Exposure to the heavy metal lead induces DNA copy number alterations in zebrafish cells

DNA copy number variants (CNVs) are associated with the development of complex neurological diseases and disorders including autism spectrum disorder, schizophrenia, Alzheimer’s disease, and Parkinson’s disease. Exposure to multiple environmental chemicals including various heavy metals is suggested as a risk factor in these neurological diseases and disorders, but few studies have addressed if heavy metal exposure can result in spontaneous DNA copy number changes as a genetic mechanism contributing to these disease outcomes. In this study to further investigate the relationship between heavy metal exposure and spontaneous copy number alterations (CNAs), zebrafish fibroblast cells were exposed to the neurotoxicant lead (Pb). A crystal violet assay was first used to determine exposure concentrations with greater than 80% cell confluency. Then a zebrafish-specific array comparative genomic hybridization (CGH) platform was used to detect CNAs following a 72 hour Pb exposure (0.24, 2.4, or 24 μM). The Pb exposure resulted in 72 CNA amplifications ranging in size from 5 to 329 kb. No deletions were detected. CNAs resulted in 15 copy number alteration regions (CNARs), leaving 7 singlet CNAs. Two of the singlets were within high repeat genomic locations. The number of CNAs tended to increase in a concentration dependent manner. Several CNARs encompassed genes previously reported to have altered expression with Pb exposure, suggesting a mechanistic link. In addition, almost all genes are associated within a molecular network with the amyloid precursor protein (APP). Overall, these findings show that Pb exposure results in spontaneous CNAs that could serve as a mechanism driving adverse health outcomes associated with Pb toxicity including neurological disease pathogenesis for further study. The objective of this study was to identify whether a Pb exposure will generate copy number alterations. First, a zebrafish fibroblast cell line was exposed to a range of Pb concentrations (0-100 uM) 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, 0.24, 2.4, and 24 um Pb) were chosen to represent a range from no effect on confluency to decreasing cell confluency to ~80% in this cell line. Cells were then exposed to these four treatment concentrations and array CGH was used to detect copy number alterations (CNAs) in Pb treatments compared to the control treatment (n=4). qPCR was used to confirm CNAs.