RESUMEN
Bacterial aryl sulfotransferases (ASSTs) catalyze sulfotransfer from a phenolic sulfate to a phenol. These enzymes are frequently found in pathogens and upregulated during infection. Their mechanistic understanding is very limited, and their natural substrates are unknown. Here, the crystal structures of Escherichia coli CFT073 ASST trapped in its presulfurylation state with model donor substrates bound in the active site are reported, which reveal the molecular interactions governing substrate recognition. Furthermore, spectroscopic titrations with donor substrates and sulfurylation kinetics of ASST illustrate that this enzyme binds substrates in a 1:1 stoichiometry and that the active sites of the ASST homooligomer act independently. Mass spectrometry and crystallographic experiments of ASST incubated with human urine demonstrate that urine contains a sulfuryl donor substrate. In addition, we examined the capability of the two paralogous dithiol oxidases present in uropathogenic E. coli CFT073, DsbA, and the ASST-specific enzyme DsbL, to introduce the single, conserved disulfide bond into ASST. We show that DsbA and DsbL introduce the disulfide bond into unfolded ASST at similar rates. Hence, a chaperone effect of DsbL, not present in DsbA, appears to be responsible for the dependence of efficient ASST folding on DsbL in vivo. The conservation of paralogous dithiol oxidases with different substrate specificities in certain bacterial strains may therefore be a consequence of the complex folding pathways of their substrate proteins.
Asunto(s)
Arilsulfotransferasa/química , Proteínas de Escherichia coli/química , Oxidorreductasas/química , Proteína Disulfuro Isomerasas/química , Secuencia de Aminoácidos , Arilsulfotransferasa/fisiología , Catálisis , Cristalografía por Rayos X , Disulfuros/química , Proteínas de Escherichia coli/fisiología , Humanos , Datos de Secuencia Molecular , Oxidorreductasas/fisiología , Proteína Disulfuro Isomerasas/fisiología , Pliegue de Proteína , Especificidad por Sustrato , Difracción de Rayos X/métodosRESUMEN
Benzene is a ubiquitous environmental pollutant and a confirmed human carcinogen, which requires metabolic activation, primarily by CYP2E1, for most of its biological actions. Chromosome damages in benzene-exposed workers and rodents have been observed, and in their urine sulfo- and glucuronide-conjugates of phenol and hydroquinone were present. Yet, direct evidence for human CYP2E1-activated mutagenicity of benzene and the exact significance of phase II metabolism for inactivating benzene metabolites are still missing. In the present study, benzene and its oxidized metabolites (phenol, hydroquinone, catechol, 1,2,4-trihydroxybenzene and 1,4-benzoquinone) were investigated for induction of micronuclei in a V79-derived cell line genetically engineered for expression of both human CYP2E1 and human sulfotransferase (SULT) 1A1 (indicated by active micronuclei induction by 1-hydroxymethylpyrene). The results demonstrated concentration-dependent induction of micronuclei by benzene and phenol, though with lower potency or efficacy than the other metabolites. Inhibition of CYP2E1 by 1-aminobenzotriazole did not change the effect of benzoquinone, but completely abolished that of benzene and phenol, and attenuated that of the other compounds. Moreover, inhibition of SULT1A1 by pentachlorophenol potentiated the effects of benzene, hydroquinone, catechol and trihydroxybenzene. Ascorbic acid, a reducing and free radical-scavenging agent, significantly lowered the effects of hydroquinone, catechol, trihydroxybenzene as well as N-nitrosodimethylamine (a known CYP2E1-dependent promutagen), with that of benzoquinone unaffected. These results suggest that in addition to activating benzene and phenol, human CYP2E1 may further convert hydroquinone, catechol and trihydroxybenzene to more genotoxic metabolites, and sulfo-conjugation of the multi-hydroxylated metabolites of benzene by human SULT1A1 may represent an important detoxifying pathway.
Asunto(s)
Arilsulfotransferasa/fisiología , Derivados del Benceno/toxicidad , Benceno/toxicidad , Citocromo P-450 CYP2E1/fisiología , Micronúcleos con Defecto Cromosómico , Animales , Benceno/metabolismo , Derivados del Benceno/metabolismo , Catecoles/metabolismo , Catecoles/toxicidad , Línea Celular , Cricetinae , Cricetulus , Citocromo P-450 CYP2E1/metabolismo , Humanos , Hidroquinonas/metabolismo , Hidroquinonas/toxicidad , Radical Hidroxilo/metabolismo , Hidroxilación , Inactivación Metabólica/genética , Micronúcleos con Defecto Cromosómico/inducido químicamente , Pruebas de MicronúcleosRESUMEN
Food-drug interaction studies are critical to evaluate appropriate dosing, timing, and formulation of new drug candidates. These interactions often reflect prandial-associated changes in the extent and/or rate of systemic drug exposure. Physiologic and physicochemical mechanisms underlying food effects on drug disposition are well-characterized. However, biochemical mechanisms involving drug metabolizing enzymes and transport proteins remain underexplored. Several plant-derived beverages have been shown to modulate enzymes and transporters in the intestine, leading to altered pharmacokinetic (PK) and potentially negative pharmacodynamic (PD) outcomes. Commonly consumed fruit juices, teas, and alcoholic drinks contain phytochemicals that inhibit intestinal cytochrome P450 and phase II conjugation enzymes, as well as uptake and efflux transport proteins. Whereas myriad phytochemicals have been shown to inhibit these processes in vitro, translation to the clinic has been deemed insignificant or undetermined. An overlooked prerequisite for elucidating food effects on drug PK is thorough knowledge of causative bioactive ingredients. Substantial variability in bioactive ingredient composition and activity of a given dietary substance poses a challenge in conducting robust food-drug interaction studies. This confounding factor can be addressed by identifying and characterizing specific components, which could be used as marker compounds to improve clinical trial design and quantitatively predict food effects. Interpretation and integration of data from in vitro, in vivo, and in silico studies require collaborative expertise from multiple disciplines, from botany to clinical pharmacology (i.e., plant to patient). Development of more systematic methods and guidelines is needed to address the general lack of information on examining drug-dietary substance interactions prospectively.
Asunto(s)
Interacciones Alimento-Droga , Mucosa Intestinal/metabolismo , Animales , Arilsulfotransferasa/fisiología , Bebidas , Transporte Biológico , Citrus paradisi , Citocromo P-450 CYP3A/fisiología , Glucuronosiltransferasa/fisiología , HumanosRESUMEN
Soluble sulfotransferases (SULTs) generate electrophilically reactive metabolites from numerous food-borne compounds, environmental contaminants and drugs, often resulting in mutagenicity and carcinogenicity. Substrate specificity, regulation and tissue distribution of SULTs show large interspecies differences. In humans, therefore, SULTs may be involved in the induction of cancer in different tissues than in standard animal models. To construct a rodent model taking some species differences into account, we transferred a 68.5 kb human (h) genomic sequence that comprised the transcribed and long flanking regions of SULT1A1 and 1A2 into murine oocytes. This approach resulted in several mouse lines expressing these human genes in a copy number-dependent manner with a tissue distribution similar to that in humans. In previous in vitro studies, we had demonstrated that human SULT1A1 and 1A2 efficiently catalyze the terminal activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to a mutagen. The transgenic mice were used to study the hSULT1A1/1A2-mediated activation. Tissue distribution and levels of DNA adducts were determined in hSULT1A1/1A2 transgenic and wild-type mice after an oral dosage of PhIP. Transgenic mice exhibited significantly elevated PhIP-DNA adduct levels compared with the wild-type in liver (13-fold), lung (3.8-fold), colon (2-fold), kidney (1.6-fold) and cecum (1.5-fold). Moreover, among the eight tissues examined, liver was the one with the lowest and highest adduct levels in wild-type and transgenic mice, respectively. Hence, expression of hSULT1A1/1A2 not only enhanced the genotoxicity but also substantially changed the organotropism of PhIP.
Asunto(s)
Arilsulfotransferasa/fisiología , Aductos de ADN/metabolismo , Imidazoles/metabolismo , Animales , Daño del ADN , Femenino , Dosificación de Gen , Genotipo , Humanos , Immunoblotting , Masculino , Ratones , Ratones Transgénicos , Distribución TisularRESUMEN
5-Hydroxymethylfurfural (HMF), a heat-induced food toxicant present in a vast number of food items, has been suggested to be genotoxic after being bioactivated by the sulfotransferase SULT1A1. The comet assay was used to evaluate the DNA damaging effect of HMF in cell lines with different activities of SULT1A1: two human cell lines (Caco-2, low activity; and HEK293, higher activity), one cell line from mouse (L5178Y, no activity) and two cell lines from Chinese hamster (V79, negligible activity; and V79-hP-PST, high activity of human SULT1A1). HMF induced significant DNA damage in all cell lines after 3 h exposure to 100 mM. Most sensitive were V79 and V79-hP-PST where HMF induced significant DNA damage at 25 mM. Consequently, in the present study we have shown that HMF is a DNA damaging agent in vitro independent of the activity of SULT1A1 in the cells. The HMF-induced DNA damage was only observed at rather high concentrations which usually was associated with a concomitant decrease in cell viability.
Asunto(s)
Arilsulfotransferasa/fisiología , Daño del ADN , Furaldehído/análogos & derivados , Animales , Células CACO-2 , Ensayo Cometa , Cricetinae , Cricetulus , Furaldehído/toxicidad , Humanos , RatonesRESUMEN
Sulfation, catalysed by members of the cytosolic sulfotransferase (SULT) enzyme family, is important in xenobiotic detoxification and in the biosynthesis and homeostasis of many hormones and neurotransmitters. The major human phenol sulfotransferase SULT1A1 plays a key role in chemical defence, is widely expressed in the body and is subject to a common polymorphism that results in reduced protein levels. Study of these enzymes in vitro requires robust probe substrates, and we have previously shown measurement of activity with the widely used SULT1A1 substrate, 4-nitrophenol, does not accurately reflect protein expression. Additionally, the high degree of substrate inhibition observed with this compound further reduces its value as a probe for SULT1A1. Here we show that 2-aminophenol is a more suitable probe substrate for quantifying SULT1A1 activity in human liver. This compound is sulfated at a high rate (V(max) with purified recombinant SULT1A1=121nmol/(minmg) and shows strong affinity for the enzyme (K(m) with purified recombinant SULT1A1=9microM) and, importantly, is a very poor substrate for the other major SULT1 enzyme expressed in liver, SULT1B1 (with V(max) and K(m) values of 17nmol/(minmg) and 114microM, respectively). Experiments with purified recombinant human SULTs and a panel of 28 human liver cytosols demonstrated that 2-aminophenol shows limited substrate inhibition with SULT1A1, and V(max) values measured in liver cytosols correlated strongly with SULT1A1 enzyme protein levels measured by a quantitative immunoblot method. We therefore suggest that 2-aminophenol is a suitable substrate to use for quantifying SULT1A1 enzyme activity.
Asunto(s)
Aminofenoles/metabolismo , Arilsulfotransferasa/fisiología , Hígado/enzimología , Citosol/metabolismo , HumanosRESUMEN
Epidemiologic evidence indicates that exposure to heterocyclic amines in the diet is an important risk factor for the development of colon cancer. Well-done cooked meats contain significant levels of heterocyclic amines, which have been shown to cause cancer in laboratory animals. To better understand the mechanisms of heterocyclic amine bioactivation in humans, the most mass abundant heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was used to assess the relationship between PhIP metabolism and DNA adduct formation. Ten human volunteers where administered a dietary relevant dose of [(14)C]PhIP 48 to 72 hours before surgery to remove colon tumors. Urine was collected for 24 hours after dosing for metabolite analysis, and DNA was extracted from colon tissue and analyzed by accelerator mass spectrometry for DNA adducts. All 10 subjects were phenotyped for cytochrome P4501A2 (CYP1A2), N-acetyltransferase 2, and sulfotransferase 1A1 enzyme activity. Twelve PhIP metabolites were detected in the urine samples. The most abundant metabolite in all volunteers was N-hydroxy-PhIP-N(2)-glucuronide. Metabolite levels varied significantly between the volunteers. Interindividual differences in colon DNA adducts levels were observed between each individual. The data showed that individuals with a rapid CYP1A2 phenotype and high levels of urinary N-hydroxy-PhIP-N(2)-glucuronide had the lowest level of colon PhIP-DNA adducts. This suggests that glucuronidation plays a significant role in detoxifying N-hydroxy-PhIP. The levels of urinary N-hydroxy-PhIP-N(2)-glucuronide were negatively correlated to colon DNA adduct levels. Although it is difficult to make definite conclusions from a small data set, the results from this pilot study have encouraged further investigations using a much larger study group.
Asunto(s)
Carcinógenos/metabolismo , Colon/metabolismo , Aductos de ADN/orina , Imidazoles/metabolismo , Arilamina N-Acetiltransferasa/fisiología , Arilsulfotransferasa/fisiología , Citocromo P-450 CYP1A2/fisiología , Glucuronosiltransferasa/fisiología , HumanosRESUMEN
Aminoflavone (AF) is entering clinical trials. We recently reported that AF induces DNA-protein cross-links (DPC) and gamma-H2AX in MCF-7 human breast cancer cells. To elucidate the mechanism of action of AF and provide biomarkers indicative of AF activity, we correlated AF activity profile (GI(50)) with gene expression patterns in the NCI-60 cell lines. Sulfotransferases (SULT) showed the highest positive correlation coefficients among approximately 14,000 probe sets analyzed (r = 0.537, P < 0.001). Stable transfection of SULT1A1 into AF-resistant MDA-MB-231 cells sensitized these cells to AF. AF produced DPCs, gamma-H2AX foci, and S-phase arrest in the SULT1A1-transfected but not in the parent MDA-MB-231 cells. Conversely, cells in which SULT1A1 was knocked down by small interfering RNA failed to induce gamma-H2AX. Inhibition of SULTs and cytochrome P450 (CYP) enzymes by natural flavonoids blocked the antiproliferative activity of AF and the formation of AF-DNA adducts. AF also induces SULT1A1 and CYP expression in MCF-7 cells, suggesting the existence of an aryl hydrocarbon receptor-mediated positive feedback for AF activation by CYP and SULT1A1. Metabolism studies showed that AF can be oxidized by CYP at two amino groups to form N-hydroxyl metabolites that are substrates for bioactivation by SULTs. We propose that both N-sulfoxy-groups can be further converted to nitrenium ions that form adducts with DNA and proteins. The results reported here show the importance of SULT1A1 and CYP for AF activation and anticancer activity. They also suggest using SULT1A1 and gamma-H2AX as biomarkers for prediction of AF activity during patient selection and monitoring of clinical trials.
Asunto(s)
Arilsulfotransferasa/fisiología , Reactivos de Enlaces Cruzados/farmacocinética , Flavonoides/farmacocinética , Histonas/biosíntesis , Proteínas de Neoplasias/biosíntesis , Profármacos/farmacocinética , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Hidrocarburo de Aril Hidroxilasas , Arilsulfotransferasa/antagonistas & inhibidores , Arilsulfotransferasa/genética , Biotransformación , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Reactivos de Enlaces Cruzados/farmacología , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1 , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Aductos de ADN , ADN de Neoplasias/efectos de los fármacos , Resistencia a Antineoplásicos , Retroalimentación Fisiológica , Femenino , Flavonoides/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histonas/genética , Humanos , Microsomas Hepáticos/enzimología , Proteínas de Neoplasias/genética , Neoplasias/enzimología , Neoplasias/patología , Profármacos/farmacología , ARN Interferente Pequeño/farmacología , Receptores de Hidrocarburo de Aril/fisiología , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
The biologically active melatonin metabolite, 6-hydroxymelatonin (6-OHMel), is conjugated to form 6-hydroxymelatonin sulfate (6-OHMelS). To elucidate the role of the sulfotransferase (SULT) enzyme 1A1, considerably expressed in normal and malignant human breast cells, we measured the formation of 6-OHMelS by ELISA in hormone-dependent MCF-7 and hormone-independent MDA-MB231 (MDA) breast cancer cell lines after stable transfection with SULT1A1. In parent MDA cells, low SULT1A1 mRNA expression was associated with moderate 6-OHMelS formation as determined after application (24 hr) of 0.1 microM 6-OHMel. As expected, overexpression of SULT1A1 in MDA cells resulted in a 2.9- and 110-fold increase in 6-OHMelS in the cytosol and cellular supernatant respectively. Furthermore, 6.3- and 115-fold increases were observed after 0.5 microM, and 12.6- and 101-fold increases after 1 microM 6-OHMel respectively. In MCF-7 cells, because of high basal SULT1A1 expression, only two- to threefold increases in 6-OHMelS were observed after transfection with the enzyme. In total, 866 and 539 pmol/mg protein 6-OHMelS were formed from 1 microM 6-OHMel in SULT1A1 overexpressing MDA and MCF-7 cells, respectively, whereas application of 1 microM melatonin produced only <1% of 6-OHMelS. Possible interactions with the SULT1A1 substrate tamoxifen (tam), an anti-estrogen applied in the therapy of breast cancer, were also studied. A concentration of 1 microM tam increased 6-OHMelS formation by approximately threefold in the presence of 1 microM melatonin or 1 microM 6-OHMel respectively. However, no alterations were detected after application of 1 microM 4-hydroxy-tamoxifen. In summary, we demonstrate the importance of SULT1A1 for the biotransformation of 6-OHMel in human breast cancer cells. Our data further suggest that tam can modulate melatonin biotransformation.
Asunto(s)
Arilsulfotransferasa/fisiología , Neoplasias de la Mama/enzimología , Melatonina/farmacocinética , Antineoplásicos Hormonales/farmacología , Arilsulfotransferasa/genética , Biotransformación , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Citosol/metabolismo , Femenino , Humanos , Melatonina/análogos & derivados , Melatonina/biosíntesis , ARN Mensajero/metabolismo , Tamoxifeno/farmacología , TransfecciónRESUMEN
BACKGROUND: Blocks of duplicated genomic DNA sequence longer than 1000 base pairs are known as low copy repeats (LCRs). Identified by their sequence similarity, LCRs are abundant in the human genome, and are interesting because they may represent recent adaptive events, or potential future adaptive opportunities within the human lineage. Sequence analysis tools are needed, however, to decide whether these interpretations are likely, whether a particular set of LCRs represents nearly neutral drift creating junk DNA, or whether the appearance of LCRs reflects assembly error. Here we investigate an LCR family containing the sulfotransferase (SULT) 1A genes involved in drug metabolism, cancer, hormone regulation, and neurotransmitter biology as a first step for defining the problems that those tools must manage. RESULTS: Sequence analysis here identified a fourth sulfotransferase gene, which may be transcriptionally active, located on human chromosome 16. Four regions of genomic sequence containing the four human SULT1A paralogs defined a new LCR family. The stem hominoid SULT1A progenitor locus was identified by comparative genomics involving complete human and rodent genomes, and a draft chimpanzee genome. SULT1A expansion in hominoid genomes was followed by positive selection acting on specific protein sites. This episode of adaptive evolution appears to be responsible for the dopamine sulfonation function of some SULT enzymes. Each of the conclusions that this bioinformatic analysis generated using data that has uncertain reliability (such as that from the chimpanzee genome sequencing project) has been confirmed experimentally or by a "finished" chromosome 16 assembly, both of which were published after the submission of this manuscript. CONCLUSION: SULT1A genes expanded from one to four copies in hominoids during intra-chromosomal LCR duplications, including (apparently) one after the divergence of chimpanzees and humans. Thus, LCRs may provide a means for amplifying genes (and other genetic elements) that are adaptively useful. Being located on and among LCRs, however, could make the human SULT1A genes susceptible to further duplications or deletions resulting in 'genomic diseases' for some individuals. Pharmacogenomic studies of SULT1Asingle nucleotide polymorphisms, therefore, should also consider examining SULT1A copy number variability when searching for genotype-phenotype associations. The latest duplication is, however, only a substantiated hypothesis; an alternative explanation, disfavored by the majority of evidence, is that the duplication is an artifact of incorrect genome assembly.
Asunto(s)
Arilsulfotransferasa/genética , Genómica/métodos , Animales , Arilsulfotransferasa/fisiología , ADN/metabolismo , Evolución Molecular , Eliminación de Gen , Duplicación de Gen , Genoma Humano , Humanos , Modelos Biológicos , Modelos Genéticos , Modelos Moleculares , Familia de Multigenes , Pan troglodytes , Farmacogenética/métodos , Filogenia , Polimorfismo Genético , Ratas , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
Cytosolic sulfotransferases (STs) are traditionally known as Phase II drug-metabolizing or detoxifying enzymes that facilitate the removal of drugs and other xenobiotic compounds. In this study, we carried out a systematic investigation on the sulfation of drug compounds by two major human phenol STs (PSTs), the monoamine (M)-form and simple phenol (P)-form PSTs. Activity data obtained showed the differential substrate specificity of the two enzymes for the thirteen drug compounds tested. Kinetic studies revealed that the M-form PST displayed stereoselectivity for the chiral drug, isoproterenol. The effects of divalent metal cations on the activity of the M-form and P-form PSTs toward representative drug compounds were quantitatively evaluated. Results obtained indicated that the drug-sulfating activities of the two human PSTs were partially or completely inhibited or stimulated by the ten divalent metal cations tested at a 5 mM concentration. The two enzymes appeared to be less sensitive to the effects of physiologically more abundant metal cations such as Mg(2+) and Ca(2+), but more sensitive to the detrimental effects of other metal cations that may enter the body as environmental contaminants.
Asunto(s)
Arilsulfotransferasa/metabolismo , Preparaciones Farmacéuticas/metabolismo , Arilsulfotransferasa/fisiología , Cationes Bivalentes/farmacología , Resistencia a Medicamentos , Humanos , Isoproterenol/metabolismo , Cinética , Estereoisomerismo , Especificidad por Sustrato , SulfatosRESUMEN
The hypotensive agent minoxidil (6-imino-1, 2-dihydro-1-hydroxy-2-imino-4-piperidinopyrimidine) depends upon aryl sulfotransferase (SULT1)-catalyzed sulfation for its bioactivation. Previous reports suggest that glucocorticoids induce class-specific SULT1 and isoform-specific SULT1A1 gene expression in rat liver. In the present study, rats were treated with the glucocorticoid triamcinolone acetonide (TA, 5 mg/kg/day i.p. x 3 days) or its vehicle, 2% Tween-20, prior to minoxidil, and subsequent effects on mean arterial pressure (MAP), heart rate (HR), and hepatic SULT1 gene expression were characterized. Minoxidil treatment (1.5 mg/kg) resulted in a steady decline in MAP values of 16.3 to 18.6% relative to basal control levels at 35 to 60 min following minoxidil injection. Pentachlorophenol (PCP, 40 micromol/kg i.p.), an inhibitor of SULT1 enzyme activity, effectively ablated the hypotensive effects of minoxidil. By contrast, pretreatment with TA significantly enhanced minoxidil-induced hypotension. Relative to vehicle-treated controls, TA-treated rats displayed a steeper rate of decline in MAP and more profound levels of hypotension with decreases in MAP following minoxidil administration of 27.8%. TA also produced significant increases in hepatic SULT1 mRNA expression (of 271%) and SULT1A1 immunoreactive protein levels (of 273%), relative to vehicle-treated controls. These results provide physiological evidence to support the biological relevance of SULT1A1 induction by glucocorticoids. The data indicate that steroid treatment induces SULT1A1 gene expression and, as a consequence, accentuates the hypotensive effects of minoxidil.
Asunto(s)
Arilsulfotransferasa/genética , Presión Sanguínea/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Hígado/enzimología , Minoxidil/farmacología , Triamcinolona Acetonida/farmacología , Animales , Antihipertensivos/farmacología , Arilsulfotransferasa/antagonistas & inhibidores , Arilsulfotransferasa/fisiología , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Cinética , Masculino , Pentaclorofenol/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The stereoselective formation of 4'-hydroxypropranolol sulfate (HOPS) from racemic 4'-hydroxypropranolol (HOP) has been investigated using canine hepatic cytosol and partially purified phenolsulfotransferases (PSTs). Pseudo first order rate constants, Vmax/Kmapp, were significantly greater (P less than .05) for the formation of R-HOPS by canine hepatic cytosol, S/R-HOPS = 0.72. Moreover, double reciprocal replots for the sulfation of both HOP enantiomers were biphasic, suggesting the presence of multiple PST enzymes. Pentachlorophenol (PCP) inhibited the sulfation of both HOP enantiomers with an IC50 of 244 nM, suggesting that phenol (P) forms of the PST enzymes may be primarily responsible for the cytosolic sulfation. Three distinct PST fractions were partially purified from the cytosol and were found to possess unique stereoselectivities toward HOP. Fraction 1 was most stereoselective, S/R-HOPS = 0.51, but was not inhibited by PCP, suggesting that it is a monoamine (M) form PST. Fraction 3 was the most active fraction and mimicked the cytosol in both stereoselectivity, S/R-HOPS = 0.73 vs. 0.72, and susceptibility to PCP inhibition, IC50 = 93 nM. The S/R-HOPS ratio produced by fraction 2 was 0.63. These data demonstrate the presence of multiple PST enzymes with differing stereoselectivities for the enantiomers of HOP in canine hepatic cytosol. Further studies with homogeneous PST isozymes, ideally from human tissue, and other chiral drugs are needed to define the enzymatic mechanism of this reaction and its role in chiral drug metabolism.