Profile of urinary phenanthrene metabolites in smokers and non-smokers

Phenanthrene metabolites (phenols and dihydrodiols) and 1-hydroxypyrene excreted in the 24-h urine of smokers, non-smokers and lung cancer patients, who after heavy smoking became light smokers, were determined and compared. In contrast to 1- hydroxypyrene, no significant differences of the absolute amounts of phenanthrene metabolites were found between smokers and non-smokers. A ratio phenanthrene metabolites/l-hydroxypyrene of 10.4 was observed for non-smokers and 9.9 for lung cancer patients, but 4.2 for smokers. Significantly different ratios for the regiospecific oxidation of phenanthrene were found for smokers when compared with non-smokers (1,2-oxidation vs 3,4-oxidation was 1.45 in the case of smokers, but 2.34 in the case of non-smokers) indicating a cigarette smoke - but not PAH - caused induction of CYP 1A2 in smokers. As a consequence of the degree of PAH exposure the ratio dihydrodiols/phenols depends on the total amount of metabolites excreted. Phenols predominate, equally in smokers and non-smokers after low exposure, while dihydrodiols become more prominent in highly exposed persons (coke plant workers). Both (i) the regiospecific oxidation of PAH and (ii) the ratio of dihydrodiol vs phenol formation may be recognized from the urinary phenanthrene metabolite profile. This pattern mirrors the enzymatic status (balance of the CYP isoforms and epoxide hydrolase) in individuals. Accordingly, more detailed information may be obtained from the urinary metabolite pattern than from 1- hydroxypyrene, commonly used in PAH biomonitoring.     

Biomonitoring of polycyclic aromatic hydrocarbons in human urine

Measurement of polycyclic aromatic hydrocarbons (PAH) metabolites in human urine is the method of choice to determine occupational and/or environmental exposure of an individual to PAH, in particular, when multiple routes of exposure have to be taken into account. Requirements for methods of biomonitoring PAH metabolites in urine are presented. Studies using 1-hydroxypyrene or phenanthrene metabolites including its phenols and dihydrodiols are summarized. The role of these PAH metabolites as established biomarkers and also more recent developments of PAH biomonitoring are discussed.     

Smoking alters thromboxane metabolism in man

Chronic smoking is a major risk factor of atherosclerosis and coronary heart disease. The measurement of three major thromboxane A2 metabolites, 11-dehydrothromboxane B2, 2,3-dinorthromboxane B2 and thromboxane B2, in the urines of 13 apparently healthy smokers (average 39 years, range 27-56 years) showed significantly elevated excretion rates for all thromboxane A2 metabolites as compared to 10 apparently healthy age-matched non-smokers (average 37 years, range 26-56 years). Importantly, characteristic alterations in the thromboxane A2 metabolite pattern were found in the urines of smokers. The contribution of 2,3-dinorthromboxane B2 to total measured excretion of thromboxane A2 metabolites was 59.2% in smokers (404.0 +/- 53.0 pg/mg creatinine) versus 19.4% in non-smokers (85.2 +/- 8.3 pg/mg creatinine), that of 11-dehydrothromboxane B2 35.7% in smokers (673.2 +/- 88.9 pg/mg creatinine) as compared to 75.5% in non-smokers (332.6 +/- 30.9 pg/mg creatinine). The contribution of thromboxane B2 (57.5 +/- 7.7 pg/mg creatinine in smokers versus 21.9 +/- 1.5 pg/mg creatinine in non-smokers) was similar at 5.1%. The excretion of cotinine, the major urinary metabolite of nicotine that correlates well with the reported daily cigarette consumption (r = 0.97, P less than 0.0001), showed a good correlation to thromboxane A2 metabolite excretion (2,3-dinorthromboxane B2: r = 0.92, P less than 0.0001; 11-dehydrothromboxane B2; r = 0.87, P less than 0.0001).     

Urinary excretion of the acrylonitrile metabolite 2-cyanoethylmercapturic acid is correlated with a variety of biomarkers of tobacco smoke exposure and consumption

Acrylonitrile is an IARC class 2B carcinogen present in cigarette smoke. Urinary 2-cyanoethylmercapturic acid (CEMA) is an acrylonitrile metabolite and a potential biomarker for acrylonitrile exposure. The objective of this work was to study the dose response of CEMA in urine of non-smokers and smokers of different ISO tar yield cigarettes. We observed that smokers excreted >100-fold higher amounts of urinary CEMA than non-smokers. The CEMA levels in smokers were significantly correlated with ISO tar yield, daily cigarette consumption, and urinary biomarkers of smoke exposure. In conclusion, urinary CEMA is a suitable biomarker for assessing smoking-related exposure to acrylonitrile.     

Determination of the nicotine metabolites cotinine and trans-3'-hydroxycotinine in biologic fluids of smokers and non-smokers using liquid chromatography-tandem mass spectrometry: biomarkers for tobacco smoke exposure and for phenotyping cytochrome P450 2A6 activity

The nicotine metabolite cotinine is widely used to assess the extent of tobacco use in smokers, and secondhand smoke exposure in non-smokers. The ratio of another nicotine metabolite, trans-3'-hydroxycotinine, to cotinine in biofluids is highly correlated with the rate of nicotine metabolism, which is catalyzed mainly by cytochrome P450 2A6 (CYP2A6). Consequently, this nicotine metabolite ratio is being used to phenotype individuals for CYP2A6 activity and to individualize pharmacotherapies for tobacco addiction. In this paper we describe a highly sensitive liquid chromatography-tandem mass spectrometry method for determination of the nicotine metabolites cotinine and trans-3'-hydroxycotinine in human plasma, urine, and saliva. Lower limits of quantitation range from 0.02 to 0.1ng/mL. The extraction procedure is straightforward and suitable for large-scale studies. The method has been applied to several thousand biofluid samples for pharmacogenetic studies and for studies of exposure to low levels of secondhand smoke. Concentrations of both metabolites in urine of non-smokers with different levels of secondhand smoke exposure are presented.     

Vasoactive and atherogenic effects of cigarette smoking: a study of monozygotic twins discordant for smoking.

The mechanism by which atherosclerotic disease is induced by cigarette smoking has not yet been identified unequivocally. Chronic cigarette smoking and the generation of vasoactive prostanoids and the size of carotid atherosclerotic plaques were studied in nine pairs of identical male twins discordant for smoking for over 20 years. The urinary excretion of 2,3-dinor-thromboxane B2 (thromboxane B2 metabolite) of the smoking twin was significantly higher (on average 1.8 times higher) in every pair and that of 2,3-dinor-6-keto-prostaglandin F1 alpha (prostacyclin metabolite) was significantly higher (on average 1.3 times higher) in eight of the nine pairs. The ratio of excretion of these metabolites was significantly higher, being 4.0 (95% confidence interval 2.7 to 5.4) among the smokers compared with 2.9 (2.1 to 3.8) among the non-smokers, thus favouring a mechanism of vasoconstriction. Excretion of the thromboxane B2 metabolite was related to the urinary concentrations of nicotine metabolites. Atherosclerotic plaques detected by ultrasonography in the carotid arteries were significantly larger among smokers but did not correlate with the urinary excretion of prostacyclin and thromboxane B2 metabolites or intensity of smoking. Smoking was concluded to induce activation of platelets by an effect mediated by nicotine. The increased prostacyclin production, on the other hand, suggested a compensatory mechanism for the general vasoconstrictive properties of cigarette smoking.     

Cigarette smokers have decreased lymphocyte and platelet alpha-tocopherol levels and increased excretion of the gamma-tocopherol metabolite gamma-carboxyethyl-hydroxychroman (gamma-CEHC)

Cigarette smoking is associated with increased oxidative stress and increased risk of degenerative disease. As the major lipophilic antioxidant, requirements for vitamin E may be higher in smokers due to increased utilisation. In this observational study we have compared vitamin E status in smokers and non-smokers using a holistic approach by measuring plasma, erythrocyte, lymphocyte and platelet alpha- and gamma-tocopherol, as well as the specific urinary vitamin E metabolites alpha- and gamma-carboxyethyl-hydroxychroman (CEHC). Fifteen smokers (average age 27 years, smoking time 7.5 years) and non-smokers of comparable age, gender and body mass index (BMI) were recruited. Subjects completed a 7-day food diary and on the final day they provided a 24 h urine collection and a 20 ml blood sample for measurement of urinary vitamin E metabolites and total vitamin E in blood components, respectively. No significant differences were found between plasma and erythrocyte alpha- and gamma-tocopherol in smokers and non-smokers. However, smokers had significantly lower alpha-tocopherol (mean+/-SD, 1.34+/-0.31 micromol/g protein compared with 1.94+/-0.54, P = 0.001) and gamma-tocopherol (0.19+/-0.04 micromol/g protein compared with 0.26+/-0.08, P = 0.026) levels in their lymphocytes, as well as significantly lower alpha-tocopherol levels in platelets (1.09+/-0.49 micromol/g protein compared with 1.60+/-0.55, P = 0.014; gamma-tocopherol levels were similar). Interestingly smokers also had significantly higher excretion of the urinary gamma-tocopherol metabolite, gamma-CEHC (0.49+/-0.25mg/g creatinine compared with 0.32+/-0.16, P = 0.036) compared to non-smokers, while their alpha-CEHC (metabolite of alpha-tocopherol) levels were similar. There was no significant difference between plasma ascorbate, urate and F2-isoprostane levels. Therefore in this population of cigarette smokers (mean age 27 years, mean smoking duration 7.5 years), alterations to vitamin E status can be observed even without the more characteristic changes to ascorbate and F2-isoprostanes. We suggest that the measurement of lymphocyte and platelet vitamin E may represent a valuable biomarker of vitamin E status in relation to oxidative stress conditions.     

Identification of metabolites of benzo[j]fluoranthene formed in vitro in rat liver homogenate

The metabolites of benzo[j]fluoranthene (BjF) as formed in vitro using the 9000 X g supernatant from Aroclor-pretreated rats have been identified. Two dihydrodiols, trans-4,5-dihydro-4,5-dihydroxyBjF and trans-9,10-dihydro-9,10-dihydroxyBjF have been identified as major metabolites by comparison of their spectral and chromatographic properties with those of pure synthetic standards. There was no evidence that any of the isomeric 2,3-dihydrodiol was formed as a metabolite of BjF under these incubation conditions. Neither of the metabolic dihydrodiols of BjF were formed with a high degree of stereoselectivity. The enantiomeric purity of the 4,5-dihydrodiol was 20% while that of the 9,10-dihydrodiol was 46%. At least four phenols were detected among the metabolites of BjF. These were identified as 3-, 4-, 6- and 10-hydroxyBjF based upon comparison of their UV spectra and HPLC retention times with those of synthetic reference standards. BjF-4,5-dione was also identified as a metabolite under these incubation conditions.     

Protein engineering of Bacillus megaterium CYP102.The oxidation of polycyclic aromatic hydrocarbons.

Cytochrome P450 (CYP) enzymes are involved in activating the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs) in mammals, but they are also utilized by microorganisms for the degradation of these hazardous environmental contaminants. Wild-type CYP102 (P450(BM-3)) from Bacillus megaterium has low activity for the oxidation of the PAHs phenanthrene, fluoranthene and pyrene. The double hydrophobic substitution R47L/Y51F at the entrance of the substrate access channel increased the PAH oxidation activity by up to 40-fold. Combining these mutations with the active site mutations F87A and A264G lead to order of magnitude increases in activity. Both these mutations increased the NADPH turnover rate, but the A264G mutation increased the coupling efficiency while the F87A mutation had dominant effects in product selectivity. Fast NADPH oxidation rates were observed (2250 min-1 for the R47L/Y51F/F87A mutant with phenanthrene) but the coupling efficiencies were relatively low (< 13%), resulting in a highest substrate oxidation rate of 110 min-1 for fluoranthene oxidation by the R47L/Y51F/A264G mutant. Mutation of M354 and L437 inside the substrate access channel reduced PAH oxidation activity. The PAHs were oxidized to a mixture of phenols and quinones. Notably mutants containing the A264G mutation showed some similarity to mammalian CYP enzymes in that some 9,10-phenanthrenequinone, the K-region oxidation product from phenanthrene, was formed. The results suggest that CYP102 mutants could be useful models for PAH oxidation by mammalian CYP enzymes, and also potentially for the preparation of novel PAH bioremediation systems.     

Comparative metabolism of the cis and trans isomers of N-nitroso-2-6-dimethylmorpholine in rats,hamsters and guinea pigs

The in vivo metabolism of the cis and trans isomers of N-[3,5-³H] nitroso-2,6-dimethylmorpholine (NDMM) was studied in female Fischer rats, Syrian golden hamsters and guinea pigs by analysis of urinary metabolites using high pressure liquid chromatography (HPLC). Animals were treated by gavage with 12 mg/kg body wt. of NDMM, composed of both isomers and 12 μCi/kg body wt. of either of the separated radioactive isomers (cis or trans). Control animals received 12 mg, 12 μCi/kg body wt. NDMM with both isomers labeled in their natural proportion.There was a substantial increase in the excretion of a particular metabolite, 2-(2-hydroxyl-methyl)ethoxy propanoic acid, in the urine of rats, hamsters and guinea pigs 24 h after received the trans isomer (24, 22 and 13% of the total dose excreted, respectively). A minor metabolite was determined to be 2,6-dimethylmorpholine-3-one, another product of α-oxidation. The metabolite 1-amino-2-hydroxypropanol was identified, indicating that NDMM was metabolized by both α-and β-oxidation.In all three species, animals administered the cis isomer excreted larger amounts of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) and N-nitroso-bis(2-hydroxypropyl)amine (BHP) products of beta oxidation, than those treated with the trans isomer. Hamsters and guinea pigs treated with the more carcinogenic cis isomer in these species, also excreted twice as much of two other metabolites than was found in the urine of animals given the trans isomer.The trans isomer of NDMM appeared to be preferentially metabolized by α-oxidation and from earlier studies this metabolic pathway seemed to be important in carcinogenesis by NDMM in the rat. The cis isomer might be in a conformation more favorable for β-oxidation and this pathway may be of primary importance in carcinogenesis by NDMM in hamsters and guinea pigs.     

Influence of the genetic polymorphism in the 5'-noncoding region of the CYP1A2 gene on CYP1A2 phenotype and urinary mutagenicity in smokers

The functional significance of genetic polymorphisms on tobacco smoke-induced CYP1A2 activity was examined. The influence of three polymorphisms of the cytochrome P450 1A2 gene (CYP1A2) (-3860 G-->A (allele *1C), -2467 T-->delT (allele *1D), -163C-->A (allele *1F)), located in the 5'-noncoding promoter region of the gene, on CYP1A2 activity (measured as caffeine metabolic ratio, CMR), was studied in Caucasian current smokers (n=95). Tobacco smoke intake was calculated from the number of cigarettes/day. Also, studied was the influence of these CYP1A2 genotypes on smoking-associated urinary mutagenicity, detected in Salmonella typhimurium strain YG1024 with S9 mix, considering the urinary excretion of nicotine plus its metabolites as an internal indicator of tobacco smoke exposure. Smokers with at least one of the variant alleles CYP1A2 -3860A and -2467 delT showed a significantly increased CYP1A2 CMR (-3860 G/A versus G/G, p<0.05; -2467 delT/delT versus T/delT and T/T, p<0.01). Multiple regression analysis showed that the increase in CYP1A2 CMR (ln values) was again significantly related to the presence of CYP1A2 variants -2467delT and also to variant -163A (p<0.05), but moderately to -3860A (p=0.084). No influence of the number of cigarettes smoked per day by each subject was found. Heavy smokers (n=48, with urinary nicotine plus its metabolites>or=0.69 mg/mmol creatinine) with variant allele -2467delT or -163A had significantly increased urinary mutagenicity (p<0.01 and <0.05). CYP1A2 genetic polymorphisms are shown to influence the CYP1A2 phenotype in smokers, -2467 T-->delT having the main effect. This information is of interest for future studies assessing the possible role of tobacco smoke-inducible CYP1A2 genotypes as individual susceptibility factors in exposure to carcinogens.     

The EmhABC efflux pump decreases the efficiency of phenanthrene biodegradation by Pseudomonas fluorescens strain LP6a

Pseudomonas fluorescens strain LP6a, designated here as strain WEN (wild-type PAH catabolism, efflux positive), utilizes the polycyclic aromatic hydrocarbon phenanthrene as a carbon source but also extrudes it into the extracellular medium using the efflux pump EmhABC. Because phenanthrene is considered a nontoxic carbon source for P. fluorescens WEP, its energy-dependent efflux seems counter-productive. We hypothesized that the efflux of phenanthrene would decrease the efficiency of its biodegradation. Indeed, an emhB disruptant strain, wild-type PAH catabolism, efflux negative (WEN), biodegraded 44% more phenanthrene than its parent strain WEP during a 6-day incubation. To determine whether efflux affected the degree of oxidation of phenanthrene, we quantified the conversion of ¹⁴C-phenanthrene to radiolabeled polar metabolites and ¹⁴CO₂. The emhB ^? WEN strain produced approximately twice as much ¹⁴CO₂ and radiolabeled water-soluble metabolites as the WEP strain. In contrast, the mineralization of ¹⁴C-glucose, which is not a known EmhB efflux substrate, was equivalent in both strains. An early open-ring metabolite of phenanthrene, trans-4-(1-hydroxynaphth-2-yl)-2-oxo-3-butenoic acid, also was found to be a substrate of the EmhABC pump and accumulated in the supernatant of WEP but not WEN cultures. The analogous open-ring metabolite of dibenzothiophene, a heterocyclic analog of phenanthrene, was extruded by EmhABC plus a putative alternative efflux pump, whereas the end product 3-hydroxy-2-formylbenzothiophene was not actively extruded from either WEP or WEN cells. These results indicate that the active efflux of phenanthrene and its early metabolite(s) decreases the efficiency of phenanthrene degradation by the WEP strain. This activity has implications for the bioremediation and biocatalytic transformation of polycyclic aromatic hydrocarbons and heterocycles.     

Association of 1 hydroxypyrene glucuronide in human urine with cigarette smoking and broiled or roasted meat consumption

Humans are exposed to polycyclic aromatic hydrocarbons PAHs from various occupational, dietary, environmental and medicinal sources. We measured 1 hydroxypyrene glucuronide 1 OHP gluc concentration in urines from male non smokers n = 50, smokers of blond tobacco n = 31, smokers of black tobacco n = 16, and pipe smokers n = 3 . Immunoaffinity chromatography was used as a preparative step and synchronous fluorescence spectroscopy as the quantitation method. The concentration of 1 OHP gluc in urine from smokers mean SE: 1.04 0.13 pmol ml-1 urine was significantly higher than in urine from non smokers 0.55 0.05 pmol ml-1 urine by the Wilcoxon rank sum test non smokers versus all smokers, p = 0.001; vs black tobacco smokers, p = 0.001; vs blond tobacco smokers, p = 0.007 . Urinary 1 OHP gluc concentration among subjects who had consumed roasted, grilled or broiled meat within the past 24 h was elevated compared with those who had not p = 0.025 . Multiple linear regression showed significant associations of urinary 1 OHP gluc with number of cigarettes smoked p = 0.002 and consumption of roasted, grilled or broiled meat p = 0.028 . Systemic CYP1A2 activity estimated by caffeine metabolism was significantly correlated with urinary 1 OHP gluc concentration. However, this association was probably due to cigarette smoking, since adjusting for cigarette smoking by multiple linear regression made the association between urinary 1 OHP gluc and CYP1A2 phenotype non significant. These results further support the use of urinary 1 OHP gluc as a biomarker of recent pyrene exposure through inhalation or diet.     

Association of 1 hydroxypyrene glucuronide in human urine with cigarette smoking and broiled or roasted meat consumption

Humans are exposed to polycyclic aromatic hydrocarbons PAHs from various occupational, dietary, environmental and medicinal sources. We measured 1 hydroxypyrene glucuronide 1 OHP gluc concentration in urines from male non smokers n = 50 , smokers of blond tobacco n = 31 , smokers of black tobacco n = 16 , and pipe smokers n = 3 . Immunoaffinity chromatography was used as a preparative step and synchronous fluorescence spectroscopy as the quantitation method. The concentration of 1 OHP gluc in urine from smokers mean SE: 1.04 0.13 pmol ml-1 urine was significantly higher than in urine from non smokers 0.55 0.05 pmol ml-1 urine by the Wilcoxon rank sum test non smokers versus all smokers, p = 0.001; vs black tobacco smokers, p = 0.001; vs blond tobacco smokers, p = 0.007 . Urinary 1 OHP gluc concentration among subjects who had consumed roasted, grilled or broiled meat within the past 24 h was elevated compared with those who had not p = 0.025 . Multiple linear regression showed significant associations of urinary 1 OHP gluc with number of cigarettes smoked p = 0.002 and consumption of roasted, grilled or broiled meat p = 0.028 . Systemic CYP1A2 activity estimated by caffeine metabolism was significantly correlated with urinary 1 OHP gluc concentration. However, this association was probably due to cigarette smoking, since adjusting for cigarette smoking by multiple linear regression made the association between urinary 1 OHP gluc and CYP1A2 phenotype non significant. These results further support the use of urinary 1 OHP gluc as a biomarker of recent pyrene exposure through inhalation or diet.     

Cometabolic oxidation of phenanthrene to phenanthrene trans-9,10-dihydrodiol by Mycobacterium strain S1 growing on anthracene in the presence of phenanthrene.

Mycobacterium strain S1, originally described as Rhodococcus strain S1 by chemotaxonomic criteria, was isolated by growth on anthracene, and is unable to use any of nine other polycyclic aromatic compounds as carbon source. Metabolism of phenanthrene during growth on anthracene as sole carbon source results in the accumulation of traces of a dihydrodiol metabolite in the growth medium, which, by comparison with authentic standards, has been tentatively identified as phenanthrene trans-9,10-dihydrodiol. Anthracene metabolites were ruled out on the basis of comparisons with authentic anthracene dihydrodiols from Pseudomonas fluorescens D1 and chemically synthesized anthrols. The original source of phenanthrene for dihydrodiol production was phenanthrene present as a < 1% contaminant in the anthracene used as carbon source. However, addition of further phenanthrene to the anthracene growth medium increased the level of phenanthrene trans-9,10-dihydrodiol formed. Mycobacterium strain S1 also produced phenanthrene trans-9,10-dihydrodiol when grown in a glucose-salts medium in the presence of phenanthrene. This dihydrodiol is a dead-end metabolite, and neither it nor its parent hydrocarbon are able to support the growth of Mycobacterium strain S1. Studies with metyrapone and ancimidol, which did not inhibit growth on anthracene but did inhibit formation of phenanthrene trans-9,10-dihydrodiol, suggest it is likely the product of a cytochrome P450 monooxygenase-like activity.     

Metabolism of phenanthrene by house fly CYP6D1 and dog liver cytochrome P450

Polycyclic aromatic hydrocarbons (PAHs) are a ubiquitous class of environmental contaminants. The compound phenanthrene is a model PAH. A novel fluorometric method for measuring phenanthrene metabolism in vitro was developed and verified with direct measurement of [14C]phenanthrene using dog liver microsomes. The fluorometric assay and direct measurement of [14C]phenanthrene metabolism were used to show that CYP6D1, a house fly cytochrome P450, is the major house fly P450 involved in phenanthrene metabolism. Phenanthrene was metabolized by microsomes from the LPR strain of house fly that overexpresses CYP6D1, but metabolism was not observed in the CS strain that has a lower level of CYP6D1. Furthermore, the majority of phenanthrene metabolism was inhibited by a CYP6D1-specific antibody. This study increases the number of known substrates of CYP6D1 and identifies polyaromatic hydrocarbons as potential substrates of CYP6D1. The utility of CYP6D1 as an agent in bioremediation and the utility of the new fluorometric assay for understanding PAH metabolism in insects and mammals are discussed.     

Increased urinary catechol estrogen excretion in female smokers

Premenopausal female smokers show significantly increased estrogen 2-hydroxylation, which may account in part for the anti-estrogenic effects of cigarette smoking. We have measured five major urinary estrogens, including estradiol (E2), estrone (E1), 16 alpha-hydroxyestrone (16 alpha OHE1), estriol (E3), and 2-hydroxyestrone (2OHE1), in premenopausal female smokers and non-smokers, to determine whether increased C-2 hydroxylation affected the urinary excretory patterns in these subjects. While total measured estrogen excretion in the follicular phase did not differ significantly between the two groups, urinary 2OHE1 among the smokers constituted a significantly greater proportion of the total (31.1 vs 18.2%, P less than 0.02). This difference was largely caused by significantly increased urinary 2OHE1 and decreased E3 observed in smokers. A urinary catechol estrogen index, defined by [2OHE1]/[E3], was significantly elevated in smokers compared with non-smokers (1.67 +/- 0.21 vs 0.56 +/- 0.08, P less than 0.001), and this urinary index correlated strongly with radiometrically determined estrogen 2-hydroxylation (r = 0.84, P less than 0.01). Ratios of the various estrogen metabolites did not vary substantially throughout the menstrual cycle. Urinary estrogen indices as described here may therefore be useful in demonstrating differences in estrogen metabolism, specifically 2-hydroxylation vs 16 alpha-hydroxylation, in selected populations.     

Benzo(a)pyrene oxidation, conjugation and disposition in the isolated perfused rabbit lung: role of the glutathione S-transferases

The isolated perfused rabbit lung metabolised 7--11 % of 20 mumol of [14C]-benzo(a)pyrene added in the perfusion medium in 1 h. The major metabolite formed was 3-hydroxybenzo(a)pyrene, both free (30--40 % of the total metabolites) and conjugated (4 % of total metabolites). Quinones comprised 15 % of the total and metabolism at the 9, 10 position accounted for a further 10 %. Forty per cent of the water-soluble metabolites was chromatographically identical to the glutathione conjugate of benzo(a)pyrene 4,5-oxide. Sulphate and glucuronide conjugates were formed in small but detectable amounts, principally from phenols, but also from dihydrodiols. After 1 h the more water-soluble conjugates had diffused from the lung into the perfusion medium, but the majority (60--90 %) of the metabolic products were still concentrated within the lung. The lung's limited ability to conjugate its major metabolites of benzo(a)pyrene with sulphuric or glucuronic acid, coupled with slow elimination of the products formed, particularly dihydrodiols may contribute to the susceptibility of this organ to polycyclic aromatic hydrocarbon-induced carcinogenesis.     

Effects of lifestyle and genetic polymorphisms on consumption of coffee or black tea and urinary caffeine levels

To find the cause of individual differences in caffeine intake and its metabolism, we investigated the effects of lifestyle and genetic polymorphisms of caffeine metabolic enzymes on coffee or black tea and urinary caffeine levels among 259 male Japanese. It was seen that cigarette smokers drank more coffee or black tea than non-smokers (p < 0.001). There was an inverse correlation between the amount of coffee or black tea consumed and age or the frequency of alcohol drinking (p < 0.05). Genetic polymorphisms of N -acetyltransferase 2 (NAT2), cytochrome P450 (CYP)1A1 and 2E1 did not significantly affect the habit of drinking coffee or black tea. The frequency of allele 1, the NAT2 allele of rapid acetylators, increased according to coffee or black tea consumption (0.05 < p < 0.1). Among lifestyle factors, two factors, i.e. smoking and the amount of coffee or black tea consumed, were related to urinary caffeine levels (p < 0.05). Geometric means of urinary caffeine levels were higher in the group who consumed higher amounts of coffee or black tea (p < 0.05) and those of smokers were lower than non-smokers- approximately 70% of non-smokers (p < 0.05). The genetic polymorphisms of NAT2, CYP1A1 and CYP2E1 were not significantly associated with the urinary caffeine levels according to each consumption level of coffee or black tea. This study suggests that smoking should be considered for the proper appreciation of individual differences in caffeine intake and urinary caffeine levels.     

Production of metabolites in the biodegradation of phenanthrene, fluoranthene and pyrene by the mixed culture of Mycobacterium sp. and Sphingomonas sp

The effects of the mixed culture of Mycobacterium sp. strain A1-PYR and Sphingomonas sp. strain PheB4 on the degradation characteristics of single polycyclic aromatic hydrocarbon were investigated. In the mixed bacterial culture, phenanthrene, fluoranthene and pyrene were degraded by 100% at Day 3, 71.2% and 50% at Day 7, respectively. Compared to their respective pure cultures, the degradation of phenanthrene and fluoranthene decreased, but that of pyrene increased significantly. Based on GC-MS analysis, eight and six new metabolites were produced from the biodegradation of phenanthrene and fluoranthene, respectively, while only two new metabolites were formed from pyrene. To our knowledge, this is the first report that the mixed bacterial culture could increase the diversity of metabolites from PAH, but the diverse metabolite pattern was not necessarily beneficial to the degradation of the recalcitrant PAH. The enhancement on pyrene degradation was possibly attributed to the rapid growth of strain PheB4.