Cytochrome P450 Enzymes In Liver Microsomes Research Articles

In Vivo Modulation of Rat Liver Microsomal Cytochrome P450 Activity by Antimalarial, Anti-HIV, and Antituberculosis Plant Medicines.

Drug interactions are key reasons for adverse drug reactions and attrition from market. Major infectious diseases causing morbidity/mortality in Ghana are malaria, tuberculosis, and HIV/AIDS. In this study, plant medicines commonly used to treat/manage these diseases in Ghana were investigated for their potential to modulate rat cytochrome P450 enzyme activities. Fluorescence and high-performance liquid chromatography–based assays were used to assess effects of antimalarial plant medicines, Fever (FEV), Mal-TF (MAL), and Kantinka terric (KT); anti-TB medicines, Chestico (CHES), CA + ST Pains + HWNT (TF), and Kantinka herbatic (KHB); and anti-HIV/AIDS medicines, Wabco (WAB), AD + T/AD (LIV) and Kantinka BA (KBA) on rat liver microsomal cytochrome P450 enzyme activities. Effects of medicines on rat biochemical and hematological parameters were also assessed. Generally, the medicines altered microsomal CYP1A1/1A2, CYP2B1/2B2, CYP2C9, and CYP2D6 activities. Only KBA elicited an increase (80%) in CYP1A1/1A2 activity. FEV, MAL, CHES, WAB, and LIV strongly inhibited the enzyme activity. All the medicines significantly inhibited CYP2C9 (24%-80%) activity. CYP2D6 activity increased after treatment with MAL, KBA, LIV, and TF. Also, MAL, WAB, LIV, KHB, and CHES increased CYP2B1/2B2 activity, while KT decrease the activity. Generally, the medicines altered liver function in the rats. Cholesterol levels declined after KBA treatment only. White and red blood cell counts, hemoglobin and hematocrit levels were significantly reduced in KT- and KBA-treated rats. Our results suggest that use of the medicines could have implications for drug interactions and safety, particularly if the medicines are administered over prolonged periods. Further investigations are imperative to establish clinical relevance of these results.

In Vitro Antioxidant Potential and Effect of a Glutathione-Enhancer Dietary Supplement on Selected Rat Liver Cytochrome P450 Enzyme Activity.

Background There is considerable evidence that many people take dietary supplements including those of herbal origin as an alternative therapy to improve their health. One such supplement, with an amalgam of constituents, is CellGevity®. However, the effect of this dietary supplement on drug-metabolizing enzymes is poorly understood, as it has not been studied extensively. Therefore, we investigated the effect of CellGevity dietary supplement on selected rat liver microsomal cytochrome P450 (CYP) enzymes, the most common drug-metabolizing enzymes. We also determined the total antioxidant potential of this dietary supplement in vitro. Methods To determine the antioxidant potential of CellGevity dietary supplement, 2,2-diphenyl-2-picryl-hydrazyl (DPPH), total phenolic, and flavonoid assays were used after initial preparation of a solution form of the supplement (low dose, LD; 4 mg/kg and high dose, HD; 8 mg/kg). Rats received oral administration of these doses of the supplement for 7 days, after which the effect of the supplement on selected liver CYP enzymes was assessed using probe substrates and spectroscopic and high-performance liquid chromatographic methods. Rats which received daily administration of 80 mg/kg of phenobarbitone and distilled water served as positive and negative controls, respectively. Results The IC50 value of the supplement 0.34 ± 0.07 mg/ml compared to 0.076 ± 0.03 mg/ml of the BHT (positive control). The total phenolic content of the supplement at a concentration of 2.5 mg/ml was 34.97 g gallic acid equivalent (GAE)/100 g while its total flavonoid content at a concentration of 2.5 mg/ml was 6 g quercetin equivalent (QE)/100 g. The supplement significantly inhibited rat CYP2B1/2B2 (LDT 92.4%; HDT 100%), CYP3A4 (LDT 81.2%; HDT 71.7%), and CYP2C9 (LDT 21.7%; HDT 28.5%) while it had no significant inhibitory effect on CYPs 1A1/1A2, CYP1A2, and CYP2D6. Conclusion CellGevity dietary supplement possesses moderate antioxidant activity in vitro and has an inhibitory effect on selected rat liver CYP enzymes, suggesting its potential interaction with drugs metabolized by CYP enzymes.

Caffeine 7-N-demethylation and C-8-oxidation mediated by liver microsomal cytochrome P450 enzymes in common marmosets

1. 3-N-Demethylation of caffeine (1,3,7-trimethylxanthine) is mediated by human cytochrome P450 1A2, whereas 7-N-demethylation and C-8-hydroxylation are reportedly catalyzed by monkey P450 2C9 and rat P450 1A2, respectively.2. Roles of marmoset P450 enzymes in caffeine oxidation were investigated using nine marmoset liver microsomes and 14 recombinantly expressed marmoset P450 enzymes.3. Predominant caffeine 7-N-demethylation and C-8-hydroxylation activities in marmoset liver microsomes were moderately (r = 0.78, p < 0.05) and highly (r = 0.82, p < 0.01) correlated with midazolam 1′-hydroxylation activities, respectively, while the former was not strongly affected by ketoconazole or α-naphthoflavone.4. Caffeine C-8-hydroxylation in liver microsomes was inhibited by ketoconazole and activated by α-naphthoflavone, suggesting main involvements of P450 3As.5. Recombinant marmoset P450 3As had high Vmax/Km values for C-8-hydroxylation, comparable to Km values for marmoset liver microsomes. Marmoset P450 1As efficiently mediated caffeine 3-N-demethylation and C-8-hydroxylation with apparently lower Km values than those of liver microsomes.6. These results collectively suggest highly active marmoset P450 3A enzymes toward caffeine 8-hydorxylaiton and involvement of multiple P450 isoforms including P450 1A in caffeine 7-N- and 3-N-demethylations in marmoset livers. Marmoset P450s have slightly different properties to human or monkey P450s regarding caffeine metabolic pathways.

The effects of endosulfan on cytochrome P450 enzymes and glutathione S-transferases in zebrafish (Danio rerio) livers

Endosulfan, an organochlorine pesticide, has been used worldwide in the past decades. The present study was performed to investigate the effect of endosulfan on liver microsomal cytochrome P450 (CYP) enzymes and glutathione S-transferases (GST) in zebrafish. Male and female zebrafish were separated and exposed to a control and four concentrations of endosulfan (0.01, 0.1, 1, and 10μgL−1) and were sampled on days 7, 14, 21, and 28. After exposure to endosulfan, the content of CYP increased and later gradually fell back to control level in most sampling time intervals. A similar tendency was also found in the activities of NADPH-P450 reductase (NCR), aminopyrine N-demethylase (APND) and erythromycin N-demethylase (ERND). GST activities were generally higher in treatment groups than control groups. Regarding sex-based differences, the induction degree of the activity of NCR was generally higher in males than females. Similar differences were also found on the 28th day in the activities of APND and ERND, as well as GST activity on the 7th day. Overall, the present results demonstrate the toxicity at low doses of endosulfan and indicated marked induction of CYP and GST enzymes in zebrafish liver.

Five new eudesmane-type sesquiterpenoid lactones biotransformed from atractylenolide I by rat hepatic microsomes

The work presented here is the first study performed on the biotransformation and/or metabolism of atractylenolide I (1) as a valuable anti-inflammatory and chemopreventive agent, using liver microsomes from rats pre-treated with sodium phenobarbital. Two known eudesmane-type sesquiterpenoid lactones, namely 1β-acetoxyatractylenolide I (2) and 1β-hydroxy-atractylenolide I (3), and five new ones, namely 3β-hydroxy-atractylenolide I (4), 1β,13-dihydroxy-atractylenolide I (5), 1β,2α-dihydroxy-atractylenolide I (6), 1β,3α-dihydroxy-atractylenolide I (7), and 1β,3β-dihydroxy-atractylenolide I (8) were obtained. Their chemical structures were unambiguously established by both 1D and 2D NMR as well as mass spectroscopic techniques. The result indicated that the parent prototype compound 1 could be specifically oxidized at C-1, C-2 and C-3 of A-ring, suggesting that the oxidizable of 1 may contribute to its in vivo anti-inflammatory and chemopreventive effects. And the result also provided valuable information for further investigation of relationship among metabolic activation and liver microsomal cytochrome P450 enzyme isoforms.

Effects of the aqueous extract from Salvia miltiorrhiza Bge on the pharmacokinetics of diazepam and on liver microsomal cytochrome P450 enzyme activity in rats.

The aim of this study was to determine the effects of the aqueous extract of Salvia miltiorrhiza Bge (danshen in Chinese) on the pharmacokinetics of diazepam and on liver microsomal cytochrome P450 enzyme activity in rats. Rats (n = 5) were pretreated with danshen extract (100 mg kg(-1) per day, p.o.) for 15 consecutive days. Control rats (n = 5) received saline at the same time. Each rat was then administered a single oral dose of 15 mg kg(-1) diazepam. The pharmacokinetic parameters of diazepam were significantly different between the two groups. In the danshen pretreated group, the maximum concentration of diazepam and the area under the plasma concentration-time curve were reduced to about 72.7% and 44.4%, respectively, while the total body clearance was markedly increased by 2-fold. To help explain the results, liver microsomal suspensions were obtained from rats that were randomly divided into the control group (n = 10), and the low- (20 mg kg(-1) for 15 days, p.o., n = 10) and high-dose groups (100 mg kg(-1) for 15 days, p.o., n = 10) pretreated with danshen extract. Compared with the control rats, the microsomal protein content, cytochrome P450 enzyme level and erythromycin N-demethylase activity of pretreated rats were significantly increased. These results indicate that danshen extract can stimulate the activity of cytochrome P450 isoforms, and changes in the pharmacokinetics of diazepam resulting from danshen extract are related to an increase in metabolic activity of cytochrome P450.

The effect of immunosuppressive agents (FK-506, rapamycin) on renal P450 systems in rat models.

It is well known that cyclosporin, rapamycin and FK-506 (tacrolimus) are metabolized by the liver microsomal cytochrome P450 enzyme system. Although there have been reports of interaction between these drugs and the renal P450 enzyme system, differences among these immunosuppressants has not been comprehensively demonstrated. We have studied the individual capacities of these immunosuppressants to induce renal microsomal P450 enzymes similar to CYP2B4 and CYP4A2 by examining renal function in treated rats, and have correlated the results by means of biochemical, immunological and immunohistochemical assays of renal P450 enzymes. Cyclosporin caused impairment of renal function with an increase in renal-specific P450 content, but FK-506 and rapamycin did not. Laurate omega- and (omega-1)-hydroxylase activity increased in rats treated with rapamycin but decreased in those treated with FK-506. Prostaglandin A1 (PGA1) omega-hydroxylase activity increased in rats treated with FK-506 but was reduced by treatment with cyclosporin. Aminopyrine N-demethylase activity increased in rats treated with cyclosporin or FK-506, but not in those treated with rapamycin. Western-blot analysis revealed significant induction of P450, (similar to CYP2B4 of the rabbit P450 isozyme) in kidneys from rats treated with cyclosporin but not in those from rats receiving FK-506 or rapamycin. Histochemical studies clearly demonstrated a form of P450 such as CYP4A2 in the proximal tubules of rats treated with cyclosporin, but not in those of rats treated with FK-506 or rapamycin. These results show that although cyclosporin has a strong effect on renal P450 systems and induces such a system in kidney cortex (microsomal P450), FK-506 and rapamycin have no substantial effect on the induction of renal P450. These findings might clarify the nephrotoxicity induced by these immunosuppressive drugs.

Interactions of Olomoucine II with Human Liver Microsomal Cytochromes P450

Olomoucine II is a cyclin-dependent kinase inhibitor and a potential antineoplastic agent because it can arrest animal cell cycles. This study examines its interactions with human liver microsomal cytochrome P450 (P450) enzymes. Spectroscopic and high-performance liquid chromatography (HPLC) methods were used to estimate the degree of olomoucine II-mediated inhibition of enzymatic activities of eight drug-metabolizing P450s in vitro. In addition, mass spectrometry coupled with HPLC was used to identify an olomoucine II metabolite (2,5-dihydroxyroscovitine) formed in the reaction mixtures, and CYP3A4 was found to be responsible for the hydroxylation of the N(6)-benzyl ring at position 5, leading to this compound. Olomoucine II significantly inhibited the enzymatic activities of CYP1A2, CYP2C9, and (to a lesser degree) CYP3A4. The results indicate that use of olomoucine II as a drug could affect the activities of CYP3A4, CYP1A2, and CYP2C9 in vivo. Hence, the clinical relevance of these interactions should be carefully evaluated.

Dimemorfan N-demethylation by mouse liver microsomal cytochrome P450 enzymes

Dimemorfan ( d-3-methyl- N-methylmorphinan), an analogue of dextromethorphan, is commonly used as a non-opioid antitussive. To clarify the contribution of cytochrome P450 (P450) in dimemorfan N-demethylation, effects of selective inducers and inhibitors were studied in ICR mice. Phenobarbital (PB)- and dexamethasone (Dex)-treatments caused 5-fold increases of liver microsomal dimemorfan N-demethylation activity. In untreated mouse liver microsomes, demethylation activity was strongly inhibited by a CYP3A inhibitor, ketoconazole. In PB-and Dex-treated mouse liver microsomes, ketoconazole caused strong inhibition, whereas orphenadrine caused a decrease of less than 20%. Pretreatment of control mouse liver microsomes with anti-CYP3A inhibited demethylation activity, whereas pre-treatment with anti-CYP2B had no effect. In PB-and Dex-treated mouse liver microsomes, the demethylation activity was inhibited by both anti-CYP3A and anti-CYP2B. In control mice, the intrinsic clearance of dimemorfan from N-demethylation was 5.8 μl min − 1mg protein − 1. In PB- and Dex-treated mice, the correlation coefficient of fitting using one-enzyme and two-enzyme models were similar. The intrinsic clearances of induced mouse liver microsomes were similar. These results revealed that CYP3A played a major role in hepatic demethylation in untreated mice. Both CYP3A and CYP2B were involved in this demethylation in PB- and Dex-treated mice.

P71 Metabolism of delta‐3‐Carene by human cytochrom 450 enzymes

Occupational exposure to monoterpenes occurs in saw mills, particle‐board plants, carpentry shops and other types of wood‐treating industries. The bicyclic monoterpene delta‐3‐Carene, one of the components of turpentine, may irritate the skin and muceous membranes and prolonged exposure may result in allergic contact dermatitis or chronic lung function impairment. The effects of low concentrations of delta‐3‐Carene on alveolar macrophages in vitro were examined and a dose‐dependent relationship between the cell viability and the delta‐3‐Carene concentration was found. Little is known about the metabolism of delta‐3‐Carene in mammalians. In order to determine the toxic potential of this monoterpene we studied the human metabolism of delta‐3‐Carene in vitro. Therefore we used pooled human liver S9 and human liver microsomal cytochrome P450 enzymes. By using GC‐MS analysis we found one main metabolite produced at high rates. The structure was identified by its mass spectra. The mass fragmentation indicated hydroxylation in allyl position. After synthesis of the assumed product in a four step reaction, it was characterized as delta‐3‐Carene‐10‐ol. There was a clear correlation between the concentration of the metabolite production, incubation time and enzyme concentration, respectively. Kinetic analysis showed that Km and Vmax values for the oxidation of delta‐3‐Carene by human liver microsomes were 0.39 μM and 0.2 nmol/min/nmol P450. It is the first time that delta‐3‐Carene‐10‐ol is described as human metabolite of delta‐3‐Carene.

Roles of human CYP2A6 and 2B6 and rat CYP2C11 and 2B1 in the 10-hydroxylation of (-)-verbenone by liver microsomes.

(-)-Verbenone, a monoterpene bicyclic ketone, is a component of the essential oil from rosemary species such as Rosmarinus officinalis L., Verbena triphylla, and Eucalyptus globulus and is used for an herb tea, a spice, and a perfume. In this study, (-)-verbenone was found to be converted to 10-hydroxyverbenone by rat and human liver microsomal cytochrome p450 (p450) enzymes. The product formation was determined by high-performance liquid chromatography with UV detection at 251 nm. There was a good correlation between activities of coumarin 7-hydroxylation and (-)-verbenone 10-hydroxylation catalyzed by liver microsomes of 16 human samples, indicating that CYP2A6 is a principal enzyme in (-)-verbenone 10-hydroxylation in humans. Human recombinant CYP2A6 and CYP2B6 catalyzed (-)verbenone 10-hydroxylation at Vmax values of 15 and 21 nmol/min/nmol p450 with apparent Km values of 16 and 91 microM, respectively. In contrast, rat CYP2A1 and 2A2 did not catalyze (-)-verbenone 10-hydroxylation at all, suggesting that there were species-related differences in the catalytic properties of human and rat CYP2A enzymes in the metabolism of (-)-verbenone. In the rat, recombinant CYP2C11, CYP2B1, and CYP3A2 catalyzed (-)-verbenone 10-hydroxylation with Vmax and Km ratios (ml/min/nmol p450) of 0.73, 0.20, and 0.03, respectively. Male-specific CYP2C11 was a major enzyme in (-)-verbenone 10-hydroxylation by untreated rat livers, and CYP2B1 catalyzed this reaction in liver microsomes of phenobarbital-treated rats. Rat CYP2C12, a female-specific enzyme, did not catalyze (-)verbenone 10-hydroxylation. These results suggest that human CYP2A6 and rat CYP2C11 are the major catalysts in the metabolism of (-)-verbenone by liver microsomes and that there are species-related differences in human and rat CYP2A enzymes and sex-related differences in male and female rats in the metabolism of (-)-verbenone.

Farnesol as an inhibitor and substrate for rabbit liver microsomal P450 enzymes

Farnesol and the related isoprenoids, geranylgeraniol, geranylgeranyl pyrophosphate, and farnesyl pyrophosphate, are produced in the endoplasmic reticulum of hepatocytes in mammals, and each serve important biological functions. Of these compounds, only farnesol was shown to significantly inhibit rabbit liver microsomal cytochrome P450 enzymes. The observed inhibition appeared to be reversible, and was not strictly competitive, but rather mixed in nature. Of the activities examined, ethoxycoumarin de-ethylase and diclofenac-4-hydroxylase activities were most sensitive to farnesol, with K I and K I ′ values between 11 and 40 μM . Caffeine-8-hydroxylation and taxol-6-hydroxylation were not inhibited at all by farnesol. Farnesol appeared to be a P450 substrate, as well as an inhibitor, as indicated by the NADPH-dependent decrease in farnesol concentration in microsomal incubations, and the metabolism was inhibited by CO, which pointed to the involvement of P450 isozymes.

Common multiple interactions of tricyclic antidepressants and orphenadrine with liver microsomal cytochrome P450 enzymes of the rat

1. Interactions of tricylic anti-depressants (TCA) and structurally related drugs with rat microsomal cytochromes P450 were studied including competitive inhibition of enzymatic activities and formation of P450 metabolite complexes. 2. All compounds examined that carry a methylated aminoalkyl sidechain formed metabolite complexes with microsomal P450 of the untreated male rat. The extent of complex formation is only slightly altered by rat pre-treatment with P450 inducers indicating that mainly constitutive P450 enzymes are involved. 3. The kinetics of in vitro complex formation differed for the di- and monomethylamino derivatives of the TCA showing either a sigmoidal or hyperbolic shape respectively. Considerable auto-inhibition of complex formation is observed at concentrations 100 muM only with the dimethyl derivatives. 4. Besides metabolite complex formation, a further effect of the drugs is competitive inhibition of the CYP2B-dependent pentoxyresorufin O-dealkylation. The inhibitory potential of the drugs depends on their degree of N-alkyl substitution. Correspondingly, the Ki is in the range of 2.8-7.1, 0.1-0.2 and 0.01 muM for the dimethyl-, monomethyl- and unsubstituted drugs respectively. 5. It has been shown that P450 interactions with tricyclic anti-depressants include several types of mechanisms and several P450 enzymes. It might be pharmacologically important that the dimethylamino compounds are demethylated in vivo by cytochromes P450 giving rise to more potent P450 inhibitors compared with the parent compounds.

In vitro biotransformation of atrazine by rat liver microsomal cytochrome P450 enzymes

We studied atrazine (ATZ) metabolism in male and female rat liver microsomes in vitro, and the major metabolite was deisopropylatrazine (DeiPr-ATZ) with deethylatrazine (DeEt-ATZ) and 1-hydroxyisopropylatrazine (iPrOH-ATZ) as minor metabolites in both sexes. The enzyme kinetics of ATZ biotransformation were examined by means of Eadie–Hofstee analyses. Although no remarkable sex difference of Michaelis–Menten values for each pathway was observed, Cl ints ( V max/ K m) for DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ were slightly higher in female than in male rats. The formation of DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ from ATZ was substantially inhibited by SKF-525A, metyrapone, diallyl sulfide, 7-ethoxycoumarin, benzphetamine, nicotine, testosterone and lauric acid in both sexes. Cimetidine effectively inhibited the formation of all metabolites in male rats. On the other hand, the inhibition rates of the formation of DeiPr-ATZ and iPrOH-ATZ by cimetidine in female rats were lower than those in male rats, and DeEt-ATZ was hardly affected by the chemicals. In contrast with the results for cimetidine, the inhibition of ATZ biotransformation by bufuralol was more effective in female than in male rats. Anti-rat CYP2B1 and CYP2E1 antibodies effectively inhibited DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ formations in both sexes. Anti-rat CYP2C11 antibody also inhibited the three metabolites in both sexes, with the inhibition rates higher in male than in female rats, similar to cimetidine. In the case of anti-rat CYP2D1 antibody, the inhibitory effect on ATZ biotransformation in male rats was less than that in female rats. On the other hand, anti-rat CYP1A2, CYP3A2 and CYP4A1 antibodies did not affect the ATZ biotransformation in either sex. There was no significant correlation between the formation rate of ATZ metabolites and P450 isoform levels in either sex. These results may mean that CYP2B2, CYP2C11, CYP2D1 (only iPrOH-ATZ formation) and CYP2E1 in male rats, and CYP2B2, CYP2D1 and CYP2E1 in female rats are involved ATZ metabolism in liver, and that the substrate specificity of P450 isoforms for ATZ is broad.

Differential roles of cytochromes P450 2D1, 2C11, and 1A1/2 in the hydroxylation of bufuralol by rat liver microsomes

Bufuralol hydroxylation activities of liver microsomal cytochrome P450 (P450) enzymes were studied in the rat; the reaction has been used widely in determining levels of liver microsomal P450 2D6, which shows debrisoquine-type genetic polymorphism in humans. Liver microsomes catalyzed the conversion of bufuralol to 1′-hydroxybufuralol and a structurally unidentified metabolite (termed here as M-1) in the presence of an NADPH-generating system and molecular oxygen. Bufuralol 1′-hydroxylation activities catalyzed by the liver microsomes were not increased in rats treated with several P450 inducers, whereas β-naphthoflavone treatment (and to a lesser extent that of isosafrole) caused a significant induction of M-l formation. The major role of P450 1A1/2 in M-l formation was confirmed by catalytic inhibition with anti-P450 antibodies and α-naphthoflavone in liver microsomes of β-naphthoflavone-treated rats, and by reconstitution experiments containing P450 1A1 and 1A2. Among nine forms of purified rat P450 enzymes studied in the reconstituted system, P450 2C11 displayed the highest activities for bufuralol 1′-hydroxylation, followed by P450 1A1 and P450 2D1. A female-specific form of P450 2C12 did not catalyze bufuralol 1′-hydroxylation. In liver microsomes of male rats, however, P450 2D1 was the dominant enzyme because only anti-P450 2D1 antibodies, and not anti-P450 2C11 and anti-P450 1A1, inhibited the bufuralol hydroxylation activities, and a specific P450 2D1 inhibitor, quinine, caused a dramatic decrease in the hydroxylation activities. The major contribution of P450 2D1 in the bufuralol 1′-hydroxylation activities was also supported by a kinetic analysis of the reconstituted system; P450 2D1 enzyme had a very low K m value (8.4μM) as compared with those of P450 2C11 ( K m = 83 μM) and P450 1A1 ( K m = 230 μM). Thus, the present results suggested that different P450 enzymes are involved in the hydroxylation of bufuralol in rat liver microsomes, and the kinetic analysis, as well as immunoinhibition and chemical inhibition experiments, may be of great importance for determining the major roles of P450 enzymes in drug hydroxylation reactions.

Activation of trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene to genotoxic metabolites by rat and human cytochromes P450.

In order to address the hypothesis that 6-aminochrysene (6-AC) is converted to genotoxic products by cytochrome P450 enzymes via two activation pathways (N-hydroxylation and epoxidation), the activation of 6-AC and trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene (6-AC-diol) to genotoxic metabolites was examined in rat and human liver microsomal cytochrome P450 enzymes using Salmonella typhimurium TA1535/pSK1002 and TA1535/pSK1002/pNM12 (NM2009) as tester strains. The latter bacteria, an O-acetyltransferase-overexpressing strain, was highly sensitive to metabolites derived from activation of 6-AC, but not those from 6-AC-diol, using liver microsomes from phenobarbital-treated rats or a reconstituted monooxygenase system containing P4502B1 or -2B2, thus suggesting the roles of P450 and acetyltransferase systems in the activation process. 6-AC-diol, on the other hand, was activated very efficiently by liver microsomes prepared from beta-naphthoflavone-treated rats or a reconstituted system containing P4501A1 or -1A2; the activation reaction is considered to proceed through diol-epoxide formation. The contribution of rat P4501A enzymes towards activation of 6-AC-diol was confirmed by the inhibitory effects on the activation process of alpha-naphthoflavone, a specific inhibitor of P4501A-related activities, and antibodies raised against purified P4501A1 and -1A2. In humans, P4501A2 was found to be the major enzyme involved in the activation of 6-AC-diol to genotoxic metabolites while the parent compound 6-AC was activated mainly by P4503A4. Experiments using recombinant P450 proteins expressed in human lymphoblastoid cell lines showed that human P4501A1 could also activate 6-AC-diol to reactive metabolites at almost the same rate measured with P4501A2. In addition, P4502B6 was found to efficiently catalyze the activation of 6-AC to genotoxic metabolites, and P4503A4 was active in the activation of 6-AC-diol as well as 6-AC. Addition of purified rat epoxide hydrolase to the incubation mixture containing purified rat P4501A1 or microsomes expressing human P4501A1 caused inhibition of activation of 6-AC-diol. These results suggest the existence of different enzymatic activation pathways for 6-AC and 6-AC-diol. The former carcinogen may be N-hydroxylated principally by P4502B enzymes in rats and P4503A4 and -2B6 in humans and activation to its ultimate metabolites may proceed through esterification of the N-hydroxy metabolites by an N-acetyltransferase. The 6-AC-diol is metabolized to its ultimate diolepoxide product by P4501A enzymes in rat and human liver microsomes. P4503A4 (humans) and P4503A2 (rats) may also contribute to some extent in the activation of 6-AC-diol, albeit at lower rates than those of P4501A enzymes.

Use of a newly developed tester strain Salmonella typhimurium NM2009 for the study of metabolic activation of carcinogenic aromatic amines by rat liver microsomal cytochrome P-450 enzymes

Using an O-acetyltransferase-overexpressing strain Salmonella typhimurium NM2009 we measured the activities for metabolic activation of several carcinogenic arylamines to genotoxic products by rat liver microsomal cytochrome P-450 enzymes, and compared them with the activities obtained in the original tester strain Salmonella typhimurium TA1535/pSK1002 or the O-acetyltransferase-defective strain Salmonella typhimurium NM2000. Since all of the tester strains had introduced the umuC′-′lacZ gene, we could detect the genotoxic activities by measuring bacterial β-galactosidase activity resulting from the DNA damage. In the O-acetyltransferase-defective strain NM2000 most of the arylamines tested showed weak responses in inducing umu gene expression after metabolic activation by liver microsomes. The strain NM2009, on the other hand, was found to be highly sensitive towards a variety of aromatic amines, and these activities were greater than those seen in the original tester strain S. typhimurium TA1535/pSK1002. The chemicals which marked responses in strain NM2009 include 2-aminoanthracene, 6-aminochrysene, 2-aminofluorene, 2-acetylaminofluorene, 3-methoxy-4-aminoazobenzene, O-aminoazotoluene, Glu-P-1, Trp-P-2, AαC, MeAαC, MeIQ, MeIQx and IQ. Of these procarcinogens tested MeIQ, MeIQx and IQ also showed strong cytotoxic effects in S. typhimurium NM2009 after metabolic activation by liver microsomes. Only PhIP was the substrate showing similar responses in strains TA1535/pSK1002 and NM2009. The results with the reconstituted monooxygenase system containing purified cytochrome P-450 enzymes support the above findings obtained with the liver microsomal enzyme system. Thus, the usefulness of the newly developed strain NM2009 for the detection of reactive metabolites of several carcinogenic aromatic amines after metabolism by the liver microsomal cytochrome P-450-linked monooxygenase system has been ascertained.

Metabolic deactivation of furylfuramide by cytothrome P450 in human and liver microsomes

Metabolic deactivation of furylfuramide by human and rat liver microsomal cytochrome P450 enzymes has been investigated in a system measuring induction of umu gene expression response in Salmonella typhimurium TA1535/pSK1002. Both human and rat liver microsomes catalyzed the metabolism of furylfuramide to inactive form(s) that are incapable of inducing umu gene expression in the tester strain. The reaction required an NADPH-generating system and molecular oxygen and was inhibited by carbon monoxide, suggesting that a cytochrome P450-linked mono-oxygenase system is prerequisite for the deactivation reaction. With liver microsomes from variously pretreated rats, 3-methylcholanthrene was found to be a powerful inducer for the furylfuramide-metabolizing activity, and antibodies raised against rat P450IA1(BNF-B, c) and P450IA2(ISF-G, d) inhibited the microsomal activity. Human liver microsomal furylfuramide-metabolizing activity was also inhibited significantly by anti-P450IA2 IgG but weakly by anti-P450IA1 IgG. In liver microsomes prepared from seven different human samples, the activities of deactivation of furylfuramide were found to correlate with the amounts of immunoreactive protein related to rat P450IA2 and with the monooxygenase activities of metabolic activation of 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ) and of ethoxyresorufin O-deethylation. These results suggest that P450IA1 and P450IA2 in rats, and P450PA (IA2, the phenacetin O-deethylase and ortholog of rat P450IA2) in humans are the major enzymes involved in the deactivation of furylfuramide in liver microsomes. The metabolic studies involving HPLC analysis of products followed by spectrophotometric examination have also suggested that furylfuramide can be degraded very rapidly through the aerobic metabolism by liver microsomes.

Characterization of cytochrome P-450-dependent activities in hagfish, dogfish, perch and spectacle caiman

1. 1. Owing to interest in the original function and evolution of cytochrome P-450 this enzyme system was studied in four species each at different levels on the phylogenetic tree, representing Agnatha, Schlachi, Teleostei, and Reptilia. 2. 2. All species contained considerable amounts of hepatic microsomal cytochrome P-450. The content in dogfish, perch and spectacle caiman was 0.2–0.3 while in the dogfish it was 0.6 nmol per mg protein. 3. 3. Hepatic microsomal cytochrome P-450 dependent ethylmorphine demethylase, benzo(a)pyrene hydroxylase, 7-ethoxycoumarin-O-deethylase and 7-ethoxyresorufin-O-deethylase activities have been measured and the assays were carefully characterized with respect to pH-, protein content-, incubation time- and temperature-activity relationships. 4. 4. The study revealed species-dependent differences in the liver microsomal cytochrome P-450 enzyme system, which could be due to differences in cytochrome P-450 composition or in the microenvironment around the enzyme.

Interaction of civetone with rat liver microsomal cytochrome P-450 and steroidogenic enzymes

L'interaction de la civetone (isomeres cis et trans) avec le cytochrome PU50 de microsomes hepatiques de rat est etudiee. Son activite inhibitrice vis-a-vis de l'aromatase (enzyme extraite de la fraction microsomale de tissu placentaire humain), et de steroide 5α-reductase est evaluee in vitro