Metabolism Of Hydrocarbons Research Articles

Cytochrome P450-dependent monooxygenase system mediated hydrocarbon metabolism and antioxidant enzyme responses in prawn, Macrobrachium malcolmsonii

We investigated the alteration of cytochrome P450-dependent monooxygenase enzymes and antioxidant enzymes in response to oil effluent in freshwater prawn, Macrobrachium malcolmsonii. The prawns were exposed to two sublethal (10% [0.91 ppt] and 25% [2.3 ppt] of 5-day median lethal concentration) concentrations of oil. After 30 days, treated prawns were transferred into untreated freshwater and depuration was followed for another 30 days. At 7-day intervals, hydrocarbons and detoxifying enzymes were analysed in the hepatopancreas. Accumulation of hydrocarbon in the tissues gradually increased when exposed to sublethal concentration of oil effluent associated with enhanced levels of cytochrome P450, NADPH cytochrome c reductase and cytochrome b 5. During depuration, the levels of accumulated hydrocarbons decreased due to the induction of these detoxifying enzymes. Oil derived hydrocarbon mediated oxyradical production would have occurred in M. malcolmsonii. This was confirmed by elevated levels of superoxide dismutase (SOD) and catalase (CAT). Thus, cytochrome P450-dependent monooxygenase enzymes and antioxidant enzymes in oil-exposed prawns demonstrate a well-established detoxifying mechanism in M. malcolmsonii.

CYP1A1 and GSTP1 polymorphisms in an oral cancer case-control study

CYP1A1 and GSTP1 polymorphisms have been associated with a higher risk to develop several cancers, including oral squamous cell carcinoma (OSCC), which is closely related to tobacco and alcohol consumption. Both genes code for enzymes that have an important role in activating or detoxifying carcinogenic elements found in tobacco and other compounds, and polymorphic variants of these genes may result in alterations of the enzymatic activity. The CYP1A1 gene codes for the enzyme aryl hydrocarbon hydroxylase, which is responsible for the metabolism of polycyclic aromatic hydrocarbons. The investigated polymorphism, Ile/Val, seems to increase the activity of the enzyme in homozygous individuals, leading to an accumulation of carcinogens. The Ile/Val polymorphism occurs because of an A->G transition at exon 7, resulting in the CYP1A1*2B allele. The GSTP1*B variant shows an A->G transition at exon 5, changing the amino acid Ile to Val, with a reduced catalytic activity of the enzyme. Due to this reduction, the carriers of mutant alleles lost the capability to metabolize carcinogens, which could be responsible for a higher susceptibility to cancer. We conducted a case-control study in a group of 72 cases with newly diagnosed OSCC and 60 healthy controls matched for age, gender, smoking habits, and ethnicity. We used PCR methods to identify the allelic variants CYP1A1*2B and GSTP1*B. The data obtained showed no statistically significant association of allelic or genotypic variants of CYP1A1*2B (OR = 1.06; 95% CI = 0.49-2.29) and GSTP1*B (OR = 1.40; 95% CI = 0.70-2.79) with OSCC.

CYP1A1 and GSTP1 polymorphisms in an oral cancer case-control study

CYP1A1 and GSTP1 polymorphisms have been associated with a higher risk to develop several cancers, including oral squamous cell carcinoma (OSCC), which is closely related to tobacco and alcohol consumption. Both genes code for enzymes that have an important role in activating or detoxifying carcinogenic elements found in tobacco and other compounds, and polymorphic variants of these genes may result in alterations of the enzymatic activity. The CYP1A1 gene codes for the enzyme aryl hydrocarbon hydroxylase, which is responsible for the metabolism of polycyclic aromatic hydrocarbons. The investigated polymorphism, Ile/Val, seems to increase the activity of the enzyme in homozygous individuals, leading to an accumulation of carcinogens. The Ile/Val polymorphism occurs because of an A->G transition at exon 7, resulting in the CYP1A1*2B allele. The GSTP1*B variant shows an A->G transition at exon 5, changing the amino acid Ile to Val, with a reduced catalytic activity of the enzyme. Due to this reduction, the carriers of mutant alleles lost the capability to metabolize carcinogens, which could be responsible for a higher susceptibility to cancer. We conducted a case-control study in a group of 72 cases with newly diagnosed OSCC and 60 healthy controls matched for age, gender, smoking habits, and ethnicity. We used PCR methods to identify the allelic variants CYP1A1*2B and GSTP1*B. The data obtained showed no statistically significant association of allelic or genotypic variants of CYP1A1*2B (OR = 1.06; 95% CI = 0.49-2.29) and GSTP1*B (OR = 1.40; 95% CI = 0.70-2.79) with OSCC.

Evaluation of the precision-cut liver and lung slice systems for the study of induction of CYP1, epoxide hydrolase and glutathione S-transferase activities

The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24 h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4 h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 μM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.

Comparison of Polymorphisms in Genes Involved in Polycyclic Aromatic Hydrocarbon Metabolism with Urinary Phenanthrene Metabolite Ratios in Smokers

The hypothesis that interindividual differences among smokers in the metabolism of polycyclic aromatic hydrocarbons (PAH) are related to lung cancer risk has been extensively investigated in the literature. These studies have compared lung cancer risk in groups of smokers with or without polymorphisms in genes involved in PAH metabolism. We believe that carcinogen metabolite phenotyping, involving the actual measurement of PAH metabolites, would be a better way to investigate differences in lung cancer risk. With this goal in mind, we have developed methods for quantifying phenanthrene metabolites in urine. Phenanthrene is the simplest PAH with a bay region, a feature closely associated with carcinogenicity. The urinary metabolite r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) is a measure of metabolic activation, whereas phenanthrols (HOPhe) are a measure of detoxification. In this study, we quantified urinary PheT/HOPhe ratios in 346 smokers who were also genotyped for 11 polymorphisms in genes involved in PAH metabolism: CYP1A1MspI, CYP1A1I462V, CYP1B1R48G, CYP1B1A119S, CYP1B1L432V, CYP1B1N453S, EPHX1Y113H, EPHX1H139R, GSTP1I105V, GSTP1A114V, and GSTM1 null. The geometric mean molar PheT/3-HOPhe ratio was 4.08 (95% confidence interval, 3.79-4.39). Ten percent of the smokers had PheT/3-HOPhe ratios of > or =9.90. We found a significant association between the presence of the CYP1A1I462V polymorphism and high PheT/3-HOPhe ratios (P = 0.02). This effect was particularly strong in females and in combination with the GSTM1 null polymorphism. In contrast, the CYP1B1R48G and CYP1B1A119S polymorphisms were associated with significantly lower PheT/3-HOPhe ratios, particularly in Blacks. There were no consistent significant effects of any of the other polymorphisms on PheT/3-HOPhe ratios. The highest 10% of PheT/3-HOPhe ratios could not be predicted by the presence of any of the 11 polymorphisms individually or by certain combinations. The effects of the CYP1A1I462 polymorphism observed here, particularly in combination with GSTM1 null, are quite consistent with reports in the literature. However, the results of this study indicate that genotyping is not an effective way to predict PAH metabolism at least as represented by PheT/HOPhe ratios.

Meta- and Pooled Analysis of GSTT1 and Lung Cancer: A HuGE-GSEC Review

Lung cancer is the most common malignancy in the Western world, and the main risk factor is tobacco smoking. Polymorphisms in metabolic genes may modulate the risk associated with environmental factors. The glutathione S-transferase theta 1 gene (GSTT1) is a particularly attractive candidate for lung cancer susceptibility because of its involvement in the metabolism of polycyclic aromatic hydrocarbons found in tobacco smoke and of other chemicals, pesticides, and industrial solvents. The frequency of the GSTT1 null genotype is lower among Caucasians (10-20%) than among Asians (50-60%). The authors present a meta- and a pooled analysis of case-control, genotype-based studies that examined the association between GSTT1 and lung cancer (34 studies, 7,629 cases and 10,087 controls for the meta-analysis; 34 studies, 7,044 cases and 10,000 controls for the pooled analysis). No association was observed between GSTT1 deletion and lung cancer for Caucasians (odds ratio (OR) = 0.99, 95% confidence interval (CI): 0.87, 1.12); for Asians, a positive association was found (OR = 1.28, 95% CI: 1.10, 1.49). In the pooled analysis, the odds ratios were not significant for either Asians (OR = 0.97, 95% CI: 0.83, 1.13) or Caucasians (OR = 1.09, 95% CI: 0.99, 1.21). No significant interaction was observed between GSTT1 and smoking on lung cancer, whereas GSTT1 appeared to modulate occupational-related lung cancer.

Quantitation of N-acetyl-S-(9,10-dihydro-9-hydroxy-10-phenanthryl)-L-cysteine in human urine: comparison with glutathione-S-transferase genotypes in smokers.

There are major interindividual differences in carcinogenic polycyclic aromatic hydrocarbon (PAH) metabolism in humans, and it has been hypothesized that these differences may be related to cancer risk in smokers and other exposed people. One important pathway of PAH metabolism involves the detoxification of the epoxide and diol epoxide metabolites by reaction with glutathione, catalyzed by glutathione-S-transferases (GSTs). Interindividual differences in these pathways have been examined by genotyping methods, investigating polymorphisms in GSTM1 and GSTP1. We are developing a phenotyping approach to assessing individual differences in PAH metabolism by quantifying human urinary metabolites of the ubiquitous PAH phenanthrene (1). In this study, we developed a method for quantitation of a mercapturic acid, N-acetyl-S-(9,10-dihydro-9-hydroxy-10-phenanthryl)-l-cysteine (PheO-NAC, 12), the end product of the reaction of phenanthrene-9,10-epoxide (11) with glutathione. [D(10)]PheO-NAC was added to the urine as internal standard, and the PheO-NAC fraction was enriched by solid-phase extraction. PheO-NAC was quantified by liquid chromatography electrospray ionization tandem mass spectrometry with selected reaction monitoring. The detection limit was approximately 4 fmol/mL of urine. PheO-NAC was detected in the urine of 46 of 104 smokers, mean (S.D.) 57.9 +/- 144 fmol/mL. PheO-NAC was detected significantly more frequently (P < 0.0001) in subjects who were GSTM1 positive than in those who were GSTM1 null, and the levels of PheO-NAC were significantly higher in the GSTM1 positive subjects, consistent with a role for GSTM1 in the detoxification of phenanthrene-9,10-epoxide. There were no significant relationships between PheO-NAC levels and the occurrence of two GSTP1 polymorphisms. The results of this study provide the first evidence for a PAH-derived mercapturic acid in human urine and should be useful in the development of a phenotyping approach to assess individual differences in PAH metabolism.

Solid-Phase Microextraction: Investigation of the Metabolism of Substances that May be Abused by Inhalation

Purified liquefied petroleum gas (LPG), a mixture of butane, isobutane, and propane, is commonly abused by inhalation. Little is known about the mammalian metabolism of these substances. Metabolism of other hydrocarbons, including n-hexane and cyclohexane, has been studied in vitro using a range of liver preparations, with metabolites analyzed by static headspace techniques. Solid-phase microextraction (SPME) for sampling metabolites in the headspace of incubates of volatile compounds with activated rat liver microsomes is investigated. Cyclohexanol and cyclohexanone were formed from cyclohexane and 1-, 2-, and 3-hexanol and 2-hexanone from n-hexane as predicted. Secondary alcohols are found for the other compounds studied, except for propene and isobutane, together with 2-propanone and 2-butanone from propane and n-butane, respectively. Samples from three individuals who died following LPG abuse contained a range of putative n-butane metabolites: n-butanol, 2-butanol, 2,3-butanediol, 3-hydroxy-2-butanone, and 2,3-butanedione. To our knowledge, the last three compounds have not been proposed as metabolites of n-butane in man. These might be produced through similar metabolic pathways to those of n-hexane and n-heptane. The findings indicate the value of SPME for investigating the metabolism of volatile substances and for detecting and monitoring exposure to these compounds.

Colorectal cancer and genetic polymorphisms of CYP1A1, GSTM1 and GSTT1: A case‐control study in the Grampian region of Scotland

AbstractCytochrome P‐450 CYP1A1 is involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs) that are derived from meat intake and tobacco smoking. Expression of the CYP1A1 gene is induced by compounds present in cruciferous vegetables. The glutathione S‐transferases play a central role in the detoxification of carcinogens, including PAHs. We investigated the association between colorectal cancer and three variants (CYP1A1*2A, CYP1A1*2C, CYP1A1*4) of the CYP1A1 gene, and homozygosity for the null deletion of the GSTM1 and GSTT1 genes, and the joint effects of these genotypes and smoking, meat intake and intake of green leafy vegetables in a population‐based study of 264 cases and 408 controls in Northeast Scotland. There was an inverse association with the CYP1A1*4 (m4) variant (OR 0.3, 95% CI 0.13–0.70). The OR for the CYP1A1*2C (m2) variant was 1.3 (95% CI 0.59–2.91), which is similar to a combined estimate for previous studies (OR 1.2, 95% CI 0.95–1.41). We observed no association with the CYP1A1*2A (m1) variant, or the GSTM1 and GSTT1 polymorphisms. Significant interactions between all 3 CYP1A1 variants and meat intake, and between the m1 and m2 variants and intake of green leafy vegetables, were observed. There was no evidence of interaction between CYP1A1 and smoking, and no evidence of interaction between the GSTM1 or GSTT1 polymorphisms and smoking, meat intake, green leafy vegetable intake, CYP1A1 variants or each other. © 2006 Wiley‐Liss, Inc.

For Dioxin-induced Birth Defects, Mouse or Human CYP1A2 in Maternal Liver Protects whereas Mouse CYP1A1 and CYP1B1 Are Inconsequential

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) induces cleft palate and hydronephrosis in mice, when exposed in utero; these effects are mediated by the aryl hydrocarbon receptor. The Cyp1a1, Cyp1a2, and Cyp1b1 genes are up-regulated by the aryl hydrocarbon receptor. To elucidate their roles in dioxin-induced teratogenesis, we compared Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) knock-out mice with Cyp1(+/+) wild-type mice. Dioxin was administered (25 microg/kg, gavage) on gestational day 10, and embryos were examined on gestational day 18. The incidence of cleft palate and hydronephrosis was not significantly different in fetuses from Cyp1a1(-/-), Cyp1b1(-/-), and Cyp1(+/+) wild-type mice. To fetuses carried by Cyp1a2(-/-) dams, however, this dose of dioxin was lethal; this effect was absolutely dependent on the maternal Cyp1a2 genotype and independent of the embryonic Cyp1a2 genotype. Dioxin levels were highest in adipose tissue, mammary gland, and circulating blood of Cyp1a2(-/-) mothers, compared with that in the Cyp1(+/+) mothers, who showed highest dioxin levels in liver. More dioxin reached the embryos from Cyp1a2(-/-) dams, compared with that from Cyp1(+/+) dams. Fetuses from Cyp1a2(-/-) dams exhibited a approximately 6-fold increased sensitivity to cleft palate, hydronephrosis, and lethality. Using the humanized hCYP1A1_1A2 transgenic mouse (expressing the human CYP1A1 and CYP1A2 genes in the absence of mouse Cyp1a2 gene), the teratogenic effects of dioxin reverted to the wild-type phenotype. These data indicate that maternal mouse hepatic CYP1A2, by sequestering dioxin and thus altering the pharmacokinetics, protects the embryos from toxicity and birth defects; substitution of the human CYP1A2 trans-gene provides the same protection. In contrast, neither CYP1A1 nor CYP1B1 appears to play a role in dioxin-mediated teratogenesis.

No Association between Polymorphisms in CYP2E1, GSTM1, NAT1, NAT2 and the Risk of Gastric Adenocarcinoma in the European Prospective Investigation into Cancer and Nutrition

No association between polymorphisms in CYP2E1, GSTM1, NAT1, NAT2 and the risk of gastric adenocarcinoma in the European prospective investigation into cancer and nutrition.

Light-Dependent Transformation of Aniline to Indole Esters by the Purple Bacterium Rhodobacter sphaeroides OU5

In an attempt to understand the aromatic hydrocarbon metabolism by purple bacteria that do not grow at their expense, we earlier reported 2-aminobenzoate transformation by a purple non-sulfur bacterium, Rhodobacter sphaeroides OU5 (Sunayana et al., 2005, J Ind Microbiol Biotech 32:41-45), which is extended in the present study with aniline, a major environmental pollutant. Aniline did not support photo (light anaerobic) or chemo (dark aerobic) heterotrophic growth of Rhodobacter sphaeroides OU5 either as a sole source of carbon or nitrogen. However, light-dependent aniline transformation was observed in the culture supernatants and the products were identified as indole derivatives. The transformation was dependent on a tricarboxylate intermediate, fumarate. Five intermediates of the aniline biotransformation pathway were isolated and identified as indole esters having a mass of 443, 441, 279, 189, and 167 with unstoichiometric total indole yields of 0.16 mM: from 5 mM: of aniline consumed. The pathway proposed based on these intermediates suggest a novel xenobiotic detoxification process in bacteria.

GSTM1andCYP1A1Polymorphisms, Tobacco, Air Pollution, and Lung Cancer: A Study in Rural Thailand

The Lampang Province is situated in the northern region of Thailand. Incidence rates of lung cancer are high for Asian standards, particularly in women. This study was conducted to quantify the risk of lung cancer associated with exposures prevalent in the area and to investigate possible interactions with genetic susceptibility. The presence of several large open-cast coal mines from 1955 close to electricity-generating plants was a particular focus of concern. Two-hundred and eleven cases of primary lung cancers diagnosed in 1993 to 1995 and residents in the province were recruited at the Lampang Provincial Hospital (main referral center for treatment of the disease). Two sets of controls, frequency-matched to the cases by sex and age, were recruited (a) from the resident population (202 interviewed) and (b) from patients admitted to the hospital for diseases predominantly unrelated to tobacco smoking (211 interviewed). Sociodemographic information, complete residential history, and characteristics of the household (place of cooking, cooking fuel, and heating fuels), occupational history, and history of tobacco smoking were obtained by interview. Cases and controls ( approximately 50% of the population-based series) provided a blood sample. A point source air pollution exposure index was calculated for each village/township reported in residential histories based on the linear distance from the Mae Moh Center (the area of the electricity-generating plants), the year-specific gaseous (SO(2) and NO(2)) or total suspended particulate emissions from the Mae Moh Power Plant, and the percentage of wind from the center. Odds ratios (OR) for the disease associated with categorical variables were estimated within unconditional logistic regression. Extraction of genomic DNA and genotyping of variants in CYP1A1 and GSTM1 were conducted to assess the extent of modification of risk by these genes that are involved in the metabolism of polycyclic aromatic hydrocarbons, a common component of the exposures. Overall, there was no evidence of relevant differences in the socioeconomic level of the three groups. The two control sets were similar with respect to lifelong tobacco habit and were subsequently pooled in analyses. Never-smokers were 7% of men and 33% of women. Smoking of local traditional products unfiltered and high in tar content is a common habit in the rural female population. ORs associated with smoking increased with duration of the habit and average daily amount, being 4.9 [95% confidence interval (95% CI), 2.5-9.7] for smokers of > or =7 cigarettes/d and 3.3 (95% CI, 1.7-6.2) for duration of 41 years or longer compared with nonsmokers. Smoking of local products was associated with an independent OR of 3.1 (95% CI, 1.7-5.6) adjusted for lifelong cumulative amount of tobacco smoked. Although most smokers had the habit for at least 16 years, the daily consumption was low compared with Western standards. Other potential sources of exposure to lung carcinogens (emission from the power-generating plants and domestic burning of coal and wood for cooking and heating) were not associated with increased risk of lung cancer. None of the three polymorphisms examined increased the risk of lung cancer or modified the risk associated with tobacco smoking. In this rural population, 96% of male and 64% of female lung cancer incidence were explained by tobacco smoking. None of the potential sources of air pollution deriving from the combustion of coal and wood, or polymorphisms in the CYP1A1 gene or deletion of the GSTM1 had an effect on the risk of lung cancer, either together or separately.

Polymorphisms in Polycyclic Aromatic Hydrocarbon Metabolism and Conjugation Genes, Interactions with Smoking and Prostate Cancer Risk

The relationship between cigarette smoking and prostate cancer remains unclear. Any potential association may depend on the individuals' ability to metabolize and detoxify cigarette carcinogens--such as polycyclic aromatic hydrocarbons. To investigate this, we studied the association between prostate cancer and smoking, as well as the main and modifying effects of functional polymorphisms in genes that metabolize polycyclic aromatic hydrocarbons (CYP1A1 Ile(462)Val, microsomal epoxide hydrolase His(139)Arg) and detoxify reactive derivatives (GSTM1 null deletion, GSTT1 null deletion, GSTP1 Ile(105)Val and Ala(114)Val) using a family-based case-control design (439 prostate cancer cases and 479 brother controls). Within the entire study population, there were no main effects for smoking or any of the polymorphisms. However, the nondeleted GSTM1 allele was inversely associated with prostate cancer [odds ratio (OR), 0.50; 95% confidence interval (95% CI), 0.26-0.94] among men with less aggressive disease (Gleason score < 7 and clinical tumor stage < T2c) and positively associated (OR, 1.68; 95% CI, 1.01-2.79) with prostate cancer in men with more aggressive disease (Gleason score > or = 7 or clinical tumor stage > or = T2c). We also found a statistically significant negative multiplicative interaction between the GSTM1 nondeleted allele and heavy smoking (> 20 pack-years) in the total study population (P = 0.01) and in Caucasians (P = 0.01). Among Caucasians, heavy smoking increased prostate cancer risk nearly 2-fold in those with the GSTM1 null genotype (OR, 1.73; 95% CI, 0.99-3.05) but this increased risk was not observed in heavy smokers who carried the GSTM1 nondeleted allele (OR, 0.95; 95% CI, 0.53-1.71). Our results highlight the importance of considering genetic modifiers of carcinogens when evaluating smoking in prostate cancer.

Variation in genes relevant to aromatic hydrocarbon metabolism and the risk of adult brain tumors

Genes involved in phase I and phase II regulation of aromatic hydrocarbon-induced effects exhibit sequence variability that may mediate the risk of adult brain tumors. We evaluated associations between gene variants in CYP1A1, CYP1B1, GSTM3, EPHX1, and NQO1 and adult brain tumor incidence. Cases were patients with glioma (n = 489), meningioma (n = 197), or acoustic neuroma (n = 96) diagnosed from 1994 to 1998 at three U.S. hospitals. Controls were 799 patients admitted to the same hospitals for nonmalignant conditions. DNA was extracted from blood samples collected from 1277 subjects, and genotyping was conducted for CYP1A1 I462V, CYP1B1 V432L, EPHX1 Y113H, GSTM3 *A/*B (intron 6 deletion), and NQO1 P187S. The CYP1B1 V432L homozygous variant was associated with decreased risk of meningioma (odds ratio [OR] = 0.6; 95% CI, 0.3-1.0) but not the other tumor types. The GSTM3 *B/*B genotype was associated with increased risk of glioma (OR = 2.3; 95% CI, 1.0-5.2) and meningioma (OR = 3.6; 95% CI, 1.3-9.8). Increased risks associated with GSTM3 *B/*B were observed in younger subjects (age < 50) and older subjects (age > or = 50), in men and women, and within each study site. The magnitude of association for GSTM3 with glioma and meningioma was greater among ever-smokers than among those who had never smoked. None of the other genotypes showed consistent associations with any tumor type. The association with the GSTM3 *B allele, while intriguing, requires replication, and additional research is needed to clarify the function of the GSTM3 alleles studied here.

REGULATION OFCYP1A1GENE EXPRESSION BY THE ANTIOXIDANTTERT-BUTYLHYDROQUINONE

CYP1A1, a major phase I enzyme, plays an important role in the metabolism of polycyclic aromatic hydrocarbons and in the chemical activation of xenobiotics to carcinogenic derivatives. The phenolic antioxidant tert-butylhydroquinone (tBHQ), often used as a food preservative, is generally considered to act only as a mono-functional inducer of phase II enzymes, thereby exerting chemo-protection. However, we recently observed that tBHQ elevated the activity of an aryl hydrocarbon receptor (AhR) response element (DRE)-driven luciferase reporter in human colon carcinoma cells (Caco-2). Therefore, we studied the effects of tBHQ on the activity of a DRE-driven reporter, CYP1A1 mRNA expression, and CYP1A enzyme activity in Caco-2 cells and human HepG2 hepatoma cells. We found tBHQ caused induction of reporter activity and CYP1A1 expression and activity in Caco-2 and HepG2 cells. Moreover, tBHQ combined with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased reporter activity and mRNA expression in Caco-2 cells in an additive manner. By contrast, tBHQ decreased TCDD-mediated induction of reporter activity and CYP1A1 mRNA expression in HepG2 cells. Resveratrol, an AhR antagonist, repressed the induction of CYP1A1 by tBHQ. Cotransfection of HepG2 cells with a dominant negative AhR nuclear translocator mutant abolished the tBHQ-induced CYP1A1 reporter activity. These findings indicate that CYP1A1 may be induced by the antioxidant tBHQ via an AhR-dependent mechanism.

Fungi growing on aromatic hydrocarbons: biotechnology's unexpected encounter with biohazard?

The biodegradation of aromatic hydrocarbons by fungi has traditionally been considered to be of a cometabolic nature. Recently, however, an increasing number of fungi isolated from air biofilters exposed to hydrocarbon-polluted gas streams have been shown to assimilate volatile aromatic hydrocarbons as the sole source of carbon and energy. The biosystematics, ecology, and metabolism of such fungi are reviewed here, based in part on re-evaluation of a collection of published hydrocarbon-degrading isolates obtained from authors around the world. Incorrect or outdated identifications in original publications are corrected by ribosomal DNA sequence analysis. The data show that many volatile-hydrocarbon-degrading strains are closely related to, or in some cases clearly conspecific with, the very restricted number of human-pathogenic fungal species causing severe mycoses, especially neurological infections, in immunocompetent individuals. Neurochemistry features a distinctive array of phenolic and aliphatic compounds that are related to molecules involved in the metabolism of aromatic hydrocarbons. Hence, there may be physiological connections between hydrocarbon assimilation and certain patterns of mammalian infection.

Increased formation of carcinogenic PAH metabolites in fish promoted by nitrite

Nitrite (NO 2 −), a highly reactive chemical species, accumulates in coastal waters as a result of pollution with nitrogenous waste and/or an imbalance in the bacterial processes of nitrification and denitrification. The present study probed the impact of nitrite (NO 2 −) on the metabolism of polycyclic aromatic hydrocarbons (PAHs) in fish. In a laboratory experiment, exposure of euryhaline fish, Oreochromis mossambicus to industrial effluents containing PAHs in the presence of NO 2 − enhanced the cytochrome P450-dependent biotransformation activity determined as 7-ethoxyresorufin- O-deethylase (EROD), by nearly 36% compared to the value observed in the absence of NO 2 − (50.2 ± 6.74 pmol resorufin min −1 g −1 liver). Fixed wavelength fluorescence measurements in bile revealed maximum enhancement to have occurred in the metabolites of benzo[ a]pyrene, a carcinogenic PAH. Lasting, sublethal physiological deterioration was apparent in fish exposed simultaneously to an oil refinery effluent and NO 2 −, from the unremittingly decreasing liver somatic index, even after the withdrawal of the contaminants.

Expression of cytochrome P450 enzymes CYP1A1, CYP1B1, CYP2E1 and CYP4B1 in cultured transitional cells from specimens of the human urinary tract and from urinary sediments

Expression of cytochromes P450 CYP1A1, CYP1B1, CYP2E1 and CYP4B1 was analysed on the transcript level in human urothelial cells obtained by various methods. As a source of urothelial cells, exfoliated cells in urine samples were used. Their expression profiles were determined either immediately after centrifugal enrichment (n=4) or after their cultivation and propagation (n=8). Another source of urothelial cells were ureter specimens from surgical subjects (n=4). Generally, expression was most prominent for CYP1B1 and CYP4B1 among the CYP transcripts analysed. CYP1B1 mRNA was detected in all samples investigated except for one ureter specimen. CYP4B1 mRNA was present in cell cultures from three out of eight healthy subjects, in three out of four directly investigated urinary sediments and in the cells of all five ureter specimens of four donors investigated after resection and subsequent cell culture. In most cases, CYP2E1 transcript levels were lower than those of CYP1B1 and CYP4B1. CYP2E1 mRNA was detected in cell cultures of six out of eight healthy subjects, in one out of four urinary sediments and in three out of five ureter specimens. CYP1A1 mRNA was clearly observed only in cells from resected ureters. In cell cultures the relative mRNA expression levels varied with subjects interindividually, intraindividually and also during the time of cell culture. The study demonstrates constitutive mRNA expressions of xenobiotic metabolising CYP enzymes in human urothelial cells obtained by different methods. In particular, transcripts of CYP1B1 and CYP4B1 are present, coding for enzymes which are active in the metabolism of polycyclic aromatic hydrocarbons and arylamines, respectively.

Error-prone Translesion Synthesis by Human DNA Polymerase η on DNA-containing Deoxyadenosine Adducts of 7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

When human DNA polymerase eta (pol eta) encounters N6-deoxyadenosine adducts formed by trans epoxide ring opening of the 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP DE) isomer with (+)-7R,8S,9S,10R configuration ((+)-BaP DE-2), misincorporation of A or G and incorporation of the correct T are equally likely to occur. On the other hand, the enzyme exhibits a 3-fold preference for correct T incorporation opposite adducts formed by trans ring opening of the (-)-(7S,8R,9R,10S)-DE-2 enantiomer. Adducts at dA formed by cis ring opening of these two BaP DE-2 isomers exhibit a 2-3-fold preference for A over T incorporation, with G intermediate between the two. Extension one nucleotide beyond these adducts is generally weaker than incorporation across from them, but among mismatches the (adducted A*) x A mispair is the most favored for extension. Because mutations can only occur if mispairs are extended, this observation is consistent with the occurrence of A x T to T x A transversions as common mutations in animal cells treated with BaP DE-2 isomers. Adducts with S absolute configuration at the point of attachment of the hydrocarbon to the base inhibit incorporation and extension by pol eta to a lesser extent than their R counterparts. Template-primers containing each of the four isomeric dA adducts derived from BaP DE-2 and two adducts derived from 9,10-epoxy-7,8,9,10-tetrahydrobenzo-[a]pyrene in which the 7- and 8-hydroxyl groups of the DEs are replaced with hydrogens exhibit reduced electrophoretic mobilities relative to the unadducted oligonucleotides. This effect is largely independent of DNA sequence. Decreased mobility correlates with an increased rate of incorporation by pol eta, suggesting a systematic relationship between the overall DNA structure and efficiency of the enzyme.