Differential gene expression in anthracene-exposed mummichogs ( Fundulus heteroclitus)

Abstract

The polycyclic aromatic hydrocarbon (PAH) anthracene is present in many estuarine systems at concentrations believed to cause sublethal adverse effects, although its exact mode of toxicity remains unclear. Knowledge of the induction or suppression of specific genes as a result of exposure may be useful in explaining these effects. We have generated a fingerprint of anthracene exposure using the mummichog ( Fundulus heteroclitus), a non-migratory estuarine fish species. The fish were exposed in 7-day static renewal tests to environmentally relevant concentrations of 0, 27, 50, and 80 μg/l of anthracene. Total RNA was extracted from the livers and differential display reverse transcription polymerase chain reaction (DD RT-PCR) was used to recover 26 differentially expressed cDNA fragments. These cDNAs were isolated, sequenced, and compared to sequences of known genes in order to identify possible physiological consequences of exposure to anthracene. We then constructed macroarrays using these fragments and probed them with RNA from both anthracene-exposed fish and fish from a known PAH-impacted site. Three genes appear to be good indicators of exposure to anthracene in the range of concentrations tested, which included CYP2N2 and two expressed sequence tags (ESTs) termed 15C1 and 18C2. The expression of nine genes was altered in fish collected from a site with multiple PAHs. Band 15C1 and CYP2N2 again showed statistically significant upregulation in the field-caught fish, while a trypsin precursor and fatty acid-binding protein (FABP) all showed similar trends in induction as the laboratory-exposed fish. Further insight into the mechanism of toxicity of contaminants will be gained by the ability to identify and use differentially expressed genes as markers of exposure and effects.