Selective inhibitory effects of suberosin on CYP1A2 in human liver microsomes.

Suberosin is a natural phytoconstituent isolated from Citropsis articulata, especially employed for its anticoagulant properties. Although metabolic studies assessing suberosin have been conducted, it is possible interactions with drugs and food have not yet been investigated. In the present study, we analyzed the selective inhibitory effects of suberosin on cytochrome P450 (CYP) enzymes using a cocktail probe assay. Various concentrations of suberosin (0-50 µM) were incubated with isoform-specific CYP probes in human liver microsomes (HLMs). We found that suberosin significantly inhibited CYP1A2-catalyzed phenacetin O-deethylation, exhibiting IC50 values of 9.39 ± 2.05 and 3.07 ± 0.45 µM with and without preincubation in the presence of ß-NADPH, respectively. Moreover, suberosin showed concentration-dependent, but not time-dependent, CYP1A2 inhibition in HLMs, indicating that suberosin acts as a substrate and reversible CYP1A2 inhibitor. Using a Lineweaver-Burk plot, we found that suberosin competitively inhibited CYP1A2-catalyzed phenacetin O-deethylation. Furthermore, suberosin showed similar inhibitory effects on recombinant human CYP1A1 and 1A2. In conclusion, suberosin may elicit herb-drug interactions by selectively inhibiting CYP1A2 during the concurrent administration of drugs that act as CYP1A2 substrates.

Yeast polysaccharide mitigated oxidative injury in broilers induced by mixed mycotoxins via regulating intestinal mucosal oxidative stress and hepatic metabolic enzymes.

This study was aimed to investigate the effects of yeast polysaccharides (YPS) on growth performance, intestinal health, and aflatoxin metabolism in livers of broilers fed diets naturally contaminated with mixed mycotoxins (MYCO). A total of 480 one-day-old Arbor Acre male broilers were randomly allocated into a 2 × 3 factorial arrangement of treatments (8 replicates with 10 birds per replicate) for 6 wk to assess the effects of 3 levels of YPS (0, 1, or 2 g/kg) on the broilers fed diets contaminated with or without MYCO (95 µg/kg aflatoxin B1, 1.5 mg/kg deoxynivalenol, and 490 µg/kg zearalenone). Results showed that mycotoxins contaminated diets led to significant increments in serum malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels, mRNA expressions of TLR4 and 4EBP1 associated with oxidative stress, mRNA expressions of CYP1A1, CYP1A2, CYP2A6, and CYP3A4 associated with hepatic phase Ⅰ metabolizing enzymes, mRNA expressions of p53 associated with hepatic mitochondrial apoptosis, and AFB1 residues in the liver (P < 0.05); meanwhile dietary MYCO decreased the jejunal villus height (VH), villus height/crypt depth (VH/CD), the activity of serum total antioxidant capacity (T-AOC), mRNA expressions of jejunal HIF-1α, HMOX, and XDH associated with oxidative stress, mRNA expressions of jejunal CLDN1, ZO1, and ZO2, and mRNA expression of GST associated with hepatic phase Ⅱ metabolizing enzymes of broilers (P < 0.05). Notably, the adverse effects induced by MYCO on broilers were mitigated by supplementation with YPS. Dietary YPS supplementation reduced the concentrations of serum MDA and 8-OHdG, jejunal CD, mRNA expression of jejunal TLR2, and 4EBP1, hepatic CYP1A2, and p53, and the AFB1 residues in the liver (P < 0.05), and elevated the serum T-AOC and SOD, jejunal VH, and VH/CD, and mRNA expression of jejunal XDH, hepatic GST of broilers (P < 0.05). There were significant interactions between MYCO and YPS levels on the growth performance (BW, ADFI, ADG, and F/G) at d 1 to 21, d 22 to 42, and d 1 to 42, serum GSH-Px activity, and mRNA expression of jejunal CLDN2 and hepatic ras of broilers (P < 0.05). In contrast with MYCO group, the addition of YPS increased BW, ADFI, and ADG, the serum GSH-Px activity (14.31%-46.92%), mRNA levels of jejunal CLDN2 (94.39%-103.02%), decreased F/G, and mRNA levels of hepatic ras (57.83%-63.62%) of broilers (P < 0.05). In conclusion, dietary supplements with YPS protected broilers from mixed mycotoxins toxicities meanwhile keeping normal performance of broilers, presumably via reducing intestinal oxidative stress, protecting intestinal structural integrity, and improving hepatic metabolic enzymes to minimize the AFB1 residue in the liver and enhance the performance of broilers.

The alteration of drug metabolism enzymes and pharmacokinetic parameters in nonalcoholic fatty liver disease: current animal models and clinical practice.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease. The whole concept of NAFLD has now moved into metabolic dysfunction-associated fatty liver disease (MAFLD) to emphasize the strong metabolic derangement as the basis of the disease. Several studies have suggested that hepatic gene expression was altered in NAFLD and NAFLD-related metabolic comorbidities, particularly mRNA and protein expression of phase I and II drug metabolism enzymes (DMEs). NAFLD may affect the pharmacokinetic parameters. However, there were a limited number of pharmacokinetic studies on NAFLD at present. Determining the pharmacokinetic variation in patients with NAFLD remains challenging. Common modalities for modeling NAFLD included: dietary induction, chemical induction, or genetic models. The altered expression of DMEs has been found in rodent and human samples with NAFLD and NAFLD-related metabolic comorbidities. We summarized the pharmacokinetic changes of clozapine (CYP1A2 substrate), caffeine (CYP1A2 substrate), omeprazole (Cyp2c29/CYP2C19 substrate), chlorzoxazone (CYP2E1 substrate), midazolam (Cyp3a11/CYP3A4 substrate) in NAFLD. These results led us to wonder whether current drug dosage recommendations may need to be reevaluated. More objective and rigorous studies are required to confirm these pharmacokinetic changes. We have also summarized the substrates of the DMEs aforementioned. In conclusion, DMEs play an important role in the metabolism of drugs. We hope that future investigations should focus on the effect and alteration of DMEs and pharmacokinetic parameters in this special patient population with NAFLD.

Investigation of doxorubicin combined with ciprofloxacin-induced cardiotoxicity: from molecular mechanism to fundamental heart function.

This study was designed to investigate the impacts of Doxo alone and in combination with Cipro on the hepatic and cardiac CYP1A2, CYP2J3, and CYP3A1 mRNA levels. We also aimed to analyze the cardiac function by perfusing isolated rat hearts. Rats were given Doxo and/or Cipro in chronic (3-week) and acute (single-day) dosing schedules. Cardiac CYP2J3, CYP3A1, and CYP1A2 gene expression levels were measured by quantitative reverse transcription PCR. Cardiac functions of the isolated hearts were evaluated by using the Langendorff technique. Doxo alone (2.5 mg/kg) and Doxo + Cipro (2.5 mg + 20 mg/kg) significantly decreased hepatic CYP1A2 expression compared to saline, whereas Doxo (2.5 mg/kg) and Doxo + Cipro (2.5 mg + 20 mg/kg) showed significantly higher cardiac CYP1A2 expression in comparison to control. In the liver tissue, Doxo (2.5 mg/kg) and Doxo + Cipro (2.5 + 20 mg/kg) decreased the CYP2J3 expression than the control group. The Doxo (2.5 mg/kg) and Doxo + Cipro (2.5 + 20 mg/kg)-treated group had significantly higher cardiac CYP2J3 expression compared to control. Doxo (2.5 mg/kg; cumulative dose 15 mg/kg) and Doxo + Cipro (2.5 + 20 mg/kg) showed significantly higher cardiac CYP3A1 expressions than the control. Rate-pressure product (HR × LVDP)/1000) showed an overall decrease in cardiac functions of Doxo (2.5 mg/kg) and Doxo + Cipro (2.5 + 20 mg/kg)-treated group. We found considerable effects in chronic protocol; Doxo alone high dose and plus Cipro decreased hepatic CYP1A2 and CYP2J3 mRNA. On the other hand, these treatment groups exhibited an increase in the cardiac CYP1A2, CYP2J3, and CYP3A1 expression and likewise deteriorated the overall hemodynamic parameters.

[Smoking cessation and drug interactions].

Tobacco smoke can cause drug interactions by induction of CYP1A2, which metabolizes drugs like clozapine, olanzapine and theophylline. This means that smokers need higher doses to achieve the same plasma concentrations as non-smokers. Furthermore, smoking cessation can cause an increase in plasma concentrations of drugs metabolised by CYP1A2, which in turn may lead to adverse effects. Of the drugs used for smoking cessation only bupropione has clinically relevant interactions. All of these situations may be handled by dose adjustment.

Human liver microsomes study on the inhibitory effect of plantainoside D on the activity of cytochrome P450 activity.

BACKGROUND: Plantainoside D is widely existed in the herbs and possesses various pharmacological activities, making it possible to co-administrate with other herbs. Its effect on cytochrome P450 enzymes (P450) is a risk factor for inducing adverse drug-drug interactions. To assess the effect of plantainoside D on the activity of major P450 isoenzymes in human liver microsomes. METHODS: The Cocktail method was conducted in human liver microsomes in the presence of probe substrates. The activity of P450 isoenzymes was evaluated by the production of corresponding metabolites. The concentration-dependent and time-dependent inhibition assays were performed in the presence of 0, 2.5, 5, 10, 25, 50, and 100 µM plantainoside D to characterize the inhibitory effect of plantainoside D. RESULTS: Significant inhibition was observed in the activity of CYP1A2, 2D6, and 3A, which was concentration-dependent with the IC50 values of 12.83, 8.39, and 14.66 µM, respectively. The non-competitive manner and competitive manner were observed in the CYP3A inhibition (Ki = 7.16 µM) and CYP1A2 (Ki = 6.26 µM) and 2D6 inhibition (Ki = 4.54 µM), respectively. Additionally, the inhibition of CYP3A was found to be time-dependent with the KI of 1.28 µM-1 and Kinact of 0.039 min-1. CONCLUSIONS: Weak inhibitory effects of plantainoside D on the activity of CYP1A2, 2D6, and 3A were revealed in vitro, implying its potential of inducing interactions with CYP1A2-, 2D6-, and 3A-metabolized drugs. Although further in vivo validations are needed, the feasibility of the Cocktail method in evaluating P450 activity has been verified.

CYP1A2 polymorphisms modify the association of habitual coffee consumption with appetite, macronutrient intake, and body mass index: results from an observational cohort and a cross-over randomized study.

BACKGROUND/OBJECTIVES: Evidence regarding the influence of coffee on appetite and weight control is equivocal and the influence of covariates, such as genetic variation in caffeine metabolism, remains unknown. Herein, we addressed the novel hypothesis that genetic variation in CYP1A2, a gene responsible for more than 95% of caffeine metabolism, differentially impacts the association of coffee consumption with appetite and BMI among individuals with different genetic predispositions to obesity. SUBJECTS/METHODS: A cross-over randomized intervention study involving 18 volunteers assessed the effects of coffee consumption on dietary intake, appetite, and levels of the appetite-controlling hormones asprosin and leptin. Data on habitual coffee intake, BMI, and perceived appetite were obtained from an observational cohort of 284 volunteers using validated questionnaires. Participants were stratified according to a validated genetic risk score (GRS) for obesity and to the -163C > A (rs762551) polymorphism of CYP1A2 as rapid (AA), intermediate (AC), or slow (CC) caffeine metabolizers. RESULTS: Coffee consumption led to lower energy and dietary fat intake and circulating asprosin levels (P for interaction of rs762551 genotype*coffee consumption=0.056, 0.039, and 0.043, respectively) as compared to slow/intermediate metabolizers. High coffee consumption was more prevalent in rapid compared to slow metabolizers (P = 0.008 after adjustment for age, sex, and BMI) and was associated with lower appetite perception and lower BMI only in rapid metabolizers (P for interaction of rs762551 genotype*coffee consumption = 0.002 and 0.048, respectively). This differential association of rs762551 genotype and coffee consumption with BMI was more evident in individuals at higher genetic risk of obesity (mean adjusted difference in BMI = -5.82 kg/m2 for rapid versus slow/intermediate metabolizers who consumed more than 14 cups of coffee per week). CONCLUSIONS: CYP1A2 rs762551 polymorphism modifies the association of habitual coffee consumption with BMI, in part by influencing appetite, energy intake and circulating levels of the orexigenic hormone asprosin. This association is more evident in subjects with high genetic predisposition to obesity. ClinicalTrials.gov: registered Clinical Trial NCT04514588.

[Multi-omics analysis of regulating effects of hyperoside on lipid metabolism in high-fat diet mice].

The aim of this study was to explore the regulating effects of hyperoside (Hyp) on lipid metabolism in high-fat diet mice. The high-fat diet mouse model was established by high-fat diet induction. After 5 weeks of Hyp intragastric administration in high-fat diet mice, the serum lipid levels before and after Hyp administration were measured by the corresponding kits. The tissue structure of mouse liver was observed by HE staining before and after Hyp administration. The changes of intestinal flora and transcriptome were measured by Illumina platforms. Liquid chromatography-mass spectrometry (LC-MS) was used to determine non-targeted metabolites. The results showed that Hyp significantly reduced lipid levels in the high-fat diet mice and effectively restored the external morphology and internal structure of liver tissue. Hyp changed the species composition of the intestinal flora in high-fat diet mice, increased the abundance of beneficial flora such as Ruminococcus, and decreased the abundance of harmful flora such as Sutterella. Combined multi-omics analysis revealed that the effect of retinoic acid on lipid metabolism was significant in the high-fat diet mice treated with Hyp, while the increase of retinoic acid content was significantly negatively correlated with the expression of genes such as cyp1a2 and ugt1a6b, positively correlated with AF12 abundance, and significantly negatively correlated with unidentified_Desulfovibrionaceae abundance. These results suggest that Hyp may modulate the abundance of AF12, unidentified_Desulfovibrionaceae and inhibit the expression of genes such as cyp1a2 and ugt1a6b, thus increasing the content of retinoic acid and regulating lipid metabolism in the high-fat diet mice.

CYP1A2 and tobacco interaction: a major pharmacokinetic challenge during smoking cessation.

Smoking cessation is underestimated in terms of drug interactions. Abrupt smoking cessation is common in cases of emergency hospitalization and restrictions of movement. Tobacco is a known cytochrome P450 1A2 (CYP1A2) inducer, its consumption and withdrawal can lead to major pharmacokinetic drug interactions. Nevertheless, references do exist, but may have different results between them. The objective of our work was to establish the broadest and most consensual list as possible of CYP1A2 substrates treatments and propose a pharmacological approach. We searched the widest possible list of CYP1A2 substrates based on various international references. We compared the references and defined probability and reliability scores of our results to sort the substances based on the scores. For the 245 substances identified as CYP1A2 substrates, we focused on the 63 CYP1A2 substrates with both probability and reliability scores >50%. Our work establishes adaptive pharmacological approaches for the management of patients initiating smoking cessation which must be integrated into the management of smoking cessation. Pharmacologists can now adopt adaptive pharmacological approaches to complement patient-specific clinical information about smoking cessation by considering pharmacokinetic risk. This work establishes an unprecedented list. It should guide in the care of patients initiating smoking cessation to prevent pharmacokinetic drug interactions.

The responses of the growth, cytochrome P450 isoenzymes activities and the metabolomics in earthworms to sublethal doses of dichlorvos in soil.

In this paper, earthworms (Eisenia fetida) were exposed to sublethal doses of dichlorvos (spiked concentration of 0.1, 1.0, 10 mg/kg) in soil for 14 days, the metabolomics and activities of cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP2C9 and CYP3A4) of earthworms were analyzed aiming to identify sensitive biomarkers and reveal possible mode of toxic action. The results showed that CYP1A2 and CYP2C9 activity appeared to be more sensitive than CYP3A4 activity in response to dichlorvos, and that metabolic responses based on the metabolomics depended on both of the length of exposure and exposure dose. Malate, ornithine, glucose, inosine, myo-inositol and some amino acids (glutamine, tryptophan, phenylalanine, tyrosine, leucine, histidine, glutamate, lysine) and CYP isozenzymes may be biomarkers to reveal the toxic effect of dichlorvos on earthworms. Compared to controls, when dichlorvos dose reached 1.0 and 10 mg/kg on day 14, glucose and ornithine increased significantly, malate and some amino acids (glutamine, tryptophan, phenylalanine, tyrosine, leucine) decreased significantly, and activities of CYP1A2 and CYP2C9 were inhibited significantly. The current results suggested that 1.0 and 10 mg/kg dichlorvos for 14 days of exposure blocked energy metabolism, disordered Krebs cycle, interfered amino acids metabolism and evoked toxic effects on earthworms.

PCB exposure and in vivo CYP1A2 activity among Native Americans.

Cytochrome P-450 1A2 (CYP1A2) is an enzyme involved in the metabolic activation of some carcinogens and is believed to be induced by xenobiotics. Very few studies, however, have investigated the association between environmental exposures and in vivo CYP1A2 activity in humans. To address this issue, a study was conducted of CYP1A2 activity among Native Americans exposed to polychlorinated biphenyls (PCBs) from the consumption of fish from the St. Lawrence River. At the Mohawk Nation at Akwesasne (in New York and in Ontario and Quebec, Canada), 103 adults were interviewed, and they donated blood for serum PCB analysis and underwent the caffeine breath test (CBT), a safe and noninvasive procedure that uses caffeine as a probe for CYP1A2 activity in vivo. The results supported the findings of other studies that CBT values are higher among smokers and men and lower among women who use oral contraceptives. Despite a relatively low average total PCB body burden in this population, the sum of serum levels for nine mono- or di-ortho-substituted PCB congeners showed positive associations with CBT values (p = 0.052 wet weight and p = 0.029 lipid adjusted), as did toxic equivalent quantities (TEQs; p = 0.091 for wet weight and 0.048 for lipid adjusted). Regarding individual congeners, serum levels of PCB-153, PCB-170, and PCB-180 were significantly correlated with CBT values. The results support the notion that CYP1A2 activity may be a marker of an early biological effect of exposure to PCBs in humans and that the CBT may be a useful tool to monitor such effects.

Human hepatic and renal microsomes, cytochromes P450 1A1/2, NADPH:cytochrome P450 reductase and prostaglandin H synthase mediate the formation of aristolochic acid-DNA adducts found in patients with urothelial cancer.

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Understanding which human enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual's susceptibility to this plant carcinogen. Using the (32)P postlabeling assay, we examined the ability of microsomal samples from 8 human livers and from 1 human kidney to activate AAI, the major component of the plant extract AA, to metabolites forming adducts in DNA. Microsomes of both organs generated DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed from AAI by all human hepatic and renal microsomes. To define the role of human microsomal enzymes in the activation of AAI, we investigated the modulation of AAI-DNA adduct formation by cofactors and selective inhibitors of microsomal reductases, cytochrome P450 (CYP) enzymes, NADPH:CYP reductase and NADH:cytochrome b(5) reductase. We also determined whether the activities of CYP and NADPH:CYP reductase in different human hepatic microsomal samples correlated with the levels of AAI-DNA adducts formed by the same microsomal samples. On the basis of these studies, we attribute most of the activation of AAI in human hepatic microsomes to CYP1A2. In contrast to human hepatic microsomes, in human renal microsomes NADPH:CYP reductase is more effective in AAI activation. In addition, prostaglandin H synthase is another enzyme activating AAI in renal microsomes. The results demonstrate for the first time the potential of microsomal enzymes in human liver and kidney to activate AAI by nitroreduction.

Time response of cytochrome P450 1A2 activity on cessation of heavy smoking.

BACKGROUND AND OBJECTIVE: Cytochrome P450 (CYP) 1A2 activity is induced by cigarette smoking. Thus smoking cessation in patients while they are undergoing therapy with a CYP1A2 substrate such as theophylline or clozapine increases its concentrations and may cause adverse effects. Our objective was to determine the time course of CYP1A2 activity changes after smoking cessation in heavy smokers as the basis for dosing adaptation schemes. METHODS: The study was conducted in 8 men and 4 women (all white) who smoked 20 cigarettes or more per day. Sudden smoking cessation was carried out after a 14-day run-in period. Subjects were phenotyped for CYP1A2 activity at 6, 4, and 1 day before smoking cessation and at 0, 1, 2, 3, 6, 8, 10, and 13 days thereafter by use of the paraxanthine-to-caffeine ratio in plasma 6 hours after a 148-mg caffeine test dose. A monoexponential decay of CYP1A2 activity to a residual value was fitted to the data by nonlinear regression analysis. RESULTS: On cessation of smoking, initial caffeine clearance (estimated geometric means and 95% confidence intervals) decreased significantly (P <.01), by 36.1% (30.9%-42.2%), from 2.47 mL. min(-1). kg(-1) body weight (2.03-3.00 mL. min(-1). kg(-1) body weight) to a new steady state of 1.53 mL. min(-1). kg(-1) body weight (1.24-1.89 mL. min(-1). kg(-1) body weight). The apparent half-life of CYP1A2 activity decrease was 38.6 hours (27.4-54.4 hours). CONCLUSION: Doses of CYP1A2 substrates with a narrow therapeutic range should be decreased immediately on cessation of heavy smoking. As a rule of thumb, a stepwise daily dose reduction of approximately 10% until the fourth day after smoking cessation is proposed, which should be accompanied by therapeutic drug monitoring.

CYP1A2 and NAT2 genotype/phenotype relations and urinary excretion of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in a human dietary intervention study.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagenic and carcinogenic heterocyclic amine formed during ordinary cooking, and is subsequently metabolically activated by cytochrome P4501A2 (CYP1A2) and N-acetyltransferase 2 (NAT2). Respective genes encoding for these enzymes, display polymorphic distribution in the human population and are thus believed to cause interindividual differences in cancer risk susceptibility. The present study investigated the influence of dietary exposure and CYP1A2 and NAT2 genotypes and phenotypes on differential urinary PhIP excretion levels in 71 human volunteers after consumption of either a high (7.4 ng/g) or low (1.7 ng/g) dose of PhIP. Urinary PhIP excretion levels were found to reflect recent dietary exposure levels, with average levels of 174% (high dose group) and 127% (low dose group), as compared to pre-feed levels. Urinary caffeine metabolite ratios were significantly different between the two NAT2 genotypes, whereas for CYP1A2, the apparent difference in metabolic ratios between the genotypes was statistically non-significant. Significant correlations were firstly found between the CYP1A2-164A-->C (CYP1A2*1F) polymorphism and differential urinary PhIP excretion levels. Although the found correlations are driven primarily by a small number of subjects possessing the homozygous variant constellation, the strong influence of this genotype indicates that the CYP1A2*1F polymorphism could play an important role in human cancer risk susceptibility.

Inhibition of CYP 1A2-dependent MROD activity in rat liver microsomes: an explanation of the hepatic sequestration of a limited subset of halogenated aromatic hydrocarbons.

Many classes of halogenated aromatic compounds (HACs) are highly lipophilic environmental contaminants that exert toxic effects via the Ah receptor signal transduction pathway and whose metabolism generally involves monooxygenase enzymes of the CYP 1A family. Despite their lipophilicity, a high proportion of the body burden of certain polychlorinated dibenzo-p-dioxins and coplanar polychlorinated biphenyls is sequestered in liver, a process believed to involve CYP 1A2. In this work we examined HAC-induced inhibition of the demethylation of 7-methoxyresorufin, a process that is selectively catalyzed by CYP 1A2. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 3,3',4,4'-tetrachlorobiphenyl (PCB 77) and 3,3',4,4',5-pentachlorobiphenyl (PCB 126) were found to be strong competitive inhibitors of methoxyresorufin-O-demethylase activity, consistent with the high ability of hepatic tissue to sequester these compounds selectively.

A pilot study of plasma caffeine concentrations in a US sample of smoker and nonsmoker volunteers.

Even though 85% of adults drink caffeinated beverages daily, very limited studies on plasma caffeine concentration in the US population have been published. Smoking induces cytochrome P450 1A2 (CYP1A2), which is the main enzyme involved in caffeine metabolism. The current naturalistic pilot study explores plasma caffeine concentrations in a US sample, and presents a mathematical model of the relationship between caffeine intake and plasma concentrations for smokers and nonsmokers. Caffeine intake and average plasma caffeine concentrations from morning (7:30-9:30 a.m.) and afternoon (2:00-4:00 p.m.) samples were studied in 69 volunteers (21 smokers and 48 nonsmokers). The mean caffeine intake obtained from caffeinated beverages was 3.02 mg/kg/day, which is similar to the intake in the US population. Almost all subjects in the present sample (99%; 95% confidence interval [CI]: 96-100) had detectable plasma caffeine concentrations. Smokers had significantly higher caffeine intake than nonsmokers. The ratio of concentration/dose of caffeine from caffeinated beverages was approximately four-fold higher in nonsmokers (1.33 kgxday/l) than in smokers (0.29 kgxday/l). According to the model, the median plasma caffeine concentration was two- to three-fold higher in nonsmokers for each level of caffeine intake. Our model improves our understanding of the interactions between caffeine and smoking. Additional studies are needed to replicate the model. This model may help epidemiologists to correct for the effects of smoking on caffeine intake and pharmacologists to screen for the activity of CYP1A2.

Caffeine test in predicting flutamide-induced hepatic injury in patients with prostate cancer.

The caffeine test measures the activity of cytochrome p450 (CYP1A2) which is a major enzyme involved in the activation of flutamide. The usefulness of this test in predicting flutamide-induced hepatic injury in patients with prostate cancer was examined. The subjects were: (1). five patients whose aspartate aminotransferase (AST) or alanine aminotransferase (ALT) level rose to 100 IU/l or higher following the start of flutamide (moderately injured group); (2). four patients whose AST and ALT levels were higher than normal but less than 100 IU/l (mildly injured group); and (3). two patients whose hepatic function remained normal (normal group). The subjects were each given canned coffee to drink. Urinary caffeine (137X), paraxanthine (17X) and 1, 7-dimethyluric acid (17U) levels were measured 4-5 h later. The metabolite ratio, (17U+17X)/137X, was calculated to serve as an indicator of CYP1A2 activity. The metabolite ratio for the moderately injured group (3.98+/-1.56) and the mildly injured group (5.55+/-1.42) were lower than that for the normal group (9.56). The results suggest that a decrease in CYP1A2 activity is involved in the onset of flutamide-induced hepatic injury, and that the caffeine test seems to provide a useful means of its prediction.

Exposure levels and cytochrome P450 1A2 activity, but not N-acetyltransferase, glutathione S-transferase (GST) M1 and T1, influence urinary mutagen excretion in smokers.

We investigated the polymorphic enzymes cytochrome P450 1A2 (CYP1A2), N-acetyltransferase (NAT2), glutathione S-transferase (GST) M1 (GSTM1), and T1 (GSTT1) in relation to cigarette smoking-associated urinary mutagenicity detected on YG1024 Salmonella typhimurium strain with S9 mix in 97 smokers. In each subject, cigarette smoke intake was checked by analysis of urinary nicotine plus its metabolites. NAT2 and CYP1A2 phenotypes were determined by the molar ratio of urinary caffeine metabolites detected by high-performance liquid chromatography, and GSTT1 and GSTM1 genotypes were determined by PCR. An increase in urinary mutagenicity was significantly related to levels of exposure to cigarette smoke and CYP1A2 N-hydroxylation activity (linear multiple regression analysis t = 4.51 and P < 0.001 and t = 3.09 and P = 0.003; F = 6.31, P < 0.001). Urinary mutagenicity was significantly higher in CYP1A2 extensive metabolizer smokers (n = 49) than in CYP1A2 poor metabolizer ones (n = 48; 2176 +/- 1525 versus 1384 +/- 1206 revertants/mmol creatinine, Mann-Whitney U-test, z = 2.65, P < 0.001). The highest mutagenic activity was seen in subjects CYP1A2 extensive metabolizer/NAT2 slow acetylators (n = 29) with respect to the other phenotype combinations (n = 68; 2392 +/- 1660 versus 1525 +/- 1238 revertants/mmol creatinine, Mann-Whitney U-test, z = 2.37, P = 0.017). NAT2 acetylation activity was slightly but inversely related to urinary mutagenicity, and the association was not significant. No effect of GSTM1 and GSTT1 genotypes in lowering (detoxifying) urinary mutagens was found. The significant enhancement of urinary mutagenicity associated with increased CYP1A2 activity, as already seen for diet-caused urinary mutagenicity, allows for many analogies between the process of mutagen formation derived from cooked meat and that from cigarette smoke condensate. In conclusion, the intensity of tobacco smoke exposure, modulated by CYP1A2 activity, is the major determinant of mutagenic urine among smokers, whereas GSTM1 and GSTT1 genotypes have no influence on this biomarker. This study suggests that CYP1A2 should definitely be determined in future studies involving urinary mutagenicity in cases in which smoking is a factor.

Metabolism and cytotoxicity of aflatoxin b1 in cytochrome p-450-expressing human lung cells.

The mycotoxin aflatoxin B(1) (AFB(1)) is a hepatocarcinogen in many animal models and probably a human carcinogen. Besides being a dietary carcinogen, AFB(1) has been detected in dusts generated in the processing and transportation of AFB(1)-contaminated products. Inhalation of grain dusts contaminated with AFB(1) may be a risk factor in human lung cancer. Aflatoxin B(1) requires cytochrome P-450 (CYP)-mediated activation to form cytotoxic and DNA-reactive intermediates, and this activation in human liver is mediated by the CYP 1A2 and 3A4 isoforms. Which isoforms are important in AFB(1) activation in human lung is not well understood. To investigate whether these CYPs can activate AFB(1) at low, environmentally relevant concentrations in human lung cells, SV40 immortalized human bronchial epithelial cells (BEAS-2B) that were transfected with cDNA for CYPs 3A4 (B3A4) or 1A2 (B-CMV1A2) were used. B-CMV1A2 cultured in 15 nM AFB(1) produced the AFB(1)-glutathione conjugate (AFB(1)-GSH) and aflatoxin M(1) (AFM(1)), while B3A4 cells produced only aflatoxin Q(1) (AFQ(1)) at 0.15 microM AFB(1). Nontransfected BEAS-2B cells produced no metabolites, even at 1.5 mM AFB(1). Microsomes prepared from B-CMV1A2 and B3A4 cells activated AFB(1) to AFB(1) 8,9-epoxide (AFBO), while those from BEAS-2B cells did not produce AFBO. Cytosol from all three cell types was ineffective at glutathione S-transferase (GST)-mediated trapping of enzymatically generated AFB(1) 8,9-epoxide. B-CMV1A2 cells were 100-fold more sensitive to AFB(1) compared to B3A4 cells, and were 6000-fold more sensitive than control BEAS-2B cells. Western immunoblots confirmed that only B-CMV1A2 cells expressed CYP 1A2 protein, while CYP 3A4 was only in B3A4 cells. B-CMV1A2 cells were the most sensitive to AFB(1), followed by B3A4 cells. CYP 3A4, which has been predicted to activate AFB(1) primarily at higher AFB(1) concentrations, was also responsible for significant AFB(1) toxicity at low concentrations. These data indicate that human lung cells expressing these CYP isoforms are capable of activating AFB(1), even at environmentally relevant concentrations.

Tobacco and cannabis smoking cessation can lead to intoxication with clozapine or olanzapine.

Plasma levels of clozapine and olanzapine are lower in smokers than in nonsmokers, which is mainly due to induction of cytochrome P4501A2 (CYP1A2) by some smoke constituents. Smoking cessation in patients treated with antipsychotic drugs that are CYP1A2 substrates may result in increased plasma levels of the drug and, consequently, in adverse drug effects. Two cases of patients who smoked tobacco and cannabis are reported. The first patient, who was receiving clozapine treatment, developed confusion after tobacco and cannabis smoking cessation, which was related to increased clozapine plasma levels. The second patient, who was receiving olanzapine treatment, showed important extrapyramidal motor symptoms after reducing his tobacco consumption. The clinical implication of these observations is that smoking patients treated with CYP1A2 substrate antipsychotics should regularly be monitored with regard to their smoking consumption in order to adjust doses in cases of a reduction or increase in smoking.