A double-blind placebo-controlled cross over trial of bupropion in smoking reduction in schizophrenia.
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Bupropion↗
Biomedical subjects
S E Murphy
Publications and source records attributed to S E Murphy.
Molecular modelling of CYP2A enzymes: application to metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).
1. Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a lung carcinogen in a variety of animal models and a putative human lung carcinogen. Its tumorigenic potential is unmasked via cytochrome P450 (CYP)-mediated hydroxylation of the carbon atoms adjacent to the nitroso moiety (i.e. alpha-hydroxylation). Therefore, elucidation of enzyme-substrate interactions that facilitate alpha-hydroxylation is important to gain insight into the tumorigenic mechanism of NNK and to develop potent inhibitors of this detrimental reaction. 2. Molecular models of CYP2A enzymes from mice, rats and humans that are catalysts of NNK bioactivation were constructed and used, in conjunction with docking experiments, to identify active-site residues that make important substrate contacts. 3. Docking studies revealed that hydrophobic residues at positions 117, 209, 365 and 481, among others, play critical roles in orienting NNK in the active site to effect alpha-hydroxylation. These molecular models were then used to rationalize the stereo- and regioselectivity, as well as the efficiency, of CYP2A-mediated NNK metabolism.
Amino Acid Sequence↗
Smoking behaviour and toxin exposure during six weeks use of a potential reduced exposure product: Omni.
OBJECTIVE: To determine smoking behaviour, acceptability, and toxin exposure when smokers switch to the potential reduced exposure product-Omni cigarette. DESIGN: 12 week randomised, crossover study of Omni versus own cigarettes. PARTICIPANTS: 19 light/ultralight and 15 regular smokers. OUTCOMES: Cigarettes/day, smoking topography, craving, withdrawal symptoms, urinary cotinine plus its glucuronide (total cotinine), nicotine plus its glucuronide (total nicotine), and carcinogen metabolites (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol plus its glucuronides and 1-hydroxypyrene). RESULTS: When switched to Omni, smokers smoked the same number of cigarettes/day, smoked Omni cigarettes less intensely (total puff volume = -11%) and had slightly lower total cotinine (-18%) levels than their own cigarettes, but had a slightly greater carbon monoxide boost/cig (+21%). Craving and withdrawal ratings were similar with Omni and own cigarettes. Carcinogen metabolite levels were somewhat but not significantly lower with Omni. About half of smokers rated Omni as better for their health and about two thirds stated it was weaker and worse tasting than their own cigarettes. CONCLUSIONS: Although Omni may be an adequate behavioural and pharmacological substitute for traditional cigarettes, it may not decrease carcinogen exposure and may increase carbon monoxide. Replications with larger sample sizes and longer follow up are needed. These results indicate the need for regulation of reduced exposure and reduced risk claims.
Adult↗
CYP2A13-catalysed coumarin metabolism: comparison with CYP2A5 and CYP2A6.
1. We investigated the total metabolism of coumarin by baculovirus (BV)-expressed CYP2A13 and compared it with metabolism by BV-expressed CYP2A6. The major coumarin metabolite formed by CYP2A13 was 7-hydroxycoumarin, which accounted for 43% of the total metabolism. The product of 3,4-epoxidation, o-hydroxyphenylacetaldehyde (o-HPA), accounted for 30% of the total metabolites. 2. The K(m) and V(max) for CYP2A13-mediated coumarin 7-hydroxylation were 0.48+/-0.07 micro m and 0.15+/-0.006 nmol min(-1) nmol(-1) CYP, respectively. The V(max) of coumarin 7-hydroxylation by CYP2A13 was about 16-fold lower than that of CYP2A6, whereas the K(m) was 10-fold lower. 3. In the mouse, there were two orthologues for CYP2A6: CYP2A4 and CYP2A5, which differed by only 11 amino acids. However, CYP2A5 is an efficient coumarin 7-hydroxylase, where as CYP2A4 is not. We report here that BV-expressed CYP2A4 metabolizes coumarin by 3,4-epoxidation. Two products of the 3,4-epoxidation pathway, o-HPA and o-hydroxyphenylacetic acid (o-HPAA), were detected by radioflow HPLC. 4. The K(m) and V(max) for the coumarin 3,4-epoxidation by CYP2A4 were 8.7+/-3.6 micro m and 0.20+/-0.04 nmol min(-1) nmol(-1) CYP, respectively. Coumarin 7-hydroxylation by CYP2A5 was more than 200 times more efficient than 3,4 epoxidation by CYP2A4.
Animals↗
Coumarin metabolism by rat esophageal microsomes and cytochrome P450 2A3.
The rat esophagus is strikingly sensitive to tumor induction by nitrosamines, and it has been hypothesized that this tissue contains cytochrome P450 enzymes (P450s) which catalyze the metabolic activation of these carcinogens. The metabolic capacity of the esophagus is not well characterized. In the study described here, the products of 14C-coumarin metabolism by rat esophageal microsomes were identified and quantified. Metabolite characterization was by LC/MS/MS and GC/MS and comparison to standards, quantification was by radioflow HPLC. The coumarin metabolites formed by rat esophageal microsomes were compared to those formed by P450 2A3. The major metabolites formed by esophageal microsomes were 8-hydroxycoumarin, o-hydroxyphenylacetaldehyde (o-HPA), and o-hydroxyphenylacetic acid (o-HPAA). A smaller amount of 5-hydroxycoumarin, about one-third the 8-hydroxycoumarin, was also formed. o-HPA and o-HPAA are products of coumarin 3,4-epoxidation. The relative rates of coumarin 8-hydroxylation and 3,4-epoxidation were similar. Coumarin 8-hydroxylation has not previously been reported as a major pathway in any tissue, and no P450s have yet been reported to catalyze this reaction. P450 2A3 catalyzed both the 7-hydroxylation and 3,4-epoxidation of coumarin. P450 2A3 was previously characterized as a coumarin 7-hydroxylase, however, in this study, we report that it catalyzes the formation of o-HPA more efficiently. The Km and Vmax were 1.3 +/- 0.35 microM and 0.65 +/- 0.06 nmol/min/nmol P450 for coumarin 7-hydroxylation and 1.4 +/- 0.58 microM and 3.1 +/- 0.46 nmol/min/nmol P450 for o-HPA formation.
Animals↗
Consumption of watercress fails to alter coumarin metabolism in humans.
Watercress is an excellent source of phenethyl isothiocyanate (PEITC), an effective inhibitor of nitrosamine carcinogenesis in rodents. The mechanism of inhibition is believed to be due in part to inhibition of cytochrome P450 (P450) enzymes. P450 2A6 is a catalyst for the metabolic activation of several nitrosamines. In this study, we investigated the effect of watercress consumption on coumarin 7-hydroxylation, a P450 2A6-specific reaction, in a group of 15 nonsmoking, healthy volunteers. The urinary excretion of 7-hydroxycoumarin (7OHC) was determined. For 6 of the 15 subjects, watercress consumption decreased the amount of 7OHC excreted in the first 2 h following coumarin administration. However, the mean 0- to 2-h excretion of 7OHC for all 15 subjects was not significantly lowered by the consumption of watercress, 2.8 +/- 0.78 versus 3.1 +/- 0.53 mg (+/-S.D.). The mean 7OHC excreted from 2 to 4 h (1.1 +/- 0.50 mg) was significantly higher (P = 0.027) during watercress consumption than before (0.77 +/- 0.22 mg), suggesting a delay in coumarin metabolism. Total excretion of 7OHC was unaffected by watercress consumption. Therefore, under the conditions of our study, PEITC and other constituents of watercress had at most a marginal inhibitory effect on P450 2A6-catalyzed coumarin 7-hydroxylation.
Adult↗
Characterization of cytochrome P450 2A4 and 2A5-catalyzed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism.
The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung carcinogen in the A/J mouse, and is believed to be a causative agent for human lung cancer. NNK requires metabolic activation by alpha-hydroxylation to exert its carcinogenic potential. The human P450, 2A6 is a catalyst of this reaction. There are two closely related enzymes in the mouse, P450 2A4 and 2A5, which differ from each other by only 11 amino acids. In the present study these two mouse P450s were expressed in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus. The catalysis of NNK metabolism by Sf9 microsomal fractions containing either P450 2A4 or 2A5 was determined. Both enzymes catalyzed the alpha-hydroxylation of NNK but with strikingly different efficiencies and specificities. P450 2A5 preferentially catalyzed NNK methyl hydroxylation, while P450 2A4 preferentially catalyzed methylene hydroxylation. The KM and Vmax for the former were 1.5 microM and 4.0 nmol/min/nmol P450, respectively, and for the latter 3.9 mM and 190 nmol/min/nmol P450. The mouse coumarin 7-hydroxylase, P450 2A5 is a significantly better catalyst of NNK alpha-hydroxylation than is the closely related human enzyme, P450 2A6.
Animals↗
2'-Hydroxylation of nicotine by cytochrome P450 2A6 and human liver microsomes: formation of a lung carcinogen precursor.
Smokers or people undergoing nicotine replacement therapy excrete approximately 10% of the nicotine dose as 4-oxo-4-(3-pyridyl)butanoic acid (keto acid) and 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid). Previously, these acids were thought to arise by secondary metabolism of the major nicotine metabolite cotinine, but our data did not support this mechanism. Therefore, we hypothesized that nicotine is metabolized by 2'-hydroxylation, which would ultimately yield keto acid and hydroxy acid as urinary metabolites. This pathway had not been established previously in mammalian systems and is potentially significant because the product of nicotine 2'-hydroxylation, 4-(methylamino)-1-(3-pyridyl)-1-butanone (aminoketone), can be converted to the potent tobacco-specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Incubation of nicotine with cytochrome P450 2A6 and cofactors did indeed produce aminoketone, which was identified as its N-benzoyl derivative by GC-MS. The rate was 11% of that of cotinine production. Incubation of human liver microsomes with nicotine gave keto acid by using aminoketone as an intermediate; keto acid was not formed from cotinine. In 10 human liver samples, rates of formation of keto acid were 5.7% of those of cotinine and production of these metabolites correlated. These results provide definitive evidence for mammalian 2'-hydroxylation of nicotine and elucidate a pathway by which endogenous formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone could occur in humans.
Aryl Hydrocarbon Hydroxylases↗
Specificity of cytochrome P450 2A3-catalyzed alpha-hydroxylation of N'-nitrosonornicotine enantiomers.
N'-nitrosonornicotine (NNN) induces tumors in the rat nasal cavity and esophagus and is believed to be a causative agent for esophageal cancer in tobacco users. To exert its carcinogenic potential, NNN must be metabolically activated by alpha-hydroxylation at either the 2'- or 5'-carbon. We previously reported that the human cytochrome P450 (P450), 2A6, efficiently and specifically catalyzed NNN 5'-hydroxylation. P450 2A3, which is expressed in the rat nasal cavity and to a small extent in the esophagus, is closely related to P450 2A6. P450 2A3, like 2A6, is a good catalyst of NNN alpha-hydroxylation (K(m) 7 microM; V(max) 17 nmol/min/nmol). However, in contrast to P450 2A6, 2A3 catalyzed both 5'- and 2'-hydroxylation of NNN. The ratio of 2'- to 5'-hydroxylation was 1:3. These data, both with P450 2A6 and 2A3, were obtained using racemic NNN. P450 2A3 catalyzed metabolism of (S)-NNN occurred exclusively at the 5'-position. The predominant pathway of (R)-NNN metabolism was 2'-hydroxylation, and occurred to a 3-fold greater extent than did 5'-hydroxylation. These data are in contrast to those obtained from a recent study of (R)- and (S)-NNN metabolism by cultured rat esophagus. In that study, (S)-NNN was metabolized predominantly by 2'-hydroxylation and (R)-NNN equally by 2'- and 5'-hydroxylation. Taken together, these data provide strong evidence that P450 2A3 is not the rat esophageal P450 that catalyzes the metabolic activation of NNN. P450 2A3 may be an important catalyst of NNN activation in rat nasal mucosa.
Animals↗
O-Glucuronidation of the lung carcinogen 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanol (NNAL) by human UDP-glucuronosyltransferases 2B7 and 1A9.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are potent lung carcinogens in animals. UGT-mediated O-glucuronidation of NNAL is an important detoxification pathway for these carcinogens. To better characterize this pathway in humans, we screened a series of UGT-overexpressing cell lines and baculosome preparations for their ability to O-glucuronidate NNAL and examined multiple human liver and lung specimens for NNAL-glucuronidating activity and their levels of expression of NNAL-glucuronidating UGTs. Human liver microsomal fractions exhibited significant levels of NNAL-glucuronidating activity, with the NNAL-Gluc II diastereomer formed at a rate 3.4 times that observed for NNAL-Gluc I. As with liver microsomal fractions, NNAL-Gluc II was the major diastereomer formed by homogenates from UGT2B7-overexpressing HK293 cells or UGT2B7-overexpressing baculosomes; the major diastereomer formed by homogenates from UGT1A9-overexpressing V79 cells was NNAL-Gluc I. No significant O-glucuronidating activity of NNAL was detected in UGT1A1-, UGT1A4-, UGT1A6-, UGT2B4-, or UGT2B15-overexpressing HK293 or V79 cell homogenates, or in UGT1A1-, UGT1A3-, UGT1A7-, or UGT1A10-overexpressing baculosomes. Significant levels of UGT2B7 mRNA were detected by reverse transcriptase-polymerase chain reaction in human liver and at low levels in human lung specimens. UGT1A9 mRNA was detected in liver but not in lung. These results suggest that although both UGT2B7 and UGT1A9 play an important role in the overall glucuronidation of NNAL in humans, UGT2B7 potentially plays an important role in the detoxification of NNAL in the lung.
Animals↗
Metabolism of N-nitrosobenzylmethylamine by human cytochrome P-450 enzymes.
N-Nitrosobenzylmethylamine (NBzMA) is a potent esophageal carcinogen in rodents, and has been found as a dietary contaminant in certain areas of China where esophageal cancer is endemic. To determine which cytochrome P-450 enzymes in humans are primarily responsible for NBzMA metabolism, microsomes from lymphoblastoid cell lines expressing a panel of human cytochrome P-450s (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP2C9, CYP2C19, and CYP3A4) and a panel of 10 different human liver microsomal preparations were examined for their abilities to metabolize [3H]NBzMA. In addition, the ability of human liver microsomes to form various NBzMA metabolites was correlated with the abilities of these preparations to metabolize coumarin, ethoxyresorufin, chlorzoxazone, 7-ethoxy-4-trifluoromethylcoumarin, S-mephenytoin, and nifedipine. NBzMA metabolites were quantitated by reversed-phase high-performance liquid chromatography (HPLC) coupled with flow-through radioactivity detection. Major metabolites included benzaldehyde, benzyl alcohol, benzoic acid, and several uncharacterized radioactive peaks. Of the representative P-450 activities, only CYP2E1 and CYP2A6 catalyzed substantial metabolism of NBzMA. Compared to CYP2E1, CYP2A6 metabolized NBzMA more readily. NBzMA acted as a potent inhibitor of coumarin 7-hydroxylation in CYP2A6 microsomes. Human liver microsomes metabolized NBzMA readily. NBzMA metabolite formation was most highly correlated with coumarin 7-hydroxylase activity, a marker of CYP2A6 activity. 8-Methoxypsoralen substantially inhibited NBzMA metabolism in human hepatic microsomes. When the effects of the potent isothiocyanates PEITC and PHITC were analyzed on microsomes from cell lines expressing CYP2E1 and CYP2A6, it was found that PEITC inhibited both enzymes, PHITC was the more effective inhibitor of CYP2E1, and PHITC was an ineffective inhibitor of CYP2A6. Collectively, these data indicate that CYP2A6 and, to a lesser degree, CYP2E1 are important P-450 enzymes in the activation of NBzMA in human systems.
Benzylamines↗
Quantitation of urinary metabolites of a tobacco-specific lung carcinogen after smoking cessation.
We quantified urinary levels of two metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in people who had stopped smoking: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its O-glucuronide, 4-[(methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc). Twenty-seven people completed the study. Thirteen used the nicotine patch starting at the quit date, whereas the others used no patch. Two 24-h urine samples were collected on 2 consecutive days before smoking cessation; blood was also obtained. Beginning at their quit date, subjects provided 24-h urine samples on days 7, 21, 42, 70, 98, and 126, and some subjects also provided samples at later times. The urine was analyzed for NNAL, NNAL-Gluc, nicotine plus nicotine-N-glucuronide, and cotinine plus cotinine-N-glucuronide. Some blood samples were also analyzed for NNAL. The decline of urinary NNAL and NNAL-Gluc after smoking cessation was much slower than expected. This was clearly demonstrated by comparison with cotinine and nicotine levels in urine. One week after smoking cessation, 34.5% of baseline NNAL plus NNAL-Gluc was detected in urine, whereas the corresponding values for cotinine and nicotine were 1.1 and 0.5%, respectively. Even 6 weeks after cessation, 7.6% of the original levels of NNAL plus NNAL-Gluc remained. In some subjects, NNAL plus NNAL-Gluc were detected 281 days after cessation. The distribution half-life for NNAL and NNAL-Gluc was 3-4 days, whereas the elimination half-life was 40-45 days. Total body clearance of NNAL was estimated to be 61.4 +/- 35.4 ml/min, and volume of distribution in the beta-phase was estimated to be 3800 +/- 2100 liters, indicating substantial distribution into the tissues. Parallel studies in rats treated chronically or acutely with NNK in the drinking water support the conclusion that NNAL has a large volume of distribution. There was no effect of the nicotine patch on levels of NNAL plus NNAL-Gluc, indicating that NNK is not formed endogenously from nicotine. The results of this study demonstrate that NNAL and NNAL-Gluc are slowly cleared from the body after smoking cessation, indicating the presence of a high-affinity compartment where NNK, NNAL, and/or NNAL-Gluc are retained or sequestered and slowly released.
Adult↗
Characterization of multiple products of cytochrome P450 2A6-catalyzed cotinine metabolism.
The primary metabolite of nicotine in smokers is cotinine. Cotinine is further metabolized to trans-3'-hydroxycotinine, the major urinary metabolite of nicotine in tobacco users. It was recently reported that cytochrome P450 2A6 catalyzes the conversion of cotinine to trans-3'-hydroxycotinine. In this work, we report that P450 2A6 metabolizes cotinine not only to trans-3'-hydroxycotinine but also to 5'-hydroxycotinine, norcotinine, and a fourth as yet unidentified metabolite. The products of baculovirus-expressed P450 2A6 [methyl-(3)H]cotinine metabolism were analyzed by radioflow HPLC. Three (3)H-labeled metabolites were detected and were present in approximately equal amounts. The identities of two of the metabolites were confirmed to be 5'-hydroxycotinine and trans-3'-hydroxycotinine by LC/MS/MS and LC/MS analysis and comparison to standards. The third product was not identified. A fourth product of P450 2A6-catalyzed cotinine metabolism was detected by LC/MS. It was identified by cochromatography with a standard and MS and MS/MS data to be norcotinine. An attempt was made to further characterize the unidentified (3)H-labeled metabolite by comparison to the cotinine metabolites generated by hamster liver microsomes. Hamster liver microsomes contain a P450, 2A8, which is closely related to P450 2A6, and have previously been shown to metabolize cotinine to three hydroxylated products, trans-3'-hydroxycotinine, 5'-hydroxycotinine, and N-(hydroxymethyl)norcotinine. We were unable to confirm that N-(hydroxymethyl)norcotinine was the unidentified cotinine metabolite generated by P450 2A6.
Animals↗
N-Nitrosobenzylmethylamine hydroxylation and coumarin 7-hydroxylation: catalysis by rat esophageal microsomes and cytochrome P450 2A3 and 2A6 enzymes.
N-Nitrosobenzylmethylamine (NBzMA) is a potent and selective esophageal carcinogen in the rat and may be a causative agent for human esophageal cancer. This nitrosamine, like most, must be metabolically activated to exert its carcinogenic potential. NBzMA may be metabolized by P450-catalyzed methyl or methylene hydroxylation; the latter is believed to be the activation pathway. The sensitivity of the esophagus to NBzMA-induced tumorigenesis is believed to be due, at least in part, to the presence of efficient P450 catalysts in this tissue. However, while it was reported almost 20 years ago that the rat esophagus catalyzes the methylene hydroxylation of NBzMA, the P450 that catalyzes this reaction has yet to be identified. We report here that human P450 2A6 and the closely related extrahepatic rat enzyme P450 2A3 both efficiently catalyze NBzMA methylene hydroxylation, characterized as benzaldehyde formation. The catalytic efficiency of P450 2A3 in this reaction was 3-fold greater than that of P450 2A6, 7.6 (K(m) = 0.63 +/- 0.18 microM and the V(max) = 4.8 nmol min(-)(1) nmol of P450(-)(1)) versus 2.3 (K(m) = 6.7 +/- 2.9 microM and the V(max) = 15.7 nmol min(-)(1) nmol of P450(-)(1)), respectively. Both enzymes catalyzed methylene hydroxylation at least 4-fold more efficiently than methyl hydroxylation. In addition, P450 2A6, but not P450 2A3, catalyzed benzyl ring hydroxylation, generating N-(p-hydroxybenzyl)methylamine. The identity of this metabolite was confirmed by synthesis of a standard and LC/MS and LC/MS/MS analysis. P450 2A6 is an efficient coumarin 7-hydroxylase, and we report here that P450 2A3 is an equally good catalyst of this reaction (K(m) = 1. 7 +/- 0.41 microM and V(max) = 1.7 +/- 0.08 nmol min(-)(1) nmol of P450(-)(1)). Rat esophageal microsomes (REM), like P450 2A3, were efficient catalysts of NBzMA methylene hydroxylation. However, in contrast to P450 2A3, the major product of this reaction was the product of benzaldehyde oxidation, benzoic acid. Antibody to the closely related mouse P450, 2A5, did not inhibit REM-catalyzed NBzMA metabolism, and most importantly, REM did not catalyze the 7-hydroxylation of coumarin. Therefore, P450 2A3 does not appear to be the P450 in the rat esophagus responsible for catalyzing the methylene hydroxylation of NBzMA.
Animals↗
Expression of cytochrome P450 2A3 in rat esophagus: relevance to N-nitrosobenzylmethylamine.
N-nitrosobenzylmethylamine (NBzMA) must be metabolically activated to exert its carcinogenic potential and is a potent inducer of tumors in the rat esophagus. The activation is believed to occur in the esophagus. Although the pathways of NBzMA metabolism are well studied, the principal cytochrome P450 enzyme(s) (P450) responsible for catalyzing its activation is unknown. Several preliminary studies have suggested that this enzyme may belong to the P450 2A family. We report here that P450 2A3 expressed in a baculovirus system metabolizes NBzMA, predominantly by methylene hydroxylation. To determine whether or not P450 2A3 is present in the rat esophagus, the relative level of P450 2A3 mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The mRNA levels of P450 2A3 were compared with the levels of P450 2A1 and 2A2 mRNA in the esophagus, liver, lung and nasal mucosa. P450 2A3 mRNA was detected in rat nasal mucosa, lung and esophagus, but not in liver, whereas P450 2A1 and 2A2 mRNAs were detected only in the liver. To determine the relative expression of P450 2A3 in each tissue, quantitative RT-PCR with PCR-MIMICS used as internal standards was performed. The expression level in the nasal mucosa was by far the greatest. The expression in the lung and esophagus was 60- and 1600-fold less, respectively. Using antibodies to P450 2A4/5 and P450 2A10/11 a 50 kDa immunoreactive protein was detected in all three tissues by western blot analysis. This is consistent with the expression of P450 2A3 in these tissues. However, the amount of protein detected in the nasal mucosa was much greater than that in the esophagus or lung. The expression of P450 2A protein was similar in the lung and esophagus. The rate of coumarin 7-hydroxylation in cultured rat esophagus was very low. This is a reaction efficiently catalyzed by P450 2A5, 2A6 and 2A10. In summary, our results clearly demonstrate the presence of P450 2A3 protein and mRNA in the esophagus, but the amounts are low and may not be sufficient to account for NBzMA activation in this tissue.
Animals↗
Glucuronidation of the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by rat UDP-glucuronosyltransferase 2B1.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are potent lung carcinogens in animals. UDP-glucuronosyltransferase (UGT)-mediated glucuronidation of NNAL is a potentially important detoxification pathway for these carcinogens. To identify the UGT isozyme(s) involved in this pathway, we examined the glucuronidation of NNAL in rat liver microsomes and homogenates from cell lines overexpressing specific UGT isozymes. NNAL glucuronidation was induced in liver microsomes from rats treated with family 2 UGT inducers including phenobarbitol and 3, 5-di-tert-butyl-4-hydroxytoluene, which exhibited 1.7- and 2.6-fold higher rates of glucuronidation than microsomes from control rats. The rates of NNAL glucuronidation in liver microsomes from GUNN (deficient in family 1 UGTs) and RHA parental control rats were similar. All rat liver microsomes used in the present study catalyzed the glucuronidation of (S)-NNAL at a rate between 3.5 and 5.5 times that of the glucuronidation of (R)-NNAL. Liver microsomes from Wistar rats exhibiting the low-androsterone glucuronidation phenotype characteristic of the UGT2B2-deficient genotype glucuronidated NNAL at a rate similar to microsomes from Wistar rats exhibiting the high-androsterone glucuronidation phenotype/UGT2B2 [+] genotype. Homogenates from UGT2B1-overexpressing cells catalyzed the glucuronidation of NNAL at a K(m) of 745 microM. As with rat liver microsomes, NNAL-Gluc I was the major diastereomer formed by UGT2B1. Glucuronidation of NNAL was not detected with homogenates from UGT2B12-overexpressing cells. These results suggest that UGT2B1 plays an important role in the glucuronidation of NNAL in the rat.
Animals↗
Effects of watercress consumption on urinary metabolites of nicotine in smokers.
The effects of watercress consumption on the metabolism of nicotine in smokers were examined. Watercress is a rich source of phenethyl isothiocyanate (PEITC), an effective chemopreventive agent for cancers of the lung and esophagus induced in rodents by nitrosamines, including the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. PEITC is believed to inhibit nitrosamine carcinogenesis in rodents by inhibiting specific cytochrome P450 (P450) enzymes. Among the P450s involved in the activation of these nitrosamines are members of the 2A family. P450 2A6 is believed to be involved in the metabolism of both nicotine and its major metabolite cotinine. Therefore, we hypothesized that watercress consumption might inhibit nicotine and cotinine metabolism in smokers. The urine samples analyzed in this study were the same ones that we used in an earlier study (S. S. Hecht et al., Cancer Epidemiol. Biomark. Prev., 4: 877-884, 1995), in which we showed that watercress consumption increased levels of two metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone: NNAL and its glucuronide NNAL-Gluc. This increase was attributed either to inhibition of cytochromes P450 or induction of glucuronidation. In the present study, we quantified urinary nicotine and seven of its metabolites. There were no effects of watercress consumption on levels of nicotine, cotinine, trans-3'-hydroxycotinine, 4-oxo-4-(3-pyridyl)butanoic acid, or 4-hydroxy-4-(3-pyridyl)butanoic acid, indicating either that watercress ingestion has little effect on the oxidative metabolism of nicotine (presumably by P450 2A6 or other P450 enzymes) or that these enzymes are not important for nicotine and cotinine metabolism in smokers. However, watercress consumption resulted in a significant increase compared to baseline levels of the glucuronides of cotinine (25%, P = 0.031) and trans-3'-hydroxycotinine (33%, P = 0.043) during the period when it was consumed and in a nonsignificant increase in levels of the glucuronide of nicotine. These levels returned to baseline values after the watercress consumption period. There was a correlation between increases in levels of the glucuronides of trans-3'-hydroxycotinine and NNAL in the same subjects, suggesting the involvement of a common enzyme. Thus, the results of this study suggest that PEITC or another component of watercress induces UDP-glucuronosyltransferase activity in humans.
Adult↗
Inhibition of N'-nitrosonornicotine-induced esophageal tumorigenesis by 3-phenylpropyl isothiocyanate.
The ability of dietary isothiocyanates to inhibit the esophageal metabolism of N'-nitrosonornicotine (NNN) was examined in F344 rats. Following feeding of benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), 4-phenylbutyl isothiocyanate (PBITC) or 6-phenylhexyl isothiocyanate for 2 weeks, rats were killed and the esophagi were incubated in vitro with [5-3H]NNN. While dietary BITC, PEITC and PBITC all decreased NNN metabolism, dietary PPITC had the greatest effect, yielding inhibition ranging from 55 to 91% of the control production of various NNN metabolites. To determine the chemopreventive efficacy of PPITC on NNN-induced esophageal tumorigenesis, rats were fed AIN-76A diets containing 0, 1.0 or 2.5 micromol/g PPITC and were given untreated drinking water or drinking water containing 5 p.p.m. NNN. After 87 weeks, the experiment was terminated and the esophageal tumors were counted. Rats that were given untreated drinking water developed no tumors. Rats that were given 5 p.p.m. NNN and unadulterated AIN-76A diet had an esophageal tumor incidence of 71% and a multiplicity of 1.57 tumors/animal. The two dietary concentrations of PPITC reduced the incidence and multiplicity of NNN-induced esophageal tumors by >95%. These results demonstrate the remarkable chemopreventive efficacy of PPITC in the NNN-induced esophageal tumor model.
Animals↗