Xenobiotic monooxygenase activity and the response to inducers of cytochrome P-450 during embryonic and larval development in fish
Binder, Robert L.
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Data demonstrating the presence and inducibility of the xenobiotic monooxygenase system in fish embryos and larvae are described. The ontogeny of benzo(a)pyrene monooxygenase (BPM) activity, and NADPH- and NADH-cytochrome c reductase activities, were followed in microsomes prepared from whole embryos of the estuarine killifish Fundulus heteroclitus. BPM activity was detectable as early as 4 days from fertilization, prior to the appearance of the liver rudiment, which indicates a substantial role for the extrahepatic tissues in xenobiotic metabolism in Fundulus embryos. At all stages assayed before hatching, BPM activity was uniformly low, but within 24 hours of hatching there was a 10-fold increase in specific activity. This increase was shown not to be age-dependent but required hatching, and was not an artifact of the presence of endogenous inhibitors in embryos. Both NADPH- and NADH-cytochrome c reductase activities were measurable at all stages assayed. The developmental patterns of these two reductases were distinct from each other and did not closely correlate with that of BPM activity. However, the functional involvement of the NADPH-cytochrome c reductase in monooxygenase activity was indicated by the inhibition of BPM activity by cytochrome c. The metabolism of benzo(a)pyrene by fractions prepared from whole Fundulus embryos and eleutheroembryos appears to be catalyzed by a typical cytochrome P-450 dependent monooxygenase. This activity is localized in the microsomal fraction, requires O2, NADPH and native enzyme, and is inhibited by CO. NADPH supports much higher activity than NADH. BPM activity was detectable in the livers of Fundulus eleutheroembryos, larvae and juveniles. The level of activity in Fundulus eleutheroembryo livers was about 1/4 the average adult activity. Specific activity rose continuously from the end of the embryonic period into the juvenile period when adult levels were approached. BPM activity was also measurable in the livers of brook trout (Salvelinus fontinalis) embryos and eleutheroembryos. The ontogenic pattern contrasted with that seen in Fundulus. At 6 and 1 days before hatching BPM specific activity in embryonic liver was close to the adult level. After hatching there was a 3-fold increase in activity, thus the livers of eleutheroembryos were considerably more active in metabolizing BP than those of adult brook trout. BPM activity was inducible in Fundulus embryos by both Aroclor 1254 and No.2 Fuel oii. Embryos were competent to respond to induction as early as 4 days from fertilization. In Fundulus eleutheroembryos, Aroclor 1254 induced BPM activity in both the liver and extrahepatic tissues. Aminopyrine N-demethylase activity was detectable in microsomes prepared from whole eleutheroembryos, but was not induced by Aroclor 1254. Neither NADPH- nor NADH-cytochrome c reductase activities were induced by Aroclor 1254 before hatching, but after hatching both activities were induced. A striking developmental change in the sensitivty of the induction response was observed in Fundulus. The tissue levels of PCBs necessary to produce a maximal induction of BPM activity were at least 5 times lower in post-hatching stages compared to prehatching stages. The relative insensitivity of the induction response prior to hatching may serve to protect embryos from damage from activated metabolites during organogenesis. Aroclor 1254 was also shown to induce BPM activity in brook trout embryonic liver. The data obtained with both Fundulus and brook trout indicate that levels of PCBs occurring in fish in contaminated environments are likely to induce the monooxygenase system during embryonic development. Metabolites of benzo( a) pyrene produced by microsomes prepared from adult Fundulus liver, and untreated and PCB exposed eleutheroembryos were analyzed by HPLC. Similar metabolite profiles were obtained in all cases, with a high proportion of benzo-ring dihydrodiols. The dihydrodiol peaks produced by e leutheroembryo microsomes were abolished by TCPO, indicating the presence of epoxide hydrolase. These results suggest that Fundulus embryos and eleutheroembryos can activate BP to the highly mutagenic trans-7, 8-dihydrodiol-9, 10-epoxides. Fish embryonic monooxygenase activity may play a role in pollutant-induced lesions, including teratogenic effects, by producing reactive and mutagenic metabolites during organodifferentiation.
Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution August 1981
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