Further consideration of phenobarbital effects on cytochrome P-450 activity in the killifish, Fundulus heteroclitus

1. Ethoxyresorufin O-deethylase (EROD) activity, aldrin epoxidase (AE) activity, cytochrome P-450 content, and levels of cytochrome P-450E (the major BNF-inducible P-450 form and primary EROD catalyst in scup) or its homologues were measured in hepatic microsomes isolated from Fundulus heteroclitus, scup (Stenotomus chrysops) and brook trout (Salvelinus fontinalis) treated with beta-naphthoflavone (BNF) or phenobarbital (PB). 2. In all three teleost species, BNF treatment caused expected increases in P-450 content, EROD activity and P-450E level; but either no change or a slight decrease in AE turnover rate (nmol/min/nmol P-450). 3. Polyclonal antibodies to P-450E did not inhibit AE activity in microsomes from BNF-treated scup, confirming that this major BNF-inducible P-450 form does not catalyze AE activity in fish. 4. In contrast, PB treatment did not affect hepatic AE activity, P-450 content or levels of "P-450E" in F. heteroclitus, but did variably affect EROD activity which was suppressed in one experiment and elevated in another. 5. The results indicate that (i) contrary to previous reports, neither PB nor MC-type inducers increase AE activity in F. heteroclitus, (ii) MC-type inducers do not affect AE activity in the other teleost species examined, and (iii) AE activity is not a reliable indicator of P-450 induction by environmental chemicals. 6. We emphasize the need to establish the mechanism of PB action, and the nature of any fish P-450 forms analogous to PB-inducible forms in mammals in order to conclusively evaluate PB-responses in fish.     

Specificity and cross-reactivity of monoclonal and polyclonal antibodies against cytochrome P-450E of the marine fish scup

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.     

Monoclonal antibodies to liver microsomal cytochrome P-450E of the marine fish Stenotomus chrysops (scup): cross reactivity with 3-methylcholanthrene induced rat cytochrome P-450

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.     

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.     

Effect of monooxygenase reactions catalyzed by cytochrome P-450 on the microsomal membrane

Hydroxylation of dimethylaniline in rabbit liver microsomes is accompanied by inactivation of cytochrome P-450 and the formation of products inhibiting the catalytic activity of non-inactivated cytochrome P-450. Other enzymes and electron carriers of microsomal membrane (cytochrome b5, NADH-ferricyanide reductase, NADPH-cytochrome c and NADPH-cytochrome P-450 reductases) as well as glucose-6-phosphatase were not inactivated in the course of the monooxygenase reactions. Phospholipids and microsomal membrane proteins were also unaffected thereby. Consequently, the changes in the microsomal membrane during cytochrome P-450 dependent monooxygenase system functioning are confined to the inactivation of cytochrome P-450.     

The effect of sex steroids and pregnenolone-16 alpha-carbonitrile on the hepatic microsomal monooxygenase system of rainbow trout (Salmo gairdneri)

Estradiol benzoate (EB-5 mg/kg) or testosterone propionate (TP-50 mg/kg) administration to sexually immature rainbow trout resulted in an increase in liver weight to body weight ratios, and a diminution in hepatic microsomal benzphetamine-N-demethylation (BeND), ethoxycoumarin- and ethoxyresorufin-O-deethylations (ECOD, EROD) and cytochrome(s) P-450 content when compared to corn oil-pretreated controls. A low dose of TP (2 mg/kg) caused an increase in cytochrome(s) P-450 content but had no effect on the selected monooxygenase activities. EB administration prior to treatment with 150 mg/kg beta-naphthoflavone caused a dose-dependent decrease in the magnitude of induction of cytochrome(s) P-450 and associated catalytic activities. These data suggest that the sex differences in monooxygenation observed in prespawning trout are mediated via the sex steroids and that fish may respond differently to inducers depending on their reproductive state at the time of exposure. Administration of the synthetic steroid, pregnenolone-16 alpha-carbonitrile (PCN), resulted in an increase in BeND and ECOD but had no effect on EROD or cytochrome(s) P-450 content.     

Hepatic microsomes from freshwater fish--I. In vitro cytochrome P-450 chemical interactions

1. Of 87 chemicals tested for their ability to interact with oxidized hepatic cytochrome P-450 from mature male brook trout (Salvelinus fontinalis), 21 formed detectable type I or type II binding spectra. 2. When 8 of these 21 chemicals were tested with cytochrome P-450 of nine other species of freshwater fish, wide species variation in hepatic microsomal cytochrome P-450 was evident, since the spectral size of chemical interactions as related to the carbon monoxide spectrum and the ratio of type II to type I binding were not alike. 3. These spectral data suggest that hepatic microsomal cytochrome P-450 of freshwater fish exists in different forms.     

Differential effects of fat deficiency on hepatic and pulmonary drug metabolizing enzymes in rat and mouse

In the present study, we have evaluated the effect of dietary fat deprivation on the carcinogen/drug metabolizing enzymes in rats and mice. In rats, hepatic AHH, Cyt.P-450 Cyt.b5 and Cyt.c-reductase were significantly decreased due to fat deficiency, whereas in lungs, AHH and Cyt.c-reductase were decreased without any change in Cyt.P-450 level. In mice, feeding of fat-free diet did not cause any alteration in hepatic AHH and Cyt.P-450, but the levels of Cyt.b5 and Cyt.c-reductase were significantly reduced. In contrast to liver, Cyt.P-450 and Cyt.b5 were increased in lungs. Activity of UDPGT was lowered both in liver and lungs of rats whereas GST and GSH were increased in liver only. In mice, a decrease in UDPGT and appreciable increases in GST and GSH in liver were observed. However, in lungs, UDPGT activity was enhanced by feeding fat-free diet. These observations suggest that mice and rats respond differentially to the depletion of fat in the diet.     

Regulation of three forms of cytochrome P-450 and epoxide hydrolase in rat liver microsomes. Effects of age, sex, and induction

A procedure for the preparation of monospecific antibody directed against rat liver microsomal cytochrome P-45-a is described. This antibody, together with monospecific antibodies to cytochromes P-450b and P-450c, has been used to show that these three forms of cytochrome P-450 are distinct and share no common antigenic determinants. These antibodies (a) give single immunoprecipitin bands with detergent-solubilized microsomes; (b) do not cross-react with the purified heterologous antigens in Ouchterlony double diffusion analyses; (c) have no effect on catalytic activity of the heterologous antigens but completely inhibit the enzymatic activity of the homologous antigens; and (d) remove only the homologous antigen from detergent-solubilized microsomes when covalently bound to a solid support. With radial immunodiffusion assay, we have quantitated these three forms of cytochrome P-450 in liver microsomes after treatment of rats with seven different inducers of cytochrome P-450. The levels of these cytochrome P-450 isozymes vary independently and are also regulated by the age and sex of the animal. The antibodies have also been used to assess the contribution of cytochromes P-450a, P-450b, and P-450c in the metabolism of xenobiotics by rat liver microsomes. A large proportion of benzo(a)pyrene metabolism and testosterone 16 alpha-hydroxylation in microsomes from untreated rats is not catalyzed by cytochromes P-450a, P-450b, and P-450c. Epoxide hydrolase, another microsomal enzyme involved in the metabolism of xenobiotics, was also quantitated by radial immunodiffusion after prior treatment of rats with microsomal enzyme inducers. The inductions of epoxide hydrolase varies independently of the induction of cytochromes P-450a, P-450b, and P-450c.     

Hepatic microsomes from freshwater fish--II. Reduction of benzo(a)pyrene metabolism by the fish anesthetics quinaldine sulfate and tricaine

1. A single in vivo exposure of brook trout (Salvelinus fontinalis) to a 30.0 mg/l solution of quinaldine sulfate or a 112.5 mg/l solution of tricaine for 5 min significantly reduced the in vitro hydroxylation of benzo(a)pyrene. 2. Since quinaldine sulfate and tricaine formed type I and II binding spectra, respectively, with brook trout hepatic cytochrome P-450, these chemicals probably reduced benzo(a)pyrene hydroxylase enzyme activity by altering the form(s) of cytochrome P-450 responsible for this activity. 3. Hepatic microsomal cytochrome P-450 from brook trout treated with tricaine for 5 min and then placed into fresh water for 24 hr had returned to control levels. 4. Caution should be exercised in the use of quinaldine sulfate or tricaine to anesthetize fish prior to analysis of hepatic microsomal mixed function oxidases.     

Inhibition of arylhydrocarbon hydroxylase and cytochrome P-450 by 20-methylcholanthrene and phenobarbital in guinea pig on excessive doses of ascorbic acid

Treatment of guinea pigs on adequate ascorbic acid (AA) with 20-methylcholanthrene (MCA) and phenobarbital (PB) significantly increased hepatic arylhydrocarbon hydroxylase (AHH), cytochrome P-450 and cytochrome-b5 activities. In lungs, only MCA treatment significantly enhanced the activities of AHH, cytochrome P-450 and cytochrome b5. In animals on excessive doses of AA, there was inhibition of hepatic AHH, cytochrome P-450 and cytochrome b5 levels by treatment with these xenobiotics. Also, inhibition was observed in pulmonary AHH and cytochrome P-450 levels. The relevance of these observations in excessive AA-fed guinea pigs to carcinogenesis requires further extensive investigations.     

Cloning, Sequencing, and Phylogenetic Analysis of Complementary DNA of Novel Cytochrome P-450 CYP1A in Japanese Eel (Anguilla japonica)

Complementary DNA of cytochrome P-450 CYP1A, in addition to CYP1A1, has been isolated from Japanese eel (Anguilla japonica) liver treated with 3-methylcholanthrene. The cDNA contained a 5′ untranslated region of 66 bp, an open reading frame of 1554 bp coding for 517 amino acids and a stop codon, and a 3′ untranslated region of 1166 bp. The predicted molecular weight of the Japanese eel CYP1A was approximately 58.5 kDa. The nucleotide sequence exhibited identities with the reported CYP1A1 sequences of 77% for Japanese eel, 75% for rainbow trout, 72% for scup, plaice, and butterfly fish, and 71% for toadfish. The deduced amino acid sequence exhibited identities with the reported CYP1A1 sequences of 78% for Japanese eel, 77% for rainbow trout, 75% for scup, 74% for toadfish, 73% for plaice, and 72% for butterfly fish. The novel eel CYP1A obtained had less similarity to the other teleost CYP1A1 proteins (72%–78%) than that of the eel CYP1A1 (74%–80%). When compared with mammalian CYP proteins, the novel eel CYP1A was more similar to the CYP1A1 proteins (54%–56%) than to the CYP1A2 proteins (50%–53%). The phylogenetic tree of the teleost CYP1A genes constructed using the maximum likelihood method suggested that the novel eel CYP1A is ubiquitous among the Anguilliformes. Received August 25, 2000; accepted November 30, 2000     

The effects of carbon tetrachloride on rat liver microsomes during the first hour of poisoning in vivo, and the modifying actions of promethazine

The effects of an oral administration of carbon tetrachloride on various liver microsomal and supernatant components were studied 1hr. and 2hr. after dosing. The modifications of such early changes resulting from a concomitant administration of promethazine together with the carbon tetrachloride were also investigated. The microsomal components studied were: cytochromes P-450 and b(5); inorganic pyrophosphatase; NADH- and NADPH-cytochrome c reductases; NADH- and NADPH-neotetrazolium reductases; a lipid-peroxidation system associated with the oxidation of NADPH and stimulated by ADP and Fe(2+). NAD- and NADP- DT-diaphorases were measured in the supernatant solution remaining after isolation of liver microsomes, and the distribution of RNA phosphorus between the microsomes and supernatant solution was also determined. Carbon tetrachloride produced a rapid fall in inorganic pyrophosphatase activity, a rather slower decrease in cytochrome P-450 content of the microsomes and small increases in the activities of NADH-cytochrome c reductase and neotetrazolium reductases. The activities of NADPH-cytochrome c reductase, the NADPH-ADP/Fe(2+)-linked lipid-peroxidation system, DT-diaphorases and the content of cytochrome b(5) in the microsomes were unchanged. There was also a loss of RNA phosphorus from the microsomes into the supernatant solution. The RNA phosphorus redistribution, the decrease in inorganic pyrophosphatase and the increases in neotetrazolium reductase activities were at least partially prevented by a concomitant dosing with promethazine. However, the decrease in cytochrome P-450 was not affected by promethazine treatment. These early changes are discussed in terms of the liver necrosis produced by carbon tetrachloride and which is greatly retarded in its onset by the administration of promethazine.     

An immunochemical analysis of the induction of cytochrome P-450 isoforms in the liver of fresh-water fishes by 3-methylcholanthrene, beta-naphthoflavone and aroclor 1254

The cytochrome P-450 isoforms have been studied in liver microsomes of some fish species from Lake Baikal. Using the inhibitory analysis of microsomal monooxygenase activities carried out by the specific polyclonal antibodies it has been shown that 3-methylcholanthrene, beta-naphthoflavone and arochlor 1254 induce isoforms immunologically related to cytochrome P-488c but not to the rat cytochrome P-450b in fish liver microsomes. The immunologic identity in isoforms of fish and rat cytochromes induced by methylcholanthrene has not been revealed. A possibility to use the method of the inhibitory analysis of fish microsomal activities by specific antibodies to the rat cytochrome P-450 isoforms for biomonitoring and biotesting of polycyclic hydrocarbons and polychlorinated biphenyls in aquatic systems is discussed.     

Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism

The metabolism of the dihydropyridine calcium antagonist and vasodilator nifedipine has been reported to exhibit polymorphism among individual humans (Kleinbloesem, C. H., van Brummelen, P., Faber, H., Danhof, M., Vermeulen, N. P. E., and Breimer, D.D. (1984) Biochem. Pharmacol. 33, 3721-3724). Nifedipine oxidation has been shown to be catalyzed by cytochrome P-450 (P-450) enzymes. Reconstitution, immunoinhibition, and induction studies with rat liver indicated that the forms designated P-450UT-A and P-450PCN-E are the major contributors to microsomal nifedipine oxidation. The P-450 which oxidizes nifedipine (P-450NF) was purified to electrophoretic homogeneity from several human liver samples. Antibodies raised to P-450NF were highly specific as judged by immunoblotting analysis and inhibited greater than 90% of the nifedipine oxidase activity in human liver microsomes. A monoclonal antibody raised to the human P-450 preparation reacted with both human P-450NF and rat P-450PCN-E. Immunoblotting analysis of 39 human liver microsomal samples using anti-P-450NF antibodies revealed the same 52,000-dalton polypeptide, corresponding to P-450NF, with only one of the microsomal samples showing an additional immunoreactive protein. The level of nifedipine oxidase activity was highly correlated with the amount of P-450NF thus detected using either polyclonal (r = 0.78) or monoclonal (r = 0.65) antibodies, suggesting that the amount of the P-450NF polypeptide may be a major factor in influencing the level of catalytic activity in humans as well as rats. Cytochrome b5 enhanced the catalytic activity of reconstituted P-450NF, and anti-cytochrome b5 inhibited nifedipine oxidase activity in human liver microsomes. P-450NF also appears to be a major contributor to human liver microsomal aldrin epoxidation, d-benzphetamine N-demethylation, 17 beta-estradiol 2- and 4-hydroxylation, and testosterone 6 beta-hydroxylation, the major pathway for oxidation of this androgen in human liver microsomes.     

Mixed-function oxygenase in juvenile rainbow trout exposed to hexachlorobenzene or 3,3',4,4'-tetrachlorobiphenyl.

1. 3,3',4,4'-tetrachlorobiphenyl (PCB 77), but not hexachlorobenzene, induced liver microsomal cytochrome P-450 (Cyt P-450), ethoxycoumarin-O-deethylase (ECOD) and ethoxyresorufin-O-deethylase (EROD) in rainbow trout. Maximum induction was observed in a PCB 77 injected group of fish (1.0 mg/kg, i.p. injection) 13 days after the injections being 2, 10 and 50 times the value of non-induced fish, respectively. 2. The apparent Km value of ethoxyresorufin of this induced group of fish differed only slightly from that of non-induced fish. The apparent Vmax value (EROD) was 50 times higher. 3. Freezing small pieces of liver in liquid nitrogen did not produce cytochrome P-420. 4. Fluorimetric and spectrophotometric measurements of EROD correlated.     

Use of a monoclonal antibody specific for rabbit microsomal cytochrome P-450 3b to characterize the participation of this cytochrome in the microsomal 6 beta- and 16 alpha-hydroxylation of progesterone

A monoclonal antibody was developed that is specific for the 3b electrophoretic class of rabbit liver microsomal cytochrome P-450 as judged by immunoprecipitation and subsequent electrophoretic analysis. The antibody is inhibitory of catalytically distinct, variant forms of P-450 3b prepared from New Zealand White or IIIVO/J rabbits, respectively. Peptide mapping of the immunopurified P-450 3b from NZW and IIIVO/J microsomes indicates that a characteristic difference between the variant forms is exhibited by the antigen. In addition, a competitive assay indicates that the binding properties of the antibody do not differ substantially toward the variant forms of P-450 3b. The inhibitory antibody was used to examine the contribution of P-450 3b to the microsomal 16 alpha- and 6 beta-hydroxylation of progesterone. The antibody inhibits 40-70% of the 16 alpha-hydroxylase activity of microsomes from either New Zealand White or IIIVO/J rabbits. In contrast, it does not inhibit 6 beta-hydroxylation catalyzed by microsomes prepared from strain IIIVO/J but does inhibit this reaction as catalyzed by microsomes from most New Zealand White rabbits. The antibody also inhibits the increased 16 alpha-hydroxylase activity of IIIVO/J microsomes observed in the presence of 5 beta-pregnane-3 beta,20 alpha-diol, an allosteric effector of this variant form of P-450 3b. Use of this monoclonal antibody provides a link between the observed properties of the purified, variant forms of P-450 3b and microsomal metabolism. These results indicate that the antibody can be used to phenotype variant forms of P-450 3b in microsomal fractions.     

Hepatic cytochrome P-450-dependent monooxygenase systems of the trout, frog and snake--I. Components

1. Components of the hepatic monooxygenase systems (cytochrome P-450, cytochrome b5, NADPH cytochrome P-450- or c-reductase) of the brown trout (Salmo trutta), leopard frog (Rana pipiens) and garter snake (Thamnophis) were considerably lower than those found in the rat. 2. Reactivity of snake NADPH-cytochrome P-450-reductase with cytochrome P-450 was about twice that of the rat reductase; reactivities of trout and frog reductases were similar, but lower than that of the rat. The optimal temperature for the rat, frog and snake reductase activity was 37 degrees C, but 26 C for the trout reductase, regardless of whether cytochrome P-450 or cytochrome c was the electron acceptor for the reaction. 3. A type I substrate (benzphetamine) and a type II substrate (aniline) were less reactive with P-450 cytochrome from the trout, frog and snake than with P-450 cytochrome from the rat. 4. Qualitative differences were seen in the ethylisocyanide spectrum of microsomes from the rat, trout, frog and snake; these differences reflect qualitative differences in the populations of P-450 cytochromes among each of the four species.