Biomedical subjects

R F Tyndale

Publications and source records attributed to R F Tyndale.

At least 19 recordsLinked to original sources

PubMed2007

Overview of the pharmacogenomics of cigarette smoking.

Cigarette smoking increases the risk of numerous health problems, including cancer, cardiovascular and pulmonary disorders, making smoking the leading cause of preventable death in the world. Nicotine is primarily responsible for the highly addictive properties of cigarettes. Although the majority of smokers express a desire to quit, few are successful in doing so. Twin and family studies have indicated substantial genetic contributions to smoking behaviors. One major research focus has been to elucidate the specific genes involved; this has been accomplished primarily through genome-wide linkage analyses and candidate gene association studies. Much attention has focused on genes involved in the neurotransmitter pathways for the brain reward system and genes altering nicotine metabolism. This paper reviews the current state of knowledge for genetic factors implicated in smoking behaviors, and examines how genetic variations may affect therapeutic outcomes for drugs used to assist smoking cessation.

Aryl Hydrocarbon Hydroxylases↗

PubMed2006

Inhibition of nicotine metabolism by methoxysalen: Pharmacokinetic and pharmacological studies in mice.

Studies were undertaken to examine whether methoxsalen (9-methoxyfuro[3,2-g][1]benzopyran-7-one), a specific and relatively selective inhibitor of human CYP2A6, inhibited CYP2A5-mediated nicotine metabolism in vitro. Furthermore, studies were performed in vivo to determine whether methoxsalen would modulate acute nicotine pharmacokinetics and pharmacological effects (antinociception and hypothermia) in the ICR mouse. Our results demonstrated that methoxsalen competitively inhibits in vitro nicotine metabolism in mice. The inhibition was potent, as seen in human inhibition studies, with a Ki of 0.32 microM. In addition, we found that administration of methoxsalen significantly increased the plasma half-life of nicotine (approximately doubled) and increased its area under the curve compared with saline treatment. There was a dose-dependent enhancement in the pharmacological effects of nicotine (body temperature and analgesia) after methoxsalen treatment. Methoxsalen prolonged the duration of nicotine-induced antinociception and hypothermia (2.5 mg/kg) for periods up to 180 min postnicotine administration. Furthermore, this prolongation in nicotine's effects after methoxsalen was associated with a parallel prolongation of nicotine plasma levels in mice. These data strongly suggest that variation in the rates of nicotine metabolic inactivation substantially alter nicotine's pharmacological effects. In conclusion, these results confirmed that methoxsalen did indeed inhibit the conversion of nicotine to cotinine both in vitro and in vivo. They also suggest that mice may represent a suitable model for studying variation in nicotine metabolism and its impact on mechanisms of nicotine dependence, including the use of inhibitors to reduce nicotine metabolism.

Animals↗

PubMed2006

Nicotine induces brain CYP enzymes: relevance to Parkinson's disease.

Brain expression of cytochromes P450 2B6, 2D6 and 2E1 is higher in smokers, and is induced by nicotine in animals. These enzymes can metabolize many of the neurotoxins associated with Parkinson's disease. Since smoking is known to be protective against Parkinson's disease, we hypothesise that nicotine-induced elevation of brain CYPs in smokers may contribute to neuroprotection against Parkinson's disease. This supports the therapeutic use of nicotine to delay the progress of this disease.

Animals↗

PubMed2006

Impact of CYP2A6 genotype on pretreatment smoking behaviour and nicotine levels from and usage of nicotine replacement therapy.

We investigated the effect of slow metabolism of nicotine, predicted by CYP2A6 genotypes resulting in less than or equal to 50% activity, on baseline smoking behaviours and treatment variables in an open-label nicotine replacement therapy (NRT) clinical trial. Caucasian smokers with CYP2A6 slow vs normal metabolism had lower metabolic activity, indicated by the 3-hydroxycotinine/cotinine ratio (0.23+/-0.17 vs 0.45+/-0.22, P<0.01, respectively). CYP2A6 slow metabolizers also smoked fewer cigarettes per day compared to normal metabolizers (20+/-7 vs 24+/-10, respectively, P<0.04). With nicotine patch use, slow metabolizers had higher nicotine plasma levels compared to normal metabolizers (22.8+/-4.6 vs 15.8+/-7.6 ng/ml, respectively, P=0.02) while using the same numbers of patches/week. With nicotine spray use, where like in smoking the nicotine intake can be easily adjusted to adapt to rates of metabolism, slow metabolizers achieved similar nicotine levels compared to normal metabolizers (5.8+/-4.1 vs 8.0+/-9.1 ng/ml, P=0.82), by using fewer doses of nicotine spray/day (4.8+/-3.6 vs 10.5+/-8.0, respectively, P<0.02). These findings indicate that CYP2A6 genotype influences smoking behaviour in a Caucasian treatment-seeking population and that CYP2A6 genotype affects plasma levels obtained from, and usage of, NRT.

Administration, Cutaneous↗

PubMed2006

Risk factors for tobacco dependence in adolescent smokers.

OBJECTIVE: To study the incidence of conversion to tobacco dependence (TD) and the prevalence of the TD state in relation to several potential determinants in a sample of adolescent smokers. METHODS: Questionnaires were administered every 3-4 months to document TD symptoms, amount of cigarette consumption, and depression symptoms in a prospective cohort of 1293 grade 7 students in a convenience sample of 10 schools. RESULTS: Over 54 months of follow-up, 113 of 344 novice smokers converted to TD. The referent series for the analysis of incidence comprised 823 person-surveys. The prevalence series included 1673 person-surveys, contributed by 429 smokers. Conversion to TD and TD status were associated with the intensity of recent (that is, past 3-month) cigarette consumption (adjusted incidence rate ratio (aIRR) 1.63 (95% confidence interval (CI) 1.36 to 1.97) and adjusted prevalence odds ratio (aPOR) 1.35 (95% CI 1.23 to 2.48) per 100 cigarettes per month), slowest CYP2A6 activity (aIRR 4.19 (95% CI 1.38 to 12.76) and aPOR 2.30 (95% CI 1.29 to 4.09)), depression score (aIRR 1.61 (95% CI 1.17 to 2.21) and aPOR 1.47 (95% CI 1.22, 1.75) per 1-unit change). Additional determinants included, for conversion to TD, time since onset of cigarette use (aIRR 0.76 (95% CI 0.58 to 1.00) per year) and, for the TD state, positive TD status six months ago (aPOR 3.53 (95% CI 2.41 to 5.19)). CONCLUSIONS: TD risk in adolescents is associated with intensity of recent cigarette consumption, while the role of more distant cigarette consumption appears small; subjects with slow nicotine metabolism and those with more depression symptoms are at increased risk of becoming tobacco dependent. The risk of being tobacco dependent is considerably higher in subjects who had previously developed the TD state.

Adolescent↗

PubMed2004

Genetically decreased CYP2A6 and the risk of tobacco dependence: a prospective study of novice smokers.

OBJECTIVE: Case control studies in adults suggest that defective alleles in the gene that codes for the hepatic cytochrome P450 2A6 (CYP2A6) protect against nicotine dependence (ND) and higher levels of cigarette consumption. These two hypotheses were tested in young adolescents. DESIGN: Self reports of tobacco use and ND symptoms were collected every 3-4 months in a prospective study of 1293 grade 7 students from a convenience sample of 10 schools. SUBJECTS: 281 smokers with genetic data were analysed; those who were not already tobacco dependent and who had inhaled (n = 228) were followed 29.9 months on average, until they became dependent or were censored. MAIN OUTCOME MEASURES: The association between metabolic activity, represented by CYP2A6 genotype, and conversion to dependence was analysed using Cox's proportional hazards model. RESULTS: During follow up 67 subjects (29.4%) became dependent. Relative to CYP2A6*1/*1, having 1-2 copies of the inactive CYP2A6*2 or *4 variant was a strong risk factor for developing dependence (hazard ratio 2.8, 95% confidence 1.3 to 6.3). Subjects with 1-2 partially inactive CYP2A6*9 or *12 variants were not at increased risk. Mean past-week cigarette consumption at the end of follow up (controlling for age, sex, and number of months since first inhalation) among dependent subjects was 29.1 among normal inactivators, compared to 17.2, and 12.7 among slower (1-2 copies of *9 or *12), and slowest (1-2 copies of *2 or *4) inactivators, respectively (p = 0.09). CONCLUSION: Adolescents with 1-2 copies of CYP2A6*2 or *4 are at substantially increased risk of becoming dependent but smoke less once dependent. Genetic risk for ND may need to be considered in the conceptualisation of tobacco control programmes for adolescents.

Adolescent↗

PubMed2003

The role of cytochrome P450 2C19 activity in flunitrazepam metabolism in vivo.

Flunitrazepam, a hypnotic benzodiazepine, is widely prescribed around the world for the treatment of insomnia and as a preanesthetic. In vitro studies have shown that the metabolism of flunitrazepam to desmethylflunitrazepam and 3-hydroxyflunitrazepam is mediated in part by the polymorphic enzyme CYP2C19. The objective was to examine the role of CYP2C19 activity in determining flunitrazepam kinetics in vivo. Sixteen healthy volunteers (14 genotypic extensive metabolizers and 2 poor metabolizers) were recruited who had a wide range of CYP2C19 activity (0.50-28.8), as determined by the omeprazole/ 5-hydroxyomeprazole ratio (OMR) at 3 hours following administration of omeprazole, 20 mg orally. Each subject received flunitrazepam, 1 mg orally. Blood samples were collected immediately before and up to 48 hours after drug administration and were assayed by HPLC for flunitrazepam and its metabolites, 7-aminoflunitrazepam, desmethylflunitrazepam, and 3-hydroxyflunitrazepam. Spearman correlations were determined for OMR and pharmacokinetic parameters. With increasing OMR (decreasing CYP2C19 activity), the ratio of flunitrazepam to both desmethylflunitrazepam and 3-hydroxyflunitrazepam AUCs increased ( r = 0.55, p = 0.03 and r = 0.65, p = 0.01, respectively). However, variation in CYP2C19 activity did not significantly affect the AUCs of flunitrazepam or its metabolites. The authors conclude that CYP2C19 contributes to the metabolism of flunitrazepam to desmethylflunitrazepam and 3-hydroxyflunitrazepam in vivo, but these data suggest that its role is minor and that differences in CYP2C19 activity do not likely substantially influence its clinical effects.

Adolescent↗

PubMed2002

An in vivo pilot study characterizing the new CYP2A67, 8, and *10 alleles.

We developed genotyping assays for CYP2A6*7 (Ile471Thr) and CYP2A6*8 (Arg485Leu). We found higher allelic frequencies in Japanese and Chinese versus Caucasians and identified an allele in which both substitutions occur together (CYP2A6*10). We created a homology model for predicting the impact of allelic variants on enzymatic activity and subsequently tested this in vivo in a pilot kinetic study. Consistent with our homology model predictions, we found (i) that CYP2A6*7 produces an enzyme that has decreased (not inactive) activity for metabolizing nicotine and coumarin; (ii) that CYP2A6*8 is unlikely to affect catalytic activity in vivo; and (iii) that having both substitutions together on an allele (CYP2A6*10) dramatically reduces function and may be fully inactive for some substrates. In conclusion, this study identifies, at relatively high frequency in Asians, an allele with decreased activity (may be substrate selective), a fully functional allele, and an allele containing both substitutions in which function is dramatically reduced.

Alleles↗

PubMed2002

Assessment of nicotine dependence symptoms in adolescents: a comparison of five indicators.

OBJECTIVE: To examine the psychometric properties, test-retest reliability, and convergent construct validity of five indicators of nicotine dependence (ND) symptoms in adolescents. DESIGN: Analysis of baseline data from a prospective study on the natural history of ND in 1264 adolescents aged 12-13 years. SETTING: Ten Montreal high schools. SUBJECTS: 233 grade 7 students who had smoked cigarettes one or more times in the three months preceding the baseline data collection. MAIN OUTCOME MEASURES: Five indicators of ND symptoms including two that are multi-dimensional (a proxy measure of ICD-10 criteria for tobacco dependence; the Hooked on Nicotine Checklist (HONC)) and three new indicators of "symptom clusters" that emerged from principal component analysis (ND/cravings, withdrawal symptoms, self medication). RESULTS: All five indicators demonstrated acceptable internal and test-retest reliability. The correlation between the HONC and ND/cravings was 0.910. All other correlations between indicators ranged between 0.716-0.824. There was considerable overlap in the independent correlates identified for each indicator. CONCLUSIONS: All five indicators performed well psychometrically. Until the meaning, relative importance, and usefulness of each scale is clarified in longitudinal work, decisions regarding which scale(s) are most informative will depend more on the content of the scales, the need for a multi- or unidimensional indicator, and whether or not the scale is theory based.

Adolescent↗

PubMed2001

Induction of CYP2B1/2 and nicotine metabolism by ethanol in rat liver but not rat brain.

A higher proportion of alcoholics than non-alcoholics smoke (>80 vs 30%). In animals, chronic administration of alcohol induces tolerance to some effects of nicotine. To investigate if chronic ethanol (EtOH) induces alterations in CYP2B1/2 and nicotine C-oxidation activity, male rats (N = 4-6/group) were treated once daily with saline or EtOH (0.3, 1.0, and 3.0 g/kg, p.o./by gavage) for 7 days. A quantitative immunoblotting assay was developed to detect CYP2B1/2 in the brain, where constitutive expression is low, and in the liver. Using this method, it was determined that EtOH did not alter CYP2B1/2 protein expression significantly in six brain regions (olfactory bulbs, olfactory tubercles, frontal cortex, hippocampus, cerebellum, and brainstem). However, a dose-dependent induction of CYP2B1/2 protein expression was detected in the liver. Significant induction of 2-, 3-, and 2.7-fold were observed for the 0.3, 1.0, and 3.0 g/kg doses, respectively. Increases were also observed in CYP2B1 mRNA, which was induced by 14, 38, and 43% at the same doses. Liver microsomal nicotine C-oxidation also was increased (1.3 to 4.5-fold). CYP2B selective inactivators demonstrated that approximately 70% of nicotine C-oxidation was mediated by CYP2B1/2 in both EtOH-induced and uninduced hepatic microsomes. In summary, chronic, behaviorally relevant doses of EtOH induce CYP2B1/2 protein, mRNA, and nicotine C-oxidation activity in rat liver but not in rat brain, and these increases could contribute to cross-tolerance and co-abuse of ethanol and nicotine.

Alcoholism↗

PubMed2001

Ethnic variability in the allelic distribution of human aryl hydrocarbon receptor codon 554 and assessment of variant receptor function in vitro.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcriptional regulator of several genes including the cytochrome P4501 (CYP1) family as well as genes encoding factors involved in cell growth and differentiation. In mice, several polymorphic forms of the AHR are known, some of which have altered affinity for toxic and carcinogenic ligands. Remarkably little genetic variation has been detected in the human AHR gene. In studies on human AHR, Kawajiri et al. (Pharmacogenetics 1995; 5:151-158) reported a variation at codon 554 that results in an amino acid change from arginine to lysine; the frequency of the variant allele in a Japanese population (n = 277) was 0.43. We investigated the Lys554 allele in 386 individuals of various ethnic origins and found the frequency to be: 0.58 in Ivory Coast Africans (n = 58); 0.53 in a mixed African group (n = 20); 0.39 in Caribbean-Africans (n = 55); 0.32 in Canadian Chinese (n = 41); 0.14 in North American Indians (n = 47); 0.12 in French Canadian Caucasians (n = 20); 0.11 in a mixed ethnicity North American group (n = 45); 0.09 in Canadian Inuits (n = 22); and 0.07 in German Caucasians (n = 78). We expressed the human Lys554 allele in an in-vitro transcription-translation system and found that the receptor bearing the R554L substitution had an equivalent ability to that of the wild-type receptor to bind to a dioxin-responsive element following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The Lys554 allele also was equivalent to the wild-type receptor at stimulating CYP1A1 mRNA expression when transfected into TCDD-treated receptor-deficient mouse Hepa-1 cells. It is not yet known if any of the wide variations in allele frequency at codon 554 are related to ethnic differences in susceptibility to adverse effects of environmental chemicals.

Alleles↗

PubMed2001

Cytochrome P450 2D6.1 and cytochrome P450 2D6.10 differ in catalytic activity for multiple substrates.

CYP2D6 is involved in the metabolism of several classes of drugs, including tricyclic antidepressants, selective serotonin reuptake inhibitors and various amphetamines. CYP2D6*10 is an allelic variant, producing an enzyme with Pro34Ser and Ser486Thr amino acid substitutions. Approximately 75% of Asians possess the *10 allele. We sought to further characterize CYP2D6.10 catalytically in vitro in a baculovirus expression system using various substrates and inhibitors, in comparison to CYP2D6.1 (wild-type). Using dextromethorphan (DEX), P-methoxyamphetamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and (+/-)3,4-methylenedioxymethamphetamine (MDMA), the ratios of intrinsic clearance (Vmax/Km) of *1 to *10 were 50, 34, 22 and 123, respectively. The CYP2D6 substrates amitriptyline, and (+) and (-) methamphetamine (MAMP) are both p-hydroxylated and N-demethylated (NDM). The intrinsic clearance *1/*10 ratios were 42, 30 and 67 for the p-hydroxylation; and 60, 120 and 157 for the NDM, respectively, illustrating chemical pathway and enantiomeric selectivity for MAMP. It was apparent that (+) and (-) MAMP NDM and MDMA demethylenation were most significantly different in CYP2D6.10. Using DEX as the substrate, the ratios of Ki(*10)/Ki(*1) for inhibitors were: budipine (1.3), sparteine (1.6), debrisoquine (8.1), fluoxetine (16), norfluoxetine (30), paroxetine (14), MDMA (21) and MMDA-2 (7.1), indicating that CYP2D6.10 shows drug-specific altered susceptibility to inhibition. Taken together, these data suggest that CYP2D6*10/*10 individuals may be expected to require different drug doses; and show altered susceptibility to toxicity, interaction risk and, in the case of the amphetamines, drug dependence and toxicity compared to CYP2D6*1/*1 individuals.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2001

Flunitrazepam metabolism by cytochrome P450S 2C19 and 3A4.

We have identified CYP2C19 and CYP3A4 as the principal cytochrome P450s involved in the metabolism of flunitrazepam to its major metabolites desmethylflunitrazepam and 3-hydroxyflunitrazepam. Human CYP2C19 and CYP3A4 mediated the formation of desmethylflunitrazepam with Km values of 11.1 and 108 microM, respectively, and 3-hydroxyflunitrazepam with Km values of 642 and 34.0 microM, respectively. In human liver microsomes (n = 4) formation of both metabolites followed biphasic kinetics. Desmethylflunitrazepam formation was inhibited 31% by S-mephenytoin and 78% by ketoconazole, suggesting involvement of both CYP2C19 and CYP3A4. Formation of 3-hydroxyflunitrazepam was also significantly inhibited by ketoconazole (94%) and S-mephenytoin (18%). In support of these chemical inhibition data, antibodies directed against CYP2C19 and CYP3A4 selectively inhibited formation of desmethylflunitrazepam by 26 and 45%, respectively, while anti-CYP3A4 antibodies reduced 3-hydroxyflunitrazepam formation by 80%. Our data also suggest that CYP1A2, -2B6, -2C8, -2C9, -2D6, and -2E1 are not involved in either of these metabolic pathways. We estimate that the relative contributions of CYP2C19 and CYP3A4 to the formation of desmethylflunitrazepam in vivo are 63 and 37%, respectively, at therapeutic flunitrazepam concentrations (0.03 microM). We conclude that the polymorphic enzyme CYP2C19 importantly mediates flunitrazepam demethylation, which may alter the efficacy and safety of the drug, while CYP3A4 catalyzes the formation of 3-hydroxyflunitrazepam.

Anti-Anxiety Agents↗

PubMed2001

Variable CYP2A6-mediated nicotine metabolism alters smoking behavior and risk.

Nicotine is the psychoactive substance responsible for tobacco dependence; smokers adjust their cigarette consumption to maintain brain nicotine levels. In humans, 70 to 80% of nicotine is metabolized to the inactive metabolite cotinine by the enzyme CYP2A6. CYP2A6 can also activate tobacco smoke procarcinogens [e.g., NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]. In initial studies we found that there was an under-representation of individuals carrying defective CYP2A6 alleles in a tobacco-dependent population, and that among smokers, those with deficient nicotine metabolism smoked fewer cigarettes. We have since reproduced this data in a prospective smoking study (400 male and female, heavy and light smokers) examining the role of the CYP2A6 genotype on carbon monoxide levels, plasma and urine nicotine and cotinine levels, and cigarette counts. We have also recently identified deletion and duplication variants in the CYP2A6 gene locus and have examined their impact on smoking. These data provide the impetus to examine how inhibition of CYP2A6 activity might be useful in a therapeutic context. Both kinetic and behavioral experiments in human smokers demonstrated that inhibiting CYP2A6 in vivo decreased nicotine metabolism and smoking behavior. This article summarizes the preliminary results from our studies.

Aryl Hydrocarbon Hydroxylases↗

PubMed2001

Evaluation of methoxsalen, tranylcypromine, and tryptamine as specific and selective CYP2A6 inhibitors in vitro.

CYP2A6 is the principle enzyme metabolizing nicotine to its inactive metabolite cotinine. In this study, the selective probe reactions for each major cytochrome P450 (P450) were used to evaluate the specificity and selectivity of the CYP2A6 inhibitors methoxsalen, tranylcypromine, and tryptamine in cDNA-expressing and human liver microsomes. Phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), diclofenac 4'-hydroxylation (CYP2C9), omeprazole 5-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), 7-ethoxy-4-trifluoromethylcoumarin deethylation (CYP2B6), p-nitrophenol hydroxylation (CYP2E1), and omeprazole sulfonation (CYP3A4) were used as index reactions. Apparent K(i) values for inhibition of P450s' (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) activities showed that tranylcypromine, methoxsalen, and tryptamine have high specificity and relative selectivity for CYP2A6. In cDNA-expressing microsomes, tranylcypromine inhibited CYP2A6 (K(i) = 0.08 microM) with about 60- to 5000-fold greater potency relative to other P450s. Methoxsalen inhibited CYP2A6 (K(i) = 0.8 microM) with about 3.5- 94-fold greater potency than other P450s, except for CYP1A2 (K(i) = 0.2 microM). Tryptamine inhibited CYP2A6 (K(i) = 1.7 microM) with about 6.5- 213-fold greater potency relative to other P450s, except for CYP1A2 (K(i) = 1.7 microM). Similar results were also obtained with methoxsalen and tranylcypromine in human liver microsomes. R-(+)-Tranylcypromine, (+/-)-tranylcypromine, and S-(-)-tranylcypromine competitively inhibited CYP2A6-mediated metabolism of nicotine with apparent K(i) values of 0.05, 0.08, and 2.0 microM, respectively. Tranylcypromine [particularly R-(+) isomer], tryptamine, and methoxsalen are specific and relatively selective for CYP2A6 and may be useful in vivo to decrease smoking by inhibiting nicotine metabolism with a low risk of metabolic drug interactions.

Aryl Hydrocarbon Hydroxylases↗

PubMed2001

Altered gabaa receptor subunit and splice variant expression in rats treated with chronic intermittent ethanol.

BACKGROUND: Intermittent chronic administration of ethanol to rats has been shown previously to produce a hyperexcitable, kindling-like state, accompanied by reduced inhibitory synaptic transmission in the hippocampus and changes in gamma-aminobutyric acid type A (GABAA) receptors. Further information is needed on the detailed changes in GABAA receptors and their time course and persistence, as is comparison to changes after chronic, continuous ethanol. METHODS: GABAA receptors were analyzed in the rat brain after chronic intermittent ethanol (CIE) by using radioligand binding, photoaffinity labeling of polypeptides, and estimates of messenger RNA (mRNA) levels of receptor subunits by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. RESULTS: CIE rats were confirmed to have increased GABAA receptor binding of the benzodiazepine partial inverse agonist and ethanol antidote ligand Ro15-4513, due to increased expression of the alpha6 subunit polypeptide in the cerebellum, shown by photoaffinity labeling. Estimates of mRNA levels by use of RT-PCR did not reveal any significant increase in alpha6 or in several other receptor subunits in several brain regions, but a decrease in the ratio of the long and short splice variants (L/S) of the gamma2 subunit was detected in the hippocampus, especially the CA1 region. CONCLUSIONS: Changes in GABAA receptors were found in rats given CIE. Increased alpha6 subunit in the cerebellum was demonstrated by using both the binding to diazepam-insensitive sites for [3H]Ro15-4513 and increased levels of the 57-kDa alpha6 polypeptide after photoaffinity labeling with this ligand. This increase appeared after 30 doses of ethanol and decayed to normal 1 week after ethanol was discontinued. The transient change in cerebellar alpha6 subunit-containing receptors, also reportedly seen after chronic continuous ethanol, is thus unlikely to account for the persistently hyperexcitable, kindled, seizure-susceptible state seen in CIE. However, the significant decrease in gamma2 subunit L/S splice variant ratio in the hippocampus implies changes in GABAA receptor function, possibly involving protein phosphorylation by protein kinase C. Altered receptor trafficking and turnover associated with synaptic plasticity may contribute to the observed reduced inhibition in the hippocampus and other signs of alcohol dependence produced by CIE.

Alcoholism↗

PubMed2001

Low doses of nicotine and ethanol induce CYP2E1 and chlorzoxazone metabolism in rat liver.

The use of ethanol and nicotine is strongly linked; 80 to 95% of heavy alcohol users are also smokers. In humans, cigarette smoking significantly enhances CYP2E1 activity, as measured by increased metabolism of chlorzoxazone in vivo. CYP2E1 metabolizes ethanol and can generate toxic intermediates. CYP2E1 also bioactivates tobacco smoke and other procarcinogens and several hepatotoxins. We hypothesized that, like ethanol, nicotine increases CYP2E1 activity. Rats were treated once daily with saline, ethanol (0.3, 1.0, and 3.0 g/kg p.o.), or nicotine bitartrate (0.1, 0.3, and 1.0 mg base/kg s.c.) for 7 days. After ethanol or nicotine administration, immunostaining for CYP2E1 was increased in the centrilobular regions of rat liver. Western blot analyses revealed that hepatic CYP2E1 levels were increased by ethanol (1.6-2.4-fold) and nicotine (1.3-1.7-fold). In vitro chlorzoxazone 6-hydroxylation analyses demonstrated elevated Vmax values (compared with saline-treated animals) by using hepatic microsomes from high-dose ethanol (2.27 +/- 0.12 versus 1.18 +/- 0.23 nmol/mg/min, p < 0.001) or nicotine-treated rats (2.35 +/- 0.04 versus 1.32 +/- 0.55 nmol/mg/min, p < 0.005), with no change in affinity. The magnitude of enhanced chlorzoxazone metabolism by microsomes from drug-treated animals is consistent with the observed increase in CYP2E1 protein by immunoblot. These data suggest that nicotine may increase CYP2E1-induced toxicity and contribute to cross-tolerance in smokers and people treated with nicotine (e.g., smokers, patients with Alzheimer's disease, ulcerative colitis, neuropsychiatric motor disorders).

Animals↗

PubMed2001

Cytochrome P4502C9 (CYP2C9) allele frequencies in Canadian Native Indian and Inuit populations.

CYP2C9 is the major P450 2C enzyme in human liver and contributes to the metabolism of a number of clinically important substrate drugs. This polymorphically expressed enzyme has been studied in Caucasian, Asian, and to some extent in African American populations, but little is known about the genetic variation in Native American populations. We therefore determined the 2C9*2 (Arg144Cys) and 2C9*3 (Ile359Leu) allele frequencies in 153 Native Canadian Indian (CNI) and 151 Inuit subjects by PCR-RFLP techniques. We also present genotyping data for two reference populations, 325 Caucasian (white North American) and 102 Chinese subjects. Genotyping analysis did not reveal any 2C9*4 alleles in the CNI, Inuit, Caucasian, or Chinese individuals. The 2C9*2 allele appears to be absent in Chinese and Inuit populations, but was present in CNI and Caucasian subjects at frequencies of 0.03 and 0.08-0.15, respectively. The 2C9*3 allele was not detected in the Inuit group, but occured in the CNI group (f = 0.06) at a frequency comparable to that of other ethnic groups. This group of Inuit individuals are the first population in which no 2C9*2 or *3 alleles have been detected so far. Therefore, these alleles may be extremely rare or absent, and unless other novel polymorphisms exist in this Inuit group one would not anticipate any CYP2C9 poor metabolizer subjects among this population.

Alleles↗