Hepatic Microsomal Enzyme Research Articles

In Vitro Inhibition of Rat CYP1A1 and CYP1A2 by Piceatannol, a Hydroxylated Metabolite of trans-Resveratrol

Piceatannol and its parent compound, trans-resveratrol, decreased the in vitro catalytic activity of rat CYP1A1 and CYP1A2 by mixed inhibition. trans-Resveratrol was not a mechanism-based inactivator of rat CYP1A in vitro and the administration of this compound (50 mg/kg) to rats did not affect hepatic microsomal CYP1A-mediated enzyme activity.

DOSE- AND TIME-DEPENDENT EFFECTS OF 2,3,7,8-TETRABROMODIBENZO-P-DIOXIN ON RAT LIVER

Dose- and time-dependent effects of 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) on the liver were examined by single administration of TBDD by gavage to male and female rats. Fifteen Wistar rats of each sex per group received 0, 10, 30, 100 or 300 microg TBDD/kg body weight. Rats surviving to scheduled necropsy on Day 2, 7 or 36 after the TBDD administration were examined for hepatic histopathology, activities of hepatic microsomal enzymes and serum levels of lipids, total cholesterol and transaminases and hepatic concentrations of TBDD. Tigroid basophilic cytoplasm and hepatocellular hypertrophy were observed at 10 microg/kg on Day 2 or 7 through 36, whereas degenerative and aggressive lesions such as necrosis, fibrosis, multinucleated hepatocytes and disarrangement of hepatocytes occurred later at higher dose levels. Persistently increased activities of hepatic aryl hydrocarbon hydroxylase (AHH), ethoxycoumarin O-deethylase (ECOD) and ethoxyresorufin O-deethylase (EROD), increased serum levels of total cholesterol and phospholipid and increased relative liver weight were observed in all groups dosed 10 mug/kg and above, suggesting that hepatic microsomal monooxygenases and basophilic cytoplasm of hepatocytes were early and sensitive indicators among those TBDD-induced effects. A dose-dependent increase in liver concentrations of TBDD on Day 2 was followed by logarithmic decreases in TBDD concentrations against the days elapsed after the TBDD administration. An elimination half-life (t(1/2)) of TBDD from the liver was estimated to range from 12 to 16 days. It was suggested that females were more susceptible to TBDD than males, and that acute hepatotoxicity of TBDD was as potent as that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Effects of two infectious bursal disease vaccine virus strains on hepatic microsomal enzyme activities in chickens

The influence of two infectious bursal disease vaccines on the activities of hepatic microsomal enzymes aniline hydroxylase, ethylmorphine N-demethylase, NADPH-cytochrome c reductase, aryl sulphotransferase and p-nitrophenol UDP-glucuronyltransferase was investigated in chickens. The vaccines contained attenuated Winterfield 2512 and VMG-91 strains, respectively. The activities of enzymes were determined on postvaccination days 0, 2, 5 and 7. At the same time, post-mitochondrial supernatant, cytosolic and microsomal pellet protein concentrations were determined. As expected, the antibody titres against infectious bursal disease virus in the serum were increased in both tested groups in relation to each administered vaccine. Using RT-PCR, the presence of the VP2 gene fragment of virus in the liver of chicken was demonstrated 4 and 6 h after vaccination. The results of this study suggest that the two commercial vaccines modulate the activities of five enzymes tested, and that the two attenuated vaccines applied triggered induction and/or inhibition of phases I and II of biotransformation enzyme activities.

Species difference in the induction of hepatic CYP1A subfamily enzymes, especially CYP1A2, by 2-methoxy-4-nitroaniline among rats, mice, and guinea pigs

Species difference in the induction of hepatic cytochrome P450 CYP1A subfamily enzymes by 2-methoxy-4-nitroaniline (2-MeO-4-NA) was investigated among male F344 rats, C57BL/6 Cr mice, and Hartley guinea pigs. All species of animals were treated with a single ip injection of 2-MeO-4-NA (0.44 mmol/kg body weight), and changes in levels of the mRNA and protein of hepatic cytochrome P4501A (CYP1A) subfamily enzymes were examined by the methods of RT-PCR and Western blot, respectively. In addition, hepatic microsomal enzyme activities were measured using methoxyresorufin and ethoxyresorufin as substrates of CYP1A2 and CYP1A1, respectively. The overall results of the RT-PCR, Western blot, and measurement of the enzyme activity indicated that 2-MeO-4-NA-mediated induction of hepatic CYP1A subfamily enzymes, especially CYP1A2, occurred only in rats but not any other species of animals examined and that the species difference in the CYP1A induction was not necessarily correlated with that in pharmacokinetics of 2-MeO-4-NA. Furthermore, a luciferase reporter gene assay for screening of the ligands of arylhydrocarbon receptor (AhR) using a rat hepatic cell line suggested that 2-MeO-4-NA is not an AhR ligand. The present findings demonstrate for the first time the species difference in the 2-MeO-4-NA-mediated induction of hepatic CYP1A subfamily enzymes between rats and other rodents, mice and guinea pigs, and further propose an AhR-independent pathway for 2-MeO-4-NA-mediated induction in rats.

Antiepileptic drugs and hormonal contraceptives in adolescent women with epilepsy

Both estrogen and progesterone influence seizure activity in women with epilepsy, with estrogen generally demonstrating proconvulsant and progesterone anticonvulsant effects. Women with epilepsy exhibit a variety of endocrine disturbances, probably due to a combination of factors, including the epilepsy syndrome and the effect of interictal and ictal epileptic discharges in the brain. The direct effects of some antiepileptic drugs (AEDs) further increase this risk, apparently related to a specific drug's effect on hepatic microsomal enzymes of the cytochrome P-450 system. AEDs that induce hepatic microsomal enzymes also interact with hormonal contraception to increase estrogen's metabolism and progesterone's protein binding, decreasing concentrations of both hormones and thus reducing contraceptive efficacy. Some evidence indicates that concurrent use of hormonal contraceptives and lamotrigine significantly decreases the plasma concentration of lamotrigine, suggesting that close monitoring is warranted. Nevertheless, hormonal contraception confers comparable or superior efficacy compared with such other contraceptives as the intrauterine device and barrier methods and remains an appropriate option in women with epilepsy. Importantly, concurrent use of hormonal contraception and AEDs does not adversely affect seizure control. Careful patient management, including the use of increased estrogen doses (> or =50 microg) in patients receiving enzyme-inducing AEDs, may further minimize the risk for unintended pregnancy. Special considerations in women of childbearing age include decreased compliance and disease prevention. Although adequate seizure control is the critical requirement of an AED, the potential for interactions with hormonal contraception and the increased risk for endocrine disturbances caused by drugs that alter hepatic microsomal enzymes suggest additional potential advantages for AED treatment that does not affect these enzymes. Both the constellation of physicians treating women with epilepsy and the patients themselves have a poor understanding of the spectrum of reproductive health issues involved, and increased awareness is needed to improve patient management.

A Toxicologist's Guide to the Preclinical Assessment of Hepatic Microsomal Enzyme Induction

The assessment of hepatic microsomal enzyme induction at the completion of preclinical toxicology studies in rodents and large mammals provides a wealth of information to the toxicologist and pharmacokineticist regarding how the drug-metabolizing system of the hepatocyte endoplasmic reticulum responded to high-dose levels of a xenobiotic designed for a specific pharmacological target in any of several target organs. The interpretation of these data can be greatly enhanced by a clear understanding of how this system functions and what the immediate and long-term ramifications are to organs and organ systems. This review focuses on how drugs modify the hepatic cytochrome P450 system, how those modifications are detected, the various consequences of these modifications, and some differences in the induction response among species.

ANTITUMOR DRUG ELLIPTICINE INHIBITS THE ACTIVITIES OF RAT HEPATIC CYTOCHROMES P450

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts. Here, we study the effect of ellipticine on CYP enzymes in rat hepatic microsomes, studying its binding to the enzymes and its potential to inhibit the CYP activities measured with their selective substrates. Although ellipticine was reported to be a selective and strong inhibitor of CYP1A1/2, we found that its inhibitory potential is non-specific. Ellipticine is the most potent inhibitor for CYP3A-dependent 6beta-hydroxylation of progesterone, followed by CYP1A1/2-dependent ethoxyresorufin O-deethylation and CYP2B-mediated pentoxyresorufin O-depentylation. Lower inhibition was detected for 1'-hydroxylation of bufurarol, 21-hydroxylation of progesterone and 6-hydroxylation of chlorzoxazone catalyzed by CYP2D, CYP2C and CYP2E1, respectively. Ellipticine binds to several CYPs of rat hepatic microsomes. The binding titration of ellipticine typically give reverse type I spectrum with CYPs in rat hepatic microsomes. The results indicate that inhibition of CYPs by ellipticine cannot be explained only by its differential potency to bind to individual CYPs.

The relationship among microsomal enzyme induction, liver weight, and histological change in cynomolgus monkey toxicology studies

The purpose of this investigation was to examine the relationship among hepatic microsomal enzyme induction, liver weight, histological evidence of hepatic injury, and serum clinical chemistry markers of hepatic origin in the cynomolgus monkey. We report here the results from independent toxicology studies for 10 investigative drug candidates representing four therapeutic classes. Study conditions were selected to elicit target organ toxicity. We found that six of the 10 compounds altered cytochrome P450-associated activities in both male and female monkeys, two in females only, and one altered similar activities in males only. Frequently, significant treatment-related elevations in NADPH cytochrome c reductase and ethylmorphine N-demethylase were noted. When the results from all 10 studies were pooled, 14 cytochrome P450-associated activities were significantly increased and five were decreased in males compared to 15 significantly increased and three decreased in the females. Treatment-associated liver weight increases were noted in four studies. Except for hepatocellular hypertrophy in one study, no significant treatment-related microscopic changes in liver and no elevations of serum biomarkers commonly associated with liver toxicity were observed in any of the studies that demonstrated significant hepatic enzyme induction. Compared to parallel rat studies, one compound was an inducer only in monkeys and one was an inducer only in rats. Significant elevations of microsomal drug-metabolizing enzymes in the cynomolgus monkey liver are not accompanied by substantial hepatic changes except for hepatomegaly. These alterations in the hepatic drug-metabolizing enzyme system were benign based the absence of histopathological lesions and serum biomarkers of hepatobiliary toxicity.

Genetic Polymorphisms of Flavin Monooxygenase 3 in Sulindac-Induced Regression of Colorectal Adenomas in Familial Adenomatous Polyposis

Sulindac is a nonsteroidal antiinflammatory drug with a chemopreventive effect in patients with familial adenomatous polyposis (FAP). In vivo, the active form of sulindac is sulindac sulfide, which is inactivated by the hepatic microsomal enzyme, flavin monooxygenase 3 (FMO3). In humans, numerous polymorphisms exist in FMO3, which alter enzymatic activity and subsequent substrate metabolism. We recently showed that certain polymorphic forms of FMO3 with reduced activity were associated with a more favorable response to sulindac in preventing the formation of adenomas in patients with FAP without polyps at baseline. Here, we determined whether these FMO3 polymorphisms correlated with the ability of sulindac to regress polyposis in patients with FAP who had polyps prior to treatment. Nineteen patients were treated with 150 mg sulindac twice a day for 6 months. The size and number of polyps in each patient was assessed at baseline (prior to the administration of sulindac), and at 3 and 6 months. Genotyping was done on seven established FMO3 polymorphisms with functional significance-M66I, E158K, P153L, V257M, E305X, E308G, and R492W. Statistical analyses were done with Wilcoxon rank sum test. Of the loci examined, only E158K and E308G showed polymorphic changes. Six patients exhibited polymorphisms in both E158K and E308G loci and were designated as genotype combination 1. The remaining patients were designated as genotype combination 2. Over the course of treatment, patients with genotype combination 1 had a greater reduction in both the size and number of polyps than those with genotype combination 2. These results suggest that combined polymorphic changes in the E158K and E308G alleles may protect against polyposis in patients with FAP treated with sulindac.

Phenytoin and Cognitive Decline

To the Editor: We read with great interest the article by Naidech et al1 on the association of phenytoin exposure and cognitive disability after subarachnoid hemorrhage. The authors speculate on a number of reasons for this association, to which we would like to add the possibility of a pharmacokinetic interaction compromising the protective effects of nimodipine. Phenytoin induces the hepatic microsomal enzyme system (cytochrome p-450 isozymes). Induction of the CYP isozyme system may …

Induction of endogenous pathways by antiepileptics and clinical implications

The aim of this study was to review modifications of the endogenous pathways (e.g. enzyme elevations, normal body constituent depletion or higher formation/excretion of endogenous metabolites) which could be ascribed to enzyme induction by antiepileptic drugs (AEDs). Information on older (e.g. phenobarbital, phenytoin and carbamazepine) and newer drugs (where information is available) is discussed together with clinical implications. The enzymes involved in the endogenous pathways and induced by the AEDs will not be limited to the hepatic microsomal enzymes; extrahepatic enzymes and/or enzymes present in other subcellular fractions will also be discussed, if pertinent. The induction of endogenous pathways by AEDs has been taken into account in the past, but much less emphasis has been given compared with the extensive literature on induction by AEDs of the metabolism of concomitantly administered drugs, either of the same or of different classes. Not all of the endogenous pathways examined and induced by AEDs appear to result in serious clinical consequences (e.g. induction of hepatic ALP, increased excretion of d-glucaric acid or of 6 beta-hydroxycortisol). In some cases, induction of some pathways (e.g. increase of high-density lipoprotein cholesterol or of conjugated bilirubin) might even be a beneficial side-effect, however enzyme induction is considered rather a detrimental aspect for an AED, as induction is generally a broad and a non-specific phenomenon. The new AEDs have generally less induction potential than the older agents. Yet some (felbamate, topiramate, oxcarbazepine and lamotrigine) have the potential for inducing enzymes, whereas others (levetiracetam, gabapentin and vigabatrin) appear to be completely devoid of enzyme inducing characteristics, at least as far as the enzymes investigated are concerned.

Nuclear Receptor Ligands: Rational and Effective Therapy for Chronic Cholestatic Liver Disease?

p r f c t p p f c he regulation of genes that are essential to many hepatic metabolic and transport functions are medited in large part by the action of small molecules that unction as nuclear receptor ligands.1 This process has een labeled chemical genomics.2 These ligands bind to heir specific nuclear receptors, activating the receptor hat then binds to specific elements in a gene’s promoter esulting in stimulation or inhibition of gene expresion.3 In the liver many drugs, metabolites, and herbal ompounds exert their biologic properties as ligands for uclear receptors.4,5 Some familiar examples of drugs hat activate nuclear receptors include phenobarbital and t John’s wort. Both drugs induce hepatic drug metablizing enzymes by acting as ligands for the pregnane X eceptor (PXR), which binds to specific response eleents in the promoter of cytochrome P-450 3A CYP3A), a major hepatic microsomal drug–metabolizng enzyme. This induction then indirectly affects the etabolism and systemic effects of a wide variety of other ompounds metabolized by CYP3A. An extract of 4 ifferent plants, Yin Zhi Huang, has been used tradiionally for treating neonatal jaundice in China and is a igand for the constitutive androstane receptor (CAR) hat up-regulates the expression of several different liver ransporters and enzymes involved in the hepatic clearnce of serum bilirubin.5 Indeed, more than 10% of edically useful drugs are known to exert their biologic ehavior by binding to ligand-binding domains on speific nuclear receptors.4 Table 1 shows some of the most mportant nuclear receptors and their physiologic ligands hat determine the hepatic transport and metabolism of variety of xenobiotics and endogenous substrates. In this issue of GASTROENTEROLOGY, Marschall et al6 eport that rifampicin and ursodeoxycholic acid (UDCA), wo well-known drugs used in the treatment of cholesatic liver disease, each stimulate the transcription of a istinct set of genes that together decrease bile acid ptake, enhance bile acid detoxification and excretion, nd stimulate the clearance of bilirubin. Rifampicin inreased the expression of CYP3A4, uridine 5=-glucuonosyl transferase (UGT1A1), and multidrug-resisance–associated protein (MRP2) whereas UDCA timulated the bile salt export pump (BSEP), and mulidrug resistance protein (MDR3), and MRP4. Although hese effects were observed in patients undergoing choecystectomy who otherwise were healthy, these separate ut complementary effects on gene and protein expresion are predicted to have beneficial effects if they ocurred in patients with cholestatic liver disease. These ndings thus suggest that there is therapeutic benefit rom combining drugs such as rifampicin and UDCA hat target nuclear receptors that have coordinated effects n the transcriptional regulation of hepatobiliary excreory mechanisms.7 How does this occur? The hepatic clearance of bile acids and bilirubin can be ivided into 4 phases that include the following: phase 0, epatic uptake; phase I, metabolism (eg, hydroxylation); hase II, detoxification (eg, conjugation); and phase III, xcretion. Nuclear receptors and their ligands that are he major determinants of the functional expression of enes that determine these pathways are shown in Figure , together with enzymes that control bile acid synthesis. In the enterohepatic circulation, the hepatic uptake of onjugated bile acids (phase 0) is mediated predominantly y the sodium taurocholate cotransporting polypeptide TCP (SLC10A1), whereas unconjugated bile acid uptake s facilitated by several organic anion transporting polypepides (OATPs) on the sinusoidal membrane, but predomi-

Alterations of hepatic microsomal enzymes in the early phase of murine schistosomiasis

Schistosoma mansoni has been reported to cause a downregulation of hepatic cytochrome P450 activities after granulomas are formed around worm eggs harbored in the mouse liver. Only a few studies, however, provided data on the activity of xenobiotic-biotransaformation enzymes in the early phase of S. mansoni infection. In this study, we evaluated the alterations of liver microsomal enzymes during early infection (post-infection days, PIDs, 15 and 30) when granulomas are not found in the mouse liver yet. Swiss Webster (SW) and DBA/2 mice of either sex were infected with 100 S. mansoni cercariae on postnatal day 10. Levels of total-CYPs and activities of alkoxyresorufin- O-dealkylases (EROD, MROD, PROD and BROD), N-nitrosodimethylamine- N-demethylase (NDMA-d), coumarin 7-hydroxylase (COH, DBA/2 only) and UDP-glucuronosyltransferase (UGT) were measured in liver microsomes from mice killed on PIDs 15 and 30. Age-matched (sham-infected) mice of the same sex and strain were used as controls. Neither total-CYP levels nor microsomal enzyme activities were altered in SW and DBA/2 mice on PID 15. On PID 30, total-CYP levels, and COH, PROD and UGT activities remained unaltered, while gender- and strain-specific minor changes of EROD, MROD, BROD and NDMA-d (i.e., increase in SW and reduction in DBA/2) were found. In conclusion, our results suggest that, contrasting to a consistent and almost generalized downregulation of CYPs in chronic schistosomiasis, alterations of hepatic CYPs in early (acute) infection are isoform and mouse's gender and strain specific.

Genetic Polymorphisms of Human Flavin Monooxygenase 3 in Sulindac-Mediated Primary Chemoprevention of Familial Adenomatous Polyposis

Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) effective in regressing adenomas in patients with familial adenomatous polyposis (FAP). However, a recent randomized trial showed that sulindac, when compared with placebo, failed to prevent the development of adenomatous polyps in genotypically positive but phenotypically negative FAP patients. The present study determined whether polymorphisms in the gene encoding flavin monooxygenase 3 (FMO3), a hepatic microsomal enzyme that inactivates sulindac, played a role in determining the efficacy of sulindac in preventing polyposis in this cohort of FAP patients. Genotyping was performed on seven established FMO3 polymorphisms previously shown to have functional relevance-M66I, P153L, E158K, V257M, E305X, E308G, and R492W-in 21 and 20 FAP patients, who received sulindac and placebo, respectively. None of the 41 patients exhibited heterozygous or homozygous M66I and R492W variant alleles, or homozygous P153L, V257M, and E305X variant alleles. Among sulindac-treated patients who did not develop adenomas ("responders"), 4 (33%) were homozygous for E158K and 2 (17%) were homozygous for E308G variant alleles. In contrast, none of the patients on sulindac who developed adenomas ("nonresponders") exhibited homozygosity for either of the two variant alleles. In addition, polymorphisms in the E158K or E308G allele were associated with a significant reduction in mucosal prostanoid levels in patients treated with sulindac. Polymorphisms in FMO3, particularly at the E158K and E308G loci, may reduce activity in catabolizing sulindac and result in an increased efficacy to prevent polyposis in FAP.

Characterization of recombinant CYP2C11: a vitamin D 25-hydroxylase and 24-hydroxylase

Studies were performed to further characterize the male-specific hepatic recombinant microsomal vitamin D 25-hydroxlase CYP2C11, expressed in baculovirus-infected insect cells, and determine whether it is also a vitamin D 24-hydroxylase. 25- and 24-hydroxylase activities were compared with those of 10 other recombinant hepatic microsomal cytochrome P-450 enzymes expressed in baculovirus-infected insect cells. Each of them 25-hydroxylated vitamin D2, vitamin D3, 1alpha-hydroxyvitamin D2 (1alphaOHD2), and 1alpha-hydroxyvitamin D3 (1alphaOHD3). CYP2C11 had the greatest activity with these substrates, except vitamin D3, which had the same activity as four of the other enzymes. The descending order of 25-hydroxylation by CYP2C11 was 1alphaOHD3 > 1alphaOHD2 > vitamin D2 > vitamin D3. Each of the recombinant cytochrome P-450 enzymes 24-hydroxylated 1alphaOHD2. CYP2C11 had the greatest activity. 24-Hydroxylation of 1alphaOHD3 was very low, and there was none with vitamin D3. Only CYP2C11 24-hydroxylated vitamin D2. Structures of vitamin D metabolites, including 24-hydroxyvitamin D2, 1,24(S)-dihydroxyvitamin D2, and 1,24-dihydroxyvitamin D3, were confirmed by HPLC and gas chromatography retention times and characteristic mass spectrometric fragmentation patterns. In male rats, hypophysectomy significantly reduced body weight, liver weight, hepatic CYP2C11 mRNA expression, and 24- and 25-hydroxylation of 1alphaOHD2. Expression of CYP2J3 and CYP2R1 mRNA did not change. In male rat hepatocytes, CYP2C11 mRNA expression and 24- and 25-hydroxylation were significantly reduced after culture for 24 h compared with uncultured cells. Expression of CYP2J3 and CYP2R1 either increased or did not change. It is concluded that CYP2C11 is a male-specific hepatic microsomal vitamin D 25-hydroxylase that hydroxylates vitamin D2, vitamin D3, 1alphaOHD2, and 1alphaOHD3. CYP2C11 is also a vitamin D 24-hydroxylase.

CYP3A4 Is a Vitamin D-24- and 25-Hydroxylase: Analysis of Structure Function by Site-Directed Mutagenesis

Studies were performed to identify the microsomal enzyme that 24-hydroxylates vitamin D, whether 25-hydroxylation occurs, and structure function of the enzyme. Sixteen hepatic recombinant microsomal cytochrome P450 enzymes expressed in baculovirus-infected insect cells were screened for 24-hydroxylase activity. CYP3A4, a vitamin D-25-hydroxylase, and CYP1A1 had the highest 24-hydroxylase activity with 1 alpha-hydroxyvitamin D(2) (1 alpha OHD(2)) as substrate. The ratio of rates of 24-hydroxylation of 1 alpha-hydroxyvitamin D(3) (1 alpha OHD(3)), 1 alpha OHD(2), and vitamin D(2) by CYP3A4 was 3.6/2.8/1.0. Structures of 24-hydroxyvitamin D(2), 1,24(S)-dihydroxyvitamin D(2), and 1,24-dihydroxyvitamin D(3) were confirmed by HPLC and gas chromatography retention time and mass spectroscopy. In characterized human liver microsomes, 24-hydroxylation of 1 alpha OHD(2) by CYP3A4 correlated significantly with 6 beta-hydroxylation of testosterone, a marker of CYP3A4 activity. 24-Hydroxylase activity in recombinant CYP3A4 and pooled human liver microsomes showed dose-dependent inhibition by ketoconazole, troleandomycin, alpha-naphthoflavone, and isoniazid, known inhibitors of CYP3A4. Rates of 24- and 25-hydroxylation of 1 alpha OHD(2) and 1 alpha OHD(3) were determined in recombinant wild-type CYP3A4 and site-directed mutants and naturally occurring variants expressed in Escherichia coli. Substitution of residues showed the most prominent alterations of function at residues 119, 120, 301, 305, and 479. Thus, CYP3A4 is both a 24- and 25-hydroxylase for vitamin D(2), 1 alpha OHD(2), and 1 alpha OHD(3).

Blarina brevicauda as a biological monitor of polychlorinated biphenyls: evaluation of hepatic cytochrome P450 induction.

We assessed the value of short-tailed shrews (Blarina brevicauda) as a possible biomonitor for polychlorinated biphenyl pollution through measurement of the induction of hepatic cytochrome P450 and associated enzyme activities. First, we checked the inducibility of four monooxygenases (benzyloxyresorufin-O-dealkylase [BROD], ethoxyresorufin-O-dealkylase [EROD], methoxyresorufin-O-dealkylase [MROD], and pentoxyresorufin-O-dealkylase [PROD]) by measuring the activity of these enzymes in hepatic microsomes prepared from shrews injected with beta-naphthoflavone (betaNF) or phenobarbital (PB), typical inducers of cytochrome P4501A (CYP1A) and CYP2B enzyme families, respectively. Enzyme activity was induced in shrews that received betaNF but not in shrews that received PB; PROD was not induced by either exposure. Later, shrews were exposed to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1242:1254, in 1:2 ratio) at 0.6, 9.6, and 150 ppm in food, for 31 d. Induction in these shrews was measured by specific enzyme activity (BROD, EROD, and MROD) in hepatic microsomes, by western blotting of solubilized microsomes against antibodies to CYP1A or CYP2B, and by duration of sodium pentobarbital-induced sleep. These three CYP enzymes were induced in shrews by PCBs at similar levels of exposure as in cotton rat (Sigmodon hispidus). Neither sleep time nor the amount of CYP2B family protein were affected by PCB exposure. Blarina brevicauda can be a useful biomonitor of PCBs that induce CYP1A, especially in habitats where they are the abundant small mammal.

CYP3A4 is a human microsomal vitamin D 25-hydroxylase.

The human hepatic microsomal vitamin D 25-hydroxylase protein and gene have not been identified with certainty. Sixteen hepatic recombinant microsomal enzymes were screened for 25-hydroxylase activity; 11 had some 25-hydroxylase activity, but CYP3A4 had the highest activity. In characterized liver microsomes, 25-hydroxylase activity correlated significantly with CYP3A4 testosterone 6beta-hydroxylase activity. Activity in pooled liver microsomes was inhibited by known inhibitors of CYP3A4 and by an antibody to CYP3A2. Thus, CYP3A4 is a hepatic microsomal vitamin D 25-hydroxylase. Studies were performed to identify human microsomal vitamin D-25 hydroxylase. Sixteen major hepatic microsomal recombinant enzymes derived from cytochrome P450 cDNAs expressed in baculovirus-infected insect cells were screened for 25-hydroxylase activity with 1alpha-hydroxyvitamin D2 [1alpha(OH)D2], 1alpha-hydroxyvitamin D3 [1alpha(OH)D3], vitamin D2, and vitamin D3 as substrates. Activity was correlated with known biological activities of enzymes in a panel of 12 characterized human liver microsomes. The effects of known inhibitors and specific antibodies on activity also were determined. CYP3A4, the most abundant cytochrome P450 enzyme in human liver and intestine, had 7-fold greater activity than that of any of the other enzymes with 1alpha(OH)D2 as substrate. CYP3A4 25-hydroxylase activity was four times higher with 1alpha(OH)D2 than with 1alpha(OH)D3 as substrate, was much less with vitamin D2, and was not detected with vitamin D3. 1alpha(OH)D2 was the substrate in subsequent experiments. In a panel of characterized human liver microsomes, 25-hydroxylase activity correlated with CYP3A4 testosterone 6beta-hydroxylase activity (r = 0.93, p < 0.001) and CYP2C9*1 diclofenac 4'-hydroxylase activity (r = 0.65, p < 0.05), but not with activity of any of the other enzymes. Activity in recombinant CYP3A4 and pooled liver microsomes was dose-dependently inhibited by ketoconazole, troleandomycin, isoniazid, and alpha-naphthoflavone, known inhibitors of CYP3A4. Activity in pooled liver microsomes was inhibited by antibodies to CYP3A2 that are known to inhibit CYP3A4 activity. CYP3A4 is a vitamin D 25-hydroxylase for vitamin D2 in human hepatic microsomes and hydroxylates both 1alpha(OH)D2 and 1alpha(OH)D3.

Comparison of liver mixed-function oxygenase and antioxidant enzymes in vertebrates

We performed a comparative analysis of cytochrome P450, cytochrome b 5, MFO associated enzymes and cytosolic antioxidant enzymes in hepatic microsomes and cytosolic fractions prepared from five animal species representing three vertebrate classes living in tropical conditions (Brazil). The data obtained show that rats have higher hepato-somatic index, specific cytochrome b 5 concentration, and NADPH-dependent cytochrome c (P450) activity compared to ectothermic species, SOD activity similar to those in amphibians, and specific concentration of cytochrome P450 and catalase activity lower than in a toad, but higher than in fishes and a frog. Our data indicate that tropical fishes may have reduced xenobiotic-metabolizing ability compared to the rat and amphibians. In contrast to fish and rat, amphibians have a low ratio (<0.5) of cytochrome b 5 concentration to that of P450. Most species showed cytochrome b 5 sensitivity to oxygen. Thus, the use of sodium dithionate as a reducer, rather than NADPH, may be preferential in b 5 determinations.

Spectral interactions of tetrachlorobiphenyls with hepatic microsomal cytochrome P450 enzymes

This study investigated the spectral interactions of hepatic microsomal cytochrome P450 (CYP) enzymes with four symmetrical polychlorinated biphenyls (PCBs): 2,2′,4,4′-tetrachlorobiphenyl (PCB 47); 2,2′,5,5′-tetrachlorobiphenyl (PCB 52); 2,2′,6,6′-tetrachlorobiphenyl (PCB 54); and 3,3′,4,4′-tetrachlorobiphenyl (PCB 77). The PCBs were selected to explore structure–activity relationships and the effect of the chlorination pattern on PCB–CYP spectral interactions. To examine CYP enzyme specificity, difference spectra were measured with hepatic microsomes prepared from control, phenobarbital (PB)-, 3-methylcholanthrene (MC)-, and dexamethasone (DEX)-treated rats in the absence and presence of CYP-specific antibodies. The four PCB congeners elicited a type I spectral change with all hepatic microsomal preparations. The binding efficiency of the PCBs was highest with microsomes from PB-treated rats. The largest absorbance change and highest binding efficiency were observed with PCB 54, the most non-coplanar congener tested. Antibody inhibition and CYP immunoquantitation data showed that the PCBs bind to CYP1A, CYP2B, CYP2C and CYP3A enzymes to varying degrees. For example, PCB 47, 52, and 54 bind preferentially to CYP2B and to a lesser extent to CYP3A enzymes in microsomes from PB-treated male rats; PCB 52 binds primarily to CYP3A enzymes in microsomes from DEX-treated female rats; and PCB 54 binds to CYP3A and to CYP2C enzymes in microsomes from control male rats. The study demonstrated that the extent of PCB–CYP binding interaction was dependent on the chlorination pattern of the PCB and on the relative abundance of individual CYP enzymes in hepatic microsomes.