Inhibition of phenol sulfotransferase (SULT1A1) by quercetin in human adult and foetal livers

1. Quercetin is one of the most abundant flavonoids in edible vegetables, fruit and wine. The aim was to study the type of inhibition of SULT1A1 by quercetin in the human adult and foetal livers. 2. The activity of SULT1A1 was measured with 4 microM 4-nitrophenol and 0.4 microM 3'-phosphoadenosine-5'-phosphosulphate-[(35)S], and its mean (+/-SD) and median were 769 +/- 311 and 740 pmol min(-1) mg(-1), respectively (adult liver, n = 10), and 185 +/- 98 and 201 pmol min(-1) mg(-1), respectively (foetal liver, n = 8, p < 0.0001). 3. In non-inhibited samples, K(m) for SULT1A1 (mean +/- SD) was 0.31 +/- 0.14 microM (adult liver) and 0.49 +/- 0.17 microM (foetal liver, n.s.). V(max) for SULT1A1 (mean +/- SD) was 885 +/- 135 pmol min(-1) mg(-1) (adult liver) and 267 +/- 93 pmol min(-1) mg(-1) (foetal liver, p = 0.007). 4. The IC(50) of quercetin for SULT1A1 was measured in three samples of adult and foetal livers and was 13 +/- 2.1 and 12 +/- 1.4 nM, respectively. 5. The type of inhibition was mixed non-competitive in adult and foetal livers and K(i) was 4.7 +/- 2.5 nM (adult liver) and 4.8 +/- 1.6 nM (foetal liver). 6. In the adult liver, the intrinsic clearance (mean +/- SD) was 3.3 +/- 1.5 ml min(-1) mg(-1) (non-inhibited samples), 0.9 +/- 0.4 ml min(-1) mg(-1) (12.5 nM quercetin) and 0.5 +/- 0.06 ml min(-1) mg(-1) (25 nM quercetin). In the foetal liver, the intrinsic clearance (mean +/- SD) was 0.5 +/- 0.2 ml min(-1) mg(-1) (non-inhibited samples), 0.12 +/- 0.01 ml min(-1) mg(-1) (12.5 nM quercetin) and 0.2 +/- 0.09 ml min(-1) mg(-1) (25 nM quercetin). 7. In conclusion, quercetin is a potent inhibitor of human adult and foetal liver SULT1A1. It reduces the sulphation rate and intrinsic clearance of 4-nitrophenol in both human adult and foetal livers. This suggests that quercetin may inhibit the sulfation rate of those drugs sulphated by SULT1A1. The inhibition of SULT1A1 is complex and not due solely to competition at the catalytic site of SULT1A1.     

Phenol sulfotransferase 1A1 activity in human liver: kinetic properties, interindividual variation and re-evaluation of the suitability of 4-nitrophenol as a probe substrate

Sulfation is an important metabolic pathway in humans for xenobiotics, hormones and neurotransmitters, and is catalysed by the cytosolic sulfotransferase (SULT) enzymes. Phenol SULTs, especially SULT1A1, are particularly important in xenobiotic and drug metabolism because of their broad substrate specificity and extensive tissue distribution. A common variant SULT1A1 allozyme (SULT1A1*2) exists in the population, and is less stable than the wild-type SULT1A1*1. 4-Nitrophenol is widely used as a substrate for quantifying SULT1A1 activity. However, our kinetic experiments suggest that 4-nitrophenol is not an ideal substrate when determining SULT1A1 activity in human liver. Assays with a bank of 68 human liver cytosols revealed three distinct kinetic profiles for 4-nitrophenol sulfation in the population: linear, biphasic and inhibition. Sulfation of 4-nitrophenol by purified, recombinant SULT1A1*1 and SULT1A1*2 shows marked substrate inhibition, with inhibition at 4-nitrophenol concentrations greater than 4 and 10 microM, respectively. Furthermore, sulfation of 4-nitrophenol by purified recombinant SULT1B1 was significant at concentrations of 4-nitrophenol less than 10 microM. Western blots showed that the SULT1A1 levels in liver are highly variable between liver samples and that no correlation was observed between SULT1A1 activity and protein level in liver cytosols. However, a correlation between SULT1A1 activity and protein level was observed in human placental cytosols, where SULT1B1 is not expressed. We believe that in human liver other SULT isoforms (particularly SULT1B1) contribute to the sulfation of 4-nitrophenol. Therefore, 4-nitrophenol is not an ideal substrate with which to quantitate SULT1A1 activity in human liver tissue.     

Curcumin is a potent inhibitor of phenol sulfotransferase (SULT1A1) in human liver and extrahepatic tissues

1. Curcumin has anti-carcinogen effects and is under clinical evaluation as a potential colon cancer chemopreventive agent. The first aim was to see whether curcumin inhibited phenol sulfotransferase (SULT1A1) and, if so, to study the variability of the IC(50) of curcumin for SULT1A1 in 50 human liver samples. For comparative purposes, the inhibition of catechol sulfotransferase (SULT1A3) in five human liver specimens was studied. The second aim was to measure the IC(50) of curcumin against SULT1A1 in five samples of human duodenum, colon, kidney and lung. 2. Curcumin was a potent inhibitor of SULT1A1 in human liver; the mean +/- SD and median of IC(50) were 14.1 +/- 7.3 nM and 12.8 nM, respectively. The IC(50) ranged from 6.2 to 30.6 nM between the 5th and 95th percentiles and the fold of variation was 4.9. The distribution of IC(50) was positively skewed (skewness 1.2) and deviated from normality (p = 0.0004). 3. Curcumin inhibited human SULT1A3, and the inhibition was studied in five liver specimens with an IC(50) of 4324 +/- 1026 nM. This inhibition was greater than the IC(50) of curcumin for SULT1A1 (p < 0.0001). 4. In the extrahepatic tissues, the IC(50) of curcumin for SULT1A1 was 25.9 +/- 4.8 nM (duodenum), 25.4 +/- 6.8 nM (colon), 23.4 +/- 2.2 nM (kidney) and 25.6 +/- 5.6 nM (lung). Inhibition in these tissues is greater than that of curcumin for SULT1A1 in human liver (p < 0.0001). 5. In conclusion, curcumin is a potent inhibitor of SULT1A1 in human liver, duodenum, colon, kidney and lung. The IC(50) of curcumin for SULT1A1 varied 4.9-fold in human liver. The comparison of the present data with those of the literature revealed that the IC(50) of curcumin in the liver and extrahepatic tissues is one order of magnitude lower that the peak serum concentration of curcumin after therapeutic doses of 4 g to humans.     

Relationship of phenol sulfotransferase activity (SULT1A1) genotype to sulfotransferase phenotype in platelet cytosol

Sulfation catalysed by human cytosolic sulfotransferases is generally considered to be a detoxification mechanism. Recently, it has been demonstrated that sulfation of heterocyclic aromatic amines by human phenol sulfotransferase (SULT1A1) can result in a DNA binding species. Therefore, sulfation capacity has the potential to influence chemical carcinogenesis in humans. To date, one genetic polymorphism (Arg213His) has been identified that is associated with reduced platelet sulfotransferase activity. In this study, data on age, race, gender, SULT1A1 genotype and platelet SULT1A1 activity were available for 279 individuals. A simple colorimetric phenotyping assay, in conjunction with genotyping, was employed to demonstrate a significant correlation (r = 0.23, P < 0.01) of SULT1A1 genotype and platelet sulfotransferase activity towards 2-naphthol, a marker substrate for this enzyme. There was also a difference in mean sulfotransferase activity based on gender (1.28 nmol/min/mg, females; 0.94 nmol/min/mg, males, P = 0.001). DNA binding studies using recombinant SULT1A1*1 and SULT1A1*2 revealed that SULT1A1*1 catalysed N-hydroxy-aminobiphenyl (N-OH-ABP) DNA adduct formation with substantially greater efficiency (5.4 versus 0.4 pmol bound/mg DNA/20 min) than the SULT1A1*2 variant. A similar pattern was observed with 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5b]pyridine (N-OH-PhIP) (4.6 versus 1.8 pmol bound/mg DNA/20 min).     

Frequency distribution of phenol sulfotransferase 1A1 activity in platelet cells from healthy Japanese subjects.

AIMS: To determine the distribution of sulfotransferase 1A1 (SULT1A1) activities, we used trans-4-hydroxytamoxifen (OHT) as a substrate to test samples from a Japanese population to examine whether the SULT1A1*2 allele can account for the wide distribution of OHT sulfating activity. We also studied genetic mutations other than the SULT1A1*2 allele to determine the cause of differences in SULT1A1 protein expression and activity. METHODS: The subjects were 103 healthy Japanese adults. Identification of SULT1A1 genotypes was performed using a polymerase chain reaction-restriction fragment length polymorphism method. SULT1A1 activity in platelet cytosol was assayed using OHT as a substrate. SULT1A1 protein was detected using Western blotting analysis. Mutations other than SULT1A1*2 in the SULT1A1 gene were detected using sequencing analysis. RESULTS: SULT1A1*2 allele frequency was found to be 16.5%, while SULT1A1 activity ranged from 63 to 1860pmol sulfated/h/mg platelet protein (260+/-241pmol sulfated/h/mg platelet protein, median+/-S.D.) using OHT as a substrate. The median values in subjects with SULT*1/*2 (221+/-113pmol sulfated/h/mg platelet protein, range 63-442, n=26) and SULT*2/*2 (124+/-66pmol sulfated/h/mg platelet protein, range 74-231, n=4) were significantly lower than that in subjects with SULT*1/*1 (303+/-267pmol sulfated/h/mg platelet protein, range 97-1859, n=73). A novel G148C mutation was found in one subject, who showed the lowest OHT sulfating activity, for a frequency of 0.49%. CONCLUSION: There was wide variety of OHT sulfating activities found among the present healthy Japanese subjects. The SULT1A1*2 allele was found to be a common variant allele and was associated with decreased OHT sulfating activity. These observations may be related to inter-individual variations of OHT pharmacokinetics and the pharmacologic effects of tamoxifen seen in Japanese patients with breast cancer.     

Sulfation of apomorphine by human sulfotransferases: evidence of a major role for the polymorphic phenol sulfotransferase, SULT1A1

1. The relative roles of various members of the human sulfotransferase (SULT) enzyme family in the metabolism of apomorphine, a dopamine receptor antagonist used in the treatment of Parkinson's disease and, more recently, erectile dysfunction, were examined. In humans, sulfation is the major route of metabolism of this drug. 2. Using recombinant SULTs expressed in Escherichia coli, R(--)-apomorphine sulfation was studied using the universal barium precipitation assay in the presence of [35S] 3'-phosphoadenosine 5'-phosphosulfate and SULTs 1A1, 1A2, 1A3, 1B1, 1C2, 1E1 and 2A1. It was shown that SULTs 1A1, 1A2, 1A3 and 1E1 all sulfated apomorphine to varying extents. Low activity with SULT1B1 was only seen at the highest concentration (100 microM) and no activity with SULT1C2 or SULT2A1 was observed. 3. Kinetic analysis using purified recombinant SULTs showed that 1A1, 1A3 and 1E1 all had similar Vmax/Km values, although SULT1E1 had a slightly lower Km at around 1 microM compared with approximately 4 microM for the other SULTs. 4. By correlating apomorphine sulfation (at 10 microM) in a bank of 28 liver cytosols with SULT activity towards 10 microM 4-nitrophenol (SULT1A1) and 0.2 microM 17beta-oestradiol (SULT1E1), a strong correlation with SULT1A1 activity was clearly demonstrated, suggesting this enzyme was primarily responsible for hepatic apomorphine sulfation. 5. These findings were confirmed using immuno-inhibition experiments with antibodies against SULT1A and SULT1E1, which showed preferential inhibition of apomorphine sulfation in human liver cytosol by anti-SULT1A. 6. The results strongly implicate SULT1A1 as the major enzyme responsible for hepatic apomorphine metabolism. As SULT1A1 is subject to a common functional polymorphism, sulfation phenotype may be an important determinant of susceptibility to side-effects of apomorphine and/or efficacy of treatment.     

Sulfation of apomorphine by human sulfotransferases: evidence of a major role for the polymorphic phenol sulfotransferase,SULT1A1

1. The relative roles of various members of the human sulfotransferase (SULT) enzyme family in the metabolism of apomorphine, a dopamine receptor antagonist used in the treatment of Parkinson's disease and, more recently, erectile dysfunction, were examined. In humans, sulfation is the major route of metabolism of this drug.

2. Using recombinant SULTs expressed in Escherichia coli, R(–)-apomorphine sulfation was studied using the universal barium precipitation assay in the presence of [³⁵S] 3′-phosphoadenosine 5′-phosphosulfate and SULTs 1A1, 1A2, 1A3, 1B1, 1C2, 1E1 and 2A1. It was shown that SULTs 1A1, 1A2, 1A3 and 1E1 all sulfated apomorphine to varying extents. Low activity with SULT1B1 was only seen at the highest concentration (100?μM) and no activity with SULT1C2 or SULT2A1 was observed.

3. Kinetic analysis using purified recombinant SULTs showed that 1A1, 1A3 and 1E1 all had similar V_max/K_m values, although SULT1E1 had a slightly lower K_m at around 1 μM compared with approximately 4 μM for the other SULTs.

4. By correlating apomorphine sulfation (at 10?μM) in a bank of 28 liver cytosols with SULT activity towards 10?μM 4-nitrophenol (SULT1A1) and 0.2?μM 17β-oestradiol (SULT1E1), a strong correlation with SULT1A1 activity was clearly demonstrated, suggesting this enzyme was primarily responsible for hepatic apomorphine sulfation.

5. These findings were confirmed using immuno-inhibition experiments with antibodies against SULT1A and SULT1E1, which showed preferential inhibition of apomorphine sulfation in human liver cytosol by anti-SULT1A.

6. The results strongly implicate SULT1A1 as the major enzyme responsible for hepatic apomorphine metabolism. As SULT1A1 is subject to a common functional polymorphism, sulfation phenotype may be an important determinant of susceptibility to side-effects of apomorphine and/or efficacy of treatment.     

Curcumin is a potent inhibitor of phenol sulfotransferase (SULT1A1) in human liver and extrahepatic tissues

1. Curcumin has anti-carcinogen effects and is under clinical evaluation as a potential colon cancer chemopreventive agent. The first aim was to see whether curcumin inhibited phenol sulfotransferase (SULT1A1) and, if so, to study the variability of the IC 50 of curcumin for SULT1A1 in 50 human liver samples. For comparative purposes, the inhibition of catechol sulfotransferase (SULT1A3) in five human liver specimens was studied. The second aim was to measure the IC 50 of curcumin against SULT1A1 in five samples of human duodenum, colon, kidney and lung. 2. Curcumin was a potent inhibitor of SULT1A1 in human liver; the mean ± SD and median of IC 50 were 14.1 ± 7.3 nM and 12.8 nM, respectively. The IC 50 ranged from 6.2 to 30.6 nM between the 5th and 95th percentiles and the fold of variation was 4.9. The distribution of IC 50 was positively skewed (skewness 1.2) and deviated from normality (p = 0.0004). 3. Curcumin inhibited human SULT1A3, and the inhibition was studied in five liver specimens with an IC 50 of 4324 ± 1026 nM. This inhibition was greater than the IC 50 of curcumin for SULT1A1 (p < 0.0001). 4. In the extrahepatic tissues, the IC 50 of curcumin for SULT1A1 was 25.9 ± 4.8 nM (duodenum), 25.4 ± 6.8 nM (colon), 23.4 ± 2.2 nM (kidney) and 25.6 ± 5.6 nM (lung). Inhibition in these tissues is greater than that of curcumin for SULT1A1 in human liver (p < 0.0001). 5. In conclusion, curcumin is a potent inhibitor of SULT1A1 in human liver, duodenum, colon, kidney and lung. The IC 50 of curcumin for SULT1A1 varied 4.9-fold in human liver. The comparison of the present data with those of the literature revealed that the IC 50 of curcumin in the liver and extrahepatic tissues is one order of magnitude lower that the peak serum concentration of curcumin after therapeutic doses of 4?g to humans.     

Variable sulfation of dietary polyphenols by recombinant human sulfotransferase (SULT) 1A1 genetic variants and SULT1E1.

Human cytosolic sulfotransferases (SULTs) catalyze the sulfate conjugation of several important endo- and xenobiotics. Among the superfamily of SULT enzymes, SULT1A1 catalyzes the sulfation of small planar phenolic compounds, whereas SULT1E1 has a major role in estrogen conjugation. The human SULT1A1 gene has common single nucleotide polymorphisms that define three allozymes, SULT1A1*1, *2, and *3. The enzyme kinetics of SULT1A1 allozymes and SULT1E1 were characterized for the polyphenolic substrates apigenin, chrysin, epicatechin, quercetin, and resveratrol. Purified recombinant SULT proteins were generated in a baculoviral-insect cell system, and incubated in vitro with each substrate to determine catalytic activity. The effect of polyphenol sulfation was examined in mammalian cell lines stably expressing SULT1E1. For all polyphenols investigated, "normal-activity" SULT1A1*1 allozyme had significantly greater Vmax estimates than SULT1E1, and allele-specific differences in SULT1A1-mediated sulfation were observed. The polymorphic SULT1A1*2 allozyme exhibited low activity toward apigenin, epicatechin, and resveratrol. SULT1A1*1 and *3 acted as normal-activity allozymes for these substrates. Altered cellular proliferation was observed in MCF-7 cells stably expressing SULT1E1 upon treatment with chrysin, quercetin, or resveratrol, thus suggesting inactivation of these compounds by SULT1E1. These results suggest an important role for SULT isozymes and their pharmacogenetics in polyphenol disposition.     

Common genetic polymorphisms in the 5'-flanking region of the SULT1A1 gene: haplotypes and their association with platelet enzymatic activity

SULT1A1 is a phase II detoxification enzyme involved in the biotransformation of a wide variety of endogenous and exogenous phenolic compounds. Human platelet SULT1A1 enzymatic activity shows marked inter-individual variability and a common coding polymorphism, SULT1A1*1/*2, has been described that accounts for a proportion of this variability. We examined the 5'-flanking region of the SULT1A1 gene to determine if genetic variability in this portion of the gene influenced enzymatic activity. Direct sequencing revealed five common genetic polymorphisms (-624G>C, -396G>A, -358A>C, -341C>G and -294T>C) that were present at different allele frequencies in Caucasian, African-American and Chinese groups. Platelet SULT1A1 enzymatic activity was significantly correlated with individual promoter region polymorphisms and the associations were different between African-Americans and Caucasians. Haplotypes were constructed and platelet enzymatic activity according to haplotype was examined. The haplotypes were also significantly correlated with activity; haplotypes GAACT and GGACT (accounting for 13% and 5% of inter-individual variability in platelet activity, respectively) were important in Caucasians while haplotypes GAACC, GAACT and GGACC (accounting for 8%, 5% and 4% of variability) were significantly associated with activity in African-Americans. The coding region polymorphism, SULT1A1*1/*2 was in linkage disequilibrium with the promoter region polymorphisms and showed no effect on activity when examined in the context of the 5'-flanking region polymorphisms. These studies indicate that variation in the promoter region of the SULT1A1 gene exerts a significant influence on enzymatic activity.     

Characterization of bovine phenol sulfotransferases: evidence of a major role for SULT1B1 in the liver

Abstract

1.?Cattle are an important component of the human food chain. Drugs used either legally or illegally in cattle may therefore enter the food chain and it is thus important to understand pathways of drug metabolism in this species, including sulfation catalyzed by the sulfotransferases (SULTs).

2.?In this study, we have analyzed the sulfation of 4-nitrophenol and other compounds in male and female bovine liver and characterized recombinant bovine SULT isoforms 1A1 and 1B1 expressed in Escherichia coli.

3.?We found that, in contrast to most other mammalian species, the major phenol sulfotransferase SULT1A1 is not expressed in bovine liver. Rather SULT1B1 seems to be a major form in both male and female bovine liver.

4.?We also identified kinetic differences between bovine and human SULT1A1 and, using the human SULT1A1 crystal structure, identified two amino acid positions in the active site of bovine SULT1A1 (Ile₈₉Val and Phe₂₄₇Val) that may be responsible for these differences.     

Inhibition of phenol sulfotransferase (SULT1A1) by quercetin in human adult and foetal livers

1. Quercetin is one of the most abundant flavonoids in edible vegetables, fruit and wine. The aim was to study the type of inhibition of SULT1A1 by quercetin in the human adult and foetal livers. 2. The activity of SULT1A1 was measured with 4 µM 4-nitrophenol and 0.4 µM 3'-phosphoadenosine-5'-phosphosulphate-[³⁵S], and its mean (± SD) and median were 769 ± 311 and 740 pmol min^?1 mg^?1, respectively (adult liver, n = 10), and 185 ± 98 and 201 pmol min^?1 mg^?1, respectively (foetal liver, n = 8, p < 0.0001). 3. In non-inhibited samples, K_m for SULT1A1 (mean ± SD) was 0.31 ± 0.14 µM (adult liver) and 0.49 ± 0.17 µM (foetal liver, n.s.). V_max for SULT1A1 (mean ± SD) was 885 ± 135 pmol min^?1 mg^?1 (adult liver) and 267 ± 93 pmol min^?1 mg^?1 (foetal liver, p = 0.007). 4. The IC₅₀ of quercetin for SULT1A1 was measured in three samples of adult and foetal livers and was 13 ± 2.1 and 12 ± 1.4 nM, respectively. 5. The type of inhibition was mixed non-competitive in adult and foetal livers and K_i was 4.7 ± 2.5 nM (adult liver) and 4.8 ± 1.6 nM (foetal liver). 6. In the adult liver, the intrinsic clearance (mean ± SD) was 3.3 ± 1.5 ml min^?1 mg^?1 (non-inhibited samples), 0.9 ± 0.4 ml min^?1 mg^?1 (12.5 nM quercetin) and 0.5 ± 0.06 ml min^?1 mg^?1 (25 nM quercetin). In the foetal liver, the intrinsic clearance (mean ± SD) was 0.5 ± 0.2 ml min^?1 mg^?1 (non-inhibited samples), 0.12 ± 0.01 ml min^?1 mg^?1 (12.5 nM quercetin) and 0.2 ± 0.09ml min^?1 mg^?1 (25nM quercetin). 7. In conclusion, quercetin is a potent inhibitor of human adult and foetal liver SULT1A1. It reduces the sulphation rate and intrinsic clearance of 4-nitrophenol in both human adult and foetal livers. This suggests that quercetin may inhibit the sulfation rate of those drugs sulphated by SULT1A1. The inhibition of SULT1A1 is complex and not due solely to competition at the catalytic site of SULT1A1.     

OATP1B1 polymorphism is a major determinant of serum bilirubin level but not associated with rifampicin-mediated bilirubin elevation

1. Elevated serum bilirubin levels are caused mainly by liver diseases, haematolysis, genetic defects and drug intake. Unconjugated bilirubin (UCB) is taken up into hepatocytes by human organic anion transporting polypeptide 1B1 (OATP1B1; encoded for by the SLCO1B1 gene). The present study was performed to determine the association between SLCO1B1 gene polymorphisms and serum bilirubin levels in vivo. Moreover, the effects of administration of low-dose rifampicin on serum bilirubin levels in different SLCO1B1 genotypes was examined. 2. Serum bilirubin levels were examined in 42 healthy volunteers who had been analysed for SLCO1B1 genotype (seven, 13, 14 and eight with SLCO1B1 genotypes *1a/*1a, *1b/*1b, *1b/*15 and *15/*15, respectively). Among them, 24 subjects (seven, seven, eight and two with SLCO1B1 genotypes *1a/*1a, *1b/*1b, *1b/*15 and *15/*15, respectively) were selected to participate in an open-label, two-phase clinical trial. Each was given 450 mg rifampicin orally once daily at 2000 hours for 5 consecutive days. Serum bilirubin concentrations at 0800 hours on the 1st and 6th days were compared between the different SLCO1B1 genotypes. 3. In the 42 volunteers, the mean (+/-SD) serum UCB in both SLCO1B1*1b/*15 and *15/*15 groups was significantly higher than that in the SLCO1B1*1b/*1b group (11.07 +/- 2.31, 13.01 +/- 3.87 and 8.21 +/- 2.68 micromol/L, respectively; P = 0.009 and P < 0.001). Total bilirum (T.BIL) in both the SLCO1B1*1b/*15 and *15/*15 groups was significantly higher than that in the SLCO1B1*1b/*1b group (16.69 +/- 4.09, 20.71 +/- 5.12 and 13.06 +/- 5.12 micromol/L, respectively; P = 0.029 and P < 0.001). The direct bilirubin (D.BIL) in the SLCO1B1*15/*15 group was significantly higher than that in the SLCO1B1*1b/*1b group (7.69 +/- 1.81 vs 4.85 +/- 1.81 micromol/L, respectively; P = 0.001). Rifampicin significantly increased UCB, T.BIL and D.BIL concentrations in 24 healthy volunteers (17.68 +/- 5.96 vs 13.95 +/- 4.44 micromol/L (P = 0.040), 5.72 +/- 2.01 vs 4.35 +/- 1.50 micromol/L (P = 0.028) and 12.00 +/- 4.26 vs 9.61 +/- 3.15 micromol/L (P = 0.035), respectively). However, the extent of the increase in serum bilirubin caused by 450 mg rifampicin for 5 days was not affected by SLCO1B1 genotype. 4. Genetic polymorphism in SLCO1B1 is a major determinant of interindividual variability in the serum bilirubin level. SLCO1B1*15 carriers had higher baseline serum UCB, T.BIL and D.BIL levels compared with subjects with the SLCO1B1*1a/*1a and SLCO1B1*1b/*1b genotypes. SLCO1B1*15/*15 homozygotes are more susceptible to hyperbilirubinaemia. Serum bilirubin levels could be increased by low-dose rifampicin administration, but the extent of the increase was not associated with SLCO1B1 genotype.     

Influence of the CYP3A5 and MDR1 genetic polymorphisms on the pharmacokinetics of tacrolimus in healthy Korean subjects

AIMS: To determine the frequencies of the genotypes of CYP3A5 and MDR1 and to examine the influence of the polymorphisms of these genes on tacrolimus pharmacokinetics in the Korean population. METHODS: Twenty-nine healthy Koreans who participated in the previous tacrolimus pharmacokinetic study were genotyped for CYP3A4*1B, CYP3A5*3, MDR1 c.1236C-->T, MDR1 c.2677G-->A/T and MDR1 c.3435C-->T. The relationship between the genotypes so obtained and tacrolimus pharmacokinetics observed in the previous study was examined. RESULTS: No subject in this study had the CYP3A4*1B variant. The observed frequencies of CYP3A5*1/*1, *1/*3, and *3/*3 were 0.069 [confidence interval (CI) -0.023, 0.161], 0.483 (CI 0.301, 0.665) and 0.448 (CI 0.267, 0.629), respectively. AUC(0-infinity) for the CYP3A5*1/*1 or *1/*3 genotype was 131.5 +/- 44.8 ng h ml(-1) (CI 109.6, 153.5), which was much lower compared with the CYP3A5*3/*3 genotype of 323.8 +/- 129.3 ng h ml(-1) (CI 253.5, 394.1) (P = 2.063E-07). Similarly, C(max) for the CYP3A5*1/*1 or *1/*3 genotype was 11.8 +/- 3.4 ng ml(-1) (CI 10.1, 13.5), which was also much lower compared with the CYP3A5*3/*3 genotype of 24.4 +/- 12.3 ng ml(-1) (CI 17.8, 31.1) (P = 0.0001). However, there was no significant difference in tacrolimus pharmacokinetics among the MDR1 diplotypes of CGC-CGC, CGC-TTT, CGC-TGC, TTT-TGC or TTT-TTT (P = 0.2486). CONCLUSIONS: This study shows that the CYP3A5*3 genetic polymorphisms may be associated with the individual difference in tacrolimus pharmacokinetics. An individualized dosage regimen design incorporating such genetic information would help increase clinical efficacy of the drug while reducing adverse drug reactions.     

7-OH-flavone is sulfated in the human liver and duodenum,whereas 5-OH-flavone and 3-OH-flavone are potent inhibitors of SULT1A1 activity and 7-OH-flavone sulfation rate

1. The aim of this investigation was to see whether 7-OH-flavone, 5-OH-flavone and 3-OH-flavone, which are present in edible vegetables, fruit and wine, are substrates or inhibitors of human liver and duodenum sulfotransferase. 2. An assay was set up to study the sulfation of 7-OH-flavone, and using this assay, it was observed that 7-OH-flavone was sulfated and the rate of sulfation (mean +/- SD) was 324 +/- 87 pmol min(-1) mg(-1) (liver) and 584 +/- 164 pmol min(-1) mg(-1) (duodenum; p < 0.0001). 3. 7-OH-flavone sulfotransferase followed Michaelis-Menten kinetics and the K(m) (mean +/- SD) was 0.2 +/- 0.04 microM (liver) and 1.1 +/- 0.3 microM (duodenum; p = 0.008). V(max) (mean +/- SD) was 392 +/- 134 pmol min(-1) mg(-1) (liver) and 815 +/- 233 pmol min(-1) mg(-1) (duodenum; p = 0.016). 4. 5-OH-flavone and 3-OH-flavone were not sulfated and were inhibitors of human liver and duodenum SULT1A1 activity and 7-OH-flavone sulfation rate. 5. The IC50 of 5-OH-flavone for SULT1A1 was 0.3 +/- 0.06 microM (liver) and 0.3 +/- 0.1 microM (duodenum; n.s.) and those of 3-OH-flavone were 1.0 +/- 0.1 microM (liver) and 1.6 +/- 0.03 microM (duodenum; p = 0.0006). 6. There was inhibition of 7-OH-flavone sulfation rate by 5-OH-flavone and 3-OH-flavone. The IC(50) of 5-OH-flavone for the sulfation rate of 7-OH-flavone was 3.5 +/- 0.5 microM (liver) and 69 +/- 18 microM (duodenum; p < 0.0001) and for 3-OH-flavone it was 18 +/- 3.4 microM (liver) and 213 +/- 47 microM (duodenum; p < 0.0001). 7. The position of the hydroxy group confers to the molecules of OH-flavones the quality of substrate or inhibitor of sulfotransferase.     

Inhibitory Effects of Various Beverages on Ritodrine Sulfation by Recombinant Human Sulfotransferase Isoforms SULT1A1 and SULT1A3

Purpose Ritodrine is known to undergo extensive presystemic sulfation in the intestinal mucosa, and its bioavailability is as low as 30%. Accordingly, inhibition of intestinal sulfation may lead to an increase in the bioavailability of ritodrine. In this study, we aimed to investigate the activities of ritodrine sulfation by SULT1A1, which is expressed predominantly in the liver, and SULT1A3, which is expressed predominantly in the intestine, as well as the inhibitory effects of beverages on their activities.Methods We investigated ritodrine sulfation by using recombinant human sulfotransferase (SULT) 1A1 and SULT1A3 in an in vitro study. Next, we investigated the inhibitory effects of grapefruit juice, orange juice, green tea, and black tea on ritodrine sulfation.Results Sulfation of ritodrine by SULT1A3 was much higher than that by SULT1A1, suggesting that the bioavailability of ritodrine may be limited by intestinal SULT1A3. The ritodrine sulfation activities of SULT1A1 and SULT1A3 were significantly inhibited by all beverages examined at a concentration of 10%. Green tea and black tea exhibited potent inhibition; even at a concentration of 5%, they both inhibited SULT1A1 by 100% and SULT1A3 by ≥95%.Conclusion Our results suggest that concomitant ingestion of beverages such as green tea and black tea may increase the bioavailability of orally administered ritodrine, and perhaps other β₂-agonists, and lead to an increase in the clinical effects or adverse reactions.     

Association between cancer risk and drug-metabolizing enzyme gene (CYP2A6, CYP2A13, CYP4B1, SULT1A1, GSTM1, and GSTT1) polymorphisms in cases of lung cancer in Japan

Genetic polymorphisms of enzymes involved in the metabolism of carcinogens are suggested to modify an individual's susceptibility to lung cancer. The purpose of this study was to investigate the relationship between lung cancer cases in Japan and variant alleles of cytochrome P450 (CYP) 2A6 (CYP2A6*4), CYP2A13 (CYP2A13*1-*10), CYP4B1 (CYP4B1*1-*7), sulfotransferase 1A1 (SULT1A1*2), glutathione S-transferase M1 (GSTM1 null), and glutathione S-transferase T1 (GSTT1 null). We investigated the distribution of these polymorphisms in 192 lung cancer patients and in 203 age- and sex-matched cancer-free controls. The polymorphisms were analyzed using various techniques including allele-specific PCR, hybridization probe assay, multiplex PCR, denaturing high-performance liquid chromatography (DHPLC), and direct sequencing. We also investigated allele and genotype frequencies and their association with lung cancer risk, demographic factors, and smoking status. The prevalence of the CYP2A6*4/*4 genotype in lung cancer cases was 3.6%, compared with 9.4% in the controls (adjusted OR = 0.36, 95% CI = 0.15-0.88, P = 0.025). In contrast, there was no association between the known CYP2A13, CYP4B1, SULT1A1, GSTM1, and GSTT1 polymorphisms and lung cancer. These data indicate that CYP2A6 deletions may be associated with lung cancer in the Japanese population studied.     

Sulfation of R(-)-apomorphine in the human liver and duodenum,and its inhibition by mefenamic acid,salicylic acid and quercetin

1. The aims were to study the sulfation of R-(-)-apomorphine (hereafter apomorphine) in the human liver and duodenum, and to study the rate of inhibition of apomorphine sulphation by mefenamic acid, salicylic acid and quercetin also in the human liver and duodenum. 2. A rapid and sensitive method was developed to measure the sulfation rate of apomorphine in the human liver and duodenum. The method was based on the use of 0.4 micro M 3'-phosphoadenosine-5'-phosphosulfate-[(35)S] (PAPS) and 50 micro M apomorphine. The unreacted PAPS was precipitated with barium hydroxide, barium acetate and zinc sulfate. 3. The rate of apomorphine sulfation (mean +/- SD and median) was 261 +/- 82 and 242 pmol min(-1) mg(-1), respectively (liver), and 433 +/- 157 and 443 pmol min(-1) mg(-1), respectively (duodenum). The apomorphine sulfation rate was higher in the duodenum than in the liver (p = 0.0005). 4. Apomorphine sulfation was correlated with SULT1A1 activity in the liver (r(2) = 0.363, p = 0.005) and duodenum (r(2) = 0.494, p = 0.0005), but it did not correlate with SULT1A3 activity both in the liver and duodenum. 5. The K(m) estimate of apomorphine sulfation rate was 20 +/- 3.6 (liver) and 6.5 +/- 0.2 microM (duodenum, p = 0.024), and the V(max) estimate was 248 +/- 99 (liver) and 636 +/- 104 pmol min(-1) mg(-1) (duodenum, p = 0.018). 6. Mefenamic acid, salicylic acid and quercetin were potent inhibitors of apomorphine sulfation rate in the liver, and the IC(50) estimates were 16 +/- 0.2 nM, 54 +/- 8.6 microM and 18 +/- 2.8 nM, respectively. These compounds were poor inhibitors of apomorphine sulfation in the duodenum. 7. Apomorphine is sulfated by the human liver and duodenum, the highest activity being associated with the duodenum. The K(m) of apomorphine sulfotransferase is in the order of micro M both in the liver and duodenum. The non-steroidal anti-inflammatory drug mefenamic acid and the natural flavonoid quercetin inhibit the hepatic sulfation of apomorphine with an IC(50) in the order of nM.     

Involvement of SULT1A3 in elevated sulfation of 4-hydroxypropranolol in Hep G2 cells pretreated with beta-naphthoflavone

Pretreatment of Hep G2 cells with beta-naphthoflavone (BNF 1-25microM) significantly increased cytosolic sulfation activities of 4-hydroxypropranolol (4-OH-PL) racemate. The profile was similar to those of sulfations towards dopamine and triiodothyronine in the same cytosolic fractions. Kinetic studies of 4-OH-PL sulfation in Hep G2 cytosolic fractions revealed that V(max) values increased but apparent K(m) values remained unchanged following the BNF pretreatment. Among five recombinant human SULT isoforms (SULT1A1, -1A3, -1B1, -1E1 and -2A1) examined, only SULT2A1 did not show 4-OH-PL sulfation activities under the conditions used. SULT1A3 and -1E1 exhibited an enantioselectivity of 4-OH-R-PL sulfation>4-OH-S-PL sulfation, which agreed with that of BNF-pretreated Hep G2 cells as well as of nontreated cells, whereas SULT1A1 and -1B1 showed a reversed enantioselectivity (R相似文献   

In vivo metabolic activity of CYP2C19 and CYP3A in relation to CYP2C19 genetic polymorphism in chronic liver disease

To study whether chronic liver disease (CLD) and genetic polymorphism affect the hepatic activity of cytochrome P450 (CYP) isoforms, we compared in vivo CYP2C19 and CYP3A activities using 3-hour omeprazole hydroxylation index (plasma concentration ratio of omeprazole to its 5-hydroxylated metabolite; a higher index indicates lower CYP2C19 activity) and partial formation clearance of cortisol to 6beta-hydroxycortisol (CL(cortisol-->6beta-HC)) in 31 CLD patients (9 with chronic hepatitis; 22 with cirrhosis comprising 20 Child-Pugh type A, 1 type B, and 1 type C) and 30 healthy subjects with different CYP2C19 genotypes. The mean (+/-SEM) omeprazole hydroxylation index in CLD patients with homozygous extensive metabolizer (EM) genotype (*1/*1, n = 8), heterozyous EM (*1/*2, n = 11; *1/*3, n = 6) genotypes and poor metabolizer (PM) genotypes (*2/*2, n = 3; *3/*3, n = 3) were 17.15 +/- 2.12, 20.02 +/- 2.63, and 26.04 +/- 3.15, respectively, which were significantly higher compared with control subjects with the corresponding CYP2C19 genotypes (0.81 +/- 0.09, 1.55 +/- 0.20, and 15.5 +/- 1.52). CLD patients with PM genotype had significantly (P < .05) higher omeprazole hydroxylation indexes than did those with homozygous EM genotype, and those with heterozygous EM genotypes had intermediate values. The mean CL(cortisol-->6beta-HC) decreased significantly (P < .001) in CLD patients compared with control subjects (1.19 +/- 0.12 versus 2.26 +/- 0.24 mL/min). Multiple regression analysis showed that CLD, serum albumin level, and CYP2C19 genotype correlated significantly (P < .05) with the omeprazole hydroxylation index, whereas no significant correlation was observed between CL(cortisol-->6beta-HC) and other variables, except CLD. Because CLD and genetic polymorphism of CYP2C19 act additively to reduce CYP2C19 activity, genotyping these patients may be of value in averting adverse reactions of drugs that depend on CYP2C19 for elimination.