CYP3A Subfamily Research Articles

Cytochrome P450 isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of NS-21

1. Cytochrome P450 (P450) isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of (±)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2- hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21) have been identified in rat and man. 2. Anti-CYP2C11 antibody inhibited the N-deethylation of S- and R-NS-21 in rat hepatic microsomes by 84 and 66% respectively, indicating that CYP2C11 is mainly responsible for these activities in male rats. 3. Of several human recombinant P450 isoforms, CYP3A4 had the activities for the Ndeethylation of S- and R-NS-21. In addition, triacetyloleandomycin (TAO), an inhibitor of the CYP3A subfamily, significantly inhibited the N-deethylation of S- and R-NS-21 in human hepatic microsomes by 67 and 69%, respectively. CYP3A4 therefore contributes to it in man. 4. Quinine, an inhibitor of the rat CYP2D subfamily, significantly inhibited the cyclohexane-4-cis-hydroxylation of S-NS-21 by 48% in rat hepatic microsomes. In contrast, this inhibitor had little effect on the cyclohexane-4-trans-hydroxylation of S-NS21, and the cyclohexane-4-cis-and trans-hydroxylation of R-NS-21. 5. Human recombinant CYP3A4 catalysed the cyclohexane-4-trans-hydroxylation of S-NS-21, and CYP2D6 supported the cyclohexane-4-cis- and trans-hydroxylation of S-NS-21. Quinidine, an inhibitor of human CYP2D6, had little effect on these latter activities in human hepatic microsomes. TAO significantly inhibited the cyclohexane-4-trans-hydroxylation of S-NS-21 by 75%, indicating that CYP3A4 catalyses this reaction.

Biotransformation of alprazolam by members of the human cytochrome P4503A subfamily

1. To aid in the prediction of drug interactions with alprazolam, the human CYP involved in the 1'- and 4-hydroxylation of alprazolam were characterized using human liver microsomes, expressed enzymes and selective chemical inhibitors. 2. The formation of 4-hydroxyalprazolam and 1-hydroxyalprazolam at an alprazolam concentration of 62.5 μM were reduced by the prototypic CYP3A inhibitor, troleandomycin (50 μM), by 97 and 99% respectively. Only microsomes from B-lymphoblastoid cells expressing CYP3A4 were capable of catalysing the 1'- and 4-hydroxylation of alprazolam. 3. The formation rates of 1'-hydroxyalprazolam and 4-hydroxyalprazolam at an alprazolam concentration of 1 mM were significantly correlated (n=19, r=0.95, p<0.01) indicating that the same enzyme(s) mediated these biotransformations. A significant (p<0.01) correlation was observed between alprazolam 4- and 1'-hydroxylase activity and CYP3A-mediated midazolam 4-hydroxylase, midazolam 1'-hydroxylase, dextromethorphan N-demethylase and erythromycin N-demethylase activities. 4. In conclusion, in adult human liver the CYP3A subfamily members are the principal enzymes involved in the 1'- and 4-hydroxylation of alprazolam. Thus, clinically significant drug-drug interactions between alprazolam and other CYP3A substrates are to be expected.

Cloning, Sequencing, and Tissue Expression of CYP3A27, a New Member of the CYP3A Subfamily from Embryonic and Adult Rainbow Trout Livers

Screening of λgt11 and λgt22A cDNA libraries of livers from adult females and embryos of rainbow trout (Oncorhynchus mykiss), respectively, using rabbit anti-rainbow trout cytochrome P450 LMC5 polyclonal antibodies showed that there were identical cDNAs of 1802-bp nucleotides with open reading frames coding for proteins containing 518 amino acids (59,206 Da, pI= 6.39). The cDNA was assigned CYP3A27 by the P450 Nomenclature Committee to represent the first CYP3A subfamily member reported for aquatic species. The deduced N-terminal sequence of CYP3A27 was in agreement with 8 of the first 12 confirmed amino acid residues from Edman degradation of LMC5, a P450 previously isolated from juvenile trout liver. In similarity comparisons between species by positional alignment, the deduced amino acid sequence of rainbow trout CYP3A27 was 56.4% identical with dog CYP3A12, 56.0% with monkey CYP3A8, 54.9% with human CYP3A4, 54.7% with rat CYP3A9, and 54.2% with sheep CYP3A24. Marked differences in sex, age, and tissue expression of CYP3A27 in rainbow trout were observed at the mRNA level as shown by Northern blots. The major extrahepatic expression site for CYP3A27 was upper small intestine. Females expressed considerably more CYP3A27 mRNA than male in the fish examined. Southern blot analysis of restriction enzyme-digested rainbow trout genomic DNA demonstrated that multiple CYP3A27-related genes exist in rainbow trout.

Tiagabine: a novel antiepileptic drug.

To provide a comprehensive review of tiagabine, including its pharmacology, toxicology, pharmacokinetics, drug interactions, efficacy, adverse effects, and dosing recommendations. A computerized search of the MEDLINE database from 1966 to December 1997 was used to identify publications related to tiagabine and nipecotic acid derivatives. Included in this review was information gathered from scientific meetings. Manufacturer's information was used when there was no primary literature. Tiagabine amplifies gamma-aminobutyric acid (GABA) neurotransmission, the predominant inhibitory neurotransmitter in the brain. Its mechanism of action is selective and has shown promise as an antiepileptic drug (AED) in patients with seizures refractory to other pharmaceutical products. Tiagabine exhibits dose-independent absorption, 90-95% bioavailability, high protein binding (96%), metabolism via hepatic cytochrome P450 enzymes (CYP3A subfamily), and displays first-order elimination pharmacokinetics. The mean plasma half-life is 5-8 hours. Concomitant medications that induce hepatic metabolism enhance tiagabine elimination; metabolism is reduced in patients with hepatic dysfunction. Adverse events of tiagabine typically involve the central nervous system, have been mild to moderate in intensity, and also have been transient in nature. Tiagabine has demonstrated a good safety profile and, while it has not been demonstrated to be superior to other second-line AEDs for partial seizures, its safety and select mechanism of action warrant its further evaluation in the clinical setting. Tiagabine should be a good alternative add-on agent for patients with unsatisfactory seizure control or intolerable adverse effects of traditional therapies; thus, this agent should be made available to these patients.

Molecular cloning of CYP1A from the estuarine fish Fundulus heteroclitus and phylogenetic analysis of CYP1 genes: update with new sequences

Since we published a phylogenetic analysis of the CYP1A subfamily in 1995, several additional full-length sequences have been reported, including three members of an entirely new subfamily, CYP1B. Two avian sequences were recently published, so that CYP1A sequence data are now available from three of the five major vertebrate lineages. The two new branches that have been added to the CYP1 family tree significantly add to our understanding of P450 evolution. The inclusion of the CYP1Bs to the phylogenetic analysis allows us to root inferred trees. Addition of the avian CYP1As indicates that the CYP1A1/CYP1A2 duplication present in the mammalian lineage may have occurred after the divergence of birds and mammals. The number of fish species from which full-length coding regions of CYP1A genes have been sequenced has increased from four (trout, plaice, toadfish, and scup) to nine. These include CYP1A sequences from tomcod, butterflyfish, sea bream, sea bass, and the full-length sequence of CYP1A from the killifish Fundulus heteroclitus that is reported here. Phylogenetic analyses incorporating the new fish CYP1A sequences support our original conclusion that the fish CYP1As are monophyletic and indicate that the genes are evolving at very different rates in different species.

In-vitro cytochrome P450 dependent metabolism of oxybutynin to N-deethyloxybutynin in humans.

Oxybutynin (Ditropan), a direct antagonist of acetylcholine on muscarinic receptors, is administered in children for the treatment of vesical immaturity and is responsible for atropinic adverse effects, which are more frequent in paediatric than in adult patients. Oxybutynin is metabolized by oxidation to N-desethyloxybutynin, a stable and toxic metabolite, and previous results in vivo have suggested that cytochrome P450 2D6 (CYP2D6) may be involved in this pathway of metabolism. We used human liver microsomes genotyped as extensive metabolizers for CYP2D6 to determine the kinetic parameters of N-desethyloxybutynin formation: Km was 16.5+/-5.2 microM and Vmax was 76.8+/-3.7 mmol/mg/h. Quinidine and anti-liver kidney microsome antibody type I had no inhibitory effects on N-desethyloxybutynin formation, demonstrating that CYP2D6 has no role in oxybutynin metabolism. The effects of specific inhibitors of other cytochromes P450 were also investigated. Recombinant human CYP 3A4 but not 2B6, 2D6, 2C8 and 2E1, displayed significant N-desethylation activity. Our results demonstrate that the CYP3A subfamily, and not CYP2D6, is involved in N-desethyloxybutynin formation.

Stereoselective metabolism of bisoprolol enantiomers in dogs and humans

To clarify the mechanism of the species difference in the metabolism of bisoprolol enantiomers, in vitro metabolic studies were performed using dog liver microsomes and human cytochrome P450 (CYP) isoforms. The O-deisopropylation of bisoprolol enantiomers showed biphasic kinetics in dog liver microsomes. The intrinsic clearance (Vmax/Km) for O-deisopropylation of R(+)-bisoprolol was higher than S(−)-isomer in both high-affinity and low-affinity components. The R S ratio of the intrinsic clearance in high- and low-affinity components was 1.34 and 1.65, respectively. The inhibition studies in dog liver microsomes using CYP isoform-selective inhibitors indicated that the O-deisopropylation of both bisoprolol enantiomers was mediated via the CYP2D and CYP3A subfamily, and suggested that high-affinity oxidation was dependent on CYP2D. The kinds of CYP subfamilies in dogs, which contribute to the metabolism of bisoprolol enantiomers, were the same as those in humans. The intrinsic clearance for O-deisopropylation of R(+)-bisoprolol by human recombinant CYP2D6 was also different from that of S(−)-enantiomers ( R S : 1.50). However, unlike the dog microsomes, the intrinsic clearance by the human recombinant CYP3A4 did not show a stereoselective difference. Therefore, the species difference in the R S ratio of metabolic clearance for the oxidation of bisoprolol enantiomers (dog > human) is mainly due to the species difference in the stereoselectivity of one of the cytochrome P450 subfamilies (CYP3A).

Evidence for Involvement of cAMP-Dependent Pathway in the Phenobarbital-Induced Expression of a Novel Hamster Cytochrome P450, CYP3A31

Recently, we isolated a novel Syrian hamster cDNA clone that encodes a protein which has been named CYP3A31. In primary hepatocyte cultures, CYP3A31 is dramatically induced by phenobarbital. To elucidate the mechanism of this induction, we first studied the effects of cAMP on phenobarbital-induced CYP3A31 expression using forskolin andN6,O2′-dibutyryl cAMP in hepatocyte cultures. At 100 μM, forskolin significantly inhibited both the phenobarbital-induced CYP3A31 mRNAs expression and the testosterone 6β-hydroxylation activity related to the CYP3A subfamily in rats, whereas 0.1 μM forskolin potentiated the phenobarbital induction of CYP3A31 mRNA and the testosterone 6β-hydroxylation activity. Treatment withN6,O2′-dibutyryl cAMP resulted in an inhibition of phenobarbital-inducedCYP3A31gene expression and testosterone 6β-hydroxylation activity. Increasing amounts of transfected cAMP-response element binding proteins (CREB) or CREB-binding proteins in hamster hepatocytes reduced the phenobarbital-induction of CYP3A31 mRNAs expression. These results suggest thatin vitroinduction of CYP3A31 by phenobarbital in Syrian hamster hepatocytes is regulated by a cAMP-dependent pathway.

The Roles of CYP3A and CYP2B Isoforms in Hepatic Bioactivation and Detoxification of the Pyrrolizidine Alkaloid Senecionine in Sheep and Hamsters

The roles of cytochrome CYP3A and CYP2B isozymes in the bioactivation and detoxification of the pyrrolizidine alkaloid (PA) senecionine (SN) have been investigatedin vitrowith sheep and hamster hepatic microsomes. Our results show that the rate of SN activation measured by (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) formation greatly exceeded the rate of SNN-oxide formation (detoxification) in hamsters. In contrast, SNN-oxide, a detoxification product, was the major metabolite in sheep with much lower DHP production. Immunoinhibition studies with anti-sheep CYP3A and CYP2B antibodies show that members of CYP3A subfamily play the major role in the conversion of PA to pyrrolic metabolites in both species (over 90% in sheep; 68% in hamster). These enzymes also contribute 38.8 and 41.3% of SN N-oxidation in sheep and hamsters, respectively. In contrast, CYP2B isoforms have a limited capacity toward DHP formation in both species (47% in sheep; 32% in hamster), while these enzymes catalyzed only 24.6 and 35.4% SN N-oxidation in sheep and hamster, respectively. Using triacetyloleandomycin (TAO) and gestodene, two highly selective chemical inhibitors of CYP3A isoforms, our data show that 90% of DHP formation was inhibited by either inhibitor in sheep. Gestodene appeared to be more efficient than TAO in the inhibition of DHP production in hamsters. Testosterone 6β-hydroxylase activity, a functional marker of CYP3A, was significantly inhibited by TAO and gestodene in sheep liver microsomes and by gestodene (100 μM) in hamster liver microsomes. These results suggest that CYP3A isozymes have important roles in bioactivation and detoxification of PA in both species, whereas CYP2B subfamily members are less efficient in biotransformation of PA.

OP2E147 - Effect of tobacco smoke on the expression of Cyp1a, Cyp2b, Cyp2e, and Cyp3a subfamilies in mice. Possiblerole of Cyp2e1 in pulmonary DNA single strand breaks induced by smoking

OP2E147 - Effect of tobacco smoke on the expression of Cyp1a, Cyp2b, Cyp2e, and Cyp3a subfamilies in mice. Possiblerole of Cyp2e1 in pulmonary DNA single strand breaks induced by smoking

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 β-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.

Strain Differences in CYP3A-Mediated C-8 Hydroxylation (1,3,7-Trimethyluric Acid Formation) of Caffeine in Wistar and Dark Agouti Rats: Rapid Metabolism of Caffeine in Debrisoquine Poor Metabolizer Model Rats

We observed significant strain differences [Dark Agouti (DA) > Wistar] in 1,3,7-trimethyluric acid formation (C-8 hydroxylation) during caffeine metabolism, though not in N-demethylations, in adult male DA and Wistar rats. In contrast, adult female and immature male rats of both DA and Wistar strains did not show significant differences in activity levels of C-8 hydroxylation. Kinetic studies using liver microsomes revealed that adult male DA rats have a larger V max for C-8 hydroxylation than do Wistar rats. Troleandomycin (TAO), known as a cytochrome P450 (CYP) 3A inhibitor, and an anti-rat CYP3A2 polyclonal antibody effectively reduced C-8 hydroxylation by rat liver microsomes in a concentration-dependent manner, suggesting that C-8 hydroxylation in rats is mediated largely by an isoform(s) of the CYP3A subfamily. Troleandomycin and the antibody did not inhibit the N-demethylations of caffeine by rat liver microsomes. Treatment of rats with CYP3A inducers caused a marked increase in C-8 hydroxylase activity. These results indicate that the rat CYP3A subfamily is capable of catalyzing C-8 hydroxylation of caffeine as is the case for human CYP3A4. The results of western blotting analysis using anti CYP3A antiserum showed that the staining intensity of the protein band in DA rat liver microsomes was higher than that in Wistar rat liver microsomes. We concluded that marked sex-dependent strain differences in C-8 hydroxylation of caffeine between Wistar and DA rats are due to the differences in the levels of expression of CYP3A in these strains of rats.

Molecular cloning and regulation of a novel guinea pig cytochrome P450 (CYP3A20) which differs from guinea pig CYP3A14 in only two amino acid residues.

A full length cDNA clone of cytochrome P450, encoding 503 amino acid residues, was isolated from a male guinea pig liver cDNA library. The sequence was highly homologous to other members in the CYP3A subfamily and was designated CYP3A20. CYP3A20 and CYP3A14, another guinea pig CYP3A, shared 99.4% nucleotide and 99.6% deduced amino acid sequence homology. There were only two amino acid differences between CYP3A20 and CYP3A14. No significant induction of CYP3A20 mRNA in the livers from male guinea pigs treated with dexamethasone was observed by S1 mapping analysis although CYP3A14 mRNA was induced. The expression of CYP3A20 mRNA in the livers did not change between 5 and 10 weeks after birth while that of CYP3A14 mRNA in livers significantly increased at 10 weeks. This is the first report that the two highly resembling forms of cytochrome P450 display differently regulated expression from each other.

Developmental expression of cytochrome P450 enzymes in human liver.

Drug-metabolizing cytochrome P450 enzymes, the major phase I enzymes, are active in human liver already at very early stages of intrauterine development, although presumably at fairly low concentrations and in low numbers. During maturation, these enzymes go through various developmental programmes towards adulthood. The major increase both in abundance as well as in number of different enzymes takes place after birth, probably during the first year of life. Detailed information concerning these developmental changes is still limited. The major drug-metabolizing P450 enzymes appear to be primarily members of the CYP3A subfamily in all stages of development. The balance between different members of this subfamily, however, undergoes significant switches from the foetal predominant CYP3A7 to the major adult form CYP3A4. The ontogeny of the other cytochrome P450 enzymes is less well characterized, but the major switch-on appears to occur mainly after birth. Developmental expression of P450 enzymes is one of the key factors determining the pharmacokinetic status of developing individuals both pre- and postnatally.

Cloning and Characterization of Syrian Hamster Testosterone 7α-Hydroxylase, CYP2A9

We report here the cloning of a cDNA encoding testosterone 7α-hydroxylase in the Syrian hamster, designated CYP2A9. Overlapping clones encoding Syrian hamster CYP2A9 were isolated by screening a liver cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction on the cDNA library. The sequence of the CYP2A9 cDNA contains an open reading frame of 1482 nucleotides encoding a polypeptide of 493 amino acids with a calculated molecular mass of 56,295 Da. The sequence is flanked by a 5′-untranslated region of 10 bp and a 3′-untranslated region of 178 bp including the poly(A) tail. The deduced amino acid sequence shares significant homology with members of CYP2A subfamily, notably with CYP2A1 and CYP2A12 which have testosterone 7α-hydroxylase activity. We characterized the catalytic activity of CYP2A9 using microsomes obtained by transient expression of its cDNA in transfected COS-7 cells. CYP2A9 was found to hydroxylate testosterone at position 7α. In Syrian hamster livers, a higher level of testosterone 7α-hydroxylase activity as well as the mRNA of CYP2A9 in male than in female was obtained. The testosterone 7α-hydroxylase activity and the mRNA level in liver were both decreased moderately by administration of 3-methylcholanthrene and slightly by administration of phenobarbital. In contrast, in kidney, both 3-methylcholanthrene and phenobarbital significantly decreased the mRNA level. These facts indicate that the regulation of the hamster testosterone 7α-hydroxylase (CYP2A9) expression is different from that of the rat (CYP2A1) and hamster reported previously by other workers.

Mapping of porcine genes belonging to two different cytochrome P450 subfamilies.

Three cDNA clones from a porcine small intestine cDNA library, tentatively identified as transcripts of two different cytochrome P450 (CYP) gene subfamilies, were sequenced and mapped. One of the clones was found to comprise an open reading frame of 503 amino acids and showed up to an 80% similarity to genes from the CYP3A subfamily. Translation of the other two cDNA clones revealed high similarity to genes from the CYP2C subfamily. One of these clones was truncated, lacking a part of the 5' end of the coding region, whereas the other encodes a putative pseudogene. The CYP3A gene was mapped cytogenetically by means of a pig/rodent somatic hybrid cell panel to pig chromosome 3, in the region p16-p17 or p11. The CYP2C pseudogene was mapped genetically to pig chromosome 14, using the PiGMaP shared reference pedigrees. The localisations provide information for the human/pig comparative map.

Sexually Dimorphic Expression of Rat CYP3A9 and CYP3A18 Genes Is Regulated by Growth Hormone

The cDNAs for two CYP3A genes were isolated from the livers of rats using an RT-PCR approach with CYP3A subfamily-specific primers. Sequence analysis revealed these cDNAs to be identical to CYP3A9, which had previously been isolated from rat brain and nasal epithelium and the recently described CYP3A18. The hepatic expression of both genes was sexually dimorphic. Thus CYP3A18 mRNA levels were 25-fold higher in male livers compared to females, while CYP3A9 showed a reverse pattern with 6-fold higher expression in the liver of females. Exposure of male rats to the female pattern of growth hormone secretion led to an increase in hepatic CYP3A9 mRNA expression and suppressed expression of CYP3A18. These findings indicate that the CYP3A subfamily in rats has both male- and female-specific isoforms which are regulated by growth hormone in a manner similar to some other sexually dimorphic cytochrome P450s.

Drug Metabolizing Enzymes in Primates: Focused on Cytochrome P450.

To date, it is very difficult to extrapolate the data obtained from experimental animals (for example rodents) to humans, since there are species differences in the characteristics of drug metabolising enzymes. The monkeys is often used for the preclinical study. However, the similarities for properties and capacities of drug metabolizing enzymes between monkeys and humans were not well recognized. Therefore, we started to characterize the drug metabolizing enzymes, especially cytochrome P450s, in non-human primates. Four forms of cytochrome P450 enzymes (termed P450 CMLa-d) were purified from hepatic microsomes of cynomolgus monkeys. P450 CMLa was classified into the CYP2B subfamily, in its N-terminal amino acid sequence, catalytic activities, and immunochemical properties and act as a testosterone 16, 8-hydroxylase in cynomolgus monkseys. P450 CMLa is expressed constitutively as a minor form of P450 and is one of the forms inducible by phenobarbital. The nucleotide sequence of a cDNA coding for the monkey CYP2B was also determined and is now called CYP2B17. P450 CMLb and P450 CMLc purified from hepatic microsomes of cynomolgus monkeys were classified into CYP2A and CYP3A subfamilies, respectively. The content of the protein immunoreactive with anti-P450 CMLc in cynomolgus monkeys was decreased by the administration of aztreonam. P450 CMLd was one of CYP2C enzymes and was the S-mephenytoin 4'-hydroxylase in cynomolgus monkeys. The cDNA coding for monkey CYP2C (reffered to as CYP2C37) was isolated and was expressed in yeast. The expressed protein act as S-mephenytoin 4'-hydroxylase.

3,3′-Diindolylmethane induces CYP1A2 in cultured precision-cut human liver slices

1. The effect of 3,3′-diindolylmethane (DIM), an indole derivative derived from cruciferous vegetables, on cytochrome P450 (CYP) isoforms in the CYP1A and CYP3A subfamilies has been studied in 72-h cultured human liver slices. 2. In cultured human liver slices 50 μM DIM induced 7-ethoxyresorufin O-deethylase and to a lesser extent 7-methoxyresorufin O-demethylase activities. 3. Western immunoblotting of liver slice microsomes was performed with antibodies to ratCYP1A2 and human CYP3A4. Compared with control liver slice microsomes (dimethyl sulphoxide-only treated), DIM induced levels of CYP1A2 but had little effect on levels of CYP3A4. The treatment of human liver slices with 2 μg ml of the polycholorinated biphenyl mixture Aroclor 1254 also resulted in an induction of levels of CYP1A2, but had no effect on CYP3A4. 4. These results demonstrate that DIM induces CYP1A isoforms in cultured human liver slices. Some variability in the magnitude of induction of enzyme activities by DIM was observed in four human liver samples examined. For 7-ethoxyresorufin O-deethylase, the magnitude of induction by 50 μM DIM ranged from 2.3- to 19.3-fold. 5. These results demonstrate that cultured human liver slices can be used to evaluate the effect of chemicals derived from cruciferous and other vegetables on CYP isoforms.

Novobiocin inhibits both UDP-glucuronosyltransferase and cytochrome P450-mediated enzyme activities in pig liver microsomes.

The effects of novobiocin (range 0.0125-2 mmol/L) on the hydroxylation of testosterone, the N-demethylation of erythromycin, and the glucuronidation of alpha-naphthol and paracetamol were studied using pig hepatic microsomes, pooled from five animals. The final concentrations of these substrates in the incubation mixtures were selected to meet Vmax conditions. Novobiocin caused a concentration-dependent inhibition of the glucuronidation of paracetamol; the formation of alpha-naphthol-glucuronide was reduced to a lesser degree. These results confirm and extend earlier findings in laboratory animal species that novobiocin inhibits UDP-glucuronosyltransferases (UDPGTs). Moreover, novobiocin strongly inhibited 6 beta-hydroxylation of testosterone. The microsomal N-demethylation of erythromycin and hydroxylation of testosterone at the 15 alpha position were less affected by novobiocin. These results suggest that novobiocin inhibits not only UDPGTs, but also cytochrome P450 (CYP) enzyme activities, probably those belonging to the CYP3A subfamily. More research is needed to reveal which CYPs and UDPGTs are affected by novobiocin in vivo, in order to improve the understanding the probably the predictability of potential drug interactions with this antibiotic.