The effect of stress on toxicant-dependent cytochrome p450 enzyme responses in the Arctic charr (Salvelinus alpinus).

In the present study we investigated the effect of stress and cortisol on cytochrome P450 (CYP) expression in Arctic charr exposed to benzo[a]pyrene (BaP). Expression of hepatic CYP1A and CYP3A was monitored 8 d after a single oral dose of BaP (10 mg/kg fish) and compared to that in unexposed fish. During this period the fish were subjected to one of the following stress regimes: no stress, no stress and cortisol implantation, 10 min of daily handling and confinement stress, and confinement stress during the last 6 h before sampling. In BaP-exposed fish daily stress resulted in significantly lower (53%) CYP1A protein levels as compared to those in unstressed fish. For CYP1A catalytic activity (measured as 7-ethoxyresorufin-O-deethylase [EROD] activity), the suppressive response to stress was less pronounced. These results contrast to previous findings of a potentiation by corticosteroids on xenobiotic-dependent CYP1A induction in vitro in cultured fish hepatic cells. No effects of high cortisol levels or BaP were found on the steroid-metabolizing CYP3A enzyme levels. The lack of any alterations in the CYP3A protein level indicates that CYP3A expression is not inducible by cortisol in the Arctic charr under the conditions used here. The conclusion was made that short-term stress associated with sampling (i.e., 6 h of confinement stress before sampling) of wild charr does not compromise the EROD activity as a reliable biomarker.

Cytochrome P450 enzymes in the kidney of the bobwhite quail (Colinus virginianus): induction and inhibition by ergosterol biosynthesis inhibiting fungicides.

Metabolism of testosterone and the alkoxyresorufins was examined in kidney microsomes from male Bobwhite quail (Colinus virginianus) and was compared with that in kidney microsomes prepared from the male rat. In addition, cross-reactivity studies were conducted with a number of antibodies prepared against cytochrome P450 (CYP) enzymes purified from rat and trout liver. The effects of treatment with the fungicides: propiconazole, vinclozolin, clotrimazole and ketoconazole were examined. While kidney microsomes from both quail and rat catalyzed testosterone metabolism at multiple positions, the pattern of hydroxylated metabolites differed. Treatment with vinclozolin resulted in significant induction of testosterone 2 beta- and 15 beta-hydroxylase activity in quail kidney accompanied by increases in expression of P450 enzymes cross-reactive with antibodies raised against a CYP 3A-like protein in teleost fish. In contrast, ketoconazole treatment resulted in inhibition of testosterone hydroxylation at positions 15 beta- and 6 alpha-. Propiconazole and vinclozolin significantly induced a CYP 1A1 cross-reactive P450 enzyme in quail kidney 2-3-fold unaccompanied by significant increases in alkoxyresorufin O-dealkylase activity. These activities were significantly inhibited by ketoconazole treatment. Quail kidney microsomes also expressed high levels of a CYP 4A1 cross-reactive apoprotein which was inducible 3-4-fold by ketoconazole. Thus, quail kidney possesses cytochrome P450 enzymes related to forms found in mammalian gene families 1, 3 and 4. Fungicide treatment results in mixed patterns of induction and inhibition of kidney P450 enzymes different from those previously reported in quail liver.

Isolation of a cytochrome P450 3A cDNA sequence (CYP3A30) from the marine teleost Fundulus heteroclitus and phylogenetic analyses of CYP3A genes.

A reverse transcriptase polymerase chain reaction (RT-PCR) protocol, using degenerate PCR-primers specific to highly conserved regions of mammalian CYP3A genes, was employed to amplify a 400 base pair cDNA fragment from Fundulus heteroclitus liver RNA. The 124 amino acid sequence deduced from this cDNA sequence was aligned with corresponding sequences from representative members from the CYP1, 2, 3, and 4 gene families retrieved from the GenBank database. Phylogenetic trees were constructed using distance-matrix and maximum parsimony methods. The F. heteroclitus sequence and all mammalian CYP3A sequences cluster together when compared to sequences of members of CYP gene families 1, 2, and 4. This fish sequence was 57 to 70% identical to the corresponding region of mammalian CYP3A genes. These data indicate that the sequence obtained from F. heteroclitus represents a teleost fish CYP3A gene and it has been designated CYP3A30.

Glucocorticoid potentiation of cytochrome P4501A1 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin in porcine and human endothelial cells in culture.

Cytochrome P4501A1 (CYP1A1) induction was examined in cultures of porcine aorta endothelial cells (PAEC) and of human aorta endothelial cells (HAEC) exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with or without the glucocorticoid receptor (GR) agonist cortisol or dexamethasone (DEX). In PAEC exposed to 0.1 nM TCDD + 10 microM cortisol the level of CYP1A1 protein and the degree of ethoxyresorufin-O-deethylase (EROD) activity induction were 2- to 3-fold greater than with 0.1 nM TCDD alone. A similar enhancement of EROD induction was obtained when 0.1 or 1 nM TCDD was added together with 0.1, 1, or 10 microM DEX in the media. Cultures of HAEC also showed potentiation of EROD induction when 1 nM TCDD was co-administered with 10 microM DEX. This potentiation caused by DEX was abolished by addition of 10 microM of the GR antagonist RU38486. These data suggest that potentiation of CYP1A1 induction in endothelial cells proceeds by a GR dependent mechanism.

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.

Cytochromes P450 (CYP) in the Poeciliopsis lucida hepatocellular carcinoma cell line (PLHC-1): dose- and time-dependent glucocorticoid potentiation of CYP1A induction without induction of CYP3A.

Glucocorticoids are being found to influence expression of cytochrome P450 (CYP) genes in multiple subfamilies in mammals (J.S. Sidhu, and C.J. Omiecinski (1995) Pharmacogenetics 5, 24--36). In the present study we investigated CYP1A and CYP3A expression in the fish Poeciliopsis lucida hepatocellular carcinoma cell line (PLHC-1) after coadministration of CYP1A and CYP3A inducers, including glucocorticoids. A putative CYP3A protein is expressed in PLHC-1 cells but its content was not altered by exposure of cultures to the prototypical mammalian CYP3A inducers dexamethasone (DEX), pregnenolone-16 alpha-carbonitrile (PCN), or rifampicin (RIF). However, when coadministered with 3,3', 4,4'-tetrachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), DEX but not PCN or RIF caused increases in the degree of CYP1A induction by these aryl hydrocarbon receptor (AHR) agonists. This increase was seen both in CYP1A protein content and rates of ethoxyresorufin-O-deethylase (EROD) activity. DEX alone caused no induction of CYP1A, indicating that the enhancement of CYP1A induction caused by DEX + AHR agonists was not an additive effect but rather a potentiation. The dose of DEX required for maximal potentiation was three orders of magnitude greater at 48 h than the dose required at 24 h. Moreover, the degree of potentiation of CYP1A induction was much greater at the lower doses than at the highest doses of TCDD. There was up to 20-fold potentiation of EROD induction in cultures exposed to 0.1 nM TCDD. Two other glucocorticoid receptor (GR) agonists, cortisol and prednisone, also produced a strong potentiation of CYP1A induction, but other mammalian CYP3A inducers that are not GR agonists, such as the anti-glucocorticoid PCN, the anti-mineralocorticoid spironolactone, or the macrolide antibiotics RIF and troleandomycin, did not potentiate the CYP1A induction in PLHC-1 cells. Addition of the mammalian GR antagonists PCN or RU 38486 reduced the DEX-mediated potentiation of CYP1A induction, whereas spironolactone had no effect on the potentiation. RU 38486 also potentiated the induction of EROD activity by the TCDD, which suggests that RU 38486 acts as a partial GR agonist in PLHC-1 cells. These results suggest that potentiation of CYP1A induction in this nonmammalian cell line proceeds by a classical GR-mediated pathway, independently of the expression of CYP3A. However, the complex interaction between doses of both GR and AHR agonists and duration of exposure, suggests that additional processes influence this potentiation. The unusually strong potentiation at lower doses of TCDD may make PLHC-1 cells particularly suitable in exploring further the consequences of this potentiation.

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.

Immunohistochemical localization of CYP1A and CYP3A-like isozymes in hepatic and extrahepatic tissues of Atlantic cod (Gadus morhua L.), a marine fish.

The cellular localization of inducible CYP1A and constitutive CYP3A-like forms in different organ systems of Atlantic cod (Gadus morhua) was determined in control fish and fish exposed to beta-naphthoflavone (BNF). Paraffin-embedded sections were stained with polyclonal rabbit anti-cod P450 1A IgG or rabbit anti-rainbow trout P450con (a putative CYP3A form which cross-reacts with purified cod P450b) serum by the avidin-biotin peroxidase complex method. Following BNF-exposure of cod, CYP1A induction was immunohistochemically demonstrated in hepatocytes and endothelial cells of liver, the endocardium and vascular endothelium in the atrium and ventricle, and epithelial cells of the proximal tubular segment, endothelial cells, and interrenal cells in kidney. The vascular endothelium was the main site of induction of CYP1A in gills, spleen, gut, pyloric caecae, and gonads. The CYP3A-like isozyme P450b was mainly localized to hepatocytes, renal tubular epithelium, and epithelial cells of the mucosa in the intestine. Furthermore, the distribution of P450b was not affected by BNF exposure. The localization of P450b bears interesting similarities to the localization of CYP3A in mammals supporting the CYP3A-like identity of cod P450b. Simultaneous localization of inducible CYP1A and a constitutively expressed CYP isoenzyme has not previously been reported in fish. This is also the first presentation of cellular distribution of a CYP3-like isozyme in fish. Staining of CYP1A in endothelial cells supports previous observations that endothelium is a major site of CYP1A induction following xenobiotic exposure in fish. The observation of CYP1A induction in interrenal cells has important implications for possible endocrine effects of xenobiotic exposure.

The limbic functional selectivity of amperozide is not mediated by dopamine D2 receptors as assessed by in vitro and in vivo binding.

Behavioural, biochemical and electrophysiological studies suggest that amperozide affects mesolimbic dopamine neurotransmission. Amperozide is a potent 5-HT2, receptor antagonist with only a moderate affinity for rat brain dopamine D2 receptors. The brain regional dopamine D2 receptor binding properties of amperozide were investigated by using in vitro and in vivo radioligand binding techniques. Amperozide displaced [3H]spiroperidol binding from rat striatal and limbic dopamine D2 receptors with moderate affinity (Ki = 540 +/- 118 nM and Ki = 403 +/- 84 nM, respectively). The dopamine D2 receptor antagonist l-sulpiride and the agonist dopamine did not show different affinity in the two brain regions. Amperozide potently displaced in vivo [3H]spiroperidol binding in rat frontal cortex (ID50 = 1.4 mg/kg s.c.) but was devoid of effect in striatum, olfactory tubercle and nucleus accumbens (ID50 > 100 mg/kg s.c.). Chronic administration of amperozide (5 mg/kg p.o.) for three weeks did not result in any change of maximal dopamine D2 receptor number in either striatal or limbic tissue. The effects of amperozide on dopamine neurotransmission are thus not likely to occur by a direct interaction with dopamine D2 receptors in either striatal or limbic tissue. The functional limbic selectivity might rather be mediated by serotoninergic pathways.

The cytochrome P450 system of Atlantic salmon (Salmo salar): II. Variations in hepatic catalytic activities and isozyme patterns during an annual reproductive cycle.

A group of Atlantic salmon (Salmo salar) was followed through their first year of maturation and spawning. At monthly intervals, starting with juvenile fish in December, 5-7 fish of each sex were killed, and liver and plasma were sampled. The last sampling point was of spawning fish in November a year later. Variables in the cytochrome P450 (P450) system were studied in hepatic microsomes, and estradiol 17ß was measured in the plasma of females to assess the maturational status. The P450 1A1-mediated 7-ethoxyresorufin O-deethylase (EROD) started at high levels in winter, but decreased to non-detectable activities in pre-spawning females. Decreases, but not to the same extent, were also observed during this period in total cytochrome P450, cytochrome b5, NADPH-cytochrome P450 reductase, and in the content of two immunochemically determined P450 isozymes. At the same time, LSI levels increased in maturing females (starting in July), and GSI levels increased in both sexes (starting in May). Sex specific differences were observed in pre-spawning fish in September and October, with levels of total P450, b5, NADPH-cytochrome P450 reductase, EROD and P450 isozymes significantly lower in females. At the same time, plasma estradiol-17ß levels reached peak values in females. The results point to the important role of sex steroids such as estradiol-17ß as major factors in the regulation of final sexual maturation. However, this study also indicates that there may be estradiol-17ß independent events of equal importance in the early stages of gonadal maturation that may involve the P450 system. The changes observed in the P450 system (as a major drug and steroid metabolizing system) of Atlantic salmon during sexual maturation may be of importance both in the endogenous transduction of hormonal signals, and as a pharmacological basis for designing therapeutic treatment of diseases in the aquaculture industry.

Environmental contaminants and biochemical responses in flatfish from the Hvaler Archipelago in Norway.

The levels of several environmental contaminants, including selected polyaromatic hydrocarbons (PAH), organochlorines (DDT/DDE, hexachlorobenzene), 15 polychlorinated biphenyl (PCB) congeners, and polychlorinated dibenzofurans and dibenzo-p-dioxins, PCDF/PCDD), and heavy metals (Cd, Hg, Pb, and As) were analyzed in muscle and liver of three different flatfish species (dab, Limanda limanda; flounder, Platichthys flesus; plaice, Pleuronectes platessa) caught by gill netting at different sites in the Hvaler Archipelago. Indices of biochemical effects in liver S9-fractions were studied by measuring cytochrome P450-dependent monooxygenase and UDP-glucuronyl transferase activities, and by immunoquantitating cytochrome P450 1A1 using an indirect enzyme-linked immunosorbent assay (ELISA). Only low levels of PCDD/PCDF, Cd, and Pb were observed, whereas PCB levels were significantly elevated in fish from the inner sites of the Archipelago compared to a reference site. The contaminant gradient toward the Glomma estuary was correlated with increased cytochrome P450 1A1 activity, measured as 7-ethoxyresorufin O-deethylase (EROD), and with immunoquantitated P450 1A1. In contrast, fish from the site at Idefjorden, although containing elevated contaminant levels, did not show elevated EROD activity, but apparently elevated P450 1A1 protein. These findings may reflect different pollution histories of the sites, and indicate the applicability of biochemical effect indices (i.e., EROD and P450 1A1 immunoquantitation) to monitoring studies. The integrated chemical-biochemical approach employed in this study can obviously be expanded to give fruitful information about cause-effect relationships in other contaminant situations.

Catalytic activity and immunochemical quantification of hepatic cytochrome P-450 in ß-naphthoflavone and isosafrol treated rainbow trout (Oncorhynchus mykiss).

In addition to catalytical assays, immunochemical techniques have recently been employed to measure induction of the cytochrome P-450 (P450) monooxygenase system in fish with polyaromatic hydrocarbons (PAH). In the present study, polyclonal antibodies were raised against rainbow trout P450IA1. Levels of rainbow trout P450IA1 determined using protein blotting- and ELISA procedures were compared with levels of 7-ethoxyresorufin-O-deethylase (7-EROD) activity in liver microsomes from rainbow trout. These comparisons showed that values of P450A1 were positively correlated (r=0.99 and r=0.97) with 7-EROD activities. In addition, the effects of isosafrol (ISF) or ß-naphthoflavone (ßNF) treatments on P450 levels in rainbow trout liver were investigated using immunochemical and catalytical methods. ISF treatment induced 7-EROD activity as well as 7-methoxycoumarin-O-demethylase-, 7-ethoxycoumarin-O-deethylase-, 7-propoxy-coumarin-O-depropylase and 7-butoxycoumarin-O-debutylase activities, although to a lesser extent, compared with the ßNF treatment. In contrast, immunochemical quantification of rainbow trout P450IA1 protein revealed a slightly different pattern. ISF appeared to be a weak inducer of P450IA1 in rainbow trout compared with ßNF. In addition, the degree of inhibition of 7-alkoxycoumarin-O-dealkylase activities in ISF microsomes differed from that measured in control- and ßNF microsomes. The discrepancies between catalytic and immunochemical estimates of rainbow trout P450IA1 in ISF treated fish in addition to differencs between specific inhibitory pattern by specific polyclonal antibodies raised against rainbow trout P450IA1, indicate that important differences exists between the responses induced by ßNF- and ISF treatments in the rainbow trout liver.

Cytochrome P-450 mediated O-dealkylation of 7-alkoxycoumarins in liver microsomes from rainbow trout (Salmo gairdneri).

Evidence has recently been presented for variation in the inducibility of various 7-alkoxycoumarin-O-dealkylase activities in liver microsomes from a number of mammalian species by ß-naphthoflavone (ßNF). In the present study we have investigated the inducibility of hepatic microsomal 7-methoxycoumarin-O-demethylase, 7-ethoxycoumarin-O-deethylase, 7-propoxycoumarin-O-depropylase and 7-butoxycoumarin-O-debutylase activities in rainbow trout by ßNF. O-demethylase activity was increased approximately 17-fold, O-deethylase and O-depropylase activities approximately 9-fold and O-debutylase activity approximately 25-fold. The kinetics of the various hepatic microsomal 7-alkoxycoumarin-O-dealkylase activities were investigated in control and ßNF-treated rainbow trout. The O-demethylase-, O-depropylase- and O-debutylase activities exhibited monophasic Michaelis-Menten kinetics in liver microsomes from both control and ßNF-treated rainbow trout, whereas the O-deethylase activity exhibited biphasic Michaelis-Menten kinetics in control liver microsomes and monophasic Michaelis-Menten kinetics in liver microsomes from ßNF-treated rainbow trout.

Comparison of xenobiotic biotransformation enzymes in kidney and liver of rainbow trout (Salmo gairdneri).

The microsomal cytochrome P-450 content in kidney of rainbow trout (Salmo gairdneri) was approximately 5-fold lower than the content in liver. The renal ethoxycoumarin- and ethoxyresorufin-O-deethylase activities calculated on a per-cytochrome P-450 basis were, however, found to be about 10-fold higher than the hepatic activities. The patterns of time-dependent increase and subsequent decrease of microsomal cytochrome P-450-dependent monooxygenase activities after a single injection of beta-naphthoflavone (BNF) were similar in the kidney and liver. The microsomal ethoxyresorufin- and ethoxycoumarin-O-deethylase activities were maximally induced in liver (120- and 10-fold, respectively) by a single BNF injection (50 mg/kg body wt), whereas in kidney the maximal levels of induction (135- and 21-fold, respectively) were reached after three injections with BNF. The induction of cytochrome P-450 systems was associated with synthesis of a new microsomal protein of 58,000 Da in both kidney and liver. UDP-glucuronosyl transferase activity toward p-nitrophenol was about 8-fold lower in kidney than in liver. A significant 2.5-fold elevation in microsomal UDP-glucuronosyltransferase activity was found in the kidney 14 days after a single injection with BNF (50 mg/kg). In the liver, a 2-fold increase of this activity was seen 3 days after the treatment. The results indicate that the rainbow trout kidney in addition to the liver is of great importance in biotransformation of lipophilic xenobiotics.