Cytochrome P450 1D1: a novel CYP1A-related gene that is not transcriptionally activated by PCB126 or TCDD.

Enzymes in the cytochrome P450 1 family oxidize many common environmental toxicants. We identified a new CYP1, termed CYP1D1, in zebrafish. Phylogenetically, CYP1D1 is paralogous to CYP1A and the two share 45% amino acid identity and similar gene structure. In adult zebrafish, CYP1D1 is most highly expressed in liver and is relatively highly expressed in brain. CYP1D1 transcript levels were higher at 9h post-fertilization than at later developmental times. Treatment of zebrafish with potent aryl hydrocarbon receptor (AHR) agonists (3,3',4,4',5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin) did not induce CYP1D1 transcript expression. Morpholino oligonucleotide knockdown of AHR2, which mediates induction of other CYP1s, did not affect CYP1D1 expression. Zebrafish CYP1D1 heterologously expressed in yeast exhibited ethoxyresorufin- and methoxyresorufin-O-dealkylase activities. Antibodies against a CYP1D1 peptide specifically detected a single electrophoretically-resolved protein band in zebrafish liver microsomes, distinct from CYP1A. CYP1D1 in zebrafish is a CYP1A-like gene that could have metabolic functions targeting endogenous compounds.

Catalytic and immunochemical properties of hepatic cytochrome P450 1A in three avian species treated with beta-naphthoflavone or isosafrole.

Induction of cytochrome P450 1A (CYP1A) can be used as a biomarker of exposure to planar halogenated aromatic hydrocarbons (PHAHs). Our objective was to characterize the induction of CYP1A activity and protein in three avian species following in vivo treatment with beta-naphthoflavone (BNF) and/or isosafrole. Alkoxyresorufin-O-dealkylase (alk-ROD) activities of hepatic microsomes from Herring Gulls (Larus argentatus) (HGs), Double-crested Cormorants (Phalacrocorax auritus) (DCCs) and chickens (Gallus domesticus) were measured using ethoxy-, methoxy-, pentoxy- and benzyloxy-resorufin, in the presence and absence of the inhibitors ellipticine or furafylline. Immunoreactivity of microsomal proteins with antibodies to several CYP1A proteins was investigated. CYP1A protein and alk-ROD activities of HGs and DCCs, but not chickens, were induced by isosafrole. Ellipticine was a potent and non-selective inhibitor of alk-ROD activity in all three species, while furafylline inhibition of alk-ROD activities varied among species and treatments. In all three species, BNF induced a protein immunoreactive with monoclonal antibody to CYP1A1 from the marine fish Stenotomus chrysops (scup), but a CYP1A2-like protein was not detected in avian microsomes probed with polyclonal antibodies to mouse CYP1A2. Variations in responses among avian species indicate that CYP1A proteins and substrate specificities should be characterized for each species used in PHAH biomonitoring programs.

Immunohistochemical localization of cytochrome P4501A induced by 3,3',4,4',5-pentachlorobiphenyl (PCB 126) in multiple organs of northern leopard frogs, Rana pipiens.

Monoclonal antibody 1-12-3 (MAb 1-12-3) recognizes an epitope exclusive to cytochrome P450s in subfamily 1A (CYP1A) from all vertebrates tested so far, including one amphibian species. In this study, we first tested the utility of MAb 1-12-3 for detection of presumed CYP1A proteins in hepatic microsomes of northern leopard frogs treated without or with 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Statistical analysis showed that ethoxyresorufin-O-deethylase (EROD) activities and CYP1A equivalents in treated groups were significantly increased at doses > or = 2.3 mg/kg compared with the control groups (p < 0.05), and the increases were maintained for at least four weeks. This result confirmed that MAb 1-12-3 can be used for detection of CYP1A in northern leopard frogs and indicated that CYP1A is the primary catalyst for EROD in this species. In a subsequent experiment, sections of organs of PCB 126-treated frogs were immunohistochemically stained with MAb 1-12-3 to identify localization of the CYP1A in different cell types. The CYP1A staining was seen prominently in hepatocytes and epithelium of nephronic duct, while capillaries close to gastric epithelium and submucosal vascular epithelium in both stomach and intestine exhibited moderate to strong staining. The CYP1A was immunodetected in coronary endothelium and the vascular endothelium of lung and gonad. In skin, mild staining was seen in epithelial cells of mucous glands and serous glands and in vascular endothelium, demonstrating induction of CYP1A in the dermal layer.

Induction of cytochrome P450 1A1 expression in captive river otters fed Prudhoe Bay crude oil: evaluation by immunohistochemistry and quantitative RT-PCR.

Numerous studies have explored the relationships between exposure to a variety of environmental contaminants, such as polycyclic aromatic hydrocarbons, and induction of cytochrome P450 1A (CYP1A) in different vertebrates. Few controlled studies, however, simulated chronic long-term exposure with repeated non-lethal sampling of the same individuals, which should better represent repeated exposure incidents in animals inhabiting polluted areas. In this study, we investigated the effects of chronic exposure to crude oil on levels of CYP1A1 in endothelial cells of skin biopsies and peripheral mononuclear blood cells in captive river otters (Lontracanadensis) using repeated sampling of the same individuals. We hypothesized that ingestion of oil would result in an increase in levels of CYP1A1 in both targets, and predicted that the relationship between prolonged exposure and expression of CYP1A1 would reach a plateau indicative of continuous detoxification of hydrocarbons. Fifteen wild-caught male otters were acclimated to captivity, and then fed diets containing no oil (control) or diets containing weathered crude oil at 5 mg day(-1) kg(-1) body weight (low-dose) and 50 mg day(-1) kg(-1) body weight (high-dose), at the Alaska Sealife Center in Seward, Alaska, USA. Expression of CYP1A1 was assessed with immunohistochemical analysis of CYP1A1 protein in skin biopsies and by quantitative RT-PCR analysis of CYP1A1 mRNA in mononuclear blood cells. Both assays revealed a decrease between capture and the transfer to captivity, indicating that the enclosure at the Alaska Sealife Center, and the food we offered to the otters were free of potential inducers of CYP1A1. During the exposure period, increases in CYP1A1 expression were registered by both techniques, followed by a decline in CYP1A1 after oil administration ended. Levels of endothelial CYP1A1 in the high-dose group were comparable to those recorded for wild river otters in PWS in 1996 and 1997. Levels of CPY1A1 mRNA in mononuclear blood cells, however, were well below levels recorded for river otters in Prince William Sound, and no correlation was detected between values obtained from the two methods. Thus, our results from this longitudinal study with repeated sampling of the same individuals provide support for the use of cytochrome P450 1A1 as a biomarker for hydrocarbon exposure. Nonetheless, our results also suggest that the induction process of CYP1A1 may be complicated and interacting with other processes in vivo. Such interactions may obscure our ability to describe specific, quantitative, predictable, dose-response relationships between exposure to hydrocarbons and induction of CYP1A1, which are required of reliable biomarkers. Evaluations of such interactions based on theoretical physiological models in live-animals merit further investigation.

Aryl hydrocarbon receptor function in early vertebrates: inducibility of cytochrome P450 1A in agnathan and elasmobranch fish.

The mammalian aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that controls the expression of cytochrome P450 1A (CYP1A) genes in response to halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The natural ligand and normal physiologic function of this protein are as yet unknown. One approach to understanding AHR function and significance is to determine the evolutionary history of this receptor and of processes such as CYP1A induction that are controlled by the AHR in mammals. In these studies, AHR function was evaluated in representative cartilaginous fish (little skate, Raja erinacea) and jawless fish (sea lamprey, Petromyzon marinus and Atlantic hagfish, Myxine glutinosa), using CYP1A induction as a model AHR-dependent response. Treatment of skate with beta-naphthoflavone (BNF) caused an 8-fold increase in hepatic ethoxyresorufin O-deethylase (EROD) activity as well as a 37-fold increase in the content of immunodetectable CYP1A protein. Evidence of CYP1A inducibility was also obtained for another cartilaginous fish, the smooth dogfish Mustelus canis. In contrast, hepatic EROD activity was not detected in untreated lamprey nor in lamprey treated with 3,3'4,4'-tetrachlorobiphenyl (TCB), a potent AHR agonist in teleosts. A possible CYP1A homolog was detected in lamprey hepatic microsomes by one of three antibodies to teleost CYP1A, but expression of this protein was not altered by TCB treatment. CYP1A protein and catalytic activity were measurable in hagfish, but neither was induced after treatment with TCB. These results suggest that the AHR-CYP1A signal transduction pathway is highly conserved in gnathostomes, but that there may be fundamental differences in AHR signaling or AHR-CYP1A coupling in agnathan fish. Agnathan fish such as hagfish and lamprey may be interesting model species for examining possible ancestral AHR functions not related to CYP1A regulation.

Cytochromes P450 in liver of the turtle Chrysemys picta picta and the induction and partial purification of CYP1A-like proteins.

Cytochromes P450 (CYP) in hepatic microsomes from the turtle Chrysemys picta picta and their response to inducers were examined. Freshly caught turtles had one protein (59 kDa) detected in western blot with monoclonal antibody 1-12-3 to scup CYP1A. That same band and a second band were detected with polyclonal anti-mouse Cyp1a1. Polyclonal anti-scup P450B (putative CYP2B) recognized three bands and anti-scup P450A (putative CYP3A), one band. TCB (3,3',4,4'-tetrachlorobiphenyl) at 5 mg kg-1 injected once induced EROD activity 3-fold. Repeated high-dose injections of TCB, 2,3,3',4,4'-pentachlorobiphenyl, Aroclor 1254 or beta-naphthoflavone induced CYP1A 20-fold and P450B-related proteins 2-3-fold. Rates of ethoxy- (EROD) methoxy- (MROD) and pentoxyresorufin O-dealkylases and benzo[a]pyrene (B[a]P) hydroxylase (AHH) were induced by these treatments, and were correlated with putative CYP1A content. Phenobarbital slightly elevated only MROD activity. Ethoxycoumarin (EC) O-deethylase rates were high, 1.6-2.2 nmol min-1 mg-1 in control and treated turtles, suggesting that EC is not a turtle CYP1A substrate. Highly induced EROD rates were 0.06 nmol min-1 mg-1, while AHH rates exceeded 4 nmol min-1 mg-1, suggesting that C. picta picta CYP1A may prefer PAH substrates. Induction of AHH was reflected in the formation of metabolites 3-OH-, 9-OH- and 7-OH-BP and BP-7,8-dihydrodiol (DHD). BP-4,5-DHD was not detected. Chromatographic procedures resolved the 59 kDa putative CYP1A from the second protein recognized by anti-Cyp1a1. The 59-kDa protein was also specifically and highly immunopurified by Mab 1-12-3. Thus, several CYP including two CYP1A-related proteins are expressed in turtle liver. Multiple CYP1A genes in reptiles may provide an insight into the origin of divergence in the CYP1A subfamily. Induction of a CYP1A may be a useful indicator of exposure to Ah receptor agonists in turtles.

Effects of triphenyltin and other organotins on hepatic monooxygenase system in fish.

The interaction of the organotin fungicide triphenyltin chloride (TPT) with fish microsomal monooxygenase systems has been studied in vitro and in vivo in the marine fish scup (Stenotomus chrysops). In vitro incubation of fish liver microsomes with TPT resulted in the conversion of about 40% of the native total spectral P450 to P420. In addition, a strong concentration-related inhibition of ethoxyresorufin O-deethylase (EROD) activity was observed, with a complete loss at 1.0 mM TPT. Pentoxyresorufin-O-dealkylase (PROD) activity was inhibited only at the highest concentration tested. This suggests either some specificity for the EROD catalyst CYP1A1, or a loss of reductant NADPH cytochrome c reductase as the cause. Further in vitro incubations showed that NADPH, but not NADH, cytochrome c reductase was strongly inhibited at 100 microM TPT and higher. To further investigate this effect, fish were injected with single doses of 5, 25 and 50 microM TPT (1.9, 9.6 and 19.3 mg kg-1 TPT), and 24 and 48 h later, hepatic microsomes were analyzed for total P450 content, EROD activity, NAD(P)H cytochrome c reductase, and the content of three CYP forms. EROD activity tended to be decreased in TPT-treated scup, with the response being stronger after 48 than 24 h. No significant conversion of spectrally determined P450 to cytochrome P420 was found, and cytochrome b5 was not affected. However, both NAD(P)H cytochrome c reductases were significantly inhibited at all concentrations. Immunoblot analysis showed reduction of CYP1A1 content at all doses, being significant at 25 mM after 48 h, but no decrease in CYP3A-like protein, the dominant catalyst of testosterone 6 beta-hydroxylation, nor CYP2B-like protein, the major contributor to indicates significant effects of TPT at high concentrations on fish hepatic CYP1A1 protein, EROD activity and the reductases. TPT seems to act more specifically on CYP1A1 than on other CYP forms. These findings combined with those of our previous studies (Brüschweiler BJ, Würgler FE, Fent K. Environ Toxicol Chem 1996;15:827-735; Fent K, Bucheli TD. Aquat Toxicol 1994;28:107-126; Fent K, Stegeman JJ. Aquat Toxicol 1991;20:159-168; Fent K, Stegeman JJ. Aquat Toxicol 1993;24:219-240) indicate a general degenerative effect of organotins on the fish microsomal monooxygenase system, although some differences are seen between the organotins, and between species. We conclude that these effects of organotins have consequences for use of CYP1A as a biomarker and endocrine disruption.

Cytochromes P450 (CYP) in tropical fishes: catalytic activities, expression of multiple CYP proteins and high levels of microsomal P450 in liver of fishes from Bermuda.

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b5 content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23-2.1 nmol/min/mg and 0.5-11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major (Abudefduf saxatilis) and Bermuda chub (Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with beta-naphthoflavone (BNF; 50 or 100 mg/kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6 beta- and 16 beta-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of proteins related to CYP2B. These species also had appreciable amounts of CYP3A-like proteins. Thus, many fishes in Bermuda appear to have induced levels of CYP1A; some also have unusually high levels of total P450 and of CYP2B-like and CYP3A-like proteins. These species may be good models for examining the structural, functional and regulatory properties of teleost CYP and the environmental or ecological factors contributing to high levels of expression of CYP in some fishes.

Immunochemical relationships of cytochrome P4503A-like proteins in teleost fish.

Multiple P450 proteins have been purified from several teleost species, including rainbow trout (Oncorhynchus mykiss), scup (Stenotomus chrysops) and Atlantic cod (Gadus morhua). Identity, relationships and/or functions have been established in these fish species for the cytochrome P4501 As. Information about the structure, function, regulation and relationships of other piscine cytochrome P450 (CYP) proteins is sparse. In the present study we have focused on constitutively expressed CYP forms, P450con and LMC5 isolated from rainbow trout, P450A from scup, and P450b from Atlantic cod, and we consider evidence for the relationship of these proteins to mammalian members of the CYP3A subfamily. Reciprocal western blot analysis shows that P450con and LMC5, isolated from rainbow trout in two different laboratories, are closely related and ostensibly identical proteins. These trout proteins show specific reciprocal cross-reactivity with scup P450A, and polyclonal antibodies (PAb) to the trout and scup proteins both recognize cod P450b, indicating that rainbow trout P450con/LMC5, scup P450A and cod P450b are immunochemically-related proteins. In analyses of liver microsomes of trout, scup and cod, PAb to trout P450con/LMC5 and scup P450A recognize only bands that are identical in migration to the CYP proteins purified from these species, and which were used as immunogens. These CYP proteins purified from fish are each immunochemically-related to mammalian CYP3A proteins, showing recognition by PAb to human CYP3A4 and to rat CYP3A1. PAb to the mammalian CYP3As also recognize the same bands in liver microsomes from these fish species as seen by PAb to the fish proteins. These results strongly suggest that these fish proteins are members of theCYP3 gene family and probably theCYP3A subfamily. Although sequence analysis is required before their designation in the CYP3A subfamily can be confirmed and specified, we refer to these as CYP3A-like. Immunoblot analyses of hepatic microsomes from other fish species with PAb to scup P450A and trout P450con show that multiple CYP3A-like proteins are expressed in liver of several species, including killifish (Fundulus heteroclitus) and winter flounder (Pleuronectes americanus). Important questions still remain to be addressed concerning CYP3A structure, multiplicity, physiological function, regulation and metabolism of endogenous as well as exogenous substrates in fish.

Induction of cytochrome P4501A1 by aryl hydrocarbon receptor agonists in porcine aorta endothelial cells in culture and cytochrome P4501A1 activity in intact cells.

Endothelium is a single-cell layer lining blood vessels and constituting capillaries and could be a primary site of chemical effects in the cardiovasculature and systemically. Cytochrome P4501A1 (CYP1A1) is strongly inducible in vertebrate endothelium in vivo by aryl hydrocarbon receptor (AhR) agonists [Mol. Pharmacol. 36:723-729 (1989); Mol. Pharmacol. 41:1039-1046 (1992)]. We investigated CYP1A expression and activity in porcine aorta endothelial cells (PAEC) exposed in culture to the AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4'-tetrachlorobiphenyl (TCB), benzo[a]pyrene (BP), or beta-naphthoflavone (BNF). Immunoblotting with monoclonal anti-CYP1A1 and polyclonal anti-CYP1A1 and anti-CYP1A2 antibodies showed that CYP1A1 was induced in cultures exposed to TCDD, TCB, BP, or BNF but was not detectable in untreated or dimethylsulfoxide-exposed cultures. CYP1A1 was strongly induced at intermediate concentrations (0.1 microM or 1.0 microM) of TCB, BP, or BNF, but induction was suppressed by higher concentrations, a response not due to general toxicity; cell viability (trypan blue exclusion) was > 97% with BNF or TCB at up to 10 microM. CYP1A1 induction by TCDD was maximal at 0.3-1.0 nM. ED50 values for induction of CYP1A1 by TCDD, TCB, and BP were 0.016 nM, 3-10 nM, and 180 nM, respectively. Immunohistochemical analysis confirmed CYP1A1 induction in PAEC but also showed that only some cells in the cultures were induced. Subcellular fractionation, marker enzyme analysis, and immunoblot analysis showed that PAEC had a typical complement of microsomal electron-transport components. NADPH-cytochrome P450 reductase showed comparable rates (approximately 40 nmol/min/mg) in induced and control cultures. Cultures maximally induced by 0.1 microM TCB had microsomal CYP1A1 [ethoxyresorufin-O-deethylase (EROD)] activity averaging 25 pmol/min/mg. Addition of purified rat reductase to PAEC microsomes increased the EROD rates 3-fold. EROD rates measured in intact cells maximally induced by BP, TCB, or TCDD ranged from 15 to 30 pmol/min/mg of whole-cell protein. Methoxyresorufin O-demethylase activity induced by TCDD was 2 pmol/min/mg, i.e., < 10% of the EROD activity. In cultures in which CYP1A1 was strongly induced, CYP1A2 was not detectably expressed. The CYP1A2 inducer acenaphthylene did not induce EROD or methoxyresorufin O-demethylase in intact cells. The results show that CYP1A1 but not CYP1A2 is strongly induced in mammalian endothelial cells in culture and that CYP1A1 is active in intact cells, although the catalytic rates are low.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochrome P450IA1 in hepatic lesions of a teleost fish (Fundulus heteroclitus) collected from a polycyclic aromatic hydrocarbon-contaminated site.

The expression of cytochrome P450IA1 was examined in hepatic lesions of mummichog (Fundulus heteroclitus), a small, non-migratory teleost fish collected from a site in the Elizabeth River, VA, heavily contaminated with polycyclic aromatic hydrocarbons (PAH) of creosote origin. Immunoblot ('Western' blot) analysis using monoclonal antibody (MAb 1-12-3) to P450IA1 of the marine fish Stenotomus chrysops indicated that cytochrome P450IA1 levels in hepatocellular carcinoma and in foci of cellular alteration were 28-85% lower than those of adjacent non-neoplastic tissue. P450IA1-dependent monooxygenase activity, measured as ethoxyresorufin O-deethylase (EROD), exhibited a similar trend with EROD activity in lesions being 15-77% lower than activity in non-neoplastic tissue. Immunohistochemical examination of liver sections revealed general low intensity P450IA1-associated staining in hepatocellular carcinoma, exocrine pancreatic tissue, bile ducts and cholangiocellular proliferative lesions. Staining intensity of non-neoplastic hepatic parenchyma varied considerably and was focally distributed. In one case intense staining was observed in an altered hepatocellular focus (putative preneoplastic lesion). The results indicate important similarities in the expression of P450IA1 in neoplasms of fish and mammals and suggest an adaptive response of a wild population to carcinogen exposure.

Sex differences in hepatic monooxygenases in winter flounder (Pseudopleuronectes americanus) and scup (Stenotomus chrysops) and regulation of P450 forms by estradiol.

Details concerning the endogenous regulation of hepatic cytochrome P450 monooxygenases in teleosts, and the features of this regulation common among fish species, are poorly known. Gonadally mature female winter flounder (Pseudopleuronectes americanus) have been reported to have severalfold lower levels of microsomal cytochromes P450 and b5 and NADPH-cytochrome c reductase than do males (Stegeman and Woodin ('84) Mar. Environ. Res., 14:422-425). These strong sex differences prompted more detailed study of P450 regulation in winter flounder liver, and a comparison with sex differences in another marine teleost, scup (Stenotomus chrysops). Ethoxyresorufin O-deethylase (EROD) activity/nmol P450 was less in gonadally mature females than in males of both species. Immunoblot analysis with MAb 1-12-3 to P450E (the EROD catalyst) showed that the content of P450E counterpart was also much less in females of both species. Aminopyrine N-demethylase (APND) and testosterone 6 beta-hydroxylase (6 beta-OHase) activities per nmol P450 were higher in gonadally mature female than in mature male flounder, differences not seen in scup. Polyclonal antibodies to scup P450A were shown to detect proteins in a number of teleosts. The levels of anti-P450A cross-reacting protein were greater in mature female than in male flounder, but as with 6 beta-OHase activity, the content of this protein was not sexually differentiated in scup. Estradiol treatment of winter flounder depressed the rates of EROD, APND, 6 beta-OHase, and estradiol 2-OHase activities per mg protein, but APND and 6 beta-OHase activities per nmol P450 were unchanged. Thus, E2 promotes general decreases in some hepatic P450-catalyzed activities, but in achieving sex differences there is also specific regulation of the P450E counterpart, and possibly of the 6 beta-OHase (P450A?). Other factors, temporal or hormonal, can modify the effect of E2 treatment, and may contribute to the specific regulation of P450 forms in naturally maturing fish, and to species differences in this regulation.

Feminization of the hepatic microsomal cytochrome P-450 system in brook trout by estradiol, testosterone, and pituitary factors.

The effects of estradiol, testosterone, and pituitary extract on hepatic microsomal enzymes were studied in sham-operated and gonadectomized immature brook trout. Estradiol reduced the specific content of cytochromes P-450 and b5 by 70% or more in both groups. Testosterone and pituitary extract also decreased the levels of total P-450 and b5, but to a lesser extent. These latter effects were not evident when the contents of P-450 and b5 were normalized per g liver. Immunoblot analysis with antibodies to P-450 forms from a teleost (scup) showed the presence of cross-reacting proteins in control fish, presumed counterparts to the scup forms. Levels of a trout counterpart to P-450A (a putative testosterone 6 beta-hydroxylase) were strongly suppressed in estradiol-treated fish. A trout P-450B counterpart was suppressed in estradiol-treated fish, but less strongly than was the P-450A counterpart. The trout orthologue of hydrocarbon-inducible P-450E (P-450IA1), the aryl hydrocarbon hydroxylase (AHH) catalyst, was undetectable in any group, consistent with very low levels of AHH activity in these fish. Estradiol or pituitary extract also decreased the levels of NADH-cytochrome b5 and NADPH-cytochrome P-450 reductase activities in sham-operated but not in total P-450 or b5, suggesting an influence of gonads in maintaining normal levels of the reductases. The results support a prominent suppressive role for estrogens in producing the general sex differences in microsomal enzymes in fish liver and indicate that there could be affects on several P-450 forms. However, the mechanism of hormone action in this regulation is uncertain. The results also suggest the existence of unidentified, possibly gonadal, factors which contribute to the regulation of reductases in teleost liver.

Induction of monooxygenase activity in the intestine of spot (Leiostomus xanthurus), a marine teleost, by dietary polycyclic aromatic hydrocarbons.

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)

Cytochrome P-450 isozymes from the marine teleost Stenotomus chrysops: their roles in steroid hydroxylation and the influence of cytochrome b5.

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.

Metabolite and enzyme contents of freeze-clamped liver of the marine fish Stenotomus chrysops.

Hepatic metabolites and enzymes in the marine fish, scup or porgy (Stenotomus chrysops), were determined in freeze-clamped tissue taken either within a day of removing fish from their natural habitat or after scup were held in captivity for 6-8 months. The same determinations were made for liver from fed or 48 hr-starved rats (Mus norvegicus albinus). Compared with rat liver, both groups of fish had, per gram of liver, higher contents of AMP, inorganic phosphate, glucose, glucose-6-phosphate, malate, glutamate and NH4+. ATP was lower in fish liver, and ADP, lactate and pyruvate contents were similar in rats and fish. Fish held in captivity had significantly lower pyruvate, alpha-ketoglutarate, and cytosolic free NAD+/NADH and higher cytosolic free NADPH/NADP+. These decreases were similar to those seen when starved rats were compared with fed ones. In scup liver, glucose-6-phosphate dehydrogenase was 3-8 times, malic enzyme about 2 times, and alanine aminotransferase 2-4 times higher than those activities in rat liver. Those results and a higher cytosolic free NADPH/NADP+ are consistent with the liver being the major site of lipogenesis in fish.

Patterns of benzo[a]pyrene metabolism by varied species, organs, and developmental stages of fish.

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.