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).     

Genetic polymorphisms and linkage disequilibrium of sulfotransferase SULT1A1 and SULT1A2 in a Korean population: comparison of other ethnic groups

Aims: To determine the allele frequencies of sulfotransferases (SULTs) 1A1 and 1A2 and their linkage disequilibrium in a Korean population and compare them with those of other ethnic groups. Methods: Genotypes of the SULT1A1*1, *2, and *3 and SULT1A2*1, *2, and *3 allelic variants were determined in 234 Korean subjects using polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) methods. Results: Allele frequencies for SULT1A1*1 and *2 were 0.876 [95% confidence interval (CI), 0.843–0.905] and 0.124 (95% CI, 0.096–0.157), respectively. Similarly, those for SULT1A2*1 and *2 were 0.885 (95% CI, 0.852–0.912) and 0.115 (95% CI, 0.088–0.150), respectively. However, no subject with SULT1A1*3 or SULT1A2*3 was detected. These genotype distributions are similar to those of Asian populations including the Chinese and Japanese, but quite different from other ethnic groups such as African-Americans and Caucasians. The expected allelic frequencies of SULT1A1 and SULT1A2 at Hardy–Weinberg equilibrium are quite similar to the observed distributions in the population. SULT1A1*2 and SULT1A2*2, the most common variant alleles of these two genes, are strongly and positively linked in the Korean population (D′=0.8919, χ² =343.24, P=0.0034). Conclusions: SULT1A1*2 and SULT1A2*2 are the major allelic variants in the Korean population, whereas the SULT1A1*3 and SULT1A2*3 alleles were not found. SULT1A1*2 and SULT1A2*2 are strongly linked.     

Sulfotransferase 1A1 genotype as a potential modifier of breast cancer risk among premenopausal women

SULT1A1 is involved in biotransformation of many endogenous and exogenous substrates, such as drugs, hormones and tobacco smoke carcinogens. A polymorphism in the sulfotransferase 1A1 gene (SULT1A1) results in an amino acid change from Arg to His at codon 213. The His allele (SULT1A1*2) has been shown to encode a protein with much lower catalytic activity than the protein encoded by the Arg allele (SULT1A1*1). We examined whether this polymorphism modified breast cancer risk in a Finnish-Caucasian study population consisting of 483 breast cancer patients and 482 healthy population controls. No significant genotype effects were seen in the overall breast cancer risk. However, a decreased risk of breast cancer was found among premenopausal women with at least three pregnancies and at least one SULT1A1*2 allele (odds ratio = 0.23, 95% confidence interval = 0.09-0.63) compared to women with two SULT1A1*1 alleles. Our results suggest that the SULT1A1 genotype is not an important risk factor for breast cancer in general, but may modify the risk among premenopausaul women with high parity.     

Genotype and allele frequencies of TPMT,NAT2, GST,SULT1A1 and MDR-1 in the Egyptian population

AIMS: The goal of this study was to determine the frequencies of important allelic variants in the TPMT, NAT2, GST, SULT1A1 and MDR-1 genes in the Egyptian population and compare them with the frequencies in other ethnic populations. METHODS: Genotyping was carried out in a total of 200 unrelated Egyptian subjects. TPMT*2 was detected using an allele-specific polymerase chain reaction (PCR) assay. TPMT*3C and NAT2 variants (*5,*6 and *7) were detected using an allele-specific real-time PCR assay. Detection of GSTM1 and GSTT1 null alleles was performed simultaneously using a multiplex PCR assay. Finally, a PCR-restriction fragment length polymorphism assay was applied for the determination of TPMT*3A (*3B), SULT1A1*2 and MDR-1 (3435T) variants. RESULTS: Genotyping of TPMT revealed frequencies of 0.003 and 0.013 for TPMT*3A and TPMT*3C, respectively. No TPMT*2 or *3B was detected in the analysed samples. The frequencies of specific NAT2 alleles were 0.215, 0.497, 0.260 and 0.028 for *4 (wild-type), *5 (341C), *6 (590A) and *7 (857A), respectively. GSTM1 and GSTT1 null alleles were detected in 55.5% and 29.5% of the subjects, respectively. SULT1A1*2 was detected at a frequency of 0.135. Finally, the frequencies of the wild-type allele (3435C) and the 3435T variant in the MDR-1 gene were found to be 0.6 and 0.4, respectively. CONCLUSIONS: We found that Egyptians resemble other Caucasians with regard to allelic frequencies of the tested variants of NAT2, GST and MDR-1. By contrast, this Egyptian population more closely resemble Africans with respect to the TPMT*3C allele, and shows a distinctly different frequency with regard to the SULT1A1*2 variant. The predominance of the slow acetylator genotype in the present study (60.50%) could not confirm a previously reported higher frequency of the slow acetylator phenotype in Egyptians (92.00%), indicating the possibility of the presence of other mutations not detectable as T341C, G590A and G857A. The purpose of our future studies is to investigate for new polymorphisms, which could be relatively unique to the Egyptian population.     

Sulfotransferase 1A1 (SULT1A1) polymorphism and breast cancer risk in Chinese women

BACKGROUND: Sulfonation catalyzed by sulfotransferase enzymes plays an important role in chemical defense mechanisms against various xenobiotics but also bioactivates carcinogens. A major human sulfotransferase, SULT1A1, catalyzes the sulfation of a variety of phenolic and estrogenic compounds. A functional polymorphism of the SULT1A1 gene has been implicated in a decreased activity and thermostability when the wild-type arginine (Arg) at codon 213 is substituted by a histidine (His). METHODS: We investigated the association between the His allele and the risk breast cancer in 213 cases and 430 matched controls in Chinese women, and the interaction between His allele and endogenous estrogen and dietary mutagens exposure factors were also determined by use of logistic regression analysis. RESULTS: There was no significant difference in genotypes between the cancer patients and control populations. However, the frequency of the His allele in cases (13.6%) were significant higher than that in controls (9.5%), P = 0.03. Compared with women carrying the Arg/Arg genotype, the adjusted odds ratio (OR) of Arg/His was 1.48 (95% CI = 0.59-3.31) and His/His was 2.28 (95% CI = 0.69-9.58), P trend was 0.04. The adjusted OR of Arg/His + His/His was 2.60 (95% CI = 1.12-6.05). His allele strengthen the effect of endogenous estrogen exposure with interaction index r > 1, and weaken the effect of heterocyclic amines and polycyclic aromatic hydrocarbons derived from dietary with interaction index r > 1, both were multiplicative interaction model. CONCLUSIONS: Our findings suggest that the SULT1A1 His allele was positively associated with the risk of breast cancer in Chinese women. And there was interaction between SULT1A1 polymorphism and related exposure factors.     

Functional analysis of six different polymorphic CYP1B1 enzyme variants found in an Ethiopian population

Cytochrome P450 1B1 (CYP1B1) is an extrahepatic enzyme of potential importance for the metabolism of estrogen and for metabolic activation of environmental carcinogens. We investigated an Ethiopian population for functional polymorphisms in the CYP1B1 gene using genomic DNA sequencing and detected three novel single nucleotide polymorphisms (SNPs). One of these (4360C-->G in exon 3) is present at a frequency of 7% and causes an Ala443Gly amino acid substitution. In addition, the four described previously missense mutations Arg48Gly, Ala119Ser, Leu432Val, and Asn453Ser were found with frequencies of 51, 50, 53, and 2%, respectively, yielding a total of 32 possible CYP1B1 haplotypes. Allele-specific PCR methods for haplotype analysis were developed and seven different CYP1B1 alleles were found: CYP1B1*1, *2, *3, *4, *5, *6, and *7 with frequencies of 8, 37, 39, 2, 0.7, 6, and 7%, respectively. The functional properties of different forms of CYP1B1, as well as of the Leu432Val + Asn453Ser and Leu432Val + Ala443Gly variants, were evaluated after heterologous expression of the corresponding cDNAs in Saccaromyces cerevisiae. The results revealed that CYP1B1.6 and CYP1B1.7, having the amino acid substitutions Arg48Gly, Ala119Ser, and Leu432Val in common, exhibited altered kinetics with significantly increased apparent K(m) and lowered V(max) values for both the 2- and 4-hydroxylation of 17 beta-estradiol, whereas the other constructs were indistinguishable from the CYP1B1.1 enzyme. The results emphasize the necessity of a complete haplotype analysis of enzyme variants for evaluation of functional consequences in vivo and for analyses of genetic polymorphisms in relation to, for example, cancer incidence.     

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.     

Functional analysis of three CYP1A2 variants found in a Japanese population.

Human cytochrome P450 1A2 (CYP1A2) catalyzes the metabolism of many important drugs and environmental chemicals. We previously reported three naturally occurring genetic polymorphisms (125C>G, Pro42Arg, CYP1A2*15; 1130G>A, Arg377Gln, *16; and 1367G>A, Arg456His, *8) found in a Japanese population. In this study, these variant enzymes were expressed in Chinese hamster V79 cells, and their mRNA and protein expression levels as well as catalytic activities were determined. All three variant enzymes showed reduced protein expression levels (66% for Pro42Arg and approximately 30% for Arg377Gln and Arg456His) compared with that of the wild type (WT) without any change in mRNA expression levels. Kinetic analysis for 7-ethoxyresorufin O-deethylation revealed that V(max) and V(max)/K(m) of all three variants were less than 3 and 1% of the WT, respectively, although the K(m) value was significantly increased only in the Arg377Gln variant (approximately a 9-fold increase). Markedly reduced activities of the three variants were also observed for phenacetin O-deethylation. In the reduced CO difference spectral analysis using recombinant proteins produced in the Sf21/baculovirus system, the peak at 450 nm seen in the WT protein was hardly observed in the three variants, suggesting marked reductions in their hemoprotein formation. These results suggest that Pro42, Arg377, and Arg456 are critical amino acids for the production of catalytically active CYP1A2 holoenzyme.     

Sulfation pharmacogenetics: SULT1A1 and SULT1A2 allele frequencies in Caucasian, Chinese and African-American subjects

Sulfotransferase (SULT) enzymes catalyze the sulfate conjugation of drugs, other xenobiotics, neurotransmitters and hormones. The genes for SULT1A1 and SULT1A2 contain common genetic polymorphisms that are associated with individual variations in levels of enzyme activity as well as variations in biochemical and physical properties. We set out to compare the frequencies of common SULT1A1 and SULT1A2 alleles in Caucasian, Chinese and African-American subjects. Allele frequencies for SULT1A1*1, *2 and *3 in 242 Caucasian subjects were 0.656, 0.332 and 0.012, respectively. Frequencies of those same alleles were significantly different in 290 Chinese subjects: 0.914, 0.080 and 0.006, respectively, as were frequencies in 70 African-American subjects: 0.477, 0.294 and 0.229, respectively. Ethnic variation in allele frequencies was also observed for SULT1A2, with frequencies in Caucasian subjects for SULT1A2*1, *2 and *3 of 0.507, 0.389 and 0.104; frequencies in Chinese of 0.924 and 0.076 with no *3 alleles observed; and, finally, in African-Americans frequencies of 0.637, 0.249 and 0.114, respectively. We also found that SULT1A1*2 and SULT1A2*2, the most common variant alleles for these two genes, were in positive linkage disequilibrium in all three populations studied, with D' values of 0.776 in Caucasian (P < 0.001), 0.915 in Chinese (P < 0.001) and 0.864 in African-American subjects (P < 0.001). These observations represent a step towards determining the possible functional implications for individual variations in sulfate conjugation of common genetic polymorphisms for SULT1A1 and SULT1A2.     

Phenotype-genotype relationships of SULT1A1 in human liver and variations in the IC50 of the SULT1A1 inhibitor quercetin

Human sulfotransferases catalyze sulfate conjugation and 2 polymorphic genes, SULT1A1 and SULT1A2 in this family of transferases have been identified, encoding for 2 isoenzymes with very similar properties and substrate specificities. In order to test the hypothesis that variability in sulfation is due to genetic polymorphism in SULT1A1, the sulfation rate of 4-nitrophenol, a diagnostic substrate, was measured in 50 human liver samples and the genotype at the SULT1A1 locus was analyzed. The rate of 4-nitrophenol sulfation varied from 473 - 1,405 pmol/min/mg between the 5th and 95th percentiles, with a median and a mean +/- SD of 757 and 807 +/- 292 pmol/min/mg, respectively. The activities detected among the SULT1A1*2/*2 homozygotes (5 cases) were significantly lower than those of the other 2 genotypes, SULTA1*11/*1 and SULT1A1*1/*2 (5 and 40 cases, respectively), whereas there was no significant difference found between the SULT1A1*1/*1 and SULT1A1*1/*2 genotypes. To evaluate the possible influence of SULT1A2 polymorphism, genotype assays were also performed for this locus. No SULT1A2*2/*2 carrier, 26 SULT1A2*1/*1 and 24 SULT1A2*1/*2 were detected in the population sample under study. However, no correlation between the rate of 4-nitrophenol sulfation and the SULT1A2 genotype was detected. These results confirm that the variation in the rate of 4-nitrophenol sulfation in human liver is mainly due to SULT1A. Since SULT1A1*1/*2 polymorphism accounts for no more than 10% of the phenotypic variation seen in this cohort, other factors must also contribute to the variability in the rate of 4-nitrophenol sulfation in human liver. However, on the basis of the data obtained, variations in age, gender and liver function as possible causative factors can be excluded. The IC50 of quercetin, a potent inhibitor of 4-nitrophenol sulfation, was measured in the liver samples and ranged from 4.6 to 17.3 nM between the 5th and 95th percentiles. The median and the mean +/- SD were 7.7 nM and 8.3 +/- 2.5 nM, respectively. There was a weak but significant correlation between the IC50 value and age of the liver donors (r = 0.283, p = 0.046). The observed variation did not correlate with the genotypes at the SULT1A1 and SULT1A2 loci.     

Phenol sulphotransferase SULT1A1 polymorphism in prostate cancer: lack of association

Preliminary evidence suggests that genetic polymorphisms in certain enzymes involved in xenobiotic metabolism and chemical defense could modify a susceptibility to prostate cancer. In the present study, two recently described phenol sulphotransferase SULT1A1 alleles (SULT1A1*1, SULT1A1*2) were investigated using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach. Genotyping was performed on DNA isolated from white blood cells from 134 patients with prostate cancer and 184 healthy control subjects. Both the prostate cancer patients and the controls demonstrated similar frequencies of the variant allele SULT1A1*2 (35.1% vs 39.1%). Homozygosity for the variant allele was slightly less frequent in cancer patients than controls (12.7% vs 17.4%). Our study does not support the hypothesis that the phenol sulphotransferase variant allele SULT1A1*2 with a G/A transition at nucleotide 638 is a risk modifier for prostate cancer in the Caucasian population.     

Phenol sulphotransferase SULT1A1*1 genotype is associated with reduced risk of colorectal cancer.

Sulphation is an important detoxification pathway for numerous xenobiotics; however, it also plays an important role in the metabolism and bioactivation of many dietary and environmental mutagens, including heterocyclic amines implicated in the pathogenesis of colorectal and other cancers. A major sulphotransferase (SULT) enzyme in humans, SULT1A1, is polymorphic with the most common variant allele, SULT1A1*2, occurring at a frequency of about 32% in the Caucasian population. This allele codes for an allozyme with low enzyme activity and stability compared to the wild-type (SULT1A1*1) enzyme, and therefore SULT1A1 genotype may influence susceptibility to mutagenicity following exposure to heterocyclic amines and other environmental toxins. Previously, a significant association of SULT1A1*1 genotype with old age has been observed, suggesting a 'chemoprotective' role for the high-activity phenotype. Here we have compared the frequencies of the most common SULT1A1 alleles in 226 colorectal cancer patients and 293 previously described control patients. We also assessed whether SULT1A1 genotype was related to various clinical parameters in the patient group, including Duke's classification, differentiation, site, nodal involvement and survival. There was no significant difference in allele frequency between the control and cancer patient populations, nor was there a significant association with any of the clinical parameters studied. However, when the age-related difference in allele frequency was considered, a significantly reduced risk of colorectal cancer (odds ratio = 0.47; 95% confidence interval = 0.27-0.83; P = 0.009), was associated with homozygosity for SULT1A1*1 in subjects under the age of 80 years. These results suggest that the high activity SULT1A1*1 allozyme protects against dietary and/or environmental chemicals involved in the pathogenesis of colorectal cancer.     

Sulfotransferase (SULT) 1A1 polymorphic variants *1, *2, and *3 are associated with altered enzymatic activity, cellular phenotype, and protein degradation

The superfamily of sulfotransferase (SULT) enzymes catalyzes the sulfate conjugation of several pharmacologically important endo- and xenobiotics. SULT1A1 catalyzes the sulfation of small planar phenols such as neurotransmitters, steroid hormones, acetaminophen, and p-nitrophenol (PNP). Genetic polymorphisms in the human SULT1A1 gene define three alleles, SULT1A1*1, *2, and *3. The enzyme activities of the SULT1A1 allozymes were studied with a variety of substrates, including PNP, 17beta-estradiol, 2-methoxyestradiol, catecholestrogens, the antiestrogen 4-hydroxytamoxifen (OHT), and dietary flavonoids. Using purified recombinant SULT1A1 protein, marked differences in *1, *2, and *3 activity toward every substrate studied were noted. Substrate inhibition was observed for most substrates. In general, the trend in V(max) estimates was *1 > *3 > *2; however, V(max)/K(m) estimate trends varied with substrate. In MCF-7 cells stably expressing either SULT1A1*1 or *2, the antiestrogenic response to OHT was found to be allele-specific: the cells expressing *2 exhibited a better antiproliferative response. The intracellular stability of the *1 and *2 allozymes was examined in insect as well as mammalian cells. The SULT1A1*2 protein had a shorter half-life than the *1 protein. In addition, the *2 protein was ubiquitinated to a greater extent than *1, suggesting increased degradation via a proteasome pathway. The results of this study suggest marked differences in activity of polymorphic SULT1A1 variants, including SULT1A1*3, toward a variety of substrates. These differences are potentially critical for understanding interindividual variability in drug response and toxicity, as well as cancer risk and incidence.     

Functional characterization of human xanthine oxidase allelic variants

OBJECTIVE: Xanthine oxidase (XO) catalyzes the oxidation of endogenous and exogenous purines and pyrimidines. In this study, we speculated that individual variations in XO activity are caused by genetic variations in the XO gene. METHODS: To investigate the genetic variations in XO in 96 Japanese participants, denaturing high-performance liquid chromatography was used. To assess the effects of these variations on enzymatic activity, wild-type XO and 21 types of variant XO--including those in the database and those just discovered--were transiently expressed in COS-7 cells. RESULTS: Three nonsynonymous single nucleotide polymorphisms, including 514G>A (Gly172Arg), 3326A>C (Asp1109Thr), and 3662A>G (His1221Arg) were identified in Japanese participants. Functional characterization of 21 XO variants showed a deficiency in enzyme activity in two variants (Arg149Cys and Thr910Lys); low activity (intrinsic clearance, CLint: 22-69% compared with the wild-type) in six variants (Pro555Ser, Arg607Gln, Thr623Ile, Asn909Lys, Pro1150Arg, and Cys1318Tyr); and high activity (CLint: approximately two-fold higher than that in the wild-type) in two variants (Ile703Val and His1221Arg). CONCLUSION: These results suggest that several single nucleotide polymorphisms in the XO gene are involved in individual variations in XO activity. In addition, such findings will be useful to identify xanthinuria patients.     

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.     

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.     

Identification and characterization of two amino acids critical for the substrate inhibition of human dehydroepiandrosterone sulfotransferase (SULT2A1)

Substrate inhibition is a characteristic feature of many cytosolic sulfotransferases. The differences between the complex structures of SULT2A1/DHEA and SULT2A1/PAP or SULT2A1/ADT (Protein Data Bank codes are 1J99, 1EFH, and 1OV4, respectively) have enabled us to elucidate the specific amino acids responsible for substrate inhibition. Based on the structural analyses, substitution of the smaller residue alanine for Tyr-238 (Y238A) significantly increases the K(i) value for dehydroepiandrosterone (DHEA) and totally eliminates substrate inhibition for androsterone (ADT). In addition, Met-137 was proposed to regulate the binding orientations of DHEA and ADT in SULT2A1. Complete elimination or regeneration of substrate inhibition for SULT2A1 with DHEA or ADT as substrate, respectively, was demonstrated with the mutations of Met-137 on Y238A mutant. Analysis of the Met-137 mutants and Met-137/Tyr-238 double mutants uncovered the relationship between substrate binding orientations and inhibition in SULT2A1. Our data indicate that, in the substrate inhibition mode, Tyr-238 regulates the release of bound substrate, and Met-137 controls substrate binding orientation of DHEA and ADT in SULT2A1. The proposed substrate inhibition mechanism is further confirmed by the crystal structures of SULT2A1 mutants at Met-137. We propose that both substrate binding orientations exhibited substrate inhibition. In addition, a corresponding residue in other cytosolic sulfotransferases was shown to have a function similar to that of Tyr-238 in SULT2A1.     

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.     

A series of simple detection systems for genetic variants of flavin-containing monooxygenase 3 (FMO3) with impaired function in Japanese subjects

Increasing numbers of single-nucleotide substitutions of the human flavin-containing monooxygenase 3 (FMO3) gene are being recorded in mega-databases. Phenotype–gene analyses revealed impaired FMO3 variants associated with the metabolic disorder trimethylaminuria. Here, a series of reliable FMO3 genotyping confirmation methods was assembled and developed for 45 impaired FMO3 variants, mainly found in Japanese populations, using singleplex or duplex polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) methods and singleplex, duplex, or tetraplex allele-specific PCR methods. Nine PCR-RFLP procedures with single restriction enzymes and fourteen duplex PCR-RFLP procedures (for p.Trp41Ter and p.Thr329Ala, p.Met66Val and p.Leu163Pro, p.Pro70Leu and p.Glu308Gly, p.Asn114Ser and p.Ser195Leu, p.Glu158Lys and p.Ile441Thr, p.Cys197Ter and p.Trp388Ter, p.Arg205Cys and p.Val257Met, p.Arg205His and p.Cys397Ser, p.Met211ArgfsTer10 and p.Arg492Trp, p.Arg223Gln and p.Leu473Pro, p.Met260Val and p.Thr488Ala, p.Tyr269His and p.Ala311Pro, p.Ser310Leu and p.Gly376Glu, and p.Gln470Ter and p.Arg500Ter) were newly established along with eight singleplex (for p.Pro153GlnfsTer14, p.Gly191Cys, p.Pro248Thr, p.Ile486Met, and p.Pro496Ser, among others), one duplex (p.Ile199Ser and p.Asp286Tyr), and one tetraplex (p.Ile7Thr, p.Val58Ile, p.Thr201Lys, and p.Gly421Val) allele-specific PCR systems. This series of systems should facilitate the easy detection in a clinical setting of FMO3 variants in Japanese subjects susceptible to low drug clearances or drug reactions possibly caused by impaired FMO3 function.     

Cloning of CYP2J2 gene and identification of functional polymorphisms

CYP2J2 is abundant in cardiovascular tissue and active in the metabolism of arachidonic acid to eicosanoids that possess potent anti-inflammatory, vasodilatory, and fibrinolytic properties. We cloned and sequenced the entire CYP2J2 gene (approximately 40.3 kb), which contains nine exons and eight introns. We then sequenced the CYP2J2 exons and intron-exon boundaries in 72 healthy persons representing African, Asian, and European/white populations as part of the National Institutes of Health/National Institute of Environmental Health Sciences Environmental Genome Single Nucleotide Polymorphism Program. A variety of polymorphisms were found, four of which resulted in coding changes (Arg158Cys, Ile192Asn, Asp342Asn, and Asn404Tyr). A fifth variant (Thr143Ala) was identified by screening a human heart cDNA library. All five variant cDNAs of CYP2J2 were generated by site-directed mutagenesis and expressed in Sf9 insect cells by using a baculovirus system. The recombinant wild-type and variant CYP2J2 proteins immunoreacted with peptide-based antibodies to CYP2J2 and displayed typical cytochrome P450 (P450) CO-difference spectra; however, the Asn404Tyr and Ile192Asn variants also had prominent spectral peaks at 420 nm. The ability of these variants to metabolize arachidonic acid and linoleic acid was compared with that of wild-type CYP2J2. Three variants (Asn404Tyr, Arg158Cys, and Thr143Ala) showed significantly reduced metabolism of both arachidonic acid and linoleic acid. The Ile192Asn variant showed significantly reduced activity toward arachidonic acid only. The Asp342Asn variant showed similar metabolism to wild-type CYP2J2 for both endogenous substrates. Based on these data, we conclude that allelic variants of the human CYP2J2 gene exist and that some of these variants result in a P450 protein that has reduced catalytic function. Insofar as CYP2J2 products have effects in the cardiovascular system, we speculate that these variants may be functionally relevant.