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

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.

Atrazine exposure elicits copy number alterations in the zebrafish genome

“…Genic CNAs (Supplementary Table 2) were compared to previously identified mRNA alterations in adult zebrafish (Wirbisky et al, 2015, 2016a, 2016b). In previous studies, zebrafish embryos were exposed to 0, 0.3, 3, or 30 ppb atrazine (0.0014, 0.014, or 0.14 μM, respectively) throughout embryogenesis (1-72 hours post fertilization).…”

“…qPCR was performed following similar methods as previously described (Weber et al, 2013; Wirbisky et al, 2015) following the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines (Bustin et al 2009). Similar to as performed in previous studies in our laboratory (Wirbisky et al, 2014, 2015, 2016a, 2016b) several genome regions were assessed to determine the best reference sequence to be used for this data set (data not shown). A primer targeting sequence containing the gene β-ACTIN was found to be the most consistent and least variable for this analysis.…”

“…The CNAs corresponding to a known gene (identified through the ADM2 algorithm) were compared to the lists of previously identified altered genes from adult male and female brain and gonad tissue identified through gene expression microarray analysis (Wirbisky et al, 2015, 2016a, 2016b). …”

“…Numerous laboratory studies have shown that atrazine alters the neuroendocrine system primarily through the hypothalamus-pituitary-gonadal (HPG) axis (Cooper et al, 2000; Foradori et al, 2009, 2013; Weber et al, 2013; Wirbisky et al, 2016a). In addition, genetic, epigenetic, and cellular mechanisms altered by atrazine exposure are under investigation (Karmaus and Zacharewski, 2015; Kucka et al, 2012; Pogrmic-Majkic et al, 2009, 2010, 2014; Wirbisky et al, 2016a, 2016b). …”

“…Although genotoxicity of atrazine has been investigated in past studies through the various endpoints previously mentioned, this is the first study to assess if atrazine exposure will result in genotoxicity by evaluating the generation of CNAs. Furthermore, in order to assess potential genetic mechanisms behind the developmental origins of health and disease hypothesis, which states that developmental chemical exposure can contribute to disease onset during adulthood, links between CNAs and previously identified gene expression alterations in adult male and female zebrafish exposed to atrazine during embryogenesis was assessed (Wirbisky et al, 2015, 2016a, 2016b). …”