Xenobiotic monooxygenase activity and the response to inducers of cytochrome P-450 during embryonic and larval development in fish
Citable URI
https://hdl.handle.net/1912/2414DOI
10.1575/1912/2414Keyword
Xenobiotics; Cytochrome c; Oxygenases; Fishes; Development; Larvae; Embryology; MetabolismAbstract
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.
Description
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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Suggested Citation
Thesis: Binder, Robert L., "Xenobiotic monooxygenase activity and the response to inducers of cytochrome P-450 during embryonic and larval development in fish", 1981-08, DOI:10.1575/1912/2414, https://hdl.handle.net/1912/2414Related items
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