Cytochrome P450 2E1 and 2A6 enzymes as major catalysts for metabolic activation of N-nitrosodialkylamines and tobacco-related nitrosamines in human liver microsomes.

An acetyltransferase-overexpressing strain of Salmonella typhimurium (NM2009) has been used to investigate roles of human liver microsomal cytochrome P450 (P450) enzymes in the activation of carcinogenic nitrosamine derivatives, including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines, to genotoxic products. Studies employing correlation of activities with several P450-dependent monooxygenase reactions in different human liver samples, inhibition of microsomal activities by antibodies raised against human P450 enzymes and by specific P450 inhibitors, and reconstitution of activities with purified P450 enzymes suggest that the tobacco-smoke-related nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and N-nitrosonornicotine (NNN) as well as N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) are oxidized to genotoxic products by different P450 enzymes, particularly P450 2E1 and 2A6. The activation of NDMA and NNN by liver microsomes was suggested to be catalyzed more actively by P450 2E1 than by other P450 enzymes because the activities were well correlated with NDMA N-demethylation and aniline p-hydroxylation in different human samples, and purified P450 2E1 had the highest activities in reconstituted monooxygenase systems. The relatively high contribution of P450 2A6 to the activation of NDEA and NNK was supported by the correlation seen with coumarin 7-hydroxylation in human liver microsomes, and antibodies raised against P450 2A6 inhibited both activities by approximately 50%. P450 3A4, 2D6 and 2C enzymes appear not to be extensively involved in the activation of these nitrosamines as judged by several criteria examined. Thus, this work indicates that several P450 enzymes, particularly P450 2E1 and 2A6, catalyze metabolic activation of nitrosamine derivatives including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines in human liver microsomes.     

Effects of administration of the chemoprotective agent oltipraz on CYP1A and CYP2B in rat liver and rat hepatocytes in culture

The success of oltipraz (OPZ) [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3- thione] as a chemoprotective agent against aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat is thought to depend principally on its ability to enhance detoxication by inducing phase II enzymes, especially glutathione transferases. However, in primary cultures of human hepatocytes, we recently demonstrated that OPZ also has an important inhibitory effect on the major cytochromes P450 (CYPs) of human hepatic AFB1 metabolism. This has prompted a detailed study of the effect of OPZ on some CYPs involved in metabolism of AFB1 in the rat. Primary cultures of rat hepatocytes behaved similarly to human hepatocytes and responded to OPZ by inhibition of ethoxyresorufin-O- deethylase (EROD) and pentoxyresorufin-O-depentylase (PROD) activities mainly associated, respectively, with CYP1A and CYP2B. A time-course shows that this inhibition is largely reversible, with EROD and PROD activities reaching a minimum at 12 h and tending towards control values within 24 h. As is to be expected, the incubation of isolated microsomes with OPZ also inhibits CYP1A and 2B. The effect of OPZ on CYP1A is not a phenomenon limited to cells in culture, but also occurs in vivo. Using the whole animal, we were able to demonstrate that OPZ also transiently inhibited CYP1A activity in a rat given caffeine, by measuring the amounts of methylxanthines found in the serum. However, microsomes isolated from rats, that had been treated with OPZ in vivo, show no such inhibition, presumably because, since OPZ is a reversible inhibitor, it dissociates and is lost during the course of conventional procedures of microsomal preparation. This explains some earlier failures in studies of isolated microsomes to observe the inhibition of CYPs by OPZ. In addition to inhibiting their enzymatic activity, OPZ is also an inducer of CYP1A and 2B as shown by the increased levels of their mRNAs and of caffeine metabolism in vivo after 24 h or more. It is concluded that the mechanism of chemoprotection by OPZ, of toxic chemical metabolism in the rat, is complex and involves competitive inhibition of activation succeeded by induction of the enzymes of both activation and detoxication.      

铬,汞,镉,镍四种重金属化合物对FL／P4501A1细胞酶活性的影响

利用不同浓度的重金属化合物溴化汞、三氯化铬、硫酸镉、氯化镍处理人羊膜ＦＬ／Ｐ４５０１Ａ１细胞，其半数抑制浓度（ＩＣ５０，ｍｇ／ｍｌ）分别为６．０２６０，３７．５２１３，２４．５４９１和４５．３８４５。结果表明：不同的重金属化合物对ＦＬ／Ｐ４５０１Ａ１细胞的毒性相差很大，溴化汞和硫酸镉的细胞毒性较大。ＦＬ／Ｐ４５０１Ａ１细胞带有Ｐ４５０１Ａ１基因，该基因产物为乙氧基异吩恶唑Ｏ－去乙基酶（ＥＲＯＤ）。     

Roles of human liver cytochrome P4502C and 3A enzymes in the 3-hydroxylation of benzo(a)pyrene.

The major oxidation product of the classic polycyclic hydrocarbon carcinogen benzo(a)pyrene [B(a)P] is 3-hydroxy B(a)P. Numerous studies have been concerned with the measurement of B(a)P 3-hydroxylation activity in experimental animals and human tissues. Although human liver is the main site of this reaction, systematic studies had not been carried out to define the roles of individual cytochrome P-450 (P-450) enzymes involved. Purified human P4502C8 and P4503A4 showed appreciable catalytic activity; purified human P4501A2 and yeast recombinant (human) P4502C9 and P4502C10 had less activity. No B(a)P 3-hydroxylation activity was observed with purified human P4502A6, P4502D6, P45602E1, or P4502CMP. When microsomes prepared from different human liver samples were compared, B(a)P 3-hydroxylation activity was well correlated with nifedipine oxidation (a P4503A4 marker) but not markers of other P-450s, including tolbutamide hydroxylation (P4502C9 and 2C10), chlorzoxazone 6-hydroxylation (P4502E1), (S)-mephenytoin 4'-hydroxylation (P4502CMP), and coumarin 7-hydroxylation (P4502A6). In three of the liver microsomal samples with relatively high B(a)P 3-hydroxylation activity, immunoinhibition was observed with anti-P4503A greater than anti-P4502C (and no inhibition with several other antibodies). The selective chemical inhibitors gestodene and troleandomycin (P4503A enzymes) and sulfaphenazole (P4502C enzymes) reduced the B(a)P 3-hydroxylation activity of the more active microsomal preparations to rates seen in the preparations with low activity. This residual activity (and most of the activity in the low activity samples) was refractory to all of the chemical inhibitors and antibodies. The addition of 7,8-benzoflavone dramatically stimulated B(a)P 3-hydroxylation in all of the microsomal samples (and also stimulated purified P4503A4), arguing against an important role for P4501A1 or P4501A2. We conclude that roles of human P-450 enzymes for B(a)P 3-hydroxylation follow the order P4503A4 greater than or equal to P4502C8 greater than P4502C9/10 in human liver and that the other P-450s examined here do not have major roles. P4502C8 and P4502CMP (but not P4503A4) were found to activate B(a)P to products genotoxic in Salmonella typhimurium; this pathway would appear to involve products other than 3-hydroxy B(a)P and B(a)P 7,8-dihydrodiols.     

Metabolism of aflatoxin B1 by rabbit and rat nasal mucosa microsomes and purified cytochrome P450, including isoforms 2A10 and 2A11

The nasal mucosa of some mammalian species are susceptible tothe toxicity of aflatoxin B1 (AFB1), a potent hepato-carcinogen,but little is known about the nasal enzymes involved in themetabolic activation of AFB1 or the metabolites produced. Inthe present study, the metabolism of AFB1 was studied with nasalmicrosomes from rats and rabbits and with several purified isozymesof rabbit P450 in a reconstituted enzyme system. The rates ofAFB1-N7-guanine DNA adduct formation with rabbit and rat nasalmicrosomes are over 3- and 10-fold higher, respectively, thanwith liver microsomes from the same species. On the other hand,the rates of formation of AFM1 (9a-hydroxy-AFB1) and AFQ1 (3-hydroxy-AFB1)products known to be less toxic, are lower with nasal than withliver microsomes. Of particular interest, nasal microsomes producehigh levels of six unidentified polar metabolites that are notformed by microsomes from liver or several other tissues. Thesesame products are also generated by P450 NMa purified from rabbitnasal microsomes in a reconstituted system, but not by fiveother isozymes of cytochrome P450 (1A2, 2B4, 2E1, 2G1, 3A6)that are known to be present in nasal microsomes. AFB1-DNA adductsare formed by P450 NMa at a rate 3-fold higher than that bynasal microsomes. The DNA adducts are formed at much slowerrates by P450s 2G1, 2B4, and 1A2, and adducts are not formedat measurable rates by P450s 2E1 and 3A6. Moreover, AFB1—     

Effects of phenethyl isothiocyanate, a carcinogenesis inhibitor, on xenobiotic-metabolizing enzymes and nitrosamine metabolism in rats.

Phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables, has been shown to inhibit chemical carcinogenesis, possibly due to its ability to block the activation or to enhance the detoxification of chemical carcinogens. The present study was conducted to elucidate the biochemical mechanisms involved by characterizing the effects of PEITC on phase I and phase II xenobiotic-metabolizing enzymes. A single dose of PEITC to F344 rats (1 mmol/kg) decreased the liver N-nitrosodimethylamine demethylase (NDMAd) activity (mainly due to P450 2E1) by 80% at 2 h and the activity of NDMAd remained decreased by 40% at 48 h after treatment. The liver pentoxyresorufin O-dealkylase (PROD) activity and P450 2B1 protein level were elevated 10- and 7-fold at 24 h after treatment respectively. The liver microsomal ethoxyresorufin O-dealkylase (EROD) (mainly due to P450 1A) and erythromycin N-demethylase (mainly due to P450 3A) activities were decreased at 2-12 h after treatment and recovered afterwards. The lung microsomal PROD and EROD activities were not significantly affected; whereas, the nasal microsomal PROD and EROD activities were decreased by 40-50%. After a treatment with PEITC, the rates of oxidative metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were decreased in liver microsomes by 40-60% at 2 h and recovered gradually; the rates in lung microsomes were markedly decreased by 60-70% at 2 h and remained at the decreased level at 24 h; and the rates in nasal mucosa microsomes were decreased gradually with the lowest activities observed at 18 h (50%) followed by a gradual recovery. Furthermore, the treatment with PEITC resulted in a maximal 5-fold increase of NAD(P)H:quinone oxidoreductase and 1.5-fold increase of glutathione S-transferase activities in the liver, but the activities of these two enzymes were not significantly affected in the lung and nasal mucosa. The sulfotransferase activity in the liver was decreased by 32-48% at 24-48 h after treatment; the nasal activity was increased by 1.8- to 2.5-fold, but the lung activity was not significantly changed. The hepatic UDP glucuronosyltransferase activity was slightly decreased at 2 h but slightly increased at 48 h after treatment, but no changes were observed for the lung and nasal activities. The study demonstrates that PEITC selectively affects xenobiotic-metabolizing enzymes in the liver, lung and nasal mucosa and it is especially effective in inhibiting the P450-dependent oxidation of NNK in the lung and of NDMA in the liver.     

Inhibition of rat liver cytochrome P450 isozymes by isothiocyanates and their conjugates: a structure-activity relationship study

A series of arylalkyl and alkyl isothiocyanates, and their glutathione,cysteine, and N-acetylcysteine conjugates were used to studytheir inhibitory activity toward the dealkylation of ethoxyresorufin(EROD), pentoxyresorufin (PROD), and methoxyresorufin (MROD)in liver microsomes obtained from the 3-methylcholanthrene orphenobarbital-treated rats. These reactions are predominantlymediated by cytochrome P450 (P450) isozymes 1A1 and 1A2, 2B1and 1A2, respectively. All isothiocyanates inhibited PROD morereadily than EROD. Increases in the alkyl chain length of arylalkylisothiocyanates to C₆ resulted in an increased inhibitory potencyin these assays; at longer alkyl chain lengths (C₈-C₁₀) theinhibitory potency declined. The IC₅₀s for phenethyl isothiocyanate(PEITC) were 47, 46 and 1.8 µM for EROD, MROD and PROD,respectively. Substitution of an additional phenyl group onPEITC also increased the inhibitory potency; the IC₅₀s for 1,2-diphenylethyl isothiocyanate (1, 2-DPEITC) and 2, 2-diphenylethylisothiocyanate (2,2-DPEITC) were 0.9 and 0.26 µM for EROD,and 0.045 and 0.13 µM for PROD, respectively. The relativeinhibitory potency of PEITC and its conjugates was N-acetylcysteine-PEITC(PEITC-NAC) < glutathione-PEITC (PEITC-GSH) < cysteine-PEITC(PEITC-CYS) < PEITC. The observations that the parent isothiocyanateswere more potent inhibitors than the conjugates suggest thatdissociation of the conjugate is required for activity. Naturallyoccurring alkyl isothiocyanates, sulforaphane (SFO) and allylisothiocyanate (AITC), were very weak inhibitors in the assays.These results suggest the potential of isothiocyanates as structuralprobes for studying P450 isozymes. In addition, the inhibitoryactivity of isothiocyanates for PROD correlated with the previouslydemonstrated tumor inhibitory potency in (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) induced A/J mouse lung tumor bioassays, which supportsearlier findings that P450 2B1 is one of the major isozymesinvolved in NNK activation and that inhibition of this isozymeis an important mechanism for the chemopreventive activity ofisothiocyanates.     

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in human lung and liver microsomes and cytochromes P-450 expressed in hepatoma cells.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-specific carcinogen in animals, has been linked to tobacco-related cancers in humans. The cytochrome(s) P-450 (P-450) responsible for the metabolic activation of NNK in humans has not been identified. The present work investigated the ability of human lung and liver microsomes and 12 forms of human P-450, expressed in Hep G2 (hepatoma) cells, to metabolize NNK. Of the 12 P-450 forms, P-450 1A2 had the highest activity in catalyzing the conversion of NNK to the keto alcohol, 4-hydroxy-1-(3-pyridyl)-1-butanone. P-450s 2A6, 2B7, 2E1, 2F1, and 3A5 also had measurable activities in the formation of keto alcohol. The apparent Km and Vmax for the formation of keto alcohol in the P-450 1A2-expressed Hep G2 cell lysate were 309 microM and 55 pmol/min/mg protein, respectively. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol, a reductive product, was the major metabolite formed, whereas the formation of keto alcohol and its aldehyde and acid derivatives (all alpha-hydroxylation products) constituted approximately 1% of the initial amount of NNK in P450-expressed Hep G2 cell lysate. A similar metabolite pattern was observed with human lung or liver microsomes. In human lung microsomes, the apparent Kms for the formation of 4-hydroxy-4-(3-pyridyl)butyric acid, 4-oxo-1-(3-pyridyl)-1-butanone, NNK-N-oxide, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were 526, 653, 531, and 573 microM, respectively; the formation of keto alcohol was not observed. For human lung microsomes, there was no sex-related difference in NNK metabolism. Carbon monoxide (90% atmosphere) significantly inhibited the metabolism of NNK in human lung and liver microsomes. 7,8-Benzoflavone, an inhibitor of P-450s 1A1 and 1A2, had no effect on NNK metabolism in human lung microsomes but decreased the formation of keto alcohol by 47% in human liver microsomes. Similarly, antibodies against human P-450s 1A2 and 2E1 decreased keto alcohol formation by 42% and 53%, respectively, in human liver microsomes but did not affect NNK metabolism in lung microsomes. Inhibitory antibodies against P-450s 2A1, 2C8, 2D1, or 3A4 had little or no effect on the metabolism of NNK in human liver or lung microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Participation of rat liver cytochrome P450 2E1 in the activation of N-nitrosodimethylamine and N-nitrosodiethylamine to products genotoxic in an acetyltransferase-overexpressing Salmonella typhimurium strain (NM2009).

The possible roles of cytochrome P450 (P450) enzymes in the metabolic activation of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) by rat liver microsomes have been examined in a system containing the bacterial tester strain Salmonella typhimurium NM2009, a newly developed strain showing high O-acetyltransfer activities. The DNA-damaging activity could be determined by measuring expression of the umu gene in a plasmid containing the fused umuC-lacZ gene construct in the bacteria. The following lines of evidence support the view that both NDMA and NDEA are principally oxidized to reactive products by P450 2E1 in rat liver microsomes. First, NDMA and NDEA were activated by rat liver microsomes in a protein- and substrate-dependent manner and the former chemical was more active than the latter; both activities were induced in rats treated with P450 2E1 inducers such as ethanol, acetone and isoniazid and by starvation. Second, activation of NDMA and NDEA were both inhibited significantly by antibodies raised against rat P450 2E1 and by P450 2E1 inhibitors such as diethyldithiocarbamate and 4-methylpyrazole in rat liver microsomes. Finally, in reconstituted monooxygenase systems containing purified rat P450 enzymes, P450 2E1 gave the highest rates of the activation of both NDMA and NDEA; the addition of rabbit cytochrome b5 to the system caused about a 1.5-fold increase in both reactions. In separate experiments we also found that N-nitrosomethylacethoxymethylamine, a compound that reacts with DNA after ester cleavage, is more genotoxic in S.typhimurium NM2009 than in S.typhimurium NM2000, a strain that is defective in O-acetyltransferase activity. Part of the pathway involved in the activation of nitrosamines is suggested to be acetylation of alkyldiazohydroxides formed by P450 or acetylesterase, because the genotoxic activity of N-nitrosomethylacethoxymethylamine in S.typhimurium NM2009 could be inhibited by the O-acetyltransferase inhibitor pentachlorophenol. These results indicate that NDMA and NDEA are oxidized to gentoxoic products by rat liver microsomes and that a P450 2E1 enzyme plays a major role in the activation of these two potent carcinogens. The activation pathway of N-nitrosodialkylamines through acetylation by O-acetyltransferase has been proposed. This simple bacterial system for measuring genotoxicity should facilitate studies on the activation of N-nitroso alkylamines.     

Contributions of human liver cytochrome P450 enzymes to the N-oxidation of 4,4'-methylene-bis(2-chloroaniline).

4,4'-Methylene-bis(2-chloroaniline) (MOCA) can produce tumors in rodents and dogs and an increased incidence of bladder tumors has been reported in exposed workers. It is therefore of interest to identify the human cytochrome P450 (P450) enzymes involved in MOCA N-oxidation, the primary reaction involved in the formation of an electrophilic product. Human liver microsomes were fractionated and MOCA N-oxidation activity was monitored through the procedure. The most active enzyme fraction corresponded to P450 3A4, as determined by immunochemical assays and N-terminal amino acid sequence analysis. Yeast recombinant P450 3A4 also had MOCA N-oxidation activity. Purified human liver P450 2A6 showed catalytic activity; however, anti-P450 2A6 inhibited less than 20% of the microsomal activity while anti-P450 3A4 inhibited up to 75%. Levels of marker activities of both P450 3A4 (nifedipine oxidation) and P450 2A6 (coumarin 7-hydroxylation) were measured in a set of human liver microsomes and both were correlated with MOCA N-oxidation rates. Gestodene and troleandomycin inhibited up to half of the microsomal MOCA N-hydroxylation activity but 7,8-benzoflavone showed only slight inhibition. Anti-P450 3A4 inhibited (up to 80% of) the microsomal transformation of MOCA to a product genotoxic as judged by bacterial SOS response. The work indicates that P450 3A4 makes a major contribution to human liver microsomal MOCA N-oxidation, and P450 2A6 has a minor role. P450 1A2, which catalyzes the hydroxylation of many arylamines, does not contribute to a great extent.     

Enzymes involved in the bioactivation of 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone in patas monkey lung and liver microsomes

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen in animals. Our previous studies indicated that there are differences between rodents and humans for the enzymes involved in the activation of NNK. To determine if the patas monkey is a better animal model for the activation of NNK in humans, we investigated the metabolism of NNK in patas monkey lung and liver microsomes and characterized the enzymes involved in the activation. In lung microsomes, the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK- N-oxide), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), and 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was observed, displaying apparent Km values of 10.3, 5.4, 4.9, and 902 microM, respectively. NNK metabolism in liver microsomes resulted in the formation of keto aldehyde, keto alcohol, and NNAL, displaying apparent Km values of 8.1, 8.2, and 474 microM, respectively. The low Km values for NNK oxidation in the patas monkey lung and liver microsomes are different from those in human lung and liver microsomes showing Km values of 400-653 microM, although loss of low Km forms from human tissue as a result of disease, surgery or anesthesia cannot be ruled out. Carbon monoxide (90%) significantly inhibited NNK metabolism in the patas monkey lung and liver microsomes by 38-66% and 82-91%, respectively. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) and aspirin (a cyclooxygenase inhibitor) decreased the rate of formation of keto aldehyde and keto alcohol by 10-20 % in the monkey lung microsomes. Alpha-Napthoflavone and coumarin markedly decreased the oxidation of NNK in monkey lung and liver microsomes, suggesting the involvement of P450s 1A and 2A6. An antibody against human P450 2A6 decreased the oxidation of NNK by 12-16% and 22-24% in the patas monkey lung and liver microsomes, respectively. These results are comparable to that obtained with human lung and liver microsomes. Coumarin hydroxylation was observed in the patas monkey lung and liver microsomes at a rate of 16 and 4000 pmol/min/mg protein, respectively, which was 5-fold higher than human lung and liver microsomes, respectively. Immunoblot analysis demonstrated that the P450 2A level in the individual patas monkey liver microsomal sample was 6-fold greater than in an individual human liver microsomal sample. Phenethyl isothiocyanate, an inhibitor of NNK activation in rodents and humans, decreased NNK oxidation in the monkey lung and liver microsomes displaying inhibitor concentration resulting in 50% inhibition of the activity (IC50) values of 0.28-0.8 microM and 4.2-6.8 microM, respectively. The results demonstrate the similarities and differences between species in the metabolic activation of NNK. The patas monkey microsomes appear to more closely resemble human microsomes than mouse or rat enzymes and may better reflect the activation of NNK in humans.      

Effects of plant-derived phenols on rat liver cytochrome P450 2B1 activity

Dietary constituents contain a variety of compounds that are known to modulate liver enzyme activity. In this report, the plant-derived phenols catechin, chlorogenic acid, diosmin, epigallo-catechin gallate (EGCG), naringenin, quercetin and resveratrol were studied for their effects on the activity of cytochrome P450 2B1 in liver microsomes from 6- and 20-month male Fisher F344 rats. The compounds at two concentrations (0.1 and 0.25 mM) were incubated with 0.2 mg liver microsomal protein and 50 microM 7-ethoxy-4-trifluoromethyl coumarin (EFC). O-deethylation of EFC to the fluorescence product 7-hydroxy-4-trifluoromethyl coumarin (HFC) is catalyzed by CYP450 2B1. EGCG, naringenin, quercetin and resveratrol inhibited the in vitro O-deethylation of EFC in liver microsomes from both 6- and 20-month rats. Quercetin was the most effective inhibitor. Catechin inhibited the in vitro O-deethylation of EFC only in microsomes from 6-month-old rats whereas diosmin inhibited the reaction only in microsomes from 20-month-old rats. Chlorogenic acid inhibited the in vitro O-deethylation of EFC in microsomes from both age groups at the 0.25 mM concentration only. These results suggest that plant phenols have varied effects on liver microsomal cytochrome P450 2B1 activity that may be influenced by the age of the animal.     

Identification of the human liver microsomal cytochrome P450s involved in the metabolism of N-nitrosodi-n-propylamine

The ability of human liver cytochrome P450s to metabolize the environmental carcinogen N-nitrosodi-n-propylamine (NDPA) was investigated. The maximum rate of NDPA depropylation in seven human liver microsomal samples was 1.15 nmol/min/mg (range 0.53-2.60). Troleandomycin, a P450 3A4/5 inhibitor, inhibited depropylation modestly (10-60%) in three of seven samples. Diethyldithiocarbamic acid, a potent 2E1 inhibitor, and a 2E1 inhibitory monoclonal antibody (mAb) inhibited the reaction in all samples (23 to almost 100%). No significant inhibition was observed with the 2C9 inhibitor sulfaphenazole or with mAbs to 3A4, 2A6 and 2D6. The 2C8/9/18/19 mAb inhibited depropylation in one sample by approximately 25% and approximately 25% of the activity in another sample could not be accounted for by the inhibitors. Denitrosation of NDPA by three of the microsomal samples exhibited low K(m) values (51-86 microM) while two of these also had high K(m) values (2.6 and 4.6 mM). Purified human P450 2B6 and 3A4 and human P450 2A6, 2C8, 2C9 and 2D6 membranes had high K(m) values relative to their maximum turnover rates and are unlikely to participate in NDPA metabolism at micromolar concentrations. Conversely, purified rabbit 2E1 exhibited K(m) and V(max) values for depropylation of 52 microM and 13.4 nmol propionaldehyde/min/nmol P450, respectively. Values for denitrosation were 66 microM and 1.44 nmol nitrite/min/nmol P450, respectively. The toxicity of NDPA in transfected human liver epithelial cells expressing 2E1 was dose dependent down to 50 microM. No toxicity was observed in control cells or those expressing 2A6. These results indicate that 2E1 is the major human liver microsomal isoform responsible for NDPA metabolism at low micromolar concentrations. We also show that purified P450s catalyze the denitrosation of NDPA at approximately 10-20% of the rate of depropylation and K(m) values for both reactions are the same for each isozyme. This is consistent with the formation of an initial intermediate common to both pathways, presumably an alpha-nitrosamino radical.     

Potent and non-specific inhibition of cytochrome P450 by JM216, a new oral platinum agent.

Bis-acetato-ammine-dichloro-cyclohexylamine-platinum (IV), JM216, is the first antineoplastic platinum compound that can be given to patients orally. Several phase II clinical trials of JM216 monotherapy have already been reported. However, no information on the potential drug interactions caused by JM216 is available. In this study, the capacity of JM216 to inhibit cytochrome P450 (CYP) in human liver microsomes was investigated by measuring the inhibition potential (IC50 and Ki) on prototype reactions. Specific substrates of CYP included testosterone (catalysed by CYP3A4), paclitaxel (CYP2C8), 7-ethoxyresorufin (CYP1A1, CYP1A2), coumarin (CYP2A6), aniline (CYP2E1) and (+/-)-bufuralol (CYP2D6). JM216 inhibited the catalytic activities of CYP isozymes. The IC50 values were between 0.3 microM and 10 microM, indicating strong and non-specific inhibitory effects of JM216. The inhibition occurred in a non-competitive manner, and the Ki value was 1.0 and 0.9 microM for metabolite formation of testosterone and paclitaxel respectively. Therefore, some in vivo studies should be conducted to determine whether or not there is a correlation between in vivo and in vitro results.     

Metabolism of 2-acetylaminofluorene and benzo(a)pyrene and activation of food-derived heterocyclic amine mutagens by human cytochromes P-450

The human P-450 CYP1A1 gene and a P450IA2 complementary DNA have been expressed in Cos-1 cells and the expressed proteins were assayed for their capacity to metabolize the carcinogens 2-acetylaminofluorene (AAF), benzo(a)pyrene, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was determined. The expressed human P450IA1 and P450IA2 proteins, when run on a 7.5% sodium dodecyl sulfate-polyacrylamide gel, migrated with different mobilities, with the former displaying the lower molecular weight. In human liver microsomes from 18 subjects, only a protein band corresponding to P450IA2 was detectable. Cos-1 cell-expressed P450IA1 and P450IA2 were capable of N-hydroxylating AAF and these activities were inhibited by alpha-naphthoflavone. In human liver microsomes, a correlation of r = 0.76 (P less than 0.05; n = 18) was obtained between AAF N-hydroxylase activity and P450IA2 content. AAF N-hydroxylase activity of human liver microsomes was also strongly inhibited by alpha-naphthoflavone. Except in the case of PhIP, where both proteins exhibited similar activities, P450IA2 was at least an order of magnitude more efficient than P450IA1 in activating IQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline to mutagens as measured in the Ames test. Statistically significant correlations were obtained between IQ activation and P450IA2 content (r = 0.75, r2 = 0.56) and PhIP activation and P450IA2 content (r = 0.71, r2 = 0.5) in human liver microsomes. The activation of both IQ and PhIP by expressed proteins and human liver microsomes was strongly inhibited by alpha-naphthoflavone. The above data suggest a major role for P450IA2 in activation (N-hydroxylation) of aromatic amides and amines in human liver. When benzo(a)pyrene hydroxylase activity was determined, only Cos-1 cell-expressed P450IA1 exhibited appreciable activity. While alpha-naphthoflavone inhibited Cos-1 cell-expressed P450IA1 benzo(a)pyrene hydroxylase activity, it caused a marked stimulation of this activity in human liver microsomes, which lack P450IA1 protein. The lack of a role for P450IA proteins in benzo(a)pyrene metabolism is further supported by the poor correlation (r = 0.43, P greater than 0.05) between this activity and P450IA2 content of human liver microsomes. However, when P450IIIA3 content of the above human liver microsomes was determined by using the Western blot technique and correlated with benzo(a)pyrene metabolism, an r value of 0.70 (P less than 0.5) was obtained. These data suggest that human P450IIIA proteins are involved in benzo(a)pyrene metabolism.     

Metabolic activation of aflatoxin B1 and 2-amino-3-methylimidazo[4,5-f]-quinoline by human adult and fetal livers

The mutagenic activation of promutagens by human adult and fetal livers was investigated using the umu test system. Among the promutagens studied, aflatoxin B1 (AFB1) and 2-amino-3-methyl-imidazo[4,5-f] quinoline (IQ) were efficiently activated to mutagens by both adult and fetal livers. 7,8-Benzoflavone inhibited the activation of IQ by fetal livers, but the inhibition observed in fetal livers was much less than that observed in adult livers. Antibodies to P450HM1 (P450111A4) and P450HFLa markedly inhibited the activation of AFB1 by adult and fetal livers, respectively. The formation of genotoxic product(s) from IQ in human adult livers was almost completely inhibited by anti-P448H (P4501A2) antibodies but not by anti-P450HM1 antibodies, whereas that in fetal livers was inhibited by both anti-P450HFLa and anti-P450IA2 antibodies. P450HFLa catalyzed the mutagenic activation of both AFB1 and IQ in a reconstituted system. On the contrary, P450HM1 catalyzed the mutagenic activation of AFB1 but not IQ. A preparation of cytochrome P450 partially purified from human fetal livers and cross-reactive with anti-P450IA2 antibodies was found to be active for mutagenic activation of IQ in a reconstituted system. These results indicate that P450HFLa and P450HM1 are mainly involved in the genotoxic product formation from AFB1 in fetal and adult livers, respectively, and that the metabolic activation of IQ in fetal livers is catalyzed by two forms of cytochrome P450, P450HFLa, and cytochrome P450 immunochemically related to P450IA2 but that in adult livers it is mainly catalyzed by cytochrome P450 related to P450IA2.     

Cytochrome P450 forms in the rodent lung involved in the metabolic activation of food-derived heterocyclic amines

The metabolic activation of the promutagens 2-amino-3,8-dimethylimidazo[4,5-f]quinoline(IQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx)and 2-amino-1-methyI-6-phenylimidazo[4,5-b]pyridine (PhIP) byrat and mouse lung microsomes was studied using Salmonella mutagenicity(strain TA98). Lungs from uninduced animals were found to activateall three compounds. A 4–6 fold higher mutagenic activitywas obtained with IQ compared to MeIQx and the mutagenic responseof PhIP was 1–2 orders of magnitude lower than that ofIQ. In order to characterize the forms of P450 in the lung responsiblefor the metabolic activation of these food mutagens Westernblots were performed with microsomes and partially purifiedP450 fractions from the lung. Western blots revealed the presenceof cytochrome P450 2A, 2B and 4A forms in untreated rats. Inthe lung CYP 1A1 was only detectable after BNF treatment ofrats. The CYP 4A isozymes, which have not previously been describedin the rat lung, were further identified after PCR amplificationfrom lung mRNA as 4A2 and 4A8. Antibody inhibition studies showedthat CYP 2A3 catalyzed a major part (70%) of the metabolic activationof IQ by uninduced rat lung microsomes. The metabolic activationof MeIQx was not influenced by this antibody. An antibody againstCYP 2B isozymes also partially inhibited the activation of IQby uninduced rat lung microsomes. However, since induction ofCYP 2B isozymes in the liver by phenobarbital treatment didnot increase the metabolic activation of the heterocyclic aminesover controls it is unlikely that the rat lung CYP 2B1 is participatingin the activation of heterocyclic amines. The inhibition ofthe IQ-dependent mutagenicity by the CYP 2B antibody is probablydue to cross-reaction with CYP 2A3. Alfa-naptho-flavone (ANF),considered to be a specific inhibitor of CYP 1A isozymes at10 µM, partly inhibited the activation of IQ (30–40%)and MeIQx (60–80%) by uninduced rat and mouse lung microsomes.Upon pretreatment of rats with BNF, lung microsomes activatedMeIQx at a rate that was 2–10-fold higher than controllung microsomes, whereas the increase in EROD activity was approximately100-fold in the same lung preparations. These results suggestthat CYP 1A1 may not be the enzyme responsible for the activationof MeIQx in the control rat despite the inhibition with ANF.It is likely that ANF can inhibit other P450 enzymes in thelung, including CYP 2A3. The involvement of CYP 2A3. The involvementof CYP 2A3 in the metabolic activation of IQ by uninduced ratlung shows that CYP forms that are not of major importance inthe liver may play a significant role in extra-hepatic activationof heterocyclic amines.     

Roles of different cytochrome P450 enzymes in bioactivation of the potent hepatocarcinogen 3-methoxy-4-aminoazobenzene by rat and human liver microsomes

The potent hepatocarcinogen 3-methoxy-4-aminoazobenzene (3-MeO-AAB) has been reported to be bioactivated to mutagenic intermediates by rat liver microsomal cytochrome P450 (P450) and to be a selective inducer of rat P450IA2. In this study we have further investigated the roles of individual rat and human P450 enzymes in the bioactivation of this hepatocarcinogen in a Salmonella typhimurium TA1535/pSK1002 system where umu response is indicative of DNA damage. 3-MeO-AAB was found to be bioactivated by liver microsomal enzymes from rats and humans in this assay system. The liver microsomal activities are increased by pretreatment of rats with various P450 inducers such as phenobarbital (PB), beta-naphthoflavone (BNF), dexamethasone (DEX), acetone, ethanol, isoniazid (INH), diphenylhydantoin and valproic acid, and can be inhibited considerably by SKF-525A and metyrapone. alpha-Naphthoflavone (ANF) is also an inhibitor for the reaction catalyzed in BNF-treated rats, but stimulated the microsomal activity in DEX-treated rats. Evidence has also been obtained that specific antibodies raised against P450IIB1, P450IA1 or IA2, P450IIE1, and P450IIIA2 inhibited the activation in liver microsomes from rats pretreated with PB, BNF, INH and DEX respectively, suggesting the possible roles of several P450 enzymes in the bioactivation of 3-MeO-AAB. The results obtained with reconstituted monooxygenase systems containing various rat P450 enzymes are highly supportive of this conclusion. Human liver microsomal activation of 3-MeO-AAB was also inhibited to various extents by antibodies raised against P450IA2, P450MP, P450IIE1 and P450IIIA4. In a reconstituted system containing purified forms of human P450, P450IA2 was the most active in catalyzing 3-MeO-AAB, followed by P450IIIA4 and P450MP. ANF, a known activator of P450IIIA-catalyzed reactions, caused an increase in activation of 3-MeO-AAB in human liver microsomal and P450IIIA4- and P450MP-containing reconstituted systems. From these results it is concluded that multiple P450 enzymes in rat and human liver microsomes are involved in the bioactivation of 3-MeO-AAB, regardless of its selective induction of the rat P450IA2 gene.     

St. John's wort extracts and some of their constituents potently inhibit ultimate carcinogen formation from benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1

Commercially available St. John's wort (Hypericum perforatum) preparations and some of their main constituents (hypericin, pseudohypericin, hyperforin, rutin, and quercetin) were examined for their potential to inhibit carcinogen activation by human cytochrome P450 1A1 (CYP1A1). We used a reconstituted system consisting of purified human CYP1A1, purified human NADPH-cytochrome P450 reductase, and dilaurylphosphatidylcholine as lipid component. St. John's wort extracts potently inhibited CYP1A1-catalyzed (+/-)-trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene (7,8-diol-B[a]P) epoxidation, the terminal reaction leading to the ultimate carcinogenic product (+/-)-B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide (diolepoxide 2). All constituents, except rutin, were shown to possess strong inhibitory potencies toward diolepoxide 2 formation from 7,8-diol-B[a]P, with IC(50) values of 0.5 microM (hypericin), 1.2 microM (hyperforin), 1.5 microM (quercetin), and 8 microM (pseudohypericin), respectively. Preincubation experiments revealed that their action was not mechanism based. Inhibition kinetics studies showed the anthrodianthrone compound hypericin to be a noncompetitive inhibitor, with a K(i) value of 0.6 microM, and the phloroglucinol hyperforin to be a competitive inhibitor, with a K(i) value of 1.1 microM. When the effects on NADPH-P450 reductase activity were investigated, all constituents of St. John's wort studied turned out to be rather ineffective inhibitors; quercetin was the only exception, with an IC(50) value of approximately 20 microM. These in vitro data indicate that St. John's wort extracts and some of their constituents potently inhibit the major human procarcinogen-activating enzyme CYP1A1.     

Cytochrome P450 mediated bioactivation of methyleugenol to 1'- hydroxymethyleugenol in Fischer 344 rat and human liver microsomes

Cytochrome P450 mediated metabolism of methyleugenol to the proximate carcinogen 1'-hydroxymethyleugenol has been investigated in vitro. Kinetic studies undertaken in liver microsomes from control male Fischer 344 rats revealed that this reaction is catalyzed by high affinity (Km of 74.9 +/- 9.0 microM, Vmax of 1.42 +/- 0.17 nmol/min/nmol P450) and low affinity (apparent Km several mM) enzymic components. Studies undertaken at low substrate concentration (20 microM) with microsomes from livers of rats treated with the enzyme inducers phenobarbital, dexamethasone, isosafrole and isoniazid indicated that a number of cytochrome P450 isozymes can catalyze the high affinity component. In control rat liver microsomes, 1'- hydroxylation of methyleugenol (assayed at 20 microM substrate) was inhibited significantly (P < 0.05) by diallylsulfide (40%), p- nitrophenol (55%), tolbutamide (30%) and alpha-naphthoflavone (25%) but not by troleandomycin, furafylline, quinine or cimetidine. These results suggested that the reaction is catalyzed by CYP 2E1 and by another as yet unidentified isozyme(s) (most probably CYP 2C6), but not by CYP 3A, CYP 1A2, CYP 2D1 or CYP 2C11. Administration of methyleugenol (0-300 mg/kg/day for 5 days) to rats in vivo caused dose- dependent auto-induction of 1'-hydroxylation of methyleugenol in vitro which could be attributed to induction of various cytochrome P450 isozymes, including CYP 2B and CYP 1A2. Consequently, high dose rodent carcinogenicity studies are likely to over-estimate the risk to human health posed by methyleugenol. The rate of 1'-hydroxylation of methyleugenol in vitro in 13 human liver samples varied markedly (by 37- fold), with the highest activities being similar to the activity evident in control rat liver microsomes. This suggests that the risk posed by dietary ingestion of methyleugenol could vary markedly in the human population.