Coumarin 7-hydroxylase Activity Research Articles

Identification of 5-Hydroxycotinine in the Plasma of Nicotine-Treated Mice: Implications for Cotinine Metabolism and Disposition in Vivo.

Two oxidation products of cotinine, 5-hydroxycotinine (5-HC) and cotinine N-oxide (CNO), were identified for the first time in vivo in the plasma of C57BL/6 mice after injection of nicotine (1 mg/kg) or exposure to an e-cigarette (e-cig) containing 2.4% nicotine. Liquid chromatography-mass spectrometry was used to separate 3-hydroxycotinine (3-HC), 5-HC, and CNO and to quantify each by the sensitive direct detection of their parent ion with m/z of 193.097. In nicotine-injected mice, 5-HC was as abundant as 3-HC 15 minutes postinjection, and CNO was readily detectable. In e-cig-exposed mice with plasma nicotine levels resembling that of human smokers, plasma 5-HC and CNO, as well as 3-HC, were readily quantifiable at the end of the 4-hour exposure time. In nicotine-injected mice, the combined concentration of 3-HC plus 5-HC plus CNO, all formed from cotinine by CYP2A5, was higher (P < 0.01) in females than in males, although the male-female difference in cotinine plasma level did not reach statistical significance. The result highlights the importance of considering these three oxidation products of cotinine in examining cotinine metabolism and disposition. Coumarin 7-hydroxylase activity, a specific marker of CYP2A5, measured in the hepatic microsomes of untreated mice showed that females have higher activity (P < 0.001) than males (N = 8 per sex). The abundance of plasma 5-hydroxycotinine in nicotine-treated mice raises intriguing questions about the site of its origin (hepatic or possibly kidney CYP2A5) and the routes of its disposition because urinary excretion of 5-HC has not been detected by liquid chromatography with tandem mass spectrometry in mice and is controversial in human smokers. SIGNIFICANCE STATEMENT: Nicotine is the active ingredient of tobacco, but its elimination route through its biomarker cotinine is not fully understood. By liquid chromatography-mass spectrometry, this study has identified and quantified for the first time 5-hydroxycotinine and cotinine N-oxide, which are oxidation products of cotinine, in the plasma of mice treated with nicotine or exposed to e-cigarettes. The results raise intriguing questions about nicotine disposition in vivo in this well established preclinical model of human smokers.

Development and Validation of a New Simple Liquid Chromatographic Method for Determination of Coumarin and its 7-Hydroxy Metabolite as a Marker of Cytochrome P450 2A6 in Rats

In this study, a simple and reliable high performance liquid chromatographic method with UV detection was developed and validated for rapid determination of coumarin hydroxylase activity in rat hepatic microsomes. The chromatographic separation was achieved using Zorbax Eclipse XDB C18 column (150×4.6 mm, 5 μm), which was kept at 40°C. The isocratic mobile phase consisted of methanol and 1% glacial acetic acid mixture (35:65, v/v) with a flow rate of 0.6 ml/min. The effluent was monitored at 320 nm using photodiode array detector (PDA). 6-hydroxychlorzoxazone (6-OH CZX) was used as internal standard. The method exhibited good linearity (R2 >0.999) for both coumarin (COUM) and its 7-hydroxy metabolite (7-OH COUM) over the assayed concentration range (0.025-5.0 μM) and demonstrated good intra-day and inter-day precision and accuracy (relative standard deviations and the deviation from predicted values were less than 15%). The detection limits were 0.001 and 0.005 μM for coumarin and 7-hydroxycoumarin, respectively. This method was also successfully applied for studying the effect of three phytochemicals on hepatic CYP2A6 activity in rats.

Effect of CYP2A6 genetic polymorphism on the metabolic conversion of tegafur to 5-fluorouracil and its enantioselectivity.

Tegafur (FT), a prodrug of 5-fluorouracil, is a chiral molecule, a racemate of R- and S-isomers, and CYP2A6 plays an important role in the enantioselective metabolism of FT in human liver microsomes (R-FT >> S-FT). This study examined the enantioselective metabolism of FT by microsomes prepared from Sf9 cells expressing wild-type CYP2A6 and its variants (CYP2A6*7, *8, *10, and *11) that are highly prevalent in the Asian population. We also investigated the metabolism of coumarin and nicotine, both CYP2A6 probe drugs, in these variants. Enzyme kinetic analyses showed that CYP2A6.7 (I471T) and CYP2A6.10 (I471T and R485L) had markedly lower Vmax values for both enantiomers than wild-type enzyme (CYP2A6.1) and other variant enzymes, whereas Km values were higher in most of the variant enzymes for both enantiomers than CYP2A6.1. The ratios of Vmax and Km values for R-FT to corresponding values for S-FT (R/S ratio) were similar among enzymes, indicating little difference in enantioselectivity among the wild-type and variant enzymes. Similarly, both CYP2A6.7 and CYP2A6.10 had markedly lower Vmax values for coumarin 7-hydroxylase and nicotine C-oxidase activities than CYP2A6.1 and other variant enzymes, whereas Km values were higher in most of the variant enzymes for both activities than CYP2A6.1. In conclusion, the amino acid substitutions in CYP2A6 variants generally resulted in lower affinity for substrates, while Vmax values were selectively reduced in CYP2A6.7 and CYP2A6.10. Consistent R/S ratios among CYP2A6.1 and variant enzymes indicated that the amino acid substitutions had little effect on enantioselectivity in the metabolism of FT.

Expression of Cytochrome P450 2A5 in a C57BL/6J Mouse Model of Nonalcoholic Fatty Liver Disease

Cytochrome P450 2A5 (CYP2A5) expression is induced during the hepatotoxicity caused by various hepatotoxins and hepatitis. However, CYP2A5 expression during nonalcoholic fatty liver disease (NAFLD) is still unknown. In this study, serum biochemical parameters and liver histopathological analyses found that NAFLD had developed in C57BL/6J mice via administration of a high-fat diet continuously for 8 weeks. Subsequently, CYP2A5 expression was probed in the mice diagnosed with NAFLD that were treated with or without pyrazole, the inducer of chemical liver injury. It is shown that hepatic CYP2A5 mRNA, protein expression and coumarin 7-hydroxylase activity are enhanced with high-fat feed, and that pyrazole is able to further increase CYP2A5 expression and activity in mice with NAFLD. These results revealed that CYP2A5 is elevated during NAFLD and suggested that pyrazole and NAFLD act synergistically to induce the expression of CYP2A5 via an unclear mechanism.

Cytochrome P450 2A isoenzymes in freshly prepared blood lymphocytes isolated from rats and validation as a biomarker for clinical studies in humans

1. The present study aimed to identify the expression of carcinogen metabolizing cytochrome P4502A (CYP2A) isoenzymes in freshly prepared rat peripheral blood lymphocytes (PBL) isolated from adult rats and investigate similarities in the regulation of lymphocyte CYP2A-isoenzymes with the tissue enzyme.2. qRT-PCR studies demonstrated significant constitutive mRNA expression of CYP2A-isoenzymes in PBL isolated from male and female rats which further increases significantly after pretreatment with nicotine or 3-methylcholanthrene (MC) indicating responsiveness of CYP2A-isoenzymes in PBL. This increase in the CYP2A expression was associated with an increase in the protein expression and CYP2A3-dependent coumarin hydroxylase (COH) activity in PBL.3. Clinical studies further demonstrated significant increase in the expression of CYP2A6 and associated enzyme activity in PBL isolated from lung cancer patients. Our data thus provided evidence for similarities in the regulation of carcinogen metabolizing CYP2A-isoenzymes in PBL with the tissue enzymes. Further, responsiveness of blood CYP2A6 in human blood lymphocytes isolated from lung cancer patients has led us to suggest that associating expression profiles of CYP2A6 and other polycyclic aromatic hydrocarbons (PAH)-responsive CYPs in PBL with the genotyping data could lead to the development of a possible screen to monitor and predict environment-induced diseases and toxicity in humans.

Metabolism of bilirubin by human cytochrome P450 2A6

The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14–22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated Ki of 2.23μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme.

Differential inhibition of human cytochrome P450 2A6 and 2B6 by major phytocannabinoids

Inhibitory effects of Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), and cannabinol (CBN) on the catalytic activities of human recombinant cytochrome P450 (CYP) 2A6 and CYP2B6 were investigated. Δ9-THC, CBD, and CBN noncompetitively inhibited coumarin 7-hydroxylase activity of recombinant CYP2A6 with the apparent K i values of 28.9, 55.0, and 39.8 μM, respectively. On the other hand, Δ9-THC, CBD, and CBN inhibited 7-benzoxyresorufin O-debenzylase activity of recombinant CYP2B6 in a mixed fashion with the K i values of 2.81, 0.694, and 2.55 μM, respectively. Because the inhibition of CYP2B6 by CBD was the most potent, investigation was conducted to determine which moiety of the CBD structure was responsible for the inhibition. Olivetol and d-limonene, the partial structure of CBD, inhibited the CYP2B6 activity to some extent. Inhibitory effects of CBD-2′-monomethyl ether and CBD-2′,6′-dimethyl ether attenuated with the number of methylations on the phenolic hydroxyl groups in the resorcinol moiety of CBD. Cannabidivarin, a CBD analogue having a propyl side chain, inhibited the CYP2B6 activity less potently than CBD possessing a pentyl side chain. Therefore, both structures of pentylresorcinol and terpene moieties of CBD were suggested to play important roles in the CYP2B6 inhibition. Δ9-THC, CBD, and CBN showed metabolism-dependent inhibition for CYP2A6 but not for CYP2B6. Furthermore, Δ9-THC and CBN were characterized as mechanism-based inhibitors for CYP2A6. The k inact and K I values of Δ9-THC were 0.0169 min−1 and 0.862 μM, respectively; the k inact and K I values of CBN were 0.00909 min−1 and 1.01 μM, respectively. These results indicated that Δ9-THC, CBD, and CBN showed differential inhibition against CYP2A6 and CYP2B6.

Inhibition of cytochrome P450 2A participating in coumarin 7‐hydroxylation in pig liver microsomes

Five commonly used human cytochrome P450 (CYP) inhibitors were examined for their effects on coumarin 7-hydroxylase (CYP2A) activity in pig liver microsomes. The K(m) and V(max) values for coumarin 7-hydroxylation in pig liver microsomes were estimated to be 1 μm and 0.26 nmol·mg/min, respectively. The following human CYP inhibitors caused little or no inhibition of CYP2A as defined by a K(i) > 200 μm: quinidine (CYP2D6), troleandomycin (CYP3A4), and sulfaphenazole (CYP2C9). The other two human CYP inhibitors were classified as strong inhibitors of CYP2A: 8-methoxypsoralen (CYP2A6) and α-naphthoflavone (CYP1A1/2). In the absence of a preincubation period, 8-MOP inhibited the 7-hydroxylation of coumarin with a K(i) value of 1.1 μm, which decreased to 0.1 μm when 8-MOP was preincubated with pig liver microsomes for 3 min. α-Naphthoflavone inhibited the 7-hydroxylation of coumarin with a K(i) value of 32 μm, which did not increase ability to inhibitor CYP2A when α-naphthoflavone was preincubated with pig liver microsomes for 3 min. These results of this study suggest that 8-MOP is a potent, mechanism-based inhibitor of pig CYP2A activity in pig liver microsomes.

Regional Distribution of Drug-metabolizing Enzyme Activities in the Liver and Small Intestine of Cynomolgus Monkeys

The cynomolgus monkey is an animal species widely used to study drug metabolism because of its evolutionary closeness to humans. However, drug-metabolizing enzyme activities have not been compared in various parts of the liver and small intestine in cynomolgus monkeys. In this study, therefore, drug-metabolizing enzyme activities were analyzed in the liver (the five lobes) and small intestine (six sections from the duodenum to the distal ileum). 7-Ethoxyresorufin O-deethylation, coumarin 7-hydroxylation, paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, tolbutamide methylhydroxylation, S-mephenytoin 4'-hydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, midazolam 1'-hydroxylation, and testosterone 6β-, 16α-, 16β-, and 2α-hydroxylation were used as the probe reactions for this investigation. In liver, all probe reactions were detected and enzyme activity levels were similar in all lobes, whereas, in the small intestine, all enzyme activities were detected (except for coumarin 7-hydroxylase and testosterone 16α-hydroxylase activity), but from jejunum to ileum there was a decrease in the level of enzyme activity. This includes midazolam 1'-hydroxylation and testosterone 6β-hydroxylation, which are catalyzed by cynomolgus monkey cytochrome P450 (CYP) 3A4/5, orthologs of human CYP3A4/5, which are important drug-metabolizing enzymes. The data presented in this study are expected to facilitate the use of cynomolgus monkeys in drug metabolism studies.

Study of inhibition of CYP2A6 by some drugs derived from quinoline.

CYP2A6 metabolizes coumarin to 7-hydroxycoumarin showing fluorescence, as measured by fluorometry. Firstly, we measured the inhibition of coumarin 7-hydroxylase of cDNA-expressed human CYP2A6 and in bovine liver microsomes, by quinoline and fluoroquinolines (FQ). Quinoline, 5-FQ, 6-FQ and 8-FQ inhibited activity but 3-FQ showed little inhibition. This suggests that the position 3 of quinoline is a recognition site for CYP2A6. We found similar patterns of coumarin 7-hydroxylase activity with human pooled liver microsomes. The level of CYP2A6 in human and bovine microsomes is the same as that detected by immunological titration with monoclonal antibody against CYP2A6. Secondly, we studied the inhibition of CYP2A6 with clinically used drugs of quinoline compounds, such as norfloxacin as an antibacterial agent, quinidine as an antiarrhythmic agent, quinine and chloroquine as antimalaria agents and rebamipide as an anti-ulcer agent. IC50 values (concentration producing 50% inhibition in activity) of norfloxacin, rebamipide and chloroquine at mM concentrations showed them to possess almost no inhibitory activity or influence on drug interaction. Meanwhile, the IC50 value of quinidine was 1.12 mM. The IC50 value of quinine was 160 microM with weak inhibition, suggesting that quinine, at a high dose, influences the metabolism of substrates for CYP2A6 by drug-drug interaction. These results also show that CYP2A6 discriminates the structure difference between the diastereoisomers quinidine and quinine.

Deactivation of anti-cancer drug letrozole to a carbinol metabolite by polymorphic cytochrome P450 2A6 in human liver microsomes

Cytochromes P450 (P450) involved in letrozole metabolism were investigated. Among 13 recombinant P450 forms examined, only P450 2A6 and 3A4 showed activities in transforming letrozole to its carbinol metabolite with small Km and high Vmax values yielding apparent Vmax/Km values of 0.48 and 0.24 nl min−1 nmol−1 P450, respectively.The metabolic activities of individual human liver microsomes showed a significant correlation with coumarin 7-hydroxylase activities (P450 2A6 marker) at a letrozole concentration of 0.5 μM, while a good correlation was also seen with testosterone 6β-hydroxylase activities (P450 3A4 marker) at 5 μM substrate concentration with different inhibition by 8-methoxypsolaren.Significantly low carbinol-forming activities were seen in human liver microsomes from individuals possessing CYP2A6*4/*4 (whole CYP2A6 gene deletion) at a letrozole concentration of 0.5 μM. A Vmax/Km value measured for CYP2A6.7 (amino acid substitution type) in human liver microsomes, in the presence of anti-P450 3A4 antibodies, was approximately seven-fold smaller than that for CYP2A6.1 (wild-type).These results demonstrate that P450 2A6 and 3A4 catalyse the conversion of letrozole to its carbinol metabolite in vitro at low and high concentrations of letrozole. Polymorphic variation of CYP2A6 is considered to be relevant to inter-subject variation in therapeutic exposure of letrozole.

Protective effects of isothiocyanates alone or in combination with vitamin C towards N‐nitrosodibutylamine or N‐nitrosopiperidine‐induced oxidative DNA damage in the single‐cell gel electrophoresis (SCGE)/HepG2 assay

The aim of this study was to investigate the protective effect of isothiocyanates alone or in combination with vitamin C towards N-nitrosodibutylamine (NDBA) or N-nitrosopiperidine (NPIP)-induced oxidative DNA damage in the single cell gel electrophoresis (SCGE)/HepG2 assay. Phenethyl isothiocyanate (PEITC) and indole-3-carbinol (I3C) alone showed a weak protective effect towards NDBA (0.1 microm, 26-27%, respectively) or NPIP (1 microm, 26-28%, respectively)-induced oxidative DNA damage. Allyl isothiocyanate (AITC) alone did not attenuate the genotoxic effect provoked by NDBA or NPIP. In contrast, HepG2 cells simultaneously treated with PEITC, I3C and AITC in combination with vitamin C showed a stronger inhibition of oxidative DNA-damage induced by NDBA (0.1 microm, 67%, 42%, 32%, respectively) or NPIP (1 microm, 50%, 73%, 63%, respectively) than isothiocyanates (ITCs) alone. One feasible mechanism by which ITCs alone or in combination with vitamin C exert their protective effects towards N-nitrosamine-induced oxidative DNA damage could be by the inhibition of their cytochrome P450 dependent bioactivation. PEITC and I3C strongly inhibited the p-nitrophenol hydroxylation (CYP2E1) activity (0.1 microm, 66-50%, respectively), while the coumarin hydroxylase (CYP2A6) activity was slightly reduced (0.1 microm, 25-37%, respectively). However, the ethoxyresorufin O-deethylation (CYP1A1) activity was only inhibited by PEITC (1 microm, 55%). The results indicate that PEITC and I3C alone or PEITC, I3C and AITC in combination with vitamin C protects human-derived cells against the oxidative DNA damaging effects of NDBA and NPIP, two food carcinogenic compounds.

Protective effect of vitamin C towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay

The aim of this study was to investigate the protective effect of vitamin C towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay. None of the vitamin C concentrations tested (1–10 μM) in presence or absence of formamidopyrimidine-DNA glycosylase (Fpg enzyme) caused DNA damage per se. HepG2 cells simultaneously treated with vitamin C and N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR), N-nitrosodibutylamine (NDBA) or N-nitrosopiperidine (NPIP) reduced the genotoxic effects of the N-nitrosamines in a dose-dependent manner. At concentrations of 1–5 μM vitamin C, the protective effect was higher towards NPYR-induced oxidative DNA damage (78–79%) than against NDMA (39–55%), NDBA (12–14%) and NPIP (3–55%), in presence of Fpg enzyme. However, a concentration of 10 μM vitamin C led to a maximum reduction in NDBA (94%), NPYR (81%), NPIP (80%) and NDMA (61%)-induced oxidative DNA damage, in presence of Fpg enzyme. The greatest protective effect of vitamin C (10 μM) was higher towards NDBA-induced oxidative DNA damage. One feasible mechanism by which vitamin C exerted its protective effect is that may interact with the enzyme systems catalyzing the metabolic activation of the N-nitrosamines, blocking the production of genotoxic intermediates. Vitamin C (10 μM) strongly reduced the coumarin hydroxylase (82%) activity. However, the p-nitrophenol hydroxylase and the ethoxyresorufine O-deethylation activities were slightly and weakly reduced (32–19%), respectively.

Determination of microsomal CYP2A6 activity by high performance liquid chromatography

Coumarin has been reported to be a reliable probe for the determination of human microsomal CYP2A6 activity. Coumarin is converted to 7-hydroxycoumarin by CYP2A6. A high pressure liquid chromatographic assay for the estimation of CYP2A6 activity in microsomes was evaluated. A RP C-18 Novapak Waters (15 cm x 3.9 mm, 5 µm) column was used for the assay. The mobile phase composition was methanol : 1% glacial acetic acid (35:65 v/v) (pH~3.1), with a flow rate of 0.6 ml/min, injection volume of 100 µl and detection at 320 nm. The retention times for coumarin and 7-hydroxycoumarin were 8.7 min and 5.3 min, respectively. The limit of detection (LOD) was 0.05 µM, and the limit of quantitation (LOQ) was 0.1 µM, for 7-hydroxycoumarin. The percent coefficient of variation associated with 7-hydroxycoumarin determination after duplicate estimation was found to be in the range 0.03 to 3.6% for buffer matrix and 0.1 to 6.5% for microsomal matrix. The mean rate of 7-hydroxycoumarin formation in guinea pig liver microsomes was 0.084 nmol/min/nmol P450. Coumarin 7-hydroxylase activity was absent in rat liver microsomes. No interference was observed from incubation mixture components.

The Missense Genetic Polymorphisms of Human CYP2A13: Functional Significance in Carcinogen Activation and Identification of A Null Allelic Variant

Cytochrome P450 2A13 (CYP2A13), an enzyme predominantly expressed in human respiratory tissues, is highly efficient for the metabolic activation of two suspected human lung carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and aflatoxin B1 (AFB1). Functional genetic polymorphisms of CYP2A13 may therefore be an important factor in human susceptibility to related lung cancers. Among the reported CYP2A13 polymorphisms with missense variations, only CYP2A13*2 variant (containing either a single or double variation of R25Q and R257C) was studied for its NNK-metabolizing activity. The present study demonstrated that there was no remarkable difference in AFB1- and NNK-induced toxicity between the Flp-In Chinese Hamster Ovary (CHO) cells stably expressing wild-type CYP2A13 and the cells expressing the individual polymorphic variants R25Q, D158E, R257C, R25Q/R257C, V323L, F453Y, and R494C. In contrast, cells transfected with R101Q variant complementary DNA (cDNA), same as the vector control cells, showed no significant death even at highest concentrations of AFB1 (10microM) and NNK (200microM). This result correlated with the lack of CYP2A13 protein in the R101Q-CHO cells, although the genomic integration of transfected R101Q cDNA and the expression of R101Q messenger RNA were clearly demonstrated in these stable transfectants. Consistent with the possibility that the variation might reduce the protein stability, R101Q variant protein expressed in insect cells showed a loss of P450 peak and coumarin 7-hydroxylase activity as well as an increased susceptibility to limited protein digestion. Thus, the R101Q polymorphic change results in a null allelic variant of CYP2A13. Our results should be useful in designing and interpreting molecular epidemiological studies related to CYP2A13 genetic polymorphisms.

THE ROLE OF CYP2A AND CYP2E1 IN THE METABOLISM OF 3-METHYLINDOLE IN PRIMARY CULTURED PORCINE HEPATOCYTES

The accumulation of 3-methylindole (3MI) in uncastrated male pigs (boars) is a major cause of boar taint, which negatively affects the quality of meat from the animal. Previously, CYP2E1 and CYP2A have been identified as cytochrome P450 (P450) isoforms involved in the metabolism of 3MI using porcine liver microsomes. This study further examines the role of these isoforms in the metabolism of 3MI using a primary porcine hepatocyte model by examining metabolic profiles of 3MI after incubation with P450 inhibitors. Incubation of hepatocytes with 4-methylpyrazole resulted in a selective inhibition of CYP2E1 activity as determined by p-nitrophenol hydroxylase activity and an associated significant decrease in the production of the 3MI metabolites 3-hydroxy-3-methyloxindole and 3-methyloxindole. Furthermore, inhibition of CYP2A, as assayed by coumarin 7-hydroxylase activity, using 8-methoxypsoralen and diethyldithiocarbamate was not associated with any further significant inhibition of the production of 3MI metabolites. Treatment with general P450 inhibitors resulted in further decreases in CYP2E1 activity and a more dramatic decrease in the production of 3MI metabolites, suggesting that additional P450s may be involved in the phase 1 metabolism of 3-methylindole. In conclusion, CYP2E1 activity levels are more important than CYP2A activity levels for the metabolism of 3-methylindole in isolated pig hepatocytes.

Evidence for induced microsomal bilirubin degradation by cytochrome P450 2A5

Oxidative metabolism of bilirubin (BR) – a breakdown product of haem with cytoprotective and toxic properties – is an important route of detoxification in addition to glucuronidation. The major enzyme(s) involved in this oxidative degradation are not known. In this paper, we present evidence for a major role of the hepatic cytochrome P450 2A5 (Cyp2a5) in BR degradation during cadmium intoxication, where the BR levels are elevated following induction of haem oxygenase-1 (HO-1). Treatment of DBA/2J mice with CdCl 2 induced both the Cyp2a5 and HO-1, and increased the microsomal BR degradation activity. By contrast, the total cytochrome P450 (CYP) content and the expression of Cyp1a2 were down-regulated by the treatment. The induction of the HO-1 and Cyp2a5 was substantial at the mRNA, protein and enzyme activity levels. In each case, the up-regulation of HO-1 preceded that of Cyp2a5 with a 5–10 h interval. BR totally inhibited the microsomal Cyp2a5-dependent coumarin hydroxylase activity, with an IC 50 approximately equal to the substrate concentration. The 7-methoxyresorufin 7- O-demethylase (MROD) activity, catalyzed mainly by the Cyp1a2, was inhibited up to 36% by BR. The microsomal BR degradation was inhibited by coumarin and a monoclonal antibody against the Cyp2a5 by about 90%. Furthermore, 7-methoxyresorufin, a substrate for the Cyp1a2, inhibited BR degradation activity by approximately 20%. In sum, the results strongly suggest a major role for Cyp2a5 in the oxidative degradation of BR. Secondly, the coordinated up-regulation of the HO-1 and Cyp2a5 during Cd-mediated injury implicates a network of enzyme systems in the maintenance of balancing BR production and elimination.

CYP2A6 and CYP2B6 are involved in nornicotine formation from nicotine in humans: Interindividual differences in these contributions

Nornicotine is an N-demethylated metabolite of nicotine. In the present study, human cytochrome P450 (P450) isoform(s) involved in nicotine N-demethylation were identified. The Eadie-Hofstee plot of nicotine N-demethylation in human liver microsomes was biphasic with high-affinity (apparent K(m) = 173 +/- 70 microM, V(max) = 57 +/- 17 pmol/min/mg) and low-affinity (apparent K(m) = 619 +/- 68 microM, V(max) = 137 +/- 6 pmol/min/mg) components. Among 13 recombinant human P450s expressed in baculovirus-infected insect cells (Supersomes), CYP2B6 exhibited the highest nicotine N-demethylase activity, followed by CYP2A6. The apparent K(m) values of CYP2A6 (49 +/- 12 microM) and CYP2B6 (550 +/- 46 microM) were close to those of high- and low-affinity components in human liver microsomes, respectively. The intrinsic clearances of CYP2A6 and CYP2B6 Supersomes were 5.1 and 12.5 nl/min/pmol P450, respectively. In addition, the intrinsic clearance of CYP2A13 expressed in Escherichia coli (44.9 nl/min/pmol P450) was higher than that of CYP2A6 expressed in E. coli (2.6 nl/min/pmol P450). Since CYP2A13 is hardly expressed in human livers, the contribution of CYP2A13 to the nicotine N-demethylation in human liver microsomes would be negligible. The nicotine N-demethylase activity in microsomes from 15 human livers at 20 microM nicotine was significantly correlated with the CYP2A6 contents (r = 0.578, p < 0.05), coumarin 7-hydroxylase activity (r = 0.802, p < 0.001), and S-mephenytoin N-demethylase activity (r = 0.694, p < 0.005). The nicotine N-demethylase activity at 100 microM nicotine was significantly correlated with the CYP2B6 contents (r = 0.677, p < 0.05) and S-mephenytoin N-demethylase activities (r = 0.740, p < 0.005). These results as well as the inhibition analyses suggested that CYP2A6 and CYP2B6 would significantly contribute to the nicotine N-demethylation at low and high substrate concentrations, respectively. The contributions of CYP2A6 and CYP2B6 would be dependent on the expression levels of these isoforms in any human liver.

CYP2A6 IS A PRINCIPAL ENZYME INVOLVED IN HYDROXYLATION OF 1,7-DIMETHYLXANTHINE, A MAIN CAFFEINE METABOLITE, IN HUMANS

In a caffeine test previously performed with healthy Japanese volunteers, we found that the CYP1A2 index defined as urinary {5-acetylamino-6-amine-3-methyluracil (AAMU)+1-methylxanthine (1X)+1-methyluric acid (1 U)}/1,7-dimethyluric acid (17 U) was affected by the whole deleted allele of CYP2A6 (CYP2A6*4). Since the high value of the CYP1A2 index could be caused by a low urinary concentration of 17 U, we postulated that CYP2A6 was responsible for the 1,7-dimethylxanthine (17 X) metabolism to generate 17 U (17 X 8-hydroxylation). Thus, the role of CYP2A6 in the 17 X 8-hydroxylation was fully examined in the present study. Among 10 isoforms of human cytochrome P450 (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, or CYP3A5) expressed in Escherichia coli cells, CYP2A6 and CYP1A2 showed high catalytic activities for the 17 X 8-hydroxylation. The 17 X 8-hydroxylase activities significantly associated with coumarin 7-hydroxylase activities (r=0.67, p<0.01) in liver microsomes from 17 individuals, but not with ethoxyresorufin O-deethylase activities. Tranylcypromine, an inhibitor of CYP2A6, reduced the 17 X 8-hydroxylase activities of human liver microsomes. The 17 X 8-hydroxylase activities of CYP2A6.7, CYP2A6.10, and CYP2A6.11 expressed in E. coli cells were 12, 13, and 22% of that of CYP2A6.1, respectively. The 17 X 8-hydroxylase activities were found to be low in liver microsomes from individuals possessing the deletion or mutations in the CYP2A6 gene. Based on these data, we conclude that CYP2A6 is a main 17 X 8-hydroxylase and that the catalytic activities for the 17 X 8-hydroxylation are reduced by the genetic polymorphisms of the CYP2A6 gene.

5-Substituted, 6-Substituted, and Unsubstituted 3-Heteroaromatic Pyridine Analogues of Nicotine as Selective Inhibitors of Cytochrome P-450 2A6

A series of 5- and 6-substituted and unsubstituted 3-heteroaromatic analogues of nicotine were synthesized in an effort to delineate the structural requirements for selectively inhibiting human cytochrome P-450 (CYP) 2A6, the major nicotine metabolizing enzyme. Thiophene, substituted thiophene, furan, substituted furan, imidazole, substituted imidazole, pyridine, substituted pyridine, thiazole, and quinoline moieties were used to replace the N-methylpyrrolidine ring of nicotine. Bromo and methyl groups were introduced at the 5-position of the pyridine ring and fluoro, chloro, and methoxy groups were placed at the 6-position of the pyridine ring in order to explore the structure-activity relationship (SAR) of inhibition of CYP2A6. The inhibitory activity of the most potent CYP2A6 inhibitors on the functional activity of human cytochrome P450s 3A4, 2E1, 2B6, 2C9, 2C19, and 2D6 was also examined to determine inhibitor selectivity. We identified 36 compounds that were more potent than nicotine at inhibition of coumarin 7-hydroxylase (CYP2A6) activity. We also found a number of compounds to be highly selective for the inhibition of human CYP2A6 versus the other human CYPs examined.