Identification of inhibitors of the nicotine metabolising CYP2A6 enzyme--an in silico approach.

The compulsive nature of tobacco use is attributable to nicotine addiction. Nicotine is eliminated by metabolism through the cytochrome P450 2A6 (CYP2A6) enzyme in liver. Inhibition of CYP2A6 by chemical compounds may represent a potential supplement to anti-smoking therapy. The purpose of this study was to rationally design potent inhibitors of CYP2A6. 3D-QSAR models were constructed to find out which structural characteristics are important for inhibition potency. Specifically located hydrophobic and hydrogen donor features were found to affect inhibition potency. These features were used in virtual screening of over 60,000 compounds in the Maybridge chemical database. A total of 22 candidate molecules were selected and tested for inhibition potency. Four of these were potent and selective CYP2A6 inhibitors with IC(50) values lower than 1 muM. They represent novel structures of CYP2A6 inhibitors, especially N1-(4-fluorophenyl)cyclopropane-1-carboxamide. This compound can be used as a lead in the design of CYP2A6 inhibitor drugs to combat nicotine addiction.

New potent and selective cytochrome P450 2B6 (CYP2B6) inhibitors based on three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis.

BACKGROUND AND PURPOSE: The cytochrome P450 2B6 (CYP2B6) enzyme metabolises a number of clinically important drugs. Drug-drug interactions resulting from inhibition or induction of CYP2B6 activity may cause serious adverse effects. The aims of this study were to construct a three-dimensional structure-activity relationship (3D-QSAR) model of the CYP2B6 protein and to identify novel potent and selective inhibitors of CYP2B6 for in vitro research purposes. EXPERIMENTAL APPROACH: The inhibition potencies (IC(50) values) of structurally diverse chemicals were determined with recombinant human CYP2B6 enzyme. Two successive models were constructed using Comparative Molecular Field Analysis (CoMFA). KEY RESULTS: Three compounds proved to be very potent and selective competitive inhibitors of CYP2B6 in vitro (IC(50)<1 microM): 4-(4-chlorobenzyl)pyridine (CBP), 4-(4-nitrobenzyl)pyridine (NBP), and 4-benzylpyridine (BP). A complete inhibition of CYP2B6 activity was achieved with 0.1 microM CBP, whereas other CYP-related activities were not affected. Forty-one compounds were selected for further testing and construction of the final CoMFA model. The created CoMFA model was of high quality and predicted accurately the inhibition potency of a test set (n=7) of structurally diverse compounds. CONCLUSIONS AND IMPLICATIONS: Two CoMFA models were created which revealed the key molecular characteristics of inhibitors of the CYP2B6 enzyme. The final model accurately predicted the inhibitory potencies of several structurally unrelated compounds. CBP, BP and NBP were identified as novel potent and selective inhibitors of CYP2B6 and CBP especially is a suitable inhibitor for in vitro screening studies.

Effect of polycyclic aromatic compounds and phorbol esters on ornithine decarboxylase and aryl hydrocarbon hydroxylase activities in mouse liver.

Single i.p. injections of 3-methylcholanthrene (MC; 50 mg/kg) administered to inbred C57BL/6 mice or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 micrograms/kg) to DBA/2 mice gave an increase in the hepatic activities of ornithine decarboxylase (ODC) and aryl hydrocarbon hydroxylase (AHH) with peaks occurring by 12 and 48 hr, respectively. A single i.p. dose of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 micrograms/kg) enhanced the activity of ODC about 70-fold within 12 hr in C57BL/6 mice and 18-fold within 24 hr in DBA/2 mice without affecting AHH activity markedly. 4-O-Methyl-12-O-tetradecanoylphorbol-13-acetate (100 micrograms/kg) raised ODC activity to about 25% of the TPA-treated value in C57BL/6 mice; in DBA/2 mice, TPA and 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate induced ODC activity to roughly the same level. Benzo(e)pyrene (50 mg/kg) failed to affect ODC and AHH activities significantly in either strain. The inducing effect of TPA on ODC activity was potentiated by a simultaneous administration of MC to C57BL/6 mice; combined TPA and TCDD to DBA/2 mice exerted an additive effect on hepatic ODC activity. Difluoromethylornithine administered i.p. effectively inhibited the induction of ODC activity elicited by TPA, MC, or TCDD either alone or in various combinations but did not interfere with AHH induction. These data indicate that different regulatory factors are involved in the ODC induction process elicited by TPA and polycyclic aromatic compounds and that MC and TCDD may induce ODC activity by different mechanisms. The results also confirm our earlier findings in rat skin and cells in culture which suggest that the ODC and AHH induction processes can occur independently of each other. Additionally, there is a strain-related difference in sensitivity with regard to ODC-inducing activity of TPA in the livers of C57BL/6 and DBA/2 mice.

Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex.

The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.

Apoptosis during breast carcinoma progression.

The purpose of this study was to investigate apoptosis, proliferation, and the expression of apoptosis-influencing proteins bcl-2 and bax and estrogen and progesterone receptors during breast carcinoma progression. The material consisted of 53 paired breast carcinoma samples representing primary and recurrent tumors and 24 control samples. The recurrent sample was located either in the breast scar tissue or at a distant metastatic site. Apoptosis was detected both morphologically and by 3' end labeling of fragmented DNA. Cell proliferation was evaluated immunohistochemically by the MIB index. The expressions of bcl-2, bax, and estrogen and progesterone receptors were studied immunohistochemically. There was a significant increase in the extent of apoptosis and proliferation in recurrent tumors compared to the primary lesions (P = 0.015 and P = 0.038, respectively). In primary tumors with an apoptotic index of >0.50%, the survival of the patients was significantly shorter (P = 0.015). In cases with a significant increase in apoptosis or proliferation in the recurrent tumor, the survival of the patients was significantly shorter (P = 0.009 and P = 0.003, respectively). Of the variables analyzed, bcl-2 expression and a positive estrogen receptor status were significantly associated with a low extent of apoptosis (P = 0.010 and P = 0.042, respectively). Their changes were parallel to the changes in apoptosis during tumor progression, although the associations did not reach statistical significance. The results show that increased apoptosis is associated with a worse prognosis in breast carcinoma. A significant increase in apoptosis in recurrent breast carcinoma lesions predicts a worse clinical outcome.

Independent induction and inhibition of ornithine decarboxylase and aryl hydrocarbon hydroxylase activities in rat epidermis.

Changes in the activities of ornithine decarboxylase (ODC) and aryl hydrocarbon hydroxylase (AHH) were investigated in rat epidermis after wounding the skin and application of 7,12-dimethylbenz(a)anthracene (DMBA), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and several enzyme inhibitors. Wounding of the skin by vigorous shaving led to a marked induction of ODC activity with a peak at 6 hr. Topical application of a single dose of tetradecanoylphorbol-13-acetate to wounded skin did not affect the activities of ODC and AHH. Application of single large dose (2.5 mg) of DMBA increased AHH activity 7-fold without affecting ODC activity. DL-alpha-difluoromethyl ornithine, a specific irreversible inhibitor of ODC, almost completely abolished ODC activity but did not inhibit DMBA- or TCDD-induced AHH activity. Several potential modifiers, including retinoic acid, indomethacin, 1,3-diamino-2-propranol, alpha-naphthoflavone, and SKF 525 A had unequal effects on ODC and AHH activities. These data indicate that ODC and AHH induction processes in the epidermis are independent biochemical events that are not causally related.

CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12: evidence for an allelic variant with altered catalytic activity.

OBJECTIVE: To determine the existence of mutant and variant CgammaP3A4 alleles in three racial groups and to assess functions of the variant alleles by complementary deoxyribonucleic acid (cDNA) expression. METHODS: A bacterial artificial chromosome that contains the complete CgammaP3A4 gene was isolated and the exons and surrounding introns were directly sequenced to develop primers to polymerase chain reaction (PCR) amplify and sequence the gene from lymphocyte DNA. DNA samples from Chinese, black, and white subjects were screened. Mutating the affected amino acid in the wild-type cDNA and expressing the variant enzyme with use of the baculovirus system was used to functionally evaluate the variant allele having a missense mutation. RESULTS: To investigate the existence of mutant and variant CgammaP3A4 alleles in humans, all 13 exons and the 5'-flanking region of the human CgammaP3A4 gene in three racial groups were sequenced and four alleles were identified. An A-->G point mutation in the 5'-flanking region of the human CgammaP3A4 gene, designated CgammaP3A4*1B, was found in the three different racial groups. The frequency of this allele in a white population was 4.2%, whereas it was 66.7% in black subjects. The CgammaP3A4*1B allele was not found in Chinese subjects. A second variant allele, designated CgammaP3A4*2, having a Ser222Pro change, was found at a frequency of 2.7% in the white population and was absent in the black subjects and Chinese subjects analyzed. Baculovirus-directed cDNA expression revealed that the CYP3A4*2 P450 had a lower intrinsic clearance for the CYP3A4 substrate nifedipine compared with the wild-type enzyme but was not significantly different from the wild-type enzyme for testosterone 6beta-hydroxylation. Another rare allele, designated CgammaP3A4*3, was found in a single Chinese subject who had a Met445Thr change in the conserved heme-binding region of the P450. CONCLUSIONS: These are the first examples of potential function polymorphisms resulting from missense mutations in the CgammaP3A4 gene. The CgammaP3A4*2 allele was found to encode a P450 with substrate-dependent altered kinetics compared with the wild-type P450.

Cloning and characterization of a member of the rat multidrug resistance (mdr) gene family.

The rat mdr gene family [genes encoding P-glycoprotein (Pgp)] was characterized and the complete sequence of a rat mdr cDNA was determined based on seven independent cDNA clones that correspond to the same gene. The longest of these clones contains a 4.3-kb insert which represents a full-length rat mdr cDNA. The longest open reading frame of this sequence is 3933 bp; the first ATG is at 103 bp, making the deduced protein 1277 amino acids long (141 kDa). This correlates well with previously identified Pgp. The sequence of this gene has a very high, greater than 90%, degree of identity to the mouse mdr1b gene (also known as the mdr1 gene) therefore, we designate it the rat mdr1b gene. Transcription of this gene begins at a single start point 151 nucleotides upstream from the start codon. We show here that the rat gene family is comprised of three members, which is consistent with previous data on other rodent species.

Diagnosis of polymorphisms in carcinogen-activating and inactivating enzymes and cancer susceptibility--a review.

Up to 90% of all cancers are possibly caused by environmental factors, such as tobacco smoke, diet and occupational exposures. The majority of chemical carcinogens require metabolic activation before they interact with cellular macromolecules and can cause cancer initiation. The xenobiotic-metabolising machinery contains two main types of enzymes: the phase-I cytochromes P-450 (CYP) mediating oxidative metabolism, and phase-II conjugating enzymes. Several phase-I and phase-II genes have recently been cloned and identified in humans. Many of them show polymorphism and have been suggested to contribute to individual cancer susceptibility as genetic modifiers of cancer risk. Altered phenotypes and genotypes in the CYP subfamilies CYP1A1, CYP2D6 and CYP2E1 have been associated with tobacco smoke-induced lung cancer and other cancers. Defective glutathione S-transferase (GST) and N-acetyltransferase (NAT) enzymes have been associated with an increased risk of developing lung and bladder cancer. There are also several studies in each category in which no associations have been found. The risk of developing lung cancer is dramatically (up to 40-fold) elevated in subpopulations having simultaneously high-risk genotypes in CYP1A1 and GSTM1. There are several difficulties in this area of research. First, many of the observed restriction-fragment length polymorphisms (RFLPs) are due to mutations in introns or other silent areas of DNA, raising the possibility that any associations found between RFLPs and cancer occur only by chance. Second, biologically plausible mechanisms linking genotypes and cancer are lacking in most of the observed cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotyping of human cytochrome P450 2A6 (CYP2A6), a nicotine C-oxidase.

Cytochrome P450 2A6 (CYP2A6) is a polymorphic enzyme responsible for the oxidation of certain precarcinogens and drugs and is the major nicotine C-oxidase. The role of CYP2A6 for nicotine elimination was emphasised recently by the finding that smokers carrying defective CYP2A6 alleles consumed fewer cigarettes [Pianezza et al. (1998) Nature 393, 750]. The method used for CYP2A6 genotyping has, however, been found to give erroneous results with respect to the coumarin hydroxylase phenotype, a probe reaction for the CYP2A6 enzyme. The present study describes an allele-specific PCR genotyping method that identifies the major defective CYP2A6 allele and accurately predicts the phenotype. An allele frequency of 1-3% was observed in Finnish, Spanish, and Swedish populations, much lower than described previously.

Identification and characterisation of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism.

The polymorphic human cytochrome P450 2A6 (CYP2A6) metabolises a number of drugs, activates a variety of precarcinogens and constitutes the major nicotine C-oxidase. A relationship between CYP2A6 genotype and smoking habits, as well as incidence of lung cancer, has been proposed. Two defective alleles have hitherto been identified, one of which is very common in Asian populations. Among Caucasians, an additional defective and frequently distributed allele (CYP2A6*3) has been suggested to play a protective role against nicotine addiction and cigarette consumption. Here, we have re-evaluated the genotyping method used for the CYP2A6*3 allele and found that a gene conversion in the 3' flanking region of 30-40% of CYP2A6*1 alleles results in genotype misclassification. In fact, no true CYP2A6*3 alleles were found among 100 Spaniards and 96 Chinese subjects. In one Spanish poor metaboliser of the CYP2A6 probe drug coumarin, we found two novel defective alleles. One, CYP2A6*5, encoded an unstable enzyme having a G479L substitution and the other was found to carry a novel type of CYP2A6 gene deletion (CYP2A6*4D). The results imply the presence of numerous defective as well as active CYP2A6 alleles as a consequence of CYP2A6/CYP2A7 gene conversion events. We conclude that molecular epidemiological studies concerning CYP2A6 require validated genotyping methods for accurate detection of all known defective CYP2A6 alleles.

Characterisation and PCR-based detection of a CYP2A6 gene deletion found at a high frequency in a Chinese population.

Cytochrome P450 2A6 is an important human hepatic P450 which activates pre-carcinogens, oxidises some drugs and constitutes the major nicotine C-oxidase. In fact, results have been presented in the literature which suggested a relationship between the distribution of defective CYP2A6 alleles and smoking behaviour as well as cigarette consumption. In the present report, we describe the structure of a novel CYP2A locus where the whole CYP2A6 gene has been deleted, resulting in an abolished cytochrome P450 2A6-dependent metabolism. The origin of this locus is apparently due to an unequal crossover event between the 3'-flanking region of the CYP2A6 and CYP2A7 genes. A rapid PCR-based method for the detection of the CYP2A6del allele was developed and the allele frequency was 15.1% among 96 Chinese subjects, but only 1.0% in Finns (n=100) and 0.5% in Spaniards (n=100). In the Chinese population, we did not detect any CYP2A6*2 alleles using an improved genotyping procedure, in contrast to the 11-20% previously reported. It is concluded that genotyping for the CYP2A6del allele is of great importance in studies correlating, for example, smoking behaviour, pre-carcinogen activation or drug metabolism to the CYP2A6 genotype, in particular when oriental populations are investigated.

Dissociation of 12-O-tetradecanoylphorbol-13-acetate and 3-methylcholanthrene-induced induction in ornithine decarboxylase and aryl hydrocarbon hydroxylase activities in C57BL/6 mouse dermal fibroblasts in culture.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 3-methyl-cholanthrene (MC) on ornithine decarboxylase (ODC) and aryl hydrocarbon hydroxylase (AHH) activities were studied in C57BL/6 mouse dermal fibroblasts in culture. TPA selectively induced ODC activity and MC selectively induced AHH activity in these cells. Acute (10 h) exposure of the cells to DL-alpha-difluoromethyl ornithine (DFMO) led to a marked inhibition of ODC activity without any significant effect on induced AHH activity. Chronic inhibition of ODC activity (4 days) resulted in a slight inhibition of basal AHH activity, but the inducibility of AHH was enhanced alpha-Naphtho-flavone, SKF 525 A and indomethacin exerted unequal effects on the enzyme activities. These results indicate that ODC and AHH induction processes are independent events with no causal link.

Dissociation between ornithine decarboxylase activity and aryl hydrocarbon hydroxylase induction in cell cultures treated with benz[a]anthracene and inhibitors of ornithine decarboxylase.

The induction of aryl hydrocarbon and ornithine decarboxylase by benz[a]-anthracene in the presence or absence of ornithine decarboxylase inhibitors was studied in three different cell culture systems. An almost complete abolishment of ornithine decarboxylase activity by 1,3-diamino-2-propanol or alpha-difluoremethyl ornithine before the addition of the inducer did not affect appreciably the induction of aryl hydrocarbon hydroxylase by benz[a]anthracene in human embryo, HeLa and Rueber H-II-4-E cells in culture. These results suggest that the induction of aryl hydrocarbon hydroxylase does not require ornithine decarboxylase activity per se and can be expressed in the absence of continuous polyamine synthesis.

Cytochrome P450 isoforms in human fetal tissues related to phenobarbital-inducible forms in the mouse.

Four polyclonal antibodies raised against purified mouse liver cytochrome P450s representing Cyp1a, Cyp2a, Cyp2b and Cyp2c subfamilies were used to detect their related forms in human adult and fetal tissues. In immunoblot analysis, anti-Cyp2c antibody detected two to three proteins in adult livers and one to three proteins in 70% of the 18 fetal livers studied. Anti-Cyp2a-5 antibody recognized a 50-kDa protein in 50% of the fetal adrenals. Anti-Cyp1a-2 antibody reacted with a single protein (55 kDa) in adult liver. The anti-Cyp2b-10 antibody did not detect proteins in any of the tissues. No proteins were detected in fetal kidneys. There was no coumarin 7-hydroxylase activity (COH) in fetal liver or adrenals. The 7-ethoxycoumarin O-deethylase (ECOD) activities were slightly higher in fetal adrenals (mean 6.1 pmol/mg protein/min) vs livers. The fetal adrenal ECOD activity was not inhibited by the anti-Cyp2a-5 antibody. Aryl hydrocarbon hydroxylase (AHH) activities in fetal livers were about 5% of those in adult livers. AHH activity in fetal liver was not inhibited by the anti-Cyp2c antibody. Testosterone 6 beta-hydroxylase activity was much lower in fetal liver than in adult liver (about 20 and 1700 pmol/mg protein/min, respectively). No immunoinhibition occurred in fetal adrenal progesterone hydroxylation, hepatic benzphetamine N-demethylation and hepatic ethylmorphine N-demethylation. These data suggest that members of the P450 subfamilies 1A, 2A and 2B are expressed at a very low level in fetal liver, and that fetal liver may contain members of the 2C subfamily.

Metabolic interactions of methoxsalen and coumarin in humans and mice.

Methoxsalen (8-methoxypsoralen) is a very potent inhibitor of human cytochrome P450 2A6 (CYP2A6) and mouse Cyp2a-5-mediated coumarin 7-hydroxylation in vitro. To determine the effect of methoxsalen on coumarin 7-hydroxylation in humans in vivo, five subjects were given 45 mg of methoxsalen and 5 mg of coumarin. Methoxsalen inhibited in vivo coumarin metabolism by 47 +/- 9.2% (mean +/- SEM). Methoxsalen was metabolized in human liver microsomes at the rate of 50-100 pmol/mg protein/min (approx. 30% of the activity in mouse liver microsomes). Metabolism was not inhibited by the anti-Cyp2a-5 antibody in human liver microsomes. NIH 3T3 cells stably expressing catalytically active CYP2A6 enzyme did not metabolize methoxsalen, indicating that CYP2A6 does not accept methoxsalen as a substrate. In pyrazole-induced mouse liver microsomes, methoxsalen metabolism was inhibited by the anti-Cyp2a-5 antibody. Cyp2a-5 protein expressed in the yeast Saccharomyces cerevisiae was capable of metabolizing methoxsalen, indicating that methoxsalen is a substrate of Cyp2a-5. Although kinetic studies indicated that the inhibition of coumarin 7-hydroxylation by methoxsalen is competitive in human liver microsomes, methoxsalen does not appear to be a substrate for CYP2A6. Methoxsalen and coumarin have the potential of strong metabolic interactions in man.

Induction of cytochrome P450IA1 in mouse hepatoma cells by several chemicals. Phenobarbital and TCDD induce the same form of cytochrome P450.

The mouse hepatoma cell line Hepa-1 was studied for aryl hydrocarbon hydroxylase (AHH) inducibility by sixteen compounds known to be inducers of cytochrome P450 of different "classes". Both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and sodium phenobarbital induced AHH activity. A cytochrome P450IA1-specific (P1-450) mouse cDNA probe was used to quantitate mRNA induction. There was a good correlation between the amount of cytochrome P450IA1 mRNA induced and AHH activity. Immunoblots with monoclonal antibody 1-7-1, which recognizes rat liver P450IA1 and P450IA2 (P450c and P450d, respectively), showed that both phenobarbital and TCDD increase the amount of a P450 isozyme immunorelated to P450IA1 in this cell line. Hepa-1 mutants with no AHH inducibility (no functional P450IA1 structural gene; no Ah receptor; no nuclear translocation of the inducer-receptor complex; and presence of dominant repressor) did not respond to phenobarbital. The cytosolic receptor for TCDD (Ah receptor) was characterized to see if phenobarbital induced cytochrome P450IA1 mRNA and the hydroxylase enzyme through the same mechanism as TCDD. 20 mM Phenobarbital almost completely abolished the binding of 3H-TCDD to the cytosolic receptor. These data indicate that phenobarbital can be a weak ligand for the Ah receptor and thus induce cytochrome P450IA1 and AHH activity. The observation increases the list of different P450 forms inducible by phenobarbital.

Expression of xenobiotic-metabolizing cytochrome P450 forms in human adult and fetal liver.

Expression of human cytochrome P450 (CYP) genes in human adult and fetal liver were studied using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. In adult liver mRNA of CYPs 1A1, 1A2, 2A6/2A7, 2B6/2B7, 2C8-19, 2D6, 2E1, 3A3/3A4 and 3A7 were detected while CYPs 2F1 and 4B1 were absent. In fetal liver mRNA of CYPs 2C8, 2D6, 3A3/3A4 and 3A7 were found but all other forms studied were undetectable. The results provide a comprehensive qualitative picture of the expression of CYP genes in families CYP1 through CYP4 in human adult and fetal liver.

Differential inhibition of coumarin 7-hydroxylase activity in mouse and human liver microsomes.

Coumarin is 7-hydroxylated by the P450 isoform Cyp2a-5 in mice and CYP2A6 in humans. Various drugs, endogenous substances, plant substances and carcinogens, altogether about 90 chemicals, were evaluated as possible inhibitors of coumarin 7-hydroxylase (COH) activity in mouse microsomes. The effects of selected compounds on COH activity in human liver microsomes were also tested. The furanocoumarin derivatives methoxsalen (8-methoxypsoralen) and psoralen proved to be the most potent inhibitors of mouse COH activity (IC50 values 1.0 and 3.1 microM, respectively). The furanocoumarins bergapten (5-methoxypsoralen), isopimpinellin (5,8-dimethoxypsoralen), imperatorin and sphondin also effectively inhibited mouse COH activity (IC50 values 19-40 microM). Methoxsalen, isopimpinellin and metyrapone were also inhibitors in mice in vivo. Methoxsalen was a potent inhibitor of COH activity also in human liver microsomes, (IC50 value 5.4 microM), whereas bergapten, isopimpinellin and imperatorin had no effect. The imidazole antimycotic miconazole was a potent but non-specific inhibitor of COH activity. Several known substrates and inhibitors of members in the CYP1A, CYP2B, CYP2C, CYP2D and CYP3A subfamilies were poor inhibitors of COH activity. These results suggest that (i) the coumarin-type compounds in particular interact with the active sites of Cyp2a-5 and CYP2A6, and (ii) the active sites of Cyp2a-5 and CYP2A6 are structurally different, since a number of compounds inhibited mouse, but not human COH activity.

Cerium-induced strain-dependent increase in Cyp2a-4/5 (cytochrome P4502a-4/5) expression in the liver and kidneys of inbred mice.

The murine Cyp2a-4 and Cyp2a-5 genes encode P450 isoforms catalysing testosterone 15 alpha-hydroxylase and coumarin 7-hydroxylase (COH) activities, respectively. Two days after the administration of a hepatotoxic dose of cerium chloride (2 mg/kg i.v.), COH activity was increased 3.2-fold in the liver of DBA/2 mice. Three and 4 days after the cerium treatment, coinciding with the occurrence of overt liver damage, there was a dramatic decrease in COH activity. The activities of testosterone 15 alpha-hydroxylase and the Cyp1a-1-mediated 7-ethoxyresorufin O-deethylase (EROD) were decreased in response to cerium. Much less pronounced changes in the enzyme activities occurred in the C57BL/6 mouse liver. Northern blot analysis showed a 21-fold increase in the hepatic Cyp2a-4/5 mRNA in the DBA/2 mice at day 2, whereas no increase occurred in the C57BL/6 mice. Also in the kidneys the increase in COH activity and in Cyp2a-4/5 mRNA was marked only in the DBA/2 mice. A polymerase chain reaction-mediated analysis method utilizing a unique PstI restriction site in the Cyp2a-5 cDNA was used to differentiate between the highly homologous Cyp2a-4 and Cyp2a-5 mRNAs. Cerium was found to increase the amount of hepatic and renal Cyp2a-4 and Cyp2a-5 mRNA only in the DBA/2 mice. These data indicate that the Cyp2a-4/5 complex is regulated in a different way in DBA/2 and C57BL/6 mice and that some association exists between the development of liver damage and COH induction.