Nitrosodiethylamine metabolism in the viviparous fish Poeciliopsis: evidence for the existence of liver P450pj activity and expression

Abstract

Several species of fish from the genus Poeciliopsis differ dramatically in their response to the carcinogen N-nitrosodiethylamine (NDEA). The differential induction of tumors among genotypes exposed to NDEA may, in part, result from differences in liver cytochrome P450pj activity (the piscine equivalent of mammalian P450j). Evidence for the existence of cytochrome P450pj activity and mRNA expression has been found in several Poeciliopsis genotypes (species and strains). Biochemical evidence suggests that a microsomal cytochrome P450 enzyme catalyzes the metabolism of NDEA to acetaldehyde and other intermediates in Poeciliopsis. This reaction was inhibited by carbon monoxide, and required molecular oxygen and reducing equivalents (NADPH). Differences were found in maximal activity as well as temperature optima among genotypes. Poeciliopsis, a livebearing fish from desert streams of northwestern Mexico, appears to have thermal optima for cytochrome P450pj activity between 25 and 30 degrees C depending on the genotype. Western blot analysis (using anti-rat P450IIE1 antibodies) detected a 55-60 kd band in microsomes isolated from rat and Poeciliopsis. Using a 49mer probe specific for rat cytochrome P450j, Northern blots revealed a 3.3 kb mRNA from livers of a Poeciliopsis genotype and rat, but none in muscle mRNA from either organism. S1 nuclease protection assays, using the same probe, revealed that a mRNA fragment protected by the probe against digestion was induced on exposure of the whole organism to ethanol (via uptake from the aquatic environment). The assays also demonstrated that ethanol treatments both induced and suppressed this mRNA, depending on concentration and exposure time.

Diversity and characterization of novel Cytochrome P450 2 genes in the marine teleost Fundulus Heteroclitus

“…Finally, an oligonucleotide probe specific for rat CYP2E1 hybridized with a 3.3 kb mRNA in RNA samples from Poeciliopsis sp. This mRNA also was induced by ethanol (Kaplan et al, 1991), a prototypic CYP2E inducer.…”

“…Fish liver microsomes were shown to dealkylate diethylnitrosamine (Kaplan et al, 1991), an activity associated with CYP2E enzymes in mammals (Gonzalez, 1989). Antibodies to rat CYP2E1 recognized a single protein in the liver of Poeciliopsis sp.…”

“…Unlike mammals, the majority of fish are carnivorous and encounter fewer dietary plant natural products (Horn, 1989). In fact, in the temperate carnivorous fish most studied, total CYP levels are not induced by the prototypic CYP2 inducer, phenobarbital (Kleinow et al, 1987;Kleinow et al, 1990), and this lack of induction has been postulated to reflect the lack of terrestrial plants in their diets (Stegeman, 1989;Stegeman and Hahn, 1994;Kaplan et al, 1991). As well, the lauric acid hydroxylases, CYP2K1 and CYP2M1 have been isolated from trout, and again, immunoblot analyses have shown CYP2KI-like and CYP2M1-like proteins in other fish species (Buhler et al, 1994;Yang et al, 1995 Technologies.…”

Compendium of the FY1990 and FY1992 Research Reviews for the Research Methods Branch

“…In the topminnows, Poeciliopsis spp., tumorigenic sensitivity, as indicated by type of hepatic neoplasm and by incidence, is related to species and strain (Shultz and Schultz, 1982a,b/, 1988). This differential induction of tumorigenesis among genotypes was shown to be related in part to the ability of sensitive species and strains to take the first steps toward metabolically activating DEN to its carcinogenic metabolites (Kaplan et al, 1991). Hepatic neoplasia induced by DEN in medaka appears to progress in stages Lauren et al, 1990;) somewhat similar to those described for rodent hepatic neoplasia.…”

Involvement of Cytochrome P450 1A in the toxicity of aryl hydrocarbon receptor agonists : alteration arachidonic acid metabolism and production of reactive oxygen species

“…Procedures including protein purification, immunological reactivity and amino acid sequencing have been used to identify and classify different CYP genes in fish. This has lead to the description of fish CYP genes in subfamilies 1-4: 1(A), 2(B,E,K,M,N,P), 3(A), 4(T) (Stegeman, 1987;Kaplan et al, 1991;Stegeman, 1993;Buhler et al, 1994;Yang et al, 1996;Oleksiak et al, 1998a;Oleksiak et al, 1998b). CYP from the ll(A), 17,19, and 26 subfamilies also have been identified in fish Tanaka et al, 1992;Takahashi et al, 1993;White et al, 1996).…”