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Title: Effects on specific promoter DNA methylation in zebrafish embryos and larvae following benzo[a]pyrene exposure

item CORRALES, J - University Of Mississippi
item FANG, X - University Of Florida
item THORNTON, C - University Of Mississippi
item MEI, W - University Of Florida
item BARBAZUK, B - University Of Florida
item Duke, Mary
item Scheffler, Brian
item WILLETT, K - University Of Mississippi

Submitted to: Comparative Biochemistry and Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/17/2014
Publication Date: 2/24/2014
Citation: Corrales, J., Fang, X., Thornton, C., Mei, W., Barbazuk, B.W., Duke, M.V., Scheffler, B.E., Willett, K.L. 2014. Effects on specific promoter DNA methylation in zebrafish embryos and larvae following benzo[a]pyrene exposure. Comparative Biochemistry and Physiology. Part C 163 (2014): 37-46.

Interpretive Summary: Zebrafish represents an excellent model system to study genes especially those genes responding to zenobiotic stress. Zebrafish works so well because its life cycle and genome are well characterized, it requires little lab space and has a fast reproductive cycle. In this study, the zenobiotic (foreign chemical substance) compound Benzo[a]pyrene (BaP) and its impact on specific genes was analyzed. BaP is a polycyclic aromatic hydrocarbon abundant in the environment and derived from the incomplete combustion of organic compounds, thus it can be found in waterways. In this study, 21 genes known to be associated with diseases were analyzed after fish embryos were exposed to various amounts of BaP and at two stages of growth. Specifically, modifications were examined at cytosine which is one of the four nucleotides of DNA. The type of modification is an addition of a methyl group to the nucleotide. This type of modification can in turn impact gene expression as the extra methyl group can interfere with cellular mechanisms associated with that gene’s expression, seen as RNA production. Typically the more cytosines that are methylated in a gene the greater impact on gene expression and typically it is a reduction in overall expression. Such modifications can result in epigenetic changes (heritable changes not due to changing the order of the four nucleotides of which DNA is composed of) and thus the impact of these methylation modifications can, at times, be seen in later life stages or future generations. This study used high throughput sequencing to get precise data on the overall amount of methylation for each relevant cytosine in the promoter (control region) of each of the 21 genes. The study showed that methylation levels were higher in later developmental stages for specific genes and that for six of the genes methylation changed by at least 10% at both life stages. This study now opens the door to further examine the impacted genes to determine if their expression levels have been changed in a tissue specific manner and it also means study of cytosine methylation of other genes is worthy of similar efforts.

Technical Abstract: Benzo[a]pyrene (BaP) is an established reproductive and developmental toxicant. BaP exposure in humans and animals has been linked to infertility and multigenerational health consequences. DNA methylation is the most studied epigenetic mechanism that regulates gene expression, and mapping of methylation patterns has become an important tool for understanding pathologic gene expression events. The goal of this study was to investigate aberrant changes in promoter DNA methylation in zebrafish embryos and larvae following a parental and continued embryonic waterborne BaP exposure. A total of 21 genes known for their role in human diseases were selected to measure percent methylation by multiplex deep sequencing. For the most part, percent methylation was constitutively higher at 96 hpf than at 3.3 hpf. At 96 hpf compared to 3.3 hpf dazl, nqo1, sox3, cyp1b1, and gstp1 had higher methylation percentages while c-fos and cdkn1a had decreased CG methylation. BaP exposure significantly reduced egg production and offspring survival. Moreover, BaP decreased global methylation and altered CG, CHH, and CHG methylation both at 3.3 and 96 hpf. CG methylation changed due to BaP by 10% or more in six genes (c-fos, cdkn1a, dazl, nqo1, nrf2, sox3) at 3.3 hpf and in ten genes (c-fos, cyp1b1, dazl, gstp1, mlh1, nqo1, pten, p53, sox2, and sox3) at 96 hpf. BaP also induced gene expression of cyp1b1 and gstp1 at 96 hpf which were found to be hypermethylated. Further studies are needed to link aberrant CG, CHH, and CHG methylation to heritable epigenetic consequences associated with disease in later life.