental exposure to environmental stressors; thereby altering and reprogramming normal cellular processes causing an altered physiological state. The disruption of the developing neuroendocrine system was identified in our initial gene expression assessment of zebrafish larvae and was also reported in adult female zebrafish exposed to atrazine during embryogenesis (Weber et al., 2013; Wirbisky et al., 2015, 2016). Although no significant morphological, histological, or hormonal alterations were observed in this study, transcriptomic results identified key pathways and genetic targets with altered expression from the embryonic atrazine exposure. The primary neuroendocrine target of atrazine is highlighted to be the HPG axis, but results from our recent studies demonstrate that the HPA and HPT axes may also be a target and indicate the need for further studies. Overall, results from these and previous studies provide support for a developmental atrazine exposure resulting in immediate and lasting adverse impacts on the neuroendocrine system.

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.oxfordjournals.org/.

Supplementary Data

ACKNOWLEDGMENTS

This article is part of the Center for the Environment publication series. The authors declare that they have no competing interests.

FUNDING

This work was supported by the National Institutes of Health, National Institute of Environmental Health Sciences (R15 ES019137 to J.L.F. and M.S.S.), a Purdue Center for Cancer Research Innovative Research Pilot Project (J.L.F. and M.S.S.), a Purdue Research Foundation Grant (J.L.F. and G.J.W.), and a Bilsland Dissertation Fellowship (S.E.W. and J.L.F.).

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