In certain waters, a flatfish known as the common dab develops liver tumors at an uncommonly high rate — so much so that the UK Clean Seas Environmental Monitoring Program uses liver pathology of wild-caught dab as an indicator of the effects of contaminants on the marine environment. At some sites, the prevalence of hepatocellular adenomas and other pre-neoplastic toxicopathic lesions in the fish exceeds 20 percent, according to recent monitoring reports.
The University of Birmingham's Kevin Chipman and his colleagues — who last year published an epigenomic profile of DNA methylation in zebrafish in BMC Genomics — decided to profile epigenetic changes in dab from polluted areas to see whether any might be tumor-specific.
Because common dab, or Limanda limanda, is an unsequenced species, the team quickly encountered challenges. "When we started the work, we had very little genome information," Chipman says.
After finding limited success with a variety of approaches to detect differential methylation between tumor-bearing and healthy dab — including antibody pull-down experiments and ChIP-chip using an array developed for European flounder, among others — the team partnered with researchers at BGI in Shenzhen and adopted a methylated DNA immunoprecipitation with de novo high-throughput sequencing method. "It was costly, but we feel that it was a very effective way of doing it," Chipman says.
Using this MeDIP-seq approach, the researchers found epigenetic changes that occurred consistently in the tumors, a result they had anticipated.
"What was really exciting, though, and very unexpected, is that we found changes in the methylation profiles of the apparently normal surrounding tissue," Chipman says. "If we compared the methylation profiles in the surrounding tissue with [those of] normal, non-tumor-bearing fish, we saw that there was a substantial difference."
That result suggested two possible scenarios in which tumor-associated changes had occurred in some fish: that epigenetic changes had occurred in surrounding tissue and had contributed to tumorigenesis, or that "there may well be a predisposition of these particular fish to tumors brought about by some early epigenetic changes in the [affected] tissue," Chipman says.
"We know from gene expression studies and chemical analysis that these fish are exposed to a range of pollutants," he adds. Those pollutants include polychlorinated biphenyls, polybrominated diphenyl ethers, and heavy metals, some of which are known to have hormonal effects.
"We're now looking at the ability to link the analysis of epigenetic changes to environmental pollutants," Chipman says. "We've seen a potential linkage between estrogenic effects and epigenetic changes."
Going forward, Chipman and his colleagues intend to explore the extent to which epigenetic changes are useful as indicators of wild-caught fishes' exposure to pollutants. "Very little has been done on epigenetics on species in the wild," he says. "Using this methodology, we've opened the door for future similar types of studies."
In the nearer term, Chipman says his team's MeDIP-seq project on a non-model organism allows for future studies on common dab, as it has "given us a lot of sequence information for this species."