Detoxification and mutagenic response in medaka (Oryzias latipes) exposed to MX, a drinking water carcinogen

David Roy Geter


In the early 1900's, water borne diseases were dramatically decreased due to chlorination of drinking water. However, chlorine disinfection of drinking water can produce toxic and mutagenic by-products. Of these, 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) is a major mutagen of concern. To study MX, we employed the medaka ( Oryzias latipes ), a well established small fish model for testing environmental carcinogens and drinking water contaminants. In this study, we investigated mechanisms of action of MX by examining cytochrome P450 2E1 (CYP2EI) activity, total glutathione (GSH) levels and mutation induction. Methylazoxymethanol-acetate (MAMAc), a multi-site carcinogen in medaka served as a control compound. Medaka liver microsome preparations and S-9 fractions catalyzed the hydroxylation of p -nitrophenol (PNP), indicating CYP2E1-like activity. Fasted male medaka exposed to the known CYP2E1 inducers ethanol and acetone, showed significant increases in activity. Also, fasting for 96 h significantly reduced total hepatic GSH. Therefore, teleost studies investigating compounds involving CYP2E1 or GSH should avoid fasting during acute exposure to maintain xenobiotic defense integrity. CYP2E1 specific inhibitors, diethyldithiocarbamate and diallyl sulfide, significantly reduced medaka liver S-9 PNP metabolism activity but not to the same degree as in mouse controls indicating that the enzyme responsible for PNP metabolism in medaka is not identical to mammalian CYP2E1. Although the medaka may lack a mammalian CYP2E1, this study shows that CYP2E1-like activity does occur in medaka liver. Exposure to MX and MAMAc induced significant increases in hepatic CYP2E1-like activity, demonstrating that the enzyme may be involved in the biotransformation of both compounds. However, MX exposure did not alter hepatic GSH levels or mutation frequencies indicating that in vivo liver cytochrome activity may effectively detoxify the compound at exposure concentrations up to one-half the 96 h LC50 . A dose and time dependent increase in mutational frequency occurred in MAMAc treated fish establishing a link between mutation induction and tumor development in medaka. Overall, this study strengthens the utility of the medaka as a model for examining cytochrome activity, GSH detoxification and mutation induction related to drinking water and its toxic and carcinogenic properties.