Atrazine Exposure Elicits Copy Number Alterations in the Zebrafish Genome

Atrazine is an agricultural herbicide used throughout the Midwestern United States that frequently contaminates potable water supplies resulting in human exposure. Using the zebrafish model system, an embryonic atrazine exposure was previously reported to decrease spawning rates with an increase in progesterone and ovarian follicular atresia in adult females. In addition, alterations in genes associated with distinct molecular pathways of the endocrine system were observed in brain and gonad tissue of the adult females and males. Current hypotheses for mechanistic changes in the developmental origins of health and disease include genetic (e.g., copy number alterations) or epigenetic (e.g., DNA methylation) mechanisms. As such, in the current study we investigated whether an atrazine exposure would generate copy number alterations (CNAs) in the zebrafish genome. A zebrafish fibroblast cell line was used to limit detection to CNAs caused by the chemical exposure. First, cells were exposed to a range of atrazine concentrations and a crystal violet assay was completed, showing confluency decreased by ~60% at 46.3 µM. Cells were then exposed to 0, 0.463, 4.63, or 46.3 µM atrazine and array comparative genomic hybridization completed. Results showed 34, 21, and 44 CNAs in the 0.463, 4.63, and 46.3µM treatments, respectively. Furthermore, CNAs were associated with previously reported gene expression alterations in adult male and female zebrafish. This study demonstrates that atrazine exposure can generate CNAs that are linked to gene expression alterations observed in adult zebrafish exposed to atrazine during embryogenesis providing a mechanism of the developmental origins of atrazine endocrine disruption. The objective of this study was to identify whether an atrazine exposure will generate copy number alterations. First, a zebrafish fibroblast cell line was exposed to a range of atrazine concentrations (0-1.125 mM) to determine concentrations where cell confluency would be altered (n=4 plates with 4 subsamples of each treatment concentration on each plate). From this experiment, treatment concentrations (0, 0.463, 4.63 and 46.3 um atrazine) were chosen to represent a range from no effect on confluency to decreasing cell confuency to ~60% in this cell line. Cells were then exposed to these four treatment concentrations and array CGH was used to detect copy number aberrations (CNAs) in atrazine treatments compared to the control treatment (n=3). qPCR was used to confirm CNAs. CNAs were compared to gene expression microarray data from adult male and adult female zebrafish gonad and brain tissue from individuals that had been exposed to atrazine during embryogenesis (GSE72242, GSE72243, GSE73740).