Susceptibility and exposure biomarkers in people exposed to PAHs from diesel exhaust.

Xenobiotic metabolizing enzymes, especially CYP1A1 and GSTM1, are involved in the activation and conjugation of PAHs and are controlled by polymorphic genes. PAHs released from diesel emissions in many cities of the world, especially in developing countries, contribute significantly to the toxic effects of airborne inhalable particles. We have evaluated the gene-environment interaction in Santiago of Chile, studying the contribution of CYP1A1 and GSTM1 polymorphisms on 1-OH-P urinary levels used as the PAHs exposure biomarker. The study was performed on 59 diesel exposed (38 diesel revision workers and 21 subjects working in an urban area as established street vendors) and 44 non-exposed subjects living in a rural area. The 1-OH-P urinary levels of the urban (P=0.043) and rural (P=0.040) populations showed, without considering the genotypes, significant differences between smokers and non-smokers, but no significant differences were found between smokers and non-smokers among the diesel plant workers (P=0.33). Non-smoking subjects of the diesel plants and the urban area showed similar 1-OHP levels (P=0.466) which were significantly higher than those of the subjects living in the rural area (P<0.05). When 1-OH-P levels were related with genotypes, an association was observed for the CYP1A1*2A genotype, so that the diesel-exposed workers carrying the CYP1A1*2A allele showed significantly higher 1-OH-P levels than the subjects from the rural area with the same genotype (P=0.008). On the other hand, there was no significant correlation between urinary 1-OH-P levels and GSTM1 null genotype, although higher levels of the urinary metabolite were found in individuals carrying the combined CYP1A1*2A and GSTM1 null genotype (P=0.055). These results may suggest an association between levels of the exposure biomarker 1-OH-P and presence of the CYP1A1*2A genotype, a potential genetic susceptibility biomarker which might be useful in identifying individuals at higher risk among people exposed to high PAH levels in diesel exhaust.

Influence of genetic polymorphisms of CYP1A1 and GSTM1 on the urinary levels of 1-hydroxypyrene.

The aim of the present study is to evaluate the influence of the genetic polymorphism of two enzymes involved in the biotransformation of xenobiotics, cytochrome P450 1A1 (CYP1A1) and glutathione-S-transferase M1 (GSTM1), on the urinary levels of 1-hydroxypyrene (1-OH-P) in workers exposed to polycyclic aromatic hydrocarbons (PAHs) and in unexposed workers (controls). The study group consisted of 30 controls recruited among employees of a service company and 171 PAHs-exposed workers from two electric steel plants and an iron foundry (all males, ranging between 18 and 60 years of age). Determination of airborne PAHs and urinary 1-OH-P was performed by high-performance liquid chromatography (HPLC) with fluorimetric detection. Polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) was used to determine the genetic polymorphisms of CYP1A1 (CYP1A1*2A and CYP1A1*2B) and GSTM1. No influence of the genetic polymorphism of CYP1A1 and GSTM1 on the urinary levels of 1-OH-P was observed in this study.

Biodistribution study of phosphonolipids: a class of non-viral vectors efficient in mice lung-directed gene transfer.

BACKGROUND: A multitude of cationic lipids have been synthesized since they were first proposed for use in gene therapy. Cationic lipids are able to efficiently transfect cells both in vitro and in vivo. Whereas most research groups have focused their investigations on the toxicity of these molecules, and on the location of expression of the DNA transferred by these vectors, little has been done to determine their biodistribution and elimination pathways. Our group has developed a family of cationic lipids termed phosphonolipids. Following a large in vitro screening experiment, we have selected several molecules for in vivo testing, with some of these phosphonolipids forming lipoplexes efficient in transfecting mouse lungs. It was thus of interest to study their fate after intravenous injection. METHODS: The respective biodistributions of both the GLB43 phosphonolipid and plasmid DNA were investigated and compared with DNA expression sites. Using the optimal conditions determined for phosphonolipids, we followed the gene transfer agent and plasmid DNA distributions versus time by radiolabeling them with (14)C and (32)P, respectively. Otherwise, we performed imaging by radiolabeling plasmid DNA with (99m)Tc. RESULTS: The lipoplexes appear to be directly located in the lung after administration. Secondly, the plasmid is released mainly into the lungs and the phosphonolipid vector is rapidly degraded. The hydrophilic moiety of the phosphonolipid is eliminated in the urine, as is the free plasmid. CONCLUSIONS: This study reveals that there are slight differences in the observed results depending on the technique used to label the DNA; secondly, results show that the residence time of phosphonolipids in the mouse body is related to the DNA binding time.

Hypertension after renal transplantation and polymorphism of genes involved in essential hypertension: ACE, AGT, AT1 R and ecNOS.

BACKGROUND: Arterial hypertension (HT), secondary to cyclosporine A (CsA) used as main immunosuppressive treatment in renal transplantation (RTx), is very frequent (70%), usually severe and explained mostly by vasoconstriction of the glomerular afferent arteriole with secondary sodium and water retention. MATERIAL AND METHODS: In a retrospective study, we have analyzed 294 consecutive recipients receiving a first renal cadaveric allograft and all treated with CsA (the majority with triple therapy). We studied, by molecular biology, the polymorphism of genes previously implicated in essential HT such as: angiotensin-converting enzyme (ACE: II, ID and DD), angiotensinogen (AGT: MM, MT and TT), angiotensin II type 1 receptor (AT1-R: AA, AC and CC) and endothelial constitutive nitric oxide synthase (ecNOS: aa, ab and bb), and correlated the data to the prevalence and severity of post-Tx HT. This cohort included 195 (66%) males and 99 females with a mean age of 42 years at time of Tx. The presence and severity of post-Tx HT were indicated by initial persistent blood pressure over 140/90 mmHg with the need for at least one anti-hypertensive drug and by the number of anti-HT medications required to achieve its control. RESULTS: The distribution of the specific alleles and genotypes for ACE, AGT, AT1-R, and ecNOS was not different in transplant recipients compared to 181 controls. At 5 years post-Tx, the prevalence of HT was 72% (169 out of 235) among functioning grafts. There was no significant difference for ACE, AGT, AT1R and ecNOS genotypes distribution between hypertensive vs non-HT recipients. The number of anti-hypertensive drugs prescribed was not different among ACE, AGT, and AT1-R genotypes. However, the a allele and the non-bb genotype (aa + ab) for ecNOS were significantly (p = 0.001) associated with a less severe HT, needing fewer anti-HT drugs. At 10 years post-Tx, the HT prevalence remained high 78% (67 out of 86) among functioning Tx. However, the limited numbers did not allow further correlation. CONCLUSIONS: This study produced mainly negative results except for ecNOS-a allele, which seems to protect against severe hypertension. The explanation remains speculative but probably relates to the known cyclosporine-induced upregulation of ecNOS gene and enzyme activity.

Inhibition of CYP3A, CYP1A and CYP2E1 activities by resveratrol and other non volatile red wine components.

Resveratrol (RESV), present at concentrations of about 10 microM in red wine, has been found to inhibit events associated with tumor initiation, promotion and progression. The mechanism involved could be the inhibition of activities catalyzed by cytochromes P450 (CYPs), which activate procarcinogens. This led us to investigate the inhibitory effect of RESV on CYP1A, CYP2E1 and CYP3A enzymatic activities and to compare it to that of non volatile compounds present in red wine. Red wine solids (RWS) were prepared by evaporating one volume of red wine to dryness followed by reconstitution with five volumes of buffer (20% natural strength). CYP activities were determined in microsomes from rat liver, human liver or cells containing cDNA-expressed CYPs. Testosterone, chlorzoxazone, and ethoxyresorufin were used as selective substrates for CYP3A, CYP2E1 and CYP1A1/1A2, respectively. RESV and RWS were found to be irreversible (probably mechanism-based) inhibitors for CYP3A4 and non competitive reversible inhibitors for CYP2E1. Their inhibitory potency was assessed using IC(50) values that were found within 4-150 microM for RESV and 0.3-9% natural strength for RWS. Non volatile compounds of other beverages such as white wine, grape juice or Xtra Old Cognac(R) displayed lower inhibitory effect on CYP activities than RWS. When considering the concentration of RESV in red wine (2 microM for 20% natural strength), it appears that RSW inhibitory effect was not only due to RESV, but also to other compounds whose identification would prove to be worthwhile because of their possible chemopreventive properties.

Oxidation of beta2-glycoprotein I (beta2GPI) by the hydroxyl radical alters phospholipid binding and modulates recognition by anti-beta2GPI autoantibodies.

We investigated whether beta2-glycoprotein I (beta2GPI), the key antigen in the antiphospholipid syndrome, is susceptible to oxidative modifications by the hydroxyl radical (*OH) that may influence its lipid-binding and antigenic properties. The effects on human and bovine beta2GPI of *OH free radicals generated by gamma-radiolysis of water with 137Cs were studied. Radiolytic *OH caused a dose-dependent loss of tryptophan, production of dityrosine and carbonyl groups. dimerization and/or extensive aggregation of beta2GPI. It ensued a reduction in affinity binding to cardiolipin liposomes and loss of beta2GPI-dependent autoantibody binding to immobilized cardiolipin. Patient anti-beta2GPI antibodies (n = 20) segregated into two groups based on the effect in the beta2GPI-ELISA of beta2GPI pretreatment with *OH: enhancement (group A, n = 10) or suppression (group B, n = 10) of IgG binding. The avidities of group A antibodies for fluid-phase beta2GPI were low but increased in a dose-dependent manner upon beta2GPI irradiation, in relation to protein crosslinking. Distinguishing features of group B antibodies included higher avidities for fluid-phase beta2GPI that was no longer recognized after *OH treatment, and negative anticardiolipin tests suggesting epitope location near the phospholipid binding site. The *OH scavengers thiourea and mannitol efficiently protected against all above changes. Therefore, oxidative modifications of beta2GPI via *OH attack of susceptible amino acids alter phospholipid binding, and modulate recognition by autoantibodies depending on their epitope specificities. These findings may be of clinical relevance for the generation and/or reactivity of anti-beta2GPI antibodies.

CYP1A1, CYP2E1 and GSTM1 genetic polymorphisms. The effect of single and combined genotypes on lung cancer susceptibility in Chilean people.

CYP1A1, CYP2E1 and GSTM1 polymorphisms were evaluated in Chilean healthy controls and lung cancer patients. In the Chilean healthy group, frequencies of CYP1A1 variant alleles for MspI (m2 or CYP1A1*2A) and ile/val (val or CYP1A1*2B) polymorphisms were 0.25 and 0.33, respectively. Frequencies of variant alleles C (CYP2E1*6) and c2 (CYP2E1*5B) for CYP2E1 were 0.21 and 0.16, respectively and frequency for GSTM1(-) was 0.24. The presence of variant alleles for GSTM1, MspI and Ile/val polymorphisms was more frequent in cases than in controls. However, frequencies for the c2 and C alleles were not significantly different in controls and in cases. The estimated relative risk for lung cancer associated to a single mutated allele in CYP1A1, CYP2E1 or GSTM1 was 2.41 for m2, 1.69 for val, 1.16 for C, 0.71 for c2 and 2.46 for GSTM1(-). The estimated relative risk was higher for individuals carrying combined CYP1A1 and GSTM1 mutated alleles (m2/val, OR=6.28; m2/GSTM1(-), OR=3.56) and lower in individuals carrying CYP1A1 and CYP2E1 mutated alleles (m2/C, OR=1.39; m2/c2, OR=2.00; val/C, OR=1.45; val/c2, OR=0.48; not significant). The OR values considering smoking were 4.37 for m2, 4.05 for val, 3.47 for GSTM1(-), 7.38 for m2/val and 3.68 for m2/GSTM1(-), higher values than those observed without any stratification by smoking. Taken together, these findings suggest that Chilean people carrying single or combined GSTM1 and CYP1A1 polymorphisms could be more susceptible to lung cancer induced by environmental pollutants such as polycyclic aromatic hydrocarbons.

Human cytochrome P450 2A6 is the major enzyme involved in the metabolism of three alkoxyethers used as oxyfuels.

Methyl t-butyl ether (MTBE), ethyl t-butyl ether (ETBE), and t-amyl methyl ether (TAME) are three alkoxyethers added to gasoline to improve combustion and thereby to reduce the level of carbon monoxide and aromatic hydrocarbons in automobile exhaust. Oxidative demethylation of MTBE and TAME and deethylation of ETBE by CYP enzymes results in the formation of tertiary alcohols and aldehydes, both potentially toxic. The metabolism of these three alkoxyethers was studied in a panel of 12 human liver microsomes. The relatively low apparent Km(1) was 0.25+/-0.17 (mean+/-SD), 0.11+/-0.08 and 0.10+/-0.07 mM and the high apparent Km(2) was 2.9+/-1.8, 5.0+/-2.7 and 1.7+/-1.0 mM for MTBE, ETBE and TAME, respectively. Kinetic data, correlation studies, chemical inhibition and metabolism by heterologously expressed human CYPs support the assertion that the major enzyme involved in MTBE, ETBE and TAME metabolisms is CYP2A6, with a minor contribution of CYP3A4 at low substrate concentration.

Cytochrome CYP2E1 phenotyping and genotyping in the evaluation of health risks from exposure to polluted environments.

Humans are exposed to over 70,000 man-made chemicals including drugs, food additives, herbicides, pesticides, and industrial agents. It is well established that environmental chemicals are the cause of numerous human diseases including cancer. In most cases, chemical carcinogens require metabolic activation, which is mainly achieved by P450s enzymes. CYP2E1 is of clinical relevance because it is inducible by ethanol, and it metabolizes many common organic solvents such as benzene, alcohols and halogenated solvents. Therefore, alteration in the level of CYP2E1 might influence the health effects of the environmental pollutants. This hypothesis needs to be validated by epidemiological studies and the objective of the "Biomed-2" project was to develop new tests to assess the individual metabolic capacity of workers exposed to volatile organic compounds in order to predict their occupational risk. In vivo chlorzoxazone 6-hydroxylation was validated as a non-invasive and selective test for the determination of liver CYP2E1 activity. Preliminary data in workers exposed to organic solvents indicated that chlorzoxazone metabolism may be a biomarker of occupational exposure to organic solvents. Other approaches, such as use of salicylate as catalytic probe or measurement of catalytic activity in lymphocytes, were not conclusive. Attempts to use CYP2E1 genotyping for estimating human risks from chemical exposure did not bring convincing data as genetic polymorphism of CYP2E1 could not be clearly related to its catalytic activity.

Genetic repeat polymorphism in the regulating region of CYP2E1: frequency and relationship with enzymatic activity in alcoholics.

BACKGROUND: Differences in the regulatory region of the CYP2E1 gene could be responsible for the interindividual variation in the cytochrome P-450 2E1 (CYP2E1) involved in ethanol oxidation. Recently, a polymorphic repeat sequence in the human gene was described between -2178 and -1945 base pairs. Its frequency seemed to vary among different ethnic populations, and it was suspected to be related to an increased inducibility to further ethanol intake. In the study reported here, the frequency of this polymorphism was investigated in a white French population. Its relationship with the previously described PstI/RsaI or DraI CYP2E1 polymorphisms, alcoholism, alcoholic liver disease, and inducibility of CYP2E1 by ethanol was examined. METHODS: The polymorphic region was characterized by polymerase chain reaction in 103 controls, 148 alcoholic subjects without liver diseases, and 98 others with liver cirrhosis. By using in vivo chlorzoxazone (CHZ) metabolism, CYP2E1 phenotype was assessed in 36 non-ethanol-induced subjects (17 controls and 19 withdrawn alcoholics) and in 14 ethanol-induced subjects (10 controls after ingestion of 0.8 g/kg ethanol and four alcoholics with 100 g of daily intake). This phenotype was expressed as the 6-hydroxy CHZ/CHZ ratio. RESULTS: The rare allele frequency was found to be 1.58% in whites (n = 349). Neither significant association with alcoholism or alcoholic liver diseases, nor relationship with the PstI/RsaI polymorphism, was observed. But the DraI polymorphism was more frequent among the heterozygous subjects when compared with wild-type homozygous ones (p < 0.05). The CYP2E1 phenotype was similar in wild-type homozygotes and in heterozygotes at the constitutive level, as well as after induction with ethanol. CONCLUSIONS: Our data suggest that CYP2E1 repeat polymorphism does not seem to constitute a major factor for interindividual differences in CYP2E1 expression and susceptibility to alcohol-related disorders in whites.

[Alcohol-xenobiotic interactions. Role of cytochrome P450 2E1]. / Interactions alcool-xénobiotiques. Rôle du cytochrome P450 2E1.

Alcohol and xenobiotics share the same oxidative microsomal pathway, which is mainly located in the endoplasmic reticulum of hepatocytes. This pathway involves enzymes that belong to the super family of cytochrome P450 and allows to explain a lot of pharmacokinetic or toxic interactions between alcohol and xenobiotics. Cytochrome P450 2E1 (CYP2E1) is the key enzyme of the microsomal pathway of ethanol oxidation. It is inducible by chronic ethanol consumption and its activity is increased by three to five fold in liver from alcoholics subjects. This induction involves to a lesser extent cytochromes P450 3A4 and 1A2 and contributes to the metabolic tolerance of alcohol and drugs observed in alcoholics. The metabolic tolerance persits several days after ethanol withdrawal. Furthermore, CYP2E1 has a high capacity to activate numerous xenobiotics into toxic or carcinogenic compounds. Drugs currently used such as paracetamol, anesthetics (enflurane, halothane), industrial solvents (benzene or its derivatives), halogenated solvents (CCl4, trichlorethylene) and nitrosamines which are present in food or tobacco smoke are included. Therefore, heavy consumption of alcohol, which results in CYP2E1 induction, increases individual susceptibility to the toxic or carcinogenic effects of these xenobiotics.

Cytochrome P-450 2E1 activity and oxidative stress in alcoholic patients.

As cytochrome P-450 2E1 (CYP2E1) induction was related to oxidative stress in experimental models, the aim of this study was to investigate the relationship between CYP2E1 activity and markers of oxidative stress in 40 alcoholic patients entering a rehabilitation programme. Plasma oxidized proteins, lipid peroxides (LPO) and antibodies against hydroxyethyl radical (HER) or malondialdehyde (MDA) adducts were assessed as markers of the production of free radicals, whereas vitamin E levels were evaluated as a marker of the antioxidant defence. CYP2E1 activity was determined by using the 6-hydroxychlorzoxazone:chlorzoxazone blood metabolic ratio, 2 h after drug intake. This ratio was increased by 4-fold in alcoholics, compared to non-alcoholic patients, and was correlated with daily intake of ethanol, carbohydrate-deficient transferrin, and blood alcohol level at the time of admission to hospital. Plasma levels of LPO and oxidized proteins were slightly increased (20%) in alcoholic patients when compared with the control group, whereas those of vitamin E were found to be slightly decreased (by 18%). Antibodies against HER or MDA adducts showed a very significant increase. However, when alcoholic patients were divided into two groups according to low or high CYP2E1 induction, no significant difference was observed in the variation of these parameters, except for anti-HER adducts antibodies. Therefore, our study confirms the main involvement of CYP2E1 in HER production. By contrast, CYP2E1 does not appear to be the main factor responsible for the oxidative stress occurring during human chronic alcoholism. Free radicals from other sources may therefore contribute significantly to the generation of this oxidative stress.

Adaptation to chronic ethanol administration emphasized by fatty acid hydroxylations in rat liver and kidney microsomes.

BACKGROUND: Long-term ethanol consumption in laboratory animals is associated with histological alterations of liver cells and modifications of fatty acid metabolism. AIM OF THE STUDY: The present study was aimed at investigating the effect of 1- and 2-month chronic treatment of rats with ethanol on the metabolism of two unsaturated (oleic and linoleic) fatty acids in liver and kidney microsomes, in relation to the CYP2E1 enzyme content in both tissues. METHODS: Rats were fed ethanol (14 g/Kg/d) or dextrose through a permanently implanted gastric cannula, as described in the intragastric feeding rat model for alcoholic liver disease (ALD). CYP2E1 level was immuno-quantified in both liver and kidney microsomes by Western blot, whereas fatty acid omega- and (omega-1)-hydroxylations were measured using HPLC and radiometric analytical methods. RESULTS: One- and two-month ethanol treatment led to a 3- to 4-fold rise of the CYP2E1 protein in both liver and kidney microsomes. Oleic and linoleic acid (omega-1)-hydroxylations were increased (approximately 3-fold) in liver microsomes after one-month of ethanol administration, but surprisingly such a rise was not observed after a two-month treatment; on the other hand, no effect was observed on the omega-hydroxylations of these fatty acids. Furthermore, as previously described for lauric acid, ethanol intake did not significantly act on the kidney microsome capability to hydroxylate unsaturated fatty acids. CONCLUSIONS: CYP2E1 is strongly inducible by ethanol and therefore accounts for the tolerance for this hepatotoxicant. Our results support the development of an adaptation process in the liver hydroxylating enzyme system, which occurs between one and two months of ethanol feeding. Although it is usually not appropriate to extrapolate animal findings to humans, rat and human CYP2E1s were observed to have comparable specificities and similar mechanisms of regulation. Thus, the present study allowed the acquirement of detailed information of CYP2E1 activity in patients with severe manifestations of ALD.

Interspecies variations in fatty acid hydroxylations involving cytochromes P450 2E1 and 4A.

The liver microsomal fractions of seven mammalian species including rat, dog, monkey, hamster, mouse, gerbil and humans, catalyzed the hydroxylation of saturated (lauric, myristic and palmitic) and unsaturated (oleic and linoleic) fatty acids to the corresponding omega and (omega-1)-hydroxylated derivatives, while stearic acid was not metabolized. Lauric acid was the most efficiently hydroxylated, and the rank of catalytic activity was lauric > myristic > oleic > palmitic > linoleic. Among the mammalian species studied, mouse and hamster presented the highest level of fatty acid omega and (omega-1)-hydroxylases, while the lowest activity was observed in dog and monkey. In all the animal species, the (omega-1)-hydroxylation of fatty acids correlated significantly with the immunodetectable content of CYP2E1 and the 4-nitrophenol hydroxylation activity, known to be mediated by cytochrome P450 2E1. On the contrary, only the omega-hydroxylation of lauric acid slighly correlated with the level of cytochrome P450 4A, while no significant correlation was found with the omega-hydroxylation of the other fatty acids. Furthermore, chemical and immuno-inhibitions of the hydroxylations of fatty acids led to the conclusion that fatty acid (omega-1)-hydroxylase activity is catalyzed by P450 2E1 in all the mammalian species, while the fatty acid omega-hydroxylase activity may be catalyzed by cytochromes P450 from the 4A family. Therefore, lauric acid (omega-1)-hydroxylation along with 4-nitrophenol hydroxylation can be used as a specific and sensitive method to measure the level of CYP2E1 induction in humans and various animals.

Requirement for omega and (omega;-1)-hydroxylations of fatty acids by human cytochromes P450 2E1 and 4A11.

Human liver microsomes and recombinant human P450 have been used as enzyme source in order to better understand the requirement for the optimal rate of omega and (omega;-1)-hydroxylations of fatty acids by cytochromes P450 2E1 and 4A. Three parameters were studied: alkyl chain length, presence and configuration of double bond(s) in the alkyl chain, and involvement of carboxylic function in the fatty acid binding inside the access channel of P450 active site. The total rate of metabolite formation decreased when increasing the alkyl chain length of saturated fatty acids (from C12 to C16), while no hydroxylated metabolite was detected when liver microsomes were incubated with stearic acid. However, unsaturated fatty acids, such as oleic, elaidic and linoleic acids, were omega and (omega;-1)-hydroxylated with an efficiency at least similar to palmitic acid. The (omega;-1)/omega ratio decreased from 2.8 to 1 with lauric, myristic and palmitic acids as substrates, while the reverse was observed for unsaturated C18 fatty acids which are mainly omega-hydroxylated, except for elaidic acid showing a metabolic profile quite similar to those of saturated fatty acids. The double bond configuration did not significantly modify the ability of hydroxylation of fatty acid, while the negatively charged carboxylic group allowed a configuration energetically favourable for omega and (omega;-1)-hydroxylation inside the access channel of active site.

Chlorzoxazone, a selective probe for phenotyping CYP2E1 in humans.

The ability of human cytochromes P450 other than CYP2E1 to catalyse the 6-hydroxylation of chlorzoxazone (6-OH-CHZ) was examined in vitro using human liver microsomal preparations and in vivo using chlorzoxazone as a metabolic probe. Chlorzoxazone 6-hydroxylation activity was significantly correlated with 4-nitrophenol 2-hydroxylase activity and immunodetected CYP2E1 in 14 human liver samples (r = 0.92 and 0.81, P < 0.001, respectively). Conversely, this catalytic activity was not correlated with CYP 3A or CYP1A activities. Diethyldithiocarbamate (DEDTC), a specific CYP2E1 inhibitor, reduced chlorzoxazone 6-hydroxylase activity by 92.3 +/- 7.6% (n = 14 samples) while ketoconazole, a specific CYP3A inhibitor, reduced this activity by 8.6 +/- 6.3% (n = 14). The residual activity following preincubation with DEDTC was significantly correlated with nifedipine oxidation and tamoxifen N-demethylations, both specific to CYP3A (r = 0.76 and 0.68, respectively). Genetically produced pure human CYP2E1 and 3A4 hydroxylated chlorzoxazone with turnover numbers of 19.7 and 0.14 min(-1), respectively. Furthermore, cytochrome b5 stimulated chlorzoxazone 6-hydroxylation. From examination of the relative liver content of CYP2E1 and 3A, it can be asserted that CYP2E1 is the major enzyme involved in chlorzoxazone 6-hydroxylation and that the contribution of CYP3A is very minor. CYP2E1 activity was evaluated by the plasmatic metabolic ratio 6-OH-CHZ/CHZ (CHZ-MR) measured 2 h after ingestion of 500 mg CHZ. Smoker status did not influence the rate of CHZ hydroxylation. The CHZ-MR was 0.30 +/- 0.13 (mean +/- SD) n = 39 non-smokers versus 0.32 +/- 0.15, n = 75 smokers. This result suggests that CYP1A, inducible by cigarette smoking, is not significantly involved in chlorzoxazone hydroxylation. Women exhibited a slightly lower CHZ-MR than men (0.29 +/- 0.15, n = 44 versus 0.34 +/- 0.15 n = 49, respectively). Obesity increased CHZ-MR, especially in non-insulin-dependent diabetic individuals (0.45 +/- 0.21, n = 13 versus 0.30 +/- 0.15, n = 42 control individuals, P = 0.007). Furthermore, exposure of workers to volatile organics in a shoe factory decreased CHZ-MR (0.19 +/- 0.09, n = 10 Mexican workers versus 0.34 +/- 0.12, n = 16 Mexican control individuals, P = 0.001). Concomitant administration of grapefruit juice (known to be an inhibitor of CYP3A4) with chlorzoxazone did not significantly modify the CHZ metabolic ratio: 0.29 +/- 0.1 versus 0.31 +/- 0.1, for nine control individuals without and with grapefruit juice, respectively. In conclusion, all these results demonstrate that chlorzoxazone is a very selective probe for phenotyping CYP2E1 in humans.

Ethnic susceptibility to lung cancer: differences in CYP2E1, CYP1A1 and GSTM1 genetic polymorphisms between French Caucasian and Chilean populations.

Many investigators have reported an association between genetic polymorphisms of cytochromes P-450 CYP2E1, CYP1A1 or glutathione S-transferase Mu (GSTM1) and susceptibility to lung cancer. However, pronounced interethnic variations have been described in the frequencies of these polymorphisms, especially between Asians and Caucasians. The present study was set up to establish CYP2E1 (c1, c2 and C, D), CYP1A1 (m1, m2 and Ile, Val) and GSTM1 (null) allelic frequencies in Chileans (n = 96) who are an admixture of Native Americans and Caucasians (Spaniards). The rare allele frequencies were found to be 0.15 (c2), 0.21 (C), 0.23 (m2), 0.32 (Val) and 0.21 ('null' genotype). These values are significantly higher than those of Caucasians except for the GSTM1 'null' genotype and suggest differences in susceptibility to lung cancer between both populations.

Involvement of cytochromes P-450 2E1 and 3A4 in the 5-hydroxylation of salicylate in humans.

Hydroxylation of salicylate into 2,3 and 2,5-dihydroxybenzoic acids (2,3-DHBA and 2,5-DHBA) by human liver microsomal preparations was investigated. Kinetic studies demonstrated that salicylate was 5-hydroxylated with two apparent Km: one high-affinity Km of 606 microM and one low-affinity Km greater than 2 mM. Liver microsomes prepared from 15 human samples catalyzed the formation of 2,5-DHBA at metabolic rate of 21.7 +/- 8.5 pmol/mg/min. The formation of 2, 3-DHBA was not P-450 dependent. Formation of 2,5-DHBA was inhibited by 36 +/- 14% following preincubation of microsomes with diethyldithiocarbamate, a mechanism-based selective inhibitor of P-450 2E1. Furthermore, the efficiency of inhibition was significantly correlated with four catalytic activities specific to P-450 2E1, whereas the residual activity was correlated with three P-450 3A4 catalytic activities. Troleandomycin, a mechanism-based inhibitor selective to P-450 3A4, inhibited by 30 +/- 12% the 5-hydroxylation of salicylate, and this inhibition was significantly correlated with nifedipine oxidation, specific to P-450 3A4. The capability of seven recombinant human P-450s to hydroxylate salicylate demonstrated that P-450 2E1 and 3A4 contributed to 2, 5-DHBA formation in approximately equal proportions. The Km values of recombinant P-450 2E1 and 3A4, 280 and 513 microM, respectively, are in the same range as the high-affinity Km measured with human liver microsomes. The plasmatic metabolic ratio 2,5-DHBA/salicylate, measured 2 h after ingestion of 1 g acetylsalicylate, was increased 3-fold in 12 alcoholic patients at the beginning of their withdrawal period versus 15 control subjects. These results confirm that P-450 2E1, inducible by ethanol, is involved in the 5-hydroxylation of salicylate in humans. Furthermore, this ratio was still increased by 2-fold 1 week after ethanol withdrawal. This finding suggests that P-450 3A4, known to be also inducible by alcoholic beverages, plays an important role in this increase, because P-450 2E1 returned to normal levels in less than 3 days after ethanol withdrawal. Finally, in vivo and in vitro data demonstrated that P-450 2E1 and P-450 3A4, both inducible by alcohols, catalyzed the 5-hydroxylation of salicylate.

Effects of interferon-alpha on cytochrome P-450 isoforms 1A2 and 3A activities in patients with chronic hepatitis C.

BACKGROUND/AIM: The risk of adverse drug interactions with interferon-alpha has been poorly assessed. The aim of our study was to establish whether administration of interferon-alpha at therapeutic doses in patients with chronic hepatitis C may have significant inhibitory effects on other drug metabolism. The study was focused on cytochromes P-450 1A2 and 3A, two major isoforms involved in the metabolism of numerous substrates. METHODS: Eighteen patients with chronic active hepatitis C requiring an interferon-alpha treatment were studied. Cytochrome P-450 1A2 activity was determined on the basis of an in vivo caffeine metabolism study. Cytochrome P-450 3A activity was determined according to in vivo cortisol metabolism into 6-beta-hydroxycortisol. Both activities were determined 1 month before, at initiation and 1 month after interferon-alpha therapy (3 x 10(6) units, three times a week). RESULTS: There were no significant differences in the caffeine index (CYP 1A2) and in the 6-beta-hydroxycortisol/free cortisol urinary ratio (CYP 3A) before and after alpha interferon treatment CONCLUSION: Chronic administration of interferon-alpha at therapeutic doses does not change in vivo cytochrome P-450 1A2 and 3A activities. These results support the suggestion that drugs metabolized by these isoenzymes may be used together with interferon-alpha in patients with chronic hepatitis C without significant risks of drug interactions.

Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry of omega- and (omega-1)-hydroxylated metabolites of elaidic and oleic acids in human and rat liver microsomes.

In order to characterize the nature of the active site of cytochrome P450 2E1, the metabolism of various fatty acids with cis/trans geometric configurations has been investigated. A system coupling atmospheric pressure chemical ionization-mass spectrometry detection with HPLC separation was developed as an alternative method for the characterization of hydroxylated metabolites of oleic and elaidic acids in rat and human liver microsomes. Oxidation of oleic and elaidic acids led to the formation of two main metabolites which were identified by LC-MS and GC-MS as omega and (omega-1)-hydroxylated (or 17-OH and 18-OH) fatty acids, on the basis of their pseudo-molecular mass and their fragmentation. The assay was accurate and reproducible, with a detection limit of 25 ng per injection, a linear range from 25 to 1128 ng per injection, no recorded interference, intra-day and inter-day precision with variation coefficients <14%. This LC-MS method was validated with oleic acid by using both radiometric and mass spectrometric detections. A significant correlation was found between the two methods in human (r=0.86 and 0.94 with P<0.05 and 0.01) and rat liver microsomes (r=0.90 and 0.85 with P<0.01 and 0.05) for 17-OH and 18-OH metabolites, respectively. HPLC coupled to mass spectrometry for the analysis of hydroxylated metabolites of elaidic acid offers considerable advantages since the method does not require use of a radioactive molecule, completely separates the two hydroxymetabolites, confirms the identification of each metabolite, and is as sensitive as the radiometric analysis method. This method allowed the comparative study of oleic and elaidic acid hydroxylations by both human and rat liver microsomal preparations.