Abstract
In order to explore the potential of DNA methylation to serve as a biomarker of toxicity, thus establishing a link between exposure to environmental contaminants and physiologically significant changes in gene expression, tissue- and gender-specific methylation patterns in the promoter regions of estrogen receptor (ER) and aromatase genes of Japanese medaka (Oryzias latipes) were determined. Adult male and female medaka were exposed to either 0 or 500 ng/L 17 α-ethinylestradiol (EE) for 14d via a waterborne exposure. Livers, gonads, and brains were removed and genomic DNA was extracted. Samples of genomic DNA were then analyzed by bisulfite-mediated methylation-specific polymerase chain reaction (PCR) of an ∼300-bp region containing suspected methylation sites from the two genes, amplified, cloned, and sequenced. ER protein content in exposed medaka was significantly induced in all male and female tissues compared to controls. Aromatase activity in exposed medaka was significantly increased in the male brain, testes, and female brain as compared to controls. The methylation changes described by these studies indicate the potential for anthropogenic alteration of the mechanisms controlling gene expression, as well as gender- and tissue-specific sensitivity. While methylation differences were not paralleled by changes in protein expression in this study, changes in methylation have the potential to impact the regulation of normal gene expression and these changes could be transmitted to offspring.
The authors thank Tung-chin Chiang for help with method setup, Christine Metzger for technical support during this project, and Dr. Mary Haasch for reviews of the original manuscript. This research was supported by the Environmental Toxicology Research Program, University of Mississippi, and U.S. Congressional Initiative Environmental Signals and Sensors from the Centers for Disease Control (CDC), though it has not been subjected to the CDC review and therefore does not necessarily reflect the views of the CDC and no official endorsement should be inferred.