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The effects of recombinant salmon growth hormone (sGH) on plasma sex steroid levels and gonadal function were investigated in hypophysectomized Fundulus heteroclitus. Effects of sGH were compared to those of purified chum salmon prolactin (sPRL), Atlantic salmon gonadotropin (sGTH), and salmon pituitary extract (sPE). Treatment with sGH significantly increased plasma concentrations of testosterone in males and estradiol-17 beta in females; sPRL had similar effects on testosterone levels in males. Further, treatment with these hormones prevented the decline in gonadal weight observed after hypophysectomy in both males and females. In vivo treatment of male fish with sGH also augmented testosterone and 11-ketotestosterone production by testis tissue subsequently incubated in vitro. Direct action(s) on gonadal steroidogenesis was examined by incubating gonadal tissues from hypophysectomized fish in vitro with various hormones. sGH significantly stimulated the in vitro production of testosterone and 11-ketotestosterone by testis, and estradiol-17 beta by ovary. sPE and sGTH also stimulated gonadal steroidogenesis, whereas sPRL and bovine GH had no significant effect. By comparison, rainbow trout gonads also produced increased amounts of steroids when treated with sGH in vitro. The use of a cloned GH rules out contamination by other pituitary hormones. These results, therefore, demonstrate that recombinant salmon growth hormone possesses steroidogenic and gonadotropic activity. Purified sPRL also has steroidogenic and gonadotropic actions. However, the significance of these effects of teleost GH and PRL is not known.

1. Hepatic microsomal enzymes from teleost and elasmobranch fishes catalyse a diversity of monooxygenase reactions, consistent with the presence of multiple, distinct P450 forms. Protein purification and immunological studies have confirmed that multiple microsomal P450s occur in teleosts. 2. A member of the aromatic hydrocarbon-inducible P450 IA family is present in all fish species examined to date. This protein appears to be most closely related to P450 IA1. Certain of the immunological probes for a teleost P450 IA1 (scup P450E) appear to be reagent antibodies, recognizing the homologous protein in members of all vertebrate groups examined. The nature of the epitope recognized by such antibodies is not known. 3. Based on immunological and amino acid sequence comparisons, teleost P450 IA1 appears to be orthologous to both P450 IA1 and P450 IA2 in mammals. Multiple P450 IA genes may appear in teleosts, but divergence on separate lines from that involving mammalian P450 IA2 could include additional, new members (P450 IA3?) of the P450 IA family. 4. There are greater similarities in the N-terminal amino acid sequences of different teleost (scup and trout) P450 IA1 forms, than seen in the N-terminal sequence relationships found in P450 IA1 of mammalian species. Whether this similarity extends to the rest of these teleost proteins is unknown. 5. The induction of P450 IA1 in teleosts involves transcriptional and translational events. However, the temporal patterns involved in induction of mRNA or protein are different from those in mammalian species, indicating additional aspects of the regulation in teleosts. 6. Relationships between other teleost and mammalian P450 forms, or between other P450 forms isolated from different teleosts, remain to be conclusively established. However, certain relationships are suggested, based on catalytic and other comparisons.

Monoclonal antibody (MAb) 1-12-3 generated against liver cytochrome P-450E (P-450E), an aryl hydrocarbon hydroxylase of the marine fish Stenotomus chrysops (scup), reacted only with P-450E when tested in immunoblot analysis with five P-450 fractions from scup liver. This and six other MAbs against P-450E recognized purified P-450E, as well as a single band in beta-naphthoflavone (BNF)-induced scup microsomes that comigrated with authentic P-450E. Like MAb 1-12-3, polyclonal anti-P-450E reacted with P-450E but not with other scup P-450 fractions and reacted strongly with a band coincident to P-450E in BNF-treated scup microsomes. However, the polyclonal antibody (PAb) also faintly recognized additional microsomal proteins. MAb 1-12-3 recognized P-450E induced by 3,3',4,4',5,5'-hexachlorobiphenyl and by polychlorinated biphenyl mixtures in scup, and a single band induced by BNF or 3-methylcholanthrene (MC) in microsomes of other teleosts, including two trout species, killifish and winter flounder. The content of the P-450E counterpart in these fish and also in untreated scup coincided with induced ethoxyresorufin O-deethylase (EROD) activity. Induced EROD activity in scup and trout was strongly inhibited by MAb 1-12-3, further demonstrating the relationship between P-450E and induced P-450E in trout. MAb 1-12-3, two other MAbs, and anti-P-450E PAb recognized a band comigrating with P-450c in BNF-induced rat microsomes. MAb 1-12-3 also recognized purified rat P-450c. MAb 1-12-3 and anti-P-450E PAb recognized a second band of lower molecular weight than P-450c in BNF rat microsomes which may correspond to P-450d, the MC- and isosafrole-inducible rat isozyme. The results firmly establish the identity of scup P-450E, the relationship of BNF-induced P-450 in other teleosts with P-450E, and the immunochemical relationship of P-450E with rat P-450c. Furthermore, results with untreated fish suggest that effects of environmental chemicals may be detected by immunoblotting with monoclonal anti-P-450E.

The response of intestinal monooxygenases to dietary polycyclic aromatic hydrocarbon (PAH) exposure was evaluated in spot (Leiostomus xanthurus), a marine teleost fish. Ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) activities were highest in the pyloric caeca and in the proximal half of the intestine. Intestinal microsomes from fish given control diets had very low levels of EROD and AHH activities relative to those in liver. After exposure to a diet containing 10 mg of 3-methylcholanthrene/kg of food, the levels of intestinal EROD and AHH activities increased 36-fold and 17-fold, respectively, such that intestinal monooxygenase activity exceeded that of the liver, which was not induced by this treatment. A significant increase in intestinal monooxygenase activity occurred in fish receiving dietary benzo[a]pyrene (BP) at concentrations as low as 10 micrograms of BP/kg food. A 5-fold increase in intestinal AHH and EROD activities was observed within 3 hr after administration of dietary BP. A plateau in gut monooxygenase activity occurred after approximately 3 days of PAH exposure; these activities decreased to control levels within 3 days after replacing the PAH diet with the control diet. Starvation resulted in disappearance of detectable monooxygenase activity. Monoclonal antibody (MAB 1-12-3) against the major PAH-inducible cytochrome P-450 (P-450E) in the liver of the marine teleost (Stenotomus chrysops) [Park et al. Arch. Biochem. Biophys. 249, 399 (1986)] recognized a single protein band in intestinal microsomes, with Mr near 54,000, which we conclude is the spot counterpart to cytochrome P-450E.(ABSTRACT TRUNCATED AT 250 WORDS)

The developmental patterns of aryl hydrocarbon hydroxylase (AHH) activity and NADPH-cytochrome c reductase activity were followed during embryonic development in Fundulus. AHH activity was localized in microsomal fractions prepared from whole Fundulus embryos and eleutheroembryos. On the basis of this subcellular localization, the requirements of O2 and NADPH for activity, and sensitivity to carbon monoxide and cytochrome c inhibition, the AHH activity in Fundulus embryos and eleutheroembryos appeared to be cytochrome P-450 dependent. AHH activity was measurable in stages prior to the appearance of the liver rudiment, and during subsequent embryonic development the extrahepatic tissues were likely to have contributed substantially to the AHH activity measured. At all stages assayed before hatching, microsomal AHH specific activity remained uniformly low, but within 24 hr of hatching, AHH specific activity increased about ninefold. This posthatching increase in AHH activity was not age dependent, nor developmental stage dependent, but rather required hatching, and was not due to the presence of endogenous inhibitors in prehatching stages. The levels of NADPH-cytochrome c reductase activity and AHH activity were not closely correlated in whole embryo and eleutheroembryo microsomes, but the AHH activity in these preparations apparently was not limited by the levels of the NADPH-cytochrome c (P-450) reductase. The presence of AHH activity in Fundulus embryos during the period of active organogenesis, prior to hatching, indicates that this species is likely to be susceptible to a variety of teratogens requiring metabolic activation, and this may be the case for other species of fish as well.

Estradiol (E2) metabolites formed in vitro by microsomes from the marine teleosts winter flounder (Pseudopleuronectes americanus) and scup (Stenotomus chrysops) included at least seven products detected by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The least polar metabolite was shown to be estrone by chromatographic and mass spectrometric identity with authentic estrone. Chromatographic analyses coupled with dual-label experiments also indicated formation of the catecholestrogen 2-hydroxyestradiol (2-OH-E2), which was the most prominent metabolite determined by TLC. Analysis of microsomal E2 2-hydroxylase activity by measuring the specific release of 3H2O from [2-3H]E2 indicated that it is mediated by cytochrome P-450. E2 2-hydroxylase activity normalized to microsomal protein was lower in females than in males for microsomes from both mature scup and winter flounder. Activity normalized to liver weight or body weight in female winter flounder was also lower than that in males. However, activity normalized to cytochrome P-450 content did not show sex differences in either species. E2 2-hydroxylase activity per nanomole cytochrome P-450 was reduced in scup treated with beta-naphthoflavone, which induces the hydrocarbon hydroxylase cytochrome P-450E. Studies employing reconstituted P-450E and microsomes preincubated with polyclonal antibodies against P-450E confirmed that this isozyme does not catalyze E2 2-hydroxylase activity in microsomes. However, preliminary work with scup cytochrome P-450A suggests that it may be an E2 2-hydroxylase. The studies establish that catecholestrogen formation is prominent in fish liver and that it is sexually differentiated, but further investigation is required to define the catalysts as well as the significance and regulation of this function.

The effectiveness of pyrazoles acting as inhibitors of alcohol dehydrogenase in vitro or of ethanol metabolism by intact, isolated hepatocytes is influenced both by the hydrophobicity of the pyrazole and by the electronic properties of the substituents at the 4-position of the pyrazole ring. In contrast, the binding of pyrazoles to cytochrome P450 in vitro and the induction of P450(s) in cultured hepatocytes are dependent only on hydrophobicity. The high correlation between the binding of pyrazoles in vitro and their ability to induce P450(s) in cultured liver cells suggests as a working hypothesis that the pyrazole:P450 complex has a role in the induction process.

Polychlorinated biphenyl congeners that are abundant in environmental samples, and known to induce hepatic monooxygenase isozymes in the P450IA gene subfamily in mammals, were examined for their ability to induce hepatic monooxygenase activity in scup, a marine teleost. Scup were dosed ip with 3,3',4,4'-tetrachlorobiphenyl (congener 77), 2,3,3',4,4'-pentachlorobiphenyl (congener 105), 2,3',4,4',5-pentachlorobiphenyl (congener 118), 2,2',3,4,4',5'-hexachlorobiphenyl (congener 138), 2,2',3,3',4,4'-hexachlorobiphenyl (congener 128), or beta-naphthoflavone and examined for increases in ethoxyresorufin O-deethylase (EROD) activity, immunodetectable cytochrome P450E (the EROD catalyst in scup), and in vitro translatable mRNA for P450E. Monooxygenase parameters were significantly induced only by 3,3',4,4'-tetrachlorobiphenyl (TCB). However, while translatable mRNA for P450E was induced at all doses (1, 5, and 10 mg/kg), EROD activity and P450E were decreased at the 5 and 10 mg/kg doses, relative to the response at 1 mg/kg. A strong relationship between residual TCB concentration in the liver and the decreased EROD activity was evident at the higher doses of TCB. Aminopyrine N-demethylase, a monooxygenase activity not catalyzed by P450E, was unaffected by TCB treatment, indicating a specificity in the TCB effect. Analysis in vitro revealed that TCB was a potent competitive inhibitor of EROD activity, with half-maximal inhibition at 0.3 microM, near the Km for ethoxyresorufin, suggesting one mechanism for the in vivo effect of TCB. These results demonstrate that PCB congeners with ortho-chlorine substitution, and which are effective inducers of AHH and EROD activity in mammals, are ineffective, at the doses tested, as inducers in the teleost scup.

A technique is proposed for the biological monitoring of pollutants in aquatic environments by use of a complementary DNA (cDNA) probe. The induction of hepatic cytochrome P(1)450 mRNA has been investigated utilizing pfP(1)450-3', a 3'-specific 1.5 kb cDNA clone derived from 3-methylcholanthrene-inducible mRNA of rainbow trout. A time course of induction of both the hybridizable mRNA and hepatic monooxygenase catalytic activity in rainbow trout with a known inducer in fish, beta-naphthoflavone, was studied. The cDNA probe was also shown to hybridize with induced mRNA of brook trout, scup, garter snake, painted turtle, and rat demonstrating the suitability of the probe for examining induction of mRNA in various species. The results of these experiments suggest that the cDNA probe may be useful as a biological monitoring tool for determining the presence and effects of chemical pollutants which are inducers of hepatic microsomal monooxygenase activity. The probe may have the potential to be applied as an early warning system in the monitoring of water quality.

Inhibition of liver microsomal ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities by alpha-naphthoflavone and by polyclonal antibodies to hydrocarbon-inducible cytochrome P-450E from teleost liver indicated a xenobiotic-induced origin of these activities in the deep-sea fish Coryphaenoides armatus. Specific recognition of a protein by antibodies to P-450E in an immunoblot assay further indicated xenobiotic-induced cytochrome P-450 in these animals. Levels of apparently induced cytochrome P-450 and monooxygenase activity correlated positively with the tissue content of chlorobiphenyls of known inducing activity, implicating such compounds in biochemical effects occurring in the deep ocean.

Previous studies have shown that high levels of cytochrome P450 can occur in cardiac microsomes of vertebrates [Mol. Pharmacol. 21:517-526, (1982)]. Here we identify the dominant cardiac P450 in the marine fish scup as P450E, a teleost representative of P450IA1, and we describe its restricted cellular localization in the heart. Treatment of scup with beta-naphthoflavone produced an unusually strong (10-fold) induction of spectrally measured P450 in cardiac microsomes, with specific content reaching levels (0.5 nmol/mg) similar to those induced in scup liver. Microsomal ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities, catalytic functions of scup P450E, were induced in parallel with P450 content. Similar induction was seen in both atrium and ventricle. Immunoblot analysis with monoclonal antibody 1-12-3, specific to scup P450E and other vertebrate P450IA1 proteins, showed that this hydrocarbon-inducible P450 is the dominant and possibly sole P450 form in heart microsomes of experimentally induced animals. Immunohistochemical analysis of scup heart sections (2-4-microns) with monoclonal antibody 1-12-3 revealed that P450E was detectable only in endothelial cells of the endocardium and of the coronary vasculature. A similar endothelial cell localization of the monoclonal antibody 1-12-3 epitope was observed in heart of rainbow trout, induced with beta-naphthoflavone, indicating a general nature for the endothelial localization of induced cardiac P450. Morphometric analysis showed that endothelium could constitute 8-9% of the volume of teleost heart, from which we calculate that P450IA1 could account for as much as 25% of the endothelial cell microsomal protein. Heart microsomes of untreated animals from contaminated environments also contained high levels of P450E, indicating that induction like that caused by beta-naphthoflavone could occur with chemicals in the environment. Strongly induced P450E (P450IA1) in endothelium could play a critical role in chemical-biological interactions involving xenobiotics affecting the vasculature of the heart or other organs.

Microsomal aryl hydrocarbon hydroxylase (AHH) activity was highly inducible by polychlorinated biphenyls (PCBs) in Fundulus embryos, and stages prior to the appearance of the liver rudiment were competent to respond to these inducers. Consistent with previous observations, basal AHH activity in whole eleutheroembryo microsomes was shown to increase about 9-fold within 24 h of hatching. Aminopyrine N-demethylase (APD) activity also increased with time after hatching. However, the increase in APD activity was much less than that of AHH activity, suggesting a post-hatching change in basal cytochrome P-450 isozyme composition. Also associated with hatching was an increase in the sensitivity to PCBs as inducers of AHH activity. The ED50 for induction of AHH activity in eleutheroembryos was estimated to be only one-third to one-fourth that in embryos. This developmental increase in the sensitivity to PCBs was not due to a redistribution of PCBs between the yolk and tissues with yolk absorption, and was not simply age-dependent, but appeared to require hatching. An additional change in the monooxygenase system associated with hatching was that microsomal NADPH-cytochrome c reductase activity was not inducible by PCBs prior to hatching, but was modestly inducible after hatching. High performance liquid chromatographic (HPLC) analysis of benzo[a]-pyrene (BP) metabolites formed by microsomes from control and PCB-treated eleutheroembryos demonstrated production of dihydrodiols in the 7,8- and 9,10-positions of the benzo-ring. The formation of these metabolites was completely inhibited by the epoxide hydrolase (EH) inhibitor, trichloropropene oxide, indicating the presence of EH in Fundulus eleutheroembryos. Furthermore, these results indicate the Fundulus eleutheroembryos probably can activate BP to its ultimate carcinogenic form, the 7,8-dihydrodiol-9,10-epoxide, and induction of AHH activity by PCBs is likely to increase the rate of formation of activated metabolites from BP and related compounds. However, during the most active period of organogenesis, prior to hatching, basal AHH activity was low, and prehatching stages were relatively insensitive to cytochrome P-450 inducers. The combination of these effects may help to protect these stages from damage from activated metabolites.

Microsomal preparations of liver from adults of several (teleost) fish species, including scup (Stenotomus chrysops), winter flounder (Pseudopleuronectes americanus), and killifish (Fundulus heteroclitus), metabolized the model polycyclic hydrocarbon carcinogen benzo[a]pyrene (BP) with a marked regiospecificity, forming high percentages of benzo-ring derivatives, but particularly BP-7,8-dihydrodiol and BP-9,10-dihydrodiol. The identity of these products was established by mass spectrometry of metabolites formed by scup (S. chrysops) liver microsomes compared with the spectra obtained for authentic trans-dihydrodiols. Whereas the benzo-ring dihydrodiols equaled 40-60% of the total ethyl acetate-soluble metabolites formed by liver microsomes of the various species, the K-region metabolite BP-4,5-dihydrodiol was formed in small or undetectable amounts. Microsomal preparations of kidney and gill from S. chrysops formed BP-7,8-dihydrodiol and BP-9,10-dihydrodiol in proportions like those seen with liver of the same species. Embryonic tissues of F. heteroclitus metabolized BP with a regiospecificity similar to that exhibited by adult liver microsomes. Isomeric forms of BP-7,8-dihydrodiol are potent proximate carcinogens, and activation of BP to mutagenic and carcinogenic derivatives is associated with metabolism on the benzo ring of BP. The widespread appearance of regiospecificity for this portion of the BP molecule may be reflected in structural requirements for metabolism of other hydrocarbons and indicate that fish are useful in the study of various factors that affect tumorigenesis by polycyclic hydrocarbons.

Poeciliopsis lucida and Poeciliopsis monacha are freshwater viviparous fishes susceptible to tumorigenesis by brief exposure to waterborne procarcinogens. The hepatic monooxygenase system and the uptake, toxicity, and distribution of benzo[a]pyrene (BP) were characterized as a first step in exploring relationships between xenobiotic metabolism and cancer in these fishes. Waterborne BP was lethal at a dose of 3.75 mg/liter with a 24-hr exposure. During a 24-hr exposure to 1.0 mg/liter (3.97 mumol/liter) [3H]BP, an average of 8.27 nmol of BP was taken up per fish. Of this total, 64-70% was in the gallbladder or gut, indicating rapid metabolism and excretion. Basal levels of aryl hydrocarbon hydroxylase (AHH) activity were fairly high, about 0.6 nmol/min/mg. Maximal induction by BP occurred at a dose of 1.0 mg/liter, but with AHH activities only about twice the levels in untreated fish. Sensitivity to inhibition by alpha-naphthoflavone (ANF) increased slightly in treated fish. Induced AHH and also 7-ethoxyresorufin O-deethylase (EROD) activities declined slowly after a single treatment, approaching pre-exposure levels after 7 days. Monoclonal and polyclonal antibodies to cytochrome P-450E, the AHH and EROD catalyst in the marine fish scup (Stenotomus chrysops), inhibited AHH and EROD activities in untreated Poeciliopsis, indicating structural similarity between the catalysts in these fish species. Untreated Poeciliopsis evidently contain a counterpart to scup cytochrome P-450E, the major polycyclic aromatic hydrocarbon-inducible from in scup. High levels of basal activity and the pattern of monooxygenase induction could be involved in the sensitivity of these fishes to polycyclic aromatic hydrocarbon and other procarcinogens.

A procedure to directly measure the cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase activity with a visible spectrophotometer is described and compared to the standard fluorometric method. The two assays yielded identical results with both beta-naphthoflavone-treated mammalian (rat) and fish (scup, Stenotomus chrysops) liver microsomes. The assay takes advantage of a clean distinction in visible absorption spectra obtained for highly purified 7-ethoxyresorufin (substrate) and resorufin (enzymatic product). The purification and characterization of resorufin, the enzymatic product, are detailed, and its extinction coefficient (epsilon 572 = 73 mM-1 cm-1) provides for an accurate quantitation of enzyme activity. The large visible extinction coefficient of the product chromophore provides a high sensitivity for low-activity samples. The application of this enzyme assay in a visible spectrophotometer, along with the considerable evidence that a single aromatic hydrocarbon-inducible cytochrome P-450 isozyme is responsible for the catalysis, enhances the utility of this substrate in microsomal monooxygenase assays. The utility of the visible assay is further demonstrated by the simple determinations of the coupling ratio for 7-ethoxyresorufin oxidation in scup liver microsomes and the K1 for 7,8-benzoflavone and phenylimidazole inhibition of the enzymatic reaction.

The roles of different forms of cytochrome P-450 in activation and deactivation of toxic chemicals, synthesis and breakdown of steroid hormones, and other functions, indicate the significance of these enzymes. Monooxygenase systems have been studied in species from several phyla of aquatic organisms. However, cytochrome P-450, the dominant catalyst in xenobiotic monooxygenase activity, is best studied in fish. Forms of cytochrome P-450 have been purified from several teleost species, including scup (Stenotomus chrysops), rainbow trout (Salmo gairdneri), and cod (Gadus morhua). Cytochrome P-450E from scup, cytochrome P-450 LM4b from trout, and cytochrome P-450c from cod have properties similar to each other and appear to be homologous hydrocarbon or BNF-inducible isozymes. Partially purified cytochrome DBA-P-450-I from little skate, Raja erinacea, is possibly an elasmobranch counterpart of these teleost forms. Cytochrome P-450E from scup is immunochemically related to the major BNF-inducible isozyme (cytochrome P-450c or BNF-B) in rats, indicating homology between the fish and mammalian BNF-inducible isozymes. Several other cytochrome P-450 forms with interesting or unusual properties have been purified from aquatic species. Mammalian homologs are not yet known for these isozymes. Further studies of cytochrome P-450 forms in aquatic species should establish additional homologies and the regulation of these forms by chemical and biological variables, possibly providing fundamental insights into the function and evolution of these proteins.

Levels of hepatic microsomal cytochrome P-450 were depressed by administration of estradiol-17 beta and were elevated by administration of testosterone in both male and female juvenile brook trout (Salvelinus fontinalis). Treatment-associated changes in the levels of other microsomal electron transfer components in liver did not reflect the changes in cytochrome P-450 content and were also distinct from the changes in these components in kidney. Electrophoretic analysis of hepatic microsomes revealed that estradiol treatment reduced the amounts of several proteins including some heme-staining protein at 56,000 daltons, possibly containing cytochrome P-450. Hepatic microsomal benzo[a]pyrene hydroxylase and the response to 7,8-benzoflavone in vitro were affected little by steroid treatment, and ethoxyresorufin O-deethylase activity could not be detected in any of the samples. Hepatic microsomes metabolized testosterone to a suite of products including 6 beta-hydroxytestosterone (the major metabolite) and 16 beta-hydroxytestosterone, plus as many as eleven unknown metabolites. Estradiol-17 beta treatment depressed the rates of testosterone metabolism and particularly the rates of 6 beta-hydroxylase activity but did not affect 16 beta-hydroxylase activity. Both activities were largely unaffected by testosterone. The results are consistent with the idea that both androgens and estrogens regulate the levels of hepatic cytochrome P-450 in brook trout and that the effect, at least of estradiol-17 beta, involves regulation of forms that function in specific hydroxylation of testosterone. The significance of these effects and whether factors additional to steroids are involved in this regulation of hepatic cytochromes P-450 in fish remain to be established.

Hybridomas were prepared from myeloma cells and spleen cells of BALB/c female mice immunized with hepatic cytochrome P-450E purified from the marine fish, Stenotomus chrysops (scup). Nine independent hybrid clones produced MAbs, either IgG1, IgG2b, or IgM, that bound to purified cytochrome P-450E in radioimmunoassay. Antibodies from one clone MAb (1-12-3), also strongly recognized rat cytochrome P-450MC-B (P-450BNF-B; P-450c). The nine antibodies inhibited reconstituted aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin O-deethylase of scup cytochrome P-450E to varying degrees, and inhibited AHH activity of beta-naphthoflavone-induced scup liver microsomes in a pattern similar to that in reconstitutions, indicating that cytochrome P-450E is identical to the AHH catalyst induced in this fish by beta-naphthoflavone. MAb 1-12-3 also inhibited the reconstituted AHH activity of the major BNF-induced rat isozyme. Conversely, MAb 1-7-1 to rat cytochrome P-450MC-B had little effect on AHH activity of scup cytochrome P-450E, and did not recognize cytochrome P-450E in radioimmunoassay nor in an immunoblot. Scup cytochrome P-450E and rat cytochrome P-450MC-B thus have at least one common epitope recognized by MAb 1-12-3, but the epitope recognized by Mab 1-7-1 is absent or recognized with low affinity in cytochrome P-450E. The various assays indicate that the nine MAbs against cytochrome P-450E are directed to different epitopes of the molecule. These MAbs should be useful in determining phylogenetic relationships of the BNF- or MC-inducible isozymes and their regulation by other environmental factors.

Two new cytochrome P-450 forms were purified from liver microsomes of the marine fish Stenotomus chrysops (scup). Cytochrome P-450A (Mr = 52.5K) had a CO-ligated, reduced difference spectrum lambda max at 447.5 nm, and reconstituted modest benzo[a]pyrene hydroxylase activity (0.16 nmol/min/nmol P-450) and ethoxycoumarin O-deethylase activity (0.42 nmol/min/nmol P-450). Cytochrome P-450A reconstituted under optimal conditions catalyzed hydroxylation of testosterone almost exclusively at the 6 beta position (0.8 nmol/min/nmol P-450) and also catalyzed 2-hydroxylation of estradiol. Cytochrome P-450A is active toward steroid substrates and we propose that it is a major contributor to microsomal testosterone 6 beta-hydroxylase activity. Cytochrome P-450A had a requirement for conspecific (scup) NADPH-cytochrome P-450 reductase and all reconstituted activities examined were stimulated by the addition of purified scup cytochrome b5. Cytochrome P-450B (Mr = 45.9K) had a CO-ligated, reduced difference spectrum lambda max at 449.5 nm and displayed low rates of reconstituted catalytic activities. However, cytochrome P-450B oxidized testosterone at several different sites including the 15 alpha position (0.07 nmol/min/nmol P-450). Both cytochromes P-450A and P-450B were distinct from the major benzo[a]pyrene hydroxylating form, cytochrome P-450E, by the criteria of spectroscopic properties, substrate profiles, minimum molecular weights on NaDodSO4-polyacrylamide gels, peptide mapping and lack of cross-reaction with antibody raised against cytochrome P-450E. Cytochrome P-450E shares epitopes with rat cytochrome P-450c indicating it is the equivalent enzyme, but possible homology between scup cytochromes P-450A or P-450B and known P-450 isozymes in other vertebrate groups is uncertain, although functional analogs exist.