Understanding the hypervariable effects of environmental toxicant exposure within and between species is vital for ecosystem modeling and remediation. Our aim is to discern the genetic components underlying intraspecific variation in environmental toxicant susceptibility. Zebrafish, Danio rerio, exhibit variable susceptibility across lab strains to 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126), a ubiquitous aromatic hydrocarbon with a high toxic equivalence factor. SNP haplotypes explain 24% of the phenotypic variation in the PCB-susceptibility trait, but the remaining 76% is unknown. Interestingly, 15% of the zebrafish genome is comprised of genomic copy number variants (CNV)—large deletions or duplications—and CNV loads differ between zebrafish strains. We hypothesize that CNV are significant contributors to variation in the PCB 126-susceptibility phenotype.
We assessed CNV across two biomarker genes, aryl hydrocarbon receptor 2 (ahr2) and cytochrome P450 1A (cyp1a), using publicly available microarray data (GEO: GSE28328) and measured post-exposure mRNA expression levels of the same genes in three strains of zebrafish (AB, TU, WIK). CNV presence was determined using mean log2 ratios over three consecutive probes. Hepatic mRNA expression levels in adults exposed to environmentally relevant levels of PCB 126 for 0, 3, 6, 9, 12, 24, and 48 hours (n=10/timepoint) were compared with time-matched vehicle controls. ahr2 and cyp1a expression levels were assessed in total mRNA via RT-qPCR, normalized to β-actin, and quantified using the relative fold change ΔΔCt method.
We found no evidence of CNV across ahr2, but found evidence of a deletion across WIK cyp1a exons 2-5 of 7 compared to AB and TU strains. PCR confirmation across the identified deletion in cyp1a exposed intact gene region frequencies of 0.64 in AB, 1.00 in TU, and 0.40 in WIK (n=25/strain). Post-exposure mRNA levels support increased expression in both ahr2 (4-fold) and cyp1a (14-fold) mRNA in WIK as compared to AB and TU strains. An increase in cyp1a expression in WIK may indicate that the identified deletion may be across an intronic repressor region. These results support our hypothesis that CNV are important contributors to interspecific variation in a receptor-mediated xenobiotic metabolism pathway. Alternatively, we may have identified a novel cyp1a subtype in zebrafish lacking exons 2 and 3, similar to cyp1a2 found in some mammals.