1-Methoxy-3-indolylmethyl DNA adducts in six tissues, and blood protein adducts, in mice under pak choi diet: time course and persistence.

We previously showed that purified 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary plant metabolite in Brassica species, is mutagenic in various in vitro systems and forms DNA and protein adducts in mouse models. In the present study, we administered 1-MIM glucosinolate in a natural matrix to mice, by feeding a diet containing pak choi powder and extract. Groups of animals were killed after 1, 2, 4 and 8 days of pak choi diet, directly or, in the case of the 8-day treatment, after 0, 8 and 16 days of recovery with pak choi-free diet. DNA adducts [N2-(1-MIM)-dG, N6-(1-MIM)-dA] in six tissues, as well as protein adducts [τN-(1-MIM)-His] in serum albumin (SA) and hemoglobin (Hb) were determined using UPLC-MS/MS with isotopically labeled internal standards. None of the samples from the 12 control animals under standard diet contained any 1-MIM adducts. All groups receiving pak choi diet showed DNA adducts in all six tissues (exception: lung of mice treated for a single day) as well as SA and Hb adducts. During the feeding period, all adduct levels continuously increased until day 8 (in the jejunum until day 4). During the 14-day recovery period, N2-(1-MIM)-dG in liver, kidney, lung, jejunum, cecum and colon decreased to 52, 41, 59, 11, 7 and 2%, respectively, of the peak level. The time course of N6-(1-MIM)-dA was similar. Immunohistochemical analyses indicated that cell turnover is a major mechanism of DNA adduct elimination in the intestine. In the same recovery period, protein adducts decreased more rapidly in SA than in Hb, to 0.7 and 37%, respectively, of the peak level, consistent with the differential turnover of these proteins. In conclusion, the pak choi diet lead to the formation of high levels of adducts in mice. Cell and protein turnover was a major mechanism of adduct elimination, at least in gut and blood.

Quantification of Sulfotransferases 1A1 and 1A3/4 in Tissue Fractions and Cell Lines by Multiple Reaction Monitoring Mass Spectrometry.

BACKGROUND: Within the sulfotransferase (SULT) superfamily of metabolic enzymes, SULT1A1 and 1A3/4 isoforms are of particular interest, due to their abilities to catalyze the sulfation of phenolic endobiotics and xenobiotics. Although the difference in their substrate specificity is well documented, an isoform-specific quantification method is still not available. OBJECTIVE: To detect and quantify SULT1A1 and 1A3/4 in S9 fractions and cell lines using targeted mass spectrometry-based proteomics. METHOD: Samples were tryptically digested, and signature peptides were quantified using liquid chromatography- multiple reaction monitoring mass spectrometry (LC-MRM/MS). Stable isotopelabeled (SIL) peptides were used as internal and calibration standards. SULT1A1 and SULT1A3/4 were quantified in various S9 fractions and cell line samples. RESULTS: Intraday and interday variabilities were low for relative quantification in S9 and cell line matrices (<8%). Expression profiles were validated using Western blot analysis of S9 fractions and lentiviral transduced SULT1A-overexpressing cell lines. CONCLUSION: A reproducible method for simultaneous quantification of SULT1A1 and SULT1A3/4 in S9 fractions and cell line samples was established and validated.

Surface reaction limited model for the evaluation of immobilized enzyme on planar surfaces.

Analysis of immobilized enzyme in situ is a crucial step to embed an enzyme onto the planar technology of standard integrated circuit (IC) and microelectromechanical systems (MEMS) for a bioreactor or enzyme-coupled biosensor. A surface reaction limited model, based on a systematized and standardized approach, mathematically derived from mass transfer dynamics and the Michaelis-Menten equation for the measuring the apparent K*(m) (Michaelis-Menten constant) and V*(max) (maximum reaction rate per unit surface area of catalyst) of an immobilized enzyme on a planar surface was developed. The derived equations for the kinetic model were simulated and experimentally confirmed. A platform of a microflow bioreactor with a one-sided planar catalytic surface that contained immobilized enzyme was constructed. The microfluidic bioreactor was designed to possess a channel height less than that of the diffusion layer thickness in a semi-infinite diffusion process, and K*(m) and V*(max) of rat phenol sulfotransferase (PST) immobilized on the silicon oxide surface were successfully determined in situ. Variation in kinetic constants and the possible differences in performance between free and immobilized PST are discussed.

Developmental expression of aryl, estrogen, and hydroxysteroid sulfotransferases in pre- and postnatal human liver.

Aryl- (SULT1A1), estrogen- (SULT1E1), and hydroxysteroid- (SULT2A1) sulfotransferases (SULTs) are active determinants of xenobiotic detoxication and hormone metabolism in the adult human liver. To investigate the role of these conjugating enzymes in the developing human liver, the ontogeny of immunoreactive SULT1A1, SULT1E1, and SULT2A1 expression was characterized in a series of 235 pre- and postnatal human liver cytosols ranging in age from early gestation to a postnatal age of 18 years. Interindividual variability in expression levels was apparent for all three SULTs in pre- and postnatal liver samples. Expression of the three SULTs displayed distinctly different developmental profiles. Semiquantitative Western blot analyses indicated that SULT1A1 and SULT2A1 immunoreactive protein levels were readily detectable in the majority of developmental human liver cytosols throughout the prenatal period. Whereas SULT1A1 expression did not differ significantly among the various developmental stages, SULT2A1 expression increased during the third trimester of gestation and continued to increase during postnatal life. By contrast, SULT1E1, a cardinal estrogen-inactivating enzyme, achieved the highest levels of expression during the earliest periods of gestation in prenatal male livers, indicating a requisite role for estrogen inactivation in the developing male. The present analysis suggests that divergent regulatory mechanisms are responsible for the differential patterns of hepatic SULT1A1, SULT1E1, and SULT2A1 immunoreactive protein levels that occur during pre- and postnatal human development, and implicates a major role for sulfotransferase expression in the developing fetus.

Sulfation of drugs and hormones in mid-gestation human fetus.

BACKGROUND: Sulfotransferase is an important enzyme family that catalyses the transfer of a sulfate group from a donor substrate, 3'-phosphoadenosine-5'-phosphosulphate (PAPS), to an acceptor substrate which may be a drug, a hormone or a neurotransmitter that possesses a hydroxy or an amine group. Drugs and hormones are sulfated in human fetal tissues but a review on this topic has not yet been published. AIMS: The aim of this article is to review the literature on the sulfation of drugs and hormones in human fetus and, when possible, to compare the rate of sulfation in fetal and adult human tissues. STUDY DESIGN: A medline search was performed by using the following key words: "fetal sulfotransferase" and "sulfotransferase in fetus" with the limit of "human". The literature was collected, critically read and a written note was produced. SUBJECTS: The majority of studies were conducted with mid-gestation human fetuses and limited studies include younger and older fetuses. RESULTS: The sulfation rate of dopamine (SULT1A3) was 3-fold higher in fetal than adult liver whereas the sulfation rate of 4-nitrophenol (SULT1A1) was one order of magnitude lower in fetal than adult liver. Ritodrine is sulfated at a higher rate in the fetal than adult liver. The sulfation rates of dopamine, 4-nitrophenol and ritodrine varied considerably in the fetal liver and did not correlate with the gestation age. 3,3'-T2 was sulfated in human placenta and the sulfation rate of 3,3'-T2 correlated with SULT1A1 activity. Dehydroepiandrosterone sulfotransferase activity is 6-fold higher in the fetal than adult adrenal. DISCUSSION: Sulfotransferase activity develops early in the human fetal liver and is subjected to a remarkable interindividual variability. Because of this variability, the examined enzyme activities did not correlate with the gestation age. Hormones are extensively sulfated in human fetal liver and other tissues. Sulfated hormones may serve as circulating or intracellular stores from which the free hormone can be regenerated by the action of the sulfatases.

Differentiated properties of hepatocytes induced from pancreatic cells.

Transdifferentiation of pancreas to liver is a well-recognized phenomenon and has been described in animal experiments and human pathology. We recently produced an in vitro model for the transdifferentiation (or conversion) of the pancreatic cell line AR42J-B13 to hepatocytes based on culture with dexamethasone (Dex). To determine whether the hepatocytes express markers of hepatic intermediary metabolism and detoxification, we investigated the patterns of expression of glucokinase, cytochrome P450s CYP3A1 and CYP2B1/2, testosterone/4-nitrophenol uridine diphosphate glucuronosyltransferase (UDPGT), and aryl sulfotransferase. All were expressed. We also determined the expression of 2 enzymes involved in ammonia detoxification: carbamoylphosphate synthetase I (CPS I) and glutamine synthetase (GS). These enzymes are normally strictly compartmentalized in liver in a wide periportal pattern and the last downstream perivenous hepatocytes, respectively. Following culture with Dex, CPS I and GS are expressed in 2 different cell populations, suggesting that both periportal and perivenous hepatocytes are induced. We also produced a reporter assay based on the activation of green fluorescent protein (GFP) by the transthyretin (TTR) promoter or glucose-6-phosphatase (G6Pase) promoter. After culture with Dex, transfected cells begin to express GFP, showing that hepatic promoters are activated in concert with the induction of the hepatocyte phenotype. Lastly, we examined the stability of the hepatic phenotype and found that some cells still express liver markers (transferrin or albumin) up to 14 days after removal of Dex. In conclusion, these results suggest that pancreatic hepatocytes produced by this method may offer an alternative model to primary cultures of hepatocytes for the study of liver function.

Role of ATP and glycogen reserves in both paracetamol sulfation and glucuronidation by cultured precision-cut rat liver slices.

Precision-cut rat liver slices (PCLS) were used to investigate the formation of paracetamol conjugates. The time course of biochemical markers such as ATP and GSH content, glycogen levels and protein synthesis rates was recorded over a period of time of 26 h and taken as index of slices viability. Low values of ATP (3.6 nmol/mg prot), GSH (7.1 nmol/mg prot) and protein synthesis rates (94.1 pmol leu/mg prot x min(-1)) were initially observed. Thereafter, they gradually recovered up to 6 h but decreased values were seen after 20 h. Glycogen, however, dropped rapidly during the first 6 h, being no longer detected after 20 h of incubation. The reincubation of PCLS in a fresh medium for 6 h allowed a strong recovery of GSH, ATP and protein synthesis rates, but no gluconeogenesis was observed. Meanwhile, paracetamol sulfate formation was fairly constant (about 3 microg/mg protein) while glucuronide gradually disappeared. The amount of both UGT1A1 and ST1A1 did not correlate with their respective enzymatic activities. We suggest that loss of glycogen impair glucuronide conjugation by decreasing the availability of UDPGA, and that low values of ATP are largely enough to support sulfotransferase activity.

Enzymatic characterization and interspecies difference of phenol sulfotransferases, ST1A forms.

Cytosolic sulfotransferases, which mediate activation and detoxification of both endogenous and exogenous compounds, consist of at least five different gene families (ST1 to ST5) in mammals. Several cDNAs corresponding to ST1A forms have been reported, but their functional properties are not well characterized. In addition, only a single form of ST1A sulfotransferase has been reported in each experimental animal species despite the expressions of plural forms in humans. Therefore, enzymatic properties of human ST1A3, ST1A5, rat ST1A1, mouse St1a4, and newly isolated rabbit ST1A8 have been characterized and compared by use of their recombinant proteins to clarify the functional difference between human and experimental animal ST1A forms. From the results using more than 25 phenolic chemicals, all the experimental animal ST1A forms showed substrate specificities similar to human ST1A3 rather than ST1A5. They showed high affinities toward p-nitrophenol and 6-hydroxymelatonin as found in human ST1A3. These forms also showed high activities toward umbelliferone and naringenin, but very low activities toward catecholamines, representative substrates of human ST1A5. Hepatic contents of experimental animal ST1A forms varied (66-250 pmol/mg of cytosolic protein) but showed the same order as observed with human ST1A3 (120 pmol/mg). Hepatic content of human ST1A5 was about 19-fold less than that of ST1A3. Therefore, ST1A forms identified in experimental animal species correspond to human ST1A3 functionally. For chemicals such as troglitazone and 2-amino-4'-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine, clear species differences were detected among the ST1A forms examined.

Measurement of aryl and alcohol sulfotransferase activity.

Two methods are presented for determining the catalytic activity of aryl and alcohol sulfotransferases. The first assay is a simple and rapid procedure that is based on the extraction of a paired ion formed between the product organic sulfate and methylene blue. The second method employs HPLC analysis of the substrate-dependent formation of PAP, an assay that is particularly useful when the sulfuric acid ester product is chemically unstable.

Localisation of aryl sulfotransferase expression in human tissues using hybridisation histochemistry and immunohistochemistry.

To date, the laboratory has cloned seven unique human sulfotransferases; five aryl sulfotransferases (HAST1, HAST2, HAST3, HAST4 and HAST4v), an estrogen sulfotransferase and a dehydroepiandrosterone sulfotransferase. The cellular distribution of human aryl sulfotransferases in human hepatic and extrahepatic tissues has been determined using the techniques of hybridization histochemistry and immunohistochemistry. Human aryl sulfotransferase expression was detected in liver, epithelial cells of the gastrointestinal mucosal layer, epithelial cells lining bronchioles and in mammary duct epithelial cells.

Differential expression and immunohistochemical localisation of the phenol and hydroxysteroid sulphotransferase enzyme families in the developing lung.

Reversible sulphation, catalysed by sulphotransferases and sulphatases, of biologically active compounds such as androgens and oestrogens is a sensitive mechanism for regulating their bioavailability, and we have previously hypothesised that this process plays a significant role in the regulation of human fetal lung development. Sulphation is also a major detoxification reaction, contributing significantly to the body's chemical defence mechanism. We have used qualitative and semiquantitative immunological studies to determine the temporal expression and localisation of phenol and hydroxysteroid sulphotransferases during human lung development. Our results show that in the early fetal lung, phenol sulphotransferase expression is at its highest, and is most widely distributed throughout the developing respiratory epithelium. With later development, expression levels decrease and become predominantly restricted to the more proximal airways. In contrast, hydroxysteroid sulphotransferase is present only at very low levels in the early-gestation lung but expression increases rapidly through gestation to reach an apparent peak by 1 year postnatal age. The proximal-to-distal gradients of phenol and hydroxysteroid sulphotransferase expression were similar in mature respiratory epithelium, with immunoreactivity in ciliated cells, non-ciliated secretory cells and basal cells, but with no apparent expression in mucus-secreting cells. These studies provide supporting evidence for the hypothesis that hydroxysteroid sulphotransferase, an androgen-inactivating enzyme, contributes to the role of androgens in retarding the maturation of human lung in utero.

Phenol sulfotransferase activities and localization in human nasal polyp epithelium.

Nasal polyp epithelia, which exhibit a wide variation in epithelial cell morphologies, were tested for phenol sulfotransferase (PST) enzymes. Immunohistology revealed little or no detectable PST antigen in normal ciliated pseudostratified epithelia or in simple squamous metaplastic epithelia; however, intense expression was observed in regions of non-ciliated epithelial cell hyperplasia and in squamous epithelial cells overlying such hyperplastic sites. Western blots confirmed the presence of both P-PST (32 kDa) and M-PST (34 kDa) in the tissue extracts. Bimodal distribution of PST activity as a function of 4-nitrophenol concentration was consistent with expression of these two PST isoforms. These results indicate dynamic and epithelial differentiation-dependent expression of human PSTs in the nasal mucosa and suggest that these sulfotransferases can be modulated within human airways in vivo.

Improved purification of arylsulfate sulfotransferase from human intestinal bacterium by using polyclonal antibody.

Arylsulfate sulfotransferase (ASST) from a human intestinal bacterium stoichiometrically catalyzed the transfer of the sulfate group of phenylsulfate esters to phenolic compounds. Polyclonal antibodies against ASST were obtained from rabbit sera. These antisera did not inhibit ASST activity. ASST was recognized by the IgG fraction of the antisera, but rat liver phenol sulfotransferase did not show cross-reactivity to ASST on Western blot (immunoblot) analysis. The ASST was purified by an anti-ASST immobilized affinity column chromatography to homogeneity on SDS-PAGE. The NH2-terminal amino acid and partial sequence of the purified enzyme were serine and SVKYSFEDHIINRQYEAEQAMLAKF, respectively. We corrected the previous result that the NH2-terminal of ASST was arginine.

Phenol sulfotransferase expression in the airways: enzymological and immunohistochemical demonstration.

Phenol (aryl) sulfotransferases (PSTs) provide a conjugative pathway that detoxifies hydroxylated aromatic xenobiotics by esterification with sulfate. Both human and bovine airways have been reported to use this pathway, and in this investigation the bovine system is examined. PST activity in tracheal through fourth generation bronchial mucosal cytosols was 0.1-0.35 nmol/mg protein/min. Activity was generally greater in more distal bronchi and in parenchymal extracts, which contained 0.6-3 nmol/mg/min PST activity. Comparison of the PST activities of bronchial and parenchymal cytosols indicated similar pH activity profiles, although steady state kinetic measurements revealed different Km values for the acceptor substrate 2-naphthol (13.7 microM for bronchial, 31.3 microM for parenchymal). Anion exchange chromatography indicated two PST isoforms being expressed in different ratios. Immunoblot analysis with mouse anti-bovine PST revealed a closely spaced doublet at 32 kDa in both bronchial mucosal and parenchymal cytosolic extracts; however, this doublet was unequally stained in parenchymal extracts. Immunohistochemical analyses revealed faint positive staining of the tracheobronchial epithelium. Greatest immunostaining was observed in the nonciliated secretory epithelial cells of the bronchioles, whereas surrounding smooth muscle, endothelial cells, and alveoli were immunonegative. These results are consistent with the known locations of other detoxification enzymes within the airways.

Sequence analysis and expression of the cDNA for the phenol-sulfating form of human liver phenol sulfotransferase.

A cDNA encoding the human liver phenol-sulfating form of phenol sulfotransferase (P-PST) has been isolated and characterized from a lambda Uni-Zap XR human liver cDNA library. P-PST is the major form of phenol sulfotransferase involved in drug and xenobiotic metabolism in human liver. P-PST is also responsible for the sulfation and activation of minoxidil to its therapeutically active sulfate ester. The full length cDNA, P-PST-1, is 1206 base pairs in length and encodes a 295-amino acid protein with a molecular mass of 34,097 Da. The translation sequence of P-PST-1 is 96% similar to the amino acid sequences of five peptides derived from the purified protein. In vitro transcription and translation of P-PST-1 generated a protein that comigrates with immunoreactive P-PST from human liver. Significant increases in sulfotransferase activity toward two P-PST-specific substrates, minoxidil and 4-nitrophenol, were detected in cytosol prepared from COS-7 cells transfected with P-PST-1 in the expression vector p-SV-SPORT-1. Northern blot analysis of human liver RNA detected a transcript of approximately 1300 nucleotides in length. Characterization of P-PST at the molecular level provides insight into the structure and heterogeneity of this major class of drug-metabolizing enzymes.

Hep G2 cell line as a human model for sulphate conjugation of drugs.

1. The objective of this study was to examine the usefulness of the hepatoma cell line Hep G2 as a model for human sulphoconjugation of drugs, in particular stereoselective conjugation. 2. Using the substrates p-nitrophenol and dopamine, we found sulphation activities consistent with the presence of both the phenol (P) and the monoamine (M) form of the human phenolsulphotransferases in these cells. 3. The Kmapp was 3.0 microM for the sulphation of p-nitrophenol. This activity was inhibited selectively by 2,6-dichloro-4-nitrophenol, IC50 6 microM. The Kmapp was 39 microM for the sulphation of dopamine. This activity was selectively inhibited by elevated temperature. 4. The chiral adrenergic drugs (+/-)-terbutaline and (+/-)-4-hydroxypropranolol were both sulphated stereoselectively with Kmapp and Vmaxapp values for each enantiomer virtually identical to previous observations with human liver cytosol. 5. In a direct comparison, the estimated activity of the P form of phenolsulphotransferase in the Hep G2 cell line was 30% of that in human liver, whereas, surprisingly, the activity of the M form of phenolsulphotransferase was 4.5 times higher in the Hep G2 cells than in the liver.

Chronic physical activity: hepatic hypertrophy and increased total biotransformation enzyme activity.

Does chronic voluntary physical activity alter hepatic or intestinal capacities for xenobiotic biotransformation? This question was investigated by comparing biotransformation enzyme activities in liver and small intestine of active and sedentary rats. Male rats allowed unlimited access to a running wheel and fed ad lib. for 6 weeks were weight-matched to sedentary controls; the active rats ate 22% more food than the sedentary rats (P less than 0.05). Active rats ran 2.8 +/- 0.6 miles/day. Liver weights were higher in the active rats (11.2 +/- 0.2 vs 9.8 +/- 0.2 g; P less than 0.05), as were total liver protein, and liver microsomal and cytosolic protein (P less than 0.05). As a result of liver hypertrophy, the active rats showed higher total liver activity of several biotransformation enzymes, including 2-naphthol sulfotransferase, styrene oxide hydrolase, benzphetamine N-demethylase, ethacrynic acid glutathione S-transferase and morphine UDP-glucuronosyltransferase (P less than 0.05). In contrast, there was no detectable difference in total liver N-acetyltransferase activity toward p-aminobenzoic acid, 2-naphthylamine, and 2-amino-fluorene as well as, relative hepatic enzyme activity (expressed per g liver or per mg protein) and total and relative intestinal enzyme activity. We conclude that chronic voluntary physical activity, accompanied by an increased food intake, results in liver hypertrophy and potentially increases total hepatic capacity to biotransform certain xenobiotic chemicals.

Continuous fluorometric assay of phenol sulfotransferase.

Phenol sulfotransferases (EC 2.8.2.1) catalyze the sulfation of the acceptor hydroxyl group using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the donor substrate. Previous assays of these enzymes, which exhibit varied acceptor substrate specificities, have required termination of the catalysis followed by isolation and quantitation of formed sulfate ester. In this report, the sulfation of the fluorescent compound, resorufin, is investigated. Reaction of PAPS with resorufin, catalyzed by bovine lung phenol sulfotransferase, bleaches the emission of this acceptor at the pH of the reaction (pH 6.4 optimum). It is thereby possible to continuously record the sulfation reaction. Analysis of single progress curves by integrated replot can be used to determine the initial velocities and also indicates the formation of a product inhibitor, probably resorufin sulfate ester, with Ki less than Km. Sensitivity of the reaction is less than 1 pmol/min. The maximal rate of resorufin sulfation by the bovine lung enzyme is estimated at 57 nmol/mg/min, which is 10% of the rate with an optimal substrate 2-naphthol. This assay may be most sensitive for phenol sulfotransferases with optimal activities at greater than pH 6, due to the acid-base properties of resorufin (pK alpha 6), which becomes nonfluorescent upon protonation.