Association between nonalcoholic hepatic steatosis and hepatic cytochrome P-450 3A activity.

BACKGROUND & AIMS: Comorbidities associated with nonalcoholic fatty liver often require therapy with medications (eg, statins) metabolized by cytochrome P-450 3A (CYP3A). There is significant interindividual variability in CYP3A expression. However, human studies that systematically examined the relationship between hepatic steatosis and hepatic CYP3A activity are lacking. METHODS: The relationship of hepatic CYP3A activity with several variables including hepatic steatosis, CYP3A4 protein content, CYP3A4 mRNA expression, CYP3A5 genotype, and its mRNA expression was investigated in human liver samples (n = 49). CYP3A activity was quantified from liver microsomes by using testosterone as a probe, and hepatic steatosis was defined to be present if >5% of hepatocytes had large globules of intracellular fat displacing the nucleus. RESULTS: The mean +/- standard error hepatic CYP3A activity of the study group was 3156 +/- 2794 pmol x min(-1) x mg(-1) of protein, and it was not associated with age, gender, medicinal use, CYP3A5 or pregnane xenobiotic receptor mRNA expression, or CYP3A5 genotype. Twenty-four liver samples with steatosis had significantly lower hepatic CYP3A activity than 25 liver samples without steatosis (1978 +/- 299 vs 4287 +/- 659 pmol x min(-1) x mg(-1) of protein; P = .003). This difference persisted even after controlling for relevant covariates in the multivariate analysis (P = .04). However, CYP3A4 protein content was not different between the 2 groups (6 +/- 1.3 vs 8.5 +/- 2.2 pmol/mg protein; P = .3). There was a significant negative relationship between severity of steatosis and hepatic CYP3A activity (P = .01). CONCLUSIONS: Hepatic steatosis is associated with decreased hepatic CYP3A activity in humans via post-translational mechanism. Further studies are needed to confirm our findings.

Vincristine transcriptional regulation of efflux drug transporters in carcinoma cell lines.

The increased expression of drug transporters following cancer chemotherapy contributes to resistance. This may reflect transcriptional up-regulation and/or clonal selection. We quantified the expression of mRNA for ABCB1 (mdr1), ABCC1 (mrp1), ABCC2 (mrp2) and ABCC3 (mrp3) to evaluate the potential contribution of induction. ABCB1, ABCC1-3 mRNAs were quantified by real time RT-PCR and normalized to GAPDH. We used intestinal cells that express high pregnane X receptor (LS174T), low pregnane X receptor (Caco-2) and lung cells (A549) that express glucocorticoid receptor and low pregnane X receptor. Rifampin (10 microM) caused significant induction of ABCB1 (595+/-263%, p<0.05) in LS174T cells but induction was absent in Caco-2 or A549 cells. ABCC1 was not induced in any cell at 24, 48 and 72 h following rifampin treatment. In contrast, vincristine (10 nM and 100 nM), a ligand for ABCB1 and ABCC1-3 and a potential PXR/CAR ligand, induced ABCC2 and ABCC3 expression in LS174T cells at 48 h (372+/-87% and 303+/-42%, respectively, p<0.05). A similar induction of ABCC2 and ABCC3 genes was also seen with 10 nM VCR in A549 cells following 48 h treatment. In summary, there may be a significant contribution of transcriptional activation to multi-drug resistance. However, this is cell selective and is not necessarily dependent on PXR mediated effects.

Activity of CYP2E1 and CYP3A enzymes in adults with moderate alcohol consumption: a comparison with nonalcoholics.

Alcohol consumption is known to induce hepatic CYP2E1 activity, but its effect on hepatic and intestinal CYP3A in humans is not known. We have conducted a study to compare the CYP2E1 and CYP3A activities in 20 individuals with moderate alcohol consumption and 20 gender-, race-. and body mass index (BMI)-matched nonalcoholics. Intravenous and oral midazolam (MDZ) clearances were used to measure the in vivo CYP3A activity, and chlorzoxazone (CHZ) oral clearance was used to assess in vivo CYP2E1 activity. Furthermore, we assessed the relationship between hepatic CYP2E1 and CYP3A activities and their messenger RNA (mRNA) expression in the peripheral lymphocytes. The systemic clearance (CL) of MDZ was not different between alcoholics (36.9 +/- 12 L/hr) and nonalcoholics (36.6 +/- 14.1; P = .9). The oral availability of MDZ was significantly lower in alcoholics than in the nonalcoholics (0.28 +/- .09 vs. 0.38 +/- .17, respectively, P = .03). The maximum serum concentration after oral midazolam dosing was significantly different between the 2 groups. CHZ CL was significantly higher in alcoholics than in nonalcoholics (31.5 +/- 11.9 vs. 23.4 +/- 8.7 L/hr, P < 0.05). CYP3A4 and CYP2E1 mRNA levels were not significantly different between the groups, and no correlation was observed between lymphocyte CYP mRNA and in vivo CYP activity. In conclusion, in individuals with moderate alcohol consumption, there was no alteration in the hepatic CYP3A activity, but the reduced midazolam oral bioavailability suggests that moderate alcohol consumption may cause intestinal CYP3A induction. Lymphocyte CYP2E1 and CYP3A4 mRNA levels did not correlate with CYP2E1 and CYP3A activities.

Inhibition of human intestinal wall metabolism by macrolide antibiotics: effect of clarithromycin on cytochrome P450 3A4/5 activity and expression.

BACKGROUND: Clarithromycin increases both hepatic and intestinal availability of the selective cytochrome P450 (CYP) 3A probe midazolam. This study was designed to identify determinants of variability in the extent of intestinal wall CYP3A inhibition by clarithromycin, such as CYP3A5 genotype, and the mechanism of inhibition. METHODS: Ten healthy volunteers received 500 mg oral clarithromycin twice a day for 7 days. Before and after administration of clarithromycin, small-bowel mucosal biopsy specimens were obtained endoscopically. Intestinal CYP3A activity was determined from the rate of 1'-hydroxymidazolam and 4-hydroxymidazolam formation by incubation of small-bowel homogenate with midazolam (25 micromol/L) and NADPH for 5 minutes. Intestinal CYP3A4 and CYP3A5 messenger ribonucleic acid was quantified by real-time reverse transcriptase-polymerase chain reaction. Intestinal CYP3A4 and CYP3A5 protein concentrations were determined by immunoblotting. Serum and homogenate concentrations of midazolam, clarithromycin, and metabolites were determined by liquid chromatography-mass spectrometry. CYP3A5 genotype was determined by real-time polymerase chain reaction. RESULTS: The formation of 1'-hydroxymidazolam (1.36 +/- 0.46 pmol . min(-1) . mg(-1) at baseline versus 0.35 +/- 0.16 pmol . min(-1) . mg(-1) after administration) and 4-hydroxymidazolam (0.39 +/- 0.12 pmol . min(-1) . mg(-1) at baseline versus 0.12 +/- 0.05 pmol . min(-1) . mg(-1) after administration) was significantly (P < .001) reduced after clarithromycin administration. Clarithromycin administration did not result in a significant change in intestinal CYP3A4 and CYP3A5 messenger ribonucleic acid and protein expression. All subjects had detectable serum clarithromycin concentrations after 7 days of clarithromycin (3.71 +/- 2.43 micromol/L). The mean concentration of clarithromycin in the intestinal biopsy homogenate was 1.2 +/- 0.7 nmol/L (range, 0.42-2.39 nmol/L). Compared with CYP3A5 nonexpressers, subjects with at least 1 CYP3A5*1 allele (CYP3A5 expressers) had greater inhibition of intestinal CYP3A activity after treatment with clarithromycin. There was a strong linear relationship between the decrease in intestinal CYP3A activity and baseline catalytic activity (R(2) = 0.9). CONCLUSION: Baseline intestinal activity of CYP3A4 was a key determinant of variability of the inhibitory effect of clarithromycin among individuals. CYP3A5*1 alleles were associated with greater baseline intestinal CYP3A activity and, therefore, greater extent of inhibition. The primary in vivo mechanism was not rapidly reversible competitive or irreversible inhibition but was likely formation of metabolic intermediate complexes.

Is adiponectin involved in the pathogenesis of nonalcoholic steatohepatitis? A preliminary human study.

BACKGROUND: Animal studies have suggested that adiponectin may play a role in the pathogenesis of alcoholic and nonalcoholic fatty liver disease. Studies are limited that evaluated the role of adiponectin in the pathogenesis of nonalcoholic steatohepatitis (NASH). METHODS: To further our understanding of the role of adiponectin in the pathogenesis of NASH, the following studies were conducted. Serum adiponectin was measured and correlated with anthropometric and nutritional variables in 21 patients with biopsy-proven NASH and 19 age-, gender-, body mass index-, and body fat-matched controls. The effect of a mixed meal on serum adiponectin levels in a subgroup of patients (n = 24) with NASH and controls was assessed. In a separate cohort, liver samples belonging to healthy (n = 11), steatotic (n = 12), and NASH (n = 12) patients were used to further explore the role of adiponectin by measuring the expression of adiponectin and adiponectin receptor (AdipoR2) mRNA. RESULTS: Patients with NASH had significantly lower levels of serum adiponectin than controls (4.9 +/- 2.7 vs. 7.3 +/- 3.5 microg/mL, P = 0.02). While no significant correlation existed between serum adiponectin and anthropometric or nutritional variables, it was independently associated with age, high density lipoprotein, and triglycerides. Mixed meal had no effect on serum adiponectin either in patients with NASH or in controls. There was no expression of adiponectin mRNA in any of liver samples studied. However, AdipoR2 mRNA expression was higher in NASH than in steatotic and normal liver tissue. CONCLUSION: These data show that adiponectin may have a role in the pathogenesis of human NASH and should be investigated further.

Expression and function of ABCB1 and ABCG2 in human placental tissue.

The placenta plays an important role in modulating xenobiotic passage from mother to fetus. Studies in mice have demonstrated that placental ABCB1 and ABCG2 can affect the transfer of drugs across the placental barrier, suggesting a role for these transporters in protecting the fetus from environmental toxicants or drugs ingested by the mother during pregnancy. To assess the role of these transporters in the human placenta, studies were conducted to evaluate the expression and functional activity of placental ABCB1 and ABCG2. The effect of maternal smoking on these placental transporters was also assessed. Uptake rates of [3H]vinblastine and [3H]mitoxantrone were used to measure ABCB1 and ABCG2 activity, respectively, and CYP1A1 activity was assessed using ethoxyresorufin O-deethylation as a positive control for smoking-related enzyme induction. ABCB1 and ABCG2 expression levels were measured by immunoblotting techniques. ATP-dependent uptake of [3H]vinblastine in vesicles was osmotically sensitive, suggesting intravesicular accumulation, and was inhibited by verapamil, an ABCB1 inhibitor. ATP-dependent uptake of [3H]mitoxantrone was inhibited by fumitremorgin C, an ABCG2 inhibitor, but not by verapamil, suggesting that the uptake of [3H]mitoxantrone was primarily mediated by ABCG2. Although CYP1A1 activity was greatly induced in smokers, no statistical differences (p > 0.05) were noted in ABCB1 and ABCG2 activity or expression between smokers and nonsmokers. In summary, both ABCB1 and ABCG2 are expressed at high levels in human placenta and are functionally active, suggesting a protective role with respect to fetal exposure to xenobiotics ingested by the mother.

Activation of CYP2C9-mediated metabolism by a series of dapsone analogs: kinetics and structural requirements.

Cytochrome P450 2C9-mediated metabolism has been shown to be activated in the presence of the effector dapsone. However, it has yet to be established what effector structural features are necessary to activate CYP2C9 activity. To address this question, kinetic studies were conducted with nine analogs of dapsone containing various functional properties (three sulfone compounds, three carbonyl compounds, and three sulfonamide compounds), to examine the functional groups important for enzyme activation by the effector (dapsone). Results show that phenylsulfone (dapsone without the para-amino groups) activates flurbiprofen 4'-hydroxylation comparable to dapsone but inhibits naproxen demethylation. Meanwhile, p-tolylsulfone had little effect on flurbiprofen metabolism, but activated naproxen demethylation, albeit only at high concentrations. These substrate-dependent differences in effect suggest that naproxen has a different binding orientation compared with flurbiprofen. Perhaps most interesting is that replacement of only one amino group from dapsone with a nitro group (4-(4-nitrophenylsulfonyl)-aniline) resulted in substantial inhibition of flurbiprofen 4'-hydroxylation, suggesting that electronic effects may influence activation of this substrate. Other analogs either had minor or no effect on CYP2C9-mediated metabolism. Overall, it is apparent from these studies that a sulfone group in direct association with two benzene rings with para-electron-donating groups represents the most efficient activator of CYP2C9. However, the effects of these analogs appear to be concentration- and substrate-dependent, further complicating the prediction of these types of in vitro interactions.