Induction and modulation of metallothioneinmRNA in Fundulus heteroclitus

Metallothionein (MT), a ubiquitously distributed cysteine-rich metal binding protein, is considered to play an essential role in the regulation of intracellular metals. Induction of MT in mammalian and non-mammalian tissues following heavy metal exposure may serve as a defense mechanism as well as a biomarker of environmental exposure to toxic metals. In this study, basal expression of MT mRNA was characterized in nonspawning and spawning killifish (Fundulus heteroclitus). In addition, MT mRNA expression was characterized in male and female killifish during both reproductive seasons following an eight day exposure to specific sublethal stressors which included temperature perturbation (26{dollar}spcirc{dollar}C or 10{dollar}spcirc{dollar}C), starvation, and waterborne cadmium (CdCl{dollar}sb2{dollar}) concentrations ranging from 0 to 80 ppb. Hepatic, gill, and intestinal MT mRNA, expressed as copy number per {dollar}mu{dollar}g total RNA, was assessed by reverse-transcriptase polymerase chain reaction (RT-PCR) and electrochemiluminescence (ECL) using a winter flounder MT cDNA primer donated by K.M. Chan (Hong Kong). A significant increase in gill and intestine MT mRNA induction was observed in spawning compared to nonspawning killifish (p {dollar}<{dollar} 0.05). These results indicate a reproductive seasonal difference in Fundulus basal MT expression. Liver, gill and intestine MT mRNA induction was significantly increased in nonspawning killifish exposed to 26{dollar}spcirc{dollar}C compared to 19{dollar}spcirc{dollar}C (control). Intestinal MT mRNA induction was significantly increased in nonfed compared to fed killifish (p {dollar}<{dollar} 0.05). Significant intestinal MT mRNA induction was also observed following waterborne exposure to 6, 12, 30 or 80 ppb CdCl{dollar}sb2{dollar} compared to control (0 ppb CdCl{dollar}sb2{dollar}) (p {dollar}<{dollar} 0.05). A significant increase in gill MT mRNA induction was observed in nonspawning killifish exposed to 26{dollar}spcirc{dollar}C and 6 ppb CdCl{dollar}sb2{dollar} compared to killifish exposed to 10{dollar}spcirc{dollar}C and 6 ppb CdCl{dollar}sb2{dollar} (p {dollar}<{dollar} 0.05). A significant increase in gill MT mRNA was also observed in nonspawning killifish exposed to starvation and 6 ppb CdCl{dollar}sb2{dollar} compared to starvation alone (p {dollar}<{dollar} 0.05). The results of this study demonstrate the variability of basal and induced MT mRNA expression in three killifish tissues in response to different physiological and chemical stressors. Further research is needed prior to the use of MT mRNA as a biomarker of xenobiotic exposure in Fundulus heteroclitus.