Sulfation Of P-nitrophenol Research Articles

Inhibition of human sulfotransferases (SULTs) by per- and polyfluoroalkyl substances (PFASs) and structure-activity relationship.

Per- and polyfluoroalkyl substances (PFASs) are a family of highly fluorinated aliphatic substances widely used in industrial and commercial applications. This study aims to determine the inhibition of PFASs towards sulfotransferases (SULTs) activity, and trying to explain the toxicity mechanism of PFASs. In vitro recombinant SULTs-catalyzed sulfation of p-nitrophenol (PNP) was utilized as a probe reaction. The incubation system was consisted of PFASs, SULTs, PNP, 3'-phosphoadenosine-5'-phosphosulfate, MgCl2 and Tris-HCl buffer. Ultra-performance liquid chromatography was employed for analysis of the metabolites. All tested PFASs showed inhibition towards SULTs. The longer the carbon chain length of the PFASs terminated with -COOH, the higher is its capability of inhibiting SULT1A3. PFASs with -SO3H had a relatively higher ability to inhibit SULT1A3 activity than those with -COOH, -I and -OH. The inhibition kinetic parameter was 2.16 and 1.42μM for PFOS-SULT1A1, PFTA-SULT1B1. In vitro in vivo extrapolation showed that the concentration of PFOS and PFTA in human matrices might be higher than the threshold for inducing inhibition of SULTs. Therefore, PFASs could interfere with the metabolic pathways catalyzed by SULTs in vivo. All these results will help to understand the toxicity of PFASs from the perspective of metabolism.

The Relative Importance of the Small Intestine and the Liver in Phase II Metabolic Transformations and Elimination of p-Nitrophenol Administered in Different Doses in the Rat

Intestinal and hepatic function have been investigated in phase II metabolic reactions and elimination of p-nitrophenol (PNP) in the rat. A jejunal loop was cannulated and recirculated with isotonic solutions containing PNP in different concentrations (0, 20, 100, 500, 1000 µM). Samples were obtained from the perfusate at given intervals. To investigate the metabolic and excretory functions of the liver, the bile duct was cannulated, and the bile was collected. Metabolites of PNP were determined by validated HPLC (high pressure liquid chromatography) methods. The results demonstrated the relative importance of the small intestine and the liver in phase II metabolic transformations and elimination of PNP. There were significant differences between the luminal and biliary appearances of p-nitrophenol-glucuronide (PNP-G) and p-nitrophenol–sulfate (PNP-S). The PNP-G appeared in the intestinal lumen at the lower PNP concentrations (20 µM and 100 µM) at higher rate than in the bile. No significant difference was found between the intestinal and the biliary excretion of PNP-G when PNP was administered at a concentration of 500 µM. However, a reverse ratio of these parameters was observed at the administration of 1000 µM PNP. The results indicated that both the small intestine and the liver might play an important role in phase II metabolic reactions and elimination of PNP. However, the relative importance of the small intestine and the liver can be dependent on the dose of drugs.

Heterologous expression in Pichia pastoris and biochemical characterization of the unmodified sulfatase from Fusarium proliferatum LE1.

Sulfatases are a family of enzymes (sulfuric ester hydrolases, EC 3.1.6.-) that catalyze the hydrolysis of a wide array of sulfate esters. To date, despite the discovery of many sulfatase genes and the accumulation of data on numerous sulfated molecules, the number of characterized enzymes that are key players in sulfur metabolism remains extremely limited. While mammalian sulfatases are well studied due to their involvement in a wide range of normal and pathological biological processes, lower eukaryotic sulfatases, especially fungal sulfatases, have not been thoroughly investigated at the biochemical and structural level. In this paper, we describe the molecular cloning of Fusarium proliferatum sulfatase (F.p.Sulf-6His), its recombinant expression in Pichia pastoris as a soluble and active cytosolic enzyme and its detailed characterization. Gel filtration and native electrophoretic experiments showed that this recombinant enzyme exists as a tetramer in solution. The enzyme is thermo-sensitive, with an optimal temperature of 25°C. The optimal pH value for the hydrolysis of sulfate esters and stability of the enzyme was 6.0. Despite the absence of the post-translational modification of cysteine into Cα-formylglycine, the recombinant F.p.Sulf-6His has remarkably stable catalytic activity against p-nitrophenol sulfate, with kcat = 0.28 s-1 and Km = 2.45 mM, which indicates potential use in the desulfating processes. The currently proposed enzymatic mechanisms of sulfate ester hydrolysis do not explain the appearance of catalytic activity for the unmodified enzyme. According to the available models, the unmodified enzyme is not able to perform multiple catalytic acts; therefore, the enzymatic mechanism of sulfate esters hydrolysis remains to be fully elucidated.

Heterologous expression in Pichia pastoris and biochemical characterization of the unmodified sulfatase from Fusarium proliferatum LE1.

Sulfatases are a family of enzymes (sulfuric ester hydrolases, EC 3.1.6.-) that catalyze the hydrolysis of a wide array of sulfate esters. To date, despite the discovery of many sulfatase genes and the accumulation of data on numerous sulfated molecules, the number of characterized enzymes that are key players in sulfur metabolism remains extremely limited. While mammalian sulfatases are well studied due to their involvement in a wide range of normal and pathological biological processes, lower eukaryotic sulfatases, especially fungal sulfatases, have not been thoroughly investigated at the biochemical and structural level. In this paper, we describe the molecular cloning of Fusarium proliferatum sulfatase (F.p.Sulf-6His), its recombinant expression in Pichia pastoris as a soluble and active cytosolic enzyme and its detailed characterization. Gel filtration and native electrophoretic experiments showed that this recombinant enzyme exists as a tetramer in solution. The enzyme is thermo-sensitive, with an optimal temperature of 25°C. The optimal pH value for the hydrolysis of sulfate esters and stability of the enzyme was 6.0. Despite the absence of the post-translational modification of cysteine into Cα-formylglycine, the recombinant F.p.Sulf-6His has remarkably stable catalytic activity against p-nitrophenol sulfate, with kcat = 0.28 s-1 and Km = 2.45 mM, which indicates potential use in the desulfating processes. The currently proposed enzymatic mechanisms of sulfate ester hydrolysis do not explain the appearance of catalytic activity for the unmodified enzyme. According to the available models, the unmodified enzyme is not able to perform multiple catalytic acts; therefore, the enzymatic mechanism of sulfate esters hydrolysis remains to be fully elucidated.

Investigation of drug metabolism in various segments of small intestine in the rat

In the extrahepatic drug metabolism the intestinal tract can play an important role. These experiments were designed to study the biotransformation of p-nitrophenol (PNP) in the small intestine in the rat. Various segments of the small intestine (proximal and distal jejunum, terminal ileum) were perfused with isotonic solution in vivo containing different concentrations of PNP (20-100-500-1000 μM) and the concentrations of metabolites (PNP-G: p-nitrophenol glucuronide, PNP-S: p-nitrophenol sulfate) were determined in the perfusion medium. It was found a decreasing tendency in the glucuronidation from the proximal to distal segment of the small intestine: e.g. 430 nmol, 240 nmol, and 100 nmol PNP-G appeared in the perfusion medium in the proximal, distal jejunum and in the terminal ileum, respectively, when 500 μM PNP was luminally perfused for 90 minutes. Similar ratio was found at the luminal perfusion of other PNP-concentrations, too. Luminal appearance of sulfoconjugate of PNP was considerably lower and no clear gradient tendency in the formation of PNP-S could be detected in the small intestine from the proximal to distal segment. Our results show that there are considerable differences in drug metabolism in various segments of the small intestine. We have found a gradient conjugating activity from proximal to distal segment of small intestine in the glucuronidation of PNP.

A New Arylsulfate Sulfotransferase Involved in Liponucleoside Antibiotic Biosynthesis in Streptomycetes

Sulfotransferases are involved in a variety of physiological processes and typically use 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor substrate. In contrast, microbial arylsulfate sulfotransferases (ASSTs) are PAPS-independent and utilize arylsulfates as sulfate donors. Yet, their genuine acceptor substrates are unknown. In this study we demonstrate that Cpz4 from Streptomyces sp. MK730-62F2 is an ASST-type sulfotransferase responsible for the formation of sulfated liponucleoside antibiotics. Gene deletion mutants showed that cpz4 is required for the production of sulfated caprazamycin derivatives. Cloning, overproduction, and purification of Cpz4 resulted in a 58-kDa soluble protein. The enzyme catalyzed the transfer of a sulfate group from p-nitrophenol sulfate (K(m) 48.1 microM, k(cat) 0.14 s(-1)) and methyl umbelliferone sulfate (K(m) 34.5 microM, k(cat) 0.15 s(-1)) onto phenol (K(m) 25.9 and 29.7 mM, respectively). The Cpz4 reaction proceeds by a ping pong bi-bi mechanism. Several structural analogs of intermediates of the caprazamycin biosynthetic pathway were synthesized and tested as substrates of Cpz4. Des-N-methyl-acyl-caprazol was converted with highest efficiency 100 times faster than phenol. The fatty acyl side chain and the uridyl moiety seem to be important for substrate recognition by Cpz4. Liponucleosides, partially purified from various mutant strains, were readily sulfated by Cpz4 using p-nitrophenol sulfate. No product formation could be observed with PAPS as the donor substrate. Sequence homology of Cpz4 to the previously examined ASSTs is low. However, numerous orthologs are encoded in microbial genomes and represent interesting subjects for future investigations.

Decreased sulfotransferase SULT1C2 gene expression in DPT-induced polycystic kidney

The pathogenesis of polycystic kidney disease (PKD) remains unclear despite the identification of the genes responsible for hereditary PKD. In this study, we investigated the alteration of gene expressions in an acquired PKD model induced by 2-amino-4,5-diphenylthiazole (DPT) using the differential display method. Kidney mRNA from a Sprague-Dawley rat fed with 1% DPT for 4 days and from a control rat was compared by the RT-PCR differential display method. Differentially expressed bands were re-amplified and subcloned. Using these subclones as probes, the changes in gene expressions were confirmed by Northern blot analysis. Subsequently, mouse kidney cDNA library was screened. The isolated 1.5-kb cDNA contained an open reading frame encoding 296 amino acids, which shared 94.3% identity with rat SULT1C2 sulfotransferase, and was considered to be its mouse ortholog (GenBank Accession No. AY005469). Mouse SULT1C2 mRNA was abundant in the kidney and stomach among normal mouse tissues. The expression of SULT1C2 mRNA was decreased in the rat kidney after DPT feeding but not in the stomach. Mouse SULT1C2 was expressed successfully using pET plasmid vector and E. coli. The recombinant 34-kD protein was capable of catalyzing the sulfation of p-nitrophenol at a Km of 3.1 mmol/L, by utilizing 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor. Although the physiological substrate and function of SULT1C2 have yet to be elucidated, its down-regulation could be involved in the cystic changes of tubules by decreasing the sulfation of the tubular basement membrane components.

Genetic Polymorphism of Human Tissue Phenol Sulfotransferases.

Three related forms of phenol sulfotransferase, thermostable ST1A2 and ST1A3 and a thermolabile ST1A5 are known to exist in human livers. Thermostable forms, whose activities are polymorphically distributed in human tissues, have been shown to mediate the bioactivation of carcinogenic N-hydroxy aromatic amines as well as phenolic substrates. Variant forms of ST1A3 mRNAs (i.e. Arg213His and Met223Val) have been found in human livers. In a Japanese population, allele frequencies of 213Arg and 213His were 0.83 and 0.17, respectively. No remarkable difference in [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS)-dependent sulfation of p-nitrophenol was observed between recombinant 213Arg- and 213His-type ST1A3, although it was reported that 213His homozygosity was associated with both lower (less than one-sevenths) p-nitrophenol sulfation and thermolability in human platelets. The recombinant ST1A3 (213His) did exhibit unstability at 45° and 37°C as compared with ST1A3 (213Arg). Liver cytosols from 213His homozygotes did not always show low p-nitrophenol-sulfating activities. Different molecular mechanisms in sulfation polymorphism are suggested in livers and platelets of humans.

Sulfating-activity and stability of cDNA-expressed allozymes of human phenol sulfotransferase, ST1A3*1 ((213)Arg) and ST1A3*2 ((213)His), both of which exist in Japanese as well as Caucasians.

We recently found single amino acid substitutions ((213)Arg/His and (223)Met/Val) in polymorphic human phenol-sulfating phenol sulfotransferase (SULT: cDNAs encoding ST1A3, P PST or HAST1/2) among Caucasians and African-Americans. In a Japanese population (n = 143), allele frequencies of (213)Arg and (213)His were 83.2 and 16. 8%, respectively, but the (223)Val allele was not found. (213)His homozygosity was reportedly associated with both very low (>7-fold) sulfating activities of p-nitrophenol (at 4 microM) and low thermostability in platelets. Sulfating-activity determinations using recombinant (213)Arg- and (213)His-forms (ST1A3*1 and ST1A3*2, respectively) did not, however, reveal appreciable deficiency in [(35)S]3'-phosphoadenosine 5'-phosphosulfate (PAPS)-dependent sulfation of p-nitrophenol (4 microM) by ST1A3*2 (7.5 vs. 10.2 nmol/min/nmol SULT for ST1A3). Kinetic parameters for p-nitrophenol for p-nitrophenol sulfation supported the slight decrease in sulfating activities at 4 microM (K(m), 0.82 vs. 1.75 microM; V(max), 13.2 vs. 13.1 nmol/min/nmol SULT, respectively, for ST1A3*1 and *2). p-Nitrophenyl sulfate-dependent 2-naphthol sulfation by ST1A3*2 was 69% of that by ST1A3*1 (p<0.05). However, ST1A3*2 was remarkably unstable at 45 and 37 degrees C as compared to ST1A3*1. The lower p-nitrophenol sulfating activity of ST1A3*2 may explain the lower platelet p-nitrophenol sulfation in ST1A3*2 homozygotes. Protein instability and ST1A3 gene regulation may be both involved in the polymorphism of p-nitrophenol sulfation in human tissues.

Molecular characterization of ST1C1-related human sulfotransferase.

Carcinogenic arylamines such as N-hydroxy-2-acetylaminofluorene (N-OH-AAF) are metabolically activated by mammalian sulfotransferases to form N-hydroxyarylamine O-sulfates. We previously showed that rat ST1C1 efficiently mediate these activations. These reactions occur in liver cytosols of humans as well as rats. However, the enzyme responsible for N-OH-AAF activation has not been identified in humans. In the present study, a human cDNA (ST1C2) encoding a sulfotransferase showing a high similarity with ST1C1, has been isolated from a human fetal liver cDNA library and expressed using a bacterial expression system. A clear difference was observed in the pH optima for p-nitrophenol sulfation between ST1C2 and ST1C1 expressed in Escherichia coli. In addition, ST1C2 did not mediate 3'-phosphoadenosine-5'-phosphosulfate-dependent DNA binding of N-OH-AAF. These results suggest that human ST1C2 has a clear different substrate specificity, in spite of the structural similarity, with rat ST1C1.

Genetic polymorphisms in human liver phenol sulfotransferases involved in the bioactivation of N-hydroxy derivatives of carcinogenic arylamines and heterocyclic amines

Three related forms of phenol sulfotransferase (PSULT), thermostable ST1A2 (SULT1A2hum) and ST1A3 (SULT1A1hum) and a thermolabile TL-PST (SULT1A3hum), are known to exist in human livers. Thermostable forms, whose activities are polymorphically distributed, have been shown to mediate the bioactivation of carcinogenic N-hydroxy arylamines and heterocyclic amines. To clarify the nature of the sulfation polymorphism, the study compared the expressed levels of ST1A2, ST1A3 and TL-PST mRNAs in human livers by the method of reverse-transcriptase polymerase chain reaction (RT-PCR), utilizing HindIII, BamHI and SnaBI sites which were unique to the above PSULT cDNAs, respectively. Of the PCR products derived from human liver ( n=26), 43–89, <1–29 and <1–21% showed the restriction pattern characteristic for ST1A3, ST1A2 and TL-PST cDNAs, respectively, thus indicating that ST1A3 mRNA is the major transcript. Hepatic p-nitrophenol and dopamine sulfation rates ranged from 440–2670 and <5–460 pmol/min per mg protein in the 26 individuals, respectively. The observed differences in the ST1A3 and TL-PST mRNA levels were consistent with the differences in p-nitrophenol and dopamine sulfations. Relative levels of hepatic ST1A3 mRNA were non-normally distributed and correlated significantly with p-nitrophenol sulfation. In addition, variant forms of ST1A3 mRNA encoding Arg213His and Met223Val were detected in human livers. With regard to Arg213His, 28 individuals who had homozygous 213 Arg alleles, 15 individuals who were heterozygotes and nine homozygous 213 His individuals were found by a newly established genotyping method among 52 human liver samples. Frequency of 223 Val allele was apparently lower than that of 213 His allele, as no homozygous 223Val individual and only three individuals who were heterozygotes ( 223 Met/ Val) were observed among 52 individuals. These results suggest that regulation of p-nitrophenol sulfation occurs at the level of gene transcription of ST1A3 which is the major transcript of the three PSULT mRNAs and that a polygenic basis for the apparent genetic polymorphism of sulfation was likely because of the existence of ST1A3 variants.

Sulfur metabolism in ulcerative colitis: investigation of detoxification enzymes in peripheral blood.

Two enzymes of detoxification were studied in blood samples from 27 patients with ulcerative colitis (UC) and 18 controls to determine whether there is an abnormality in sulfur metabolism in UC. Thiol methyltransferase (TMT) activity was measured in erythrocyte membranes as the extent of conversion of 2-mercaptoethanol to S-methyl-2-mercaptoethanol with [3H]methyl-S-adenosyl methionine as methyl donor. Phenol sulfotransferase (PST) activity was measured in platelet homogenates as the extent of sulfation of p-nitrophenol with 3-phosphoadenosine 5-phospho[35S]sulfate (PAPS) as sulfate donor. TMT activity was significantly higher in UC patients (27.0 vs 17.1 nmol/mg protein/hr; P < 0.005). No difference in PST activity was found. We conclude that TMT may be up-regulated in UC to detoxify excess hydrogen sulfide exposed to the peripheral blood compartment. This may arise from either increased luminal sulfide production or reduced colonic detoxification.

Endotoxin inhibits glucuronidation in the liver: An effect mediated by intercellular communication

Endotoxin [lipopolysaccharide (LPS) 50 μg/mL] added to the perfusion medium increased glucose production and inhibited the glucuronidation of p-nitrophenol in perfused mouse liver both in recirculating and non-recirculating systems, while sulfation of p-nitrophenol was unchanged. The effects of endotoxin could be prevented by the addition of cyclooxygenase inhibitors, while PGD 2 and PGE 2 also caused a decrease in p-nitrophenol glucuronidation in perfused liver. In isolated hepatocytes endotoxin failed to affect p-nitrophenol conjugation, while PGD 2 and PGE 2 decreased the rate of it. Our results suggest that endotoxin inhibits glucuronidation through an intercellular communication presumably mediated by eicosanoids.

2,6-Dichloro-4-nitrophenol (DCNP), an alternate-substrate inhibitor of phenolsulfotransferase

2,6-Dichloro-4-nitrophenol (DCNP)- 35sulfate was identified and quantified by an HPLC-radiometric assay following its biosynthesis in vitro from 35S-labeled 3'-phosphoadenosine-5'-phosphosulfate (PAP 35S) by phenolsulfotransferase (PST) of rat liver cytosol. Acid hydrolysis of DCNP- 35sulfate produced almost stoichiometric release of inorganic 35sulfate and DCNP. In two-substrate experiments of sulfation of p-nitrophenol ( p-NP) or dopamine (prototype substrates for P and M human PST forms), 10 μM DCNP inhibited the reactions by about 15 and 78%, respectively. This contrasts with its action on PST of human origin where the P-PST was more sensitive to DCNP inhibition. In all mixed bi-substrate experiments, a reciprocal relationship between the two sulfated products was observed. Kinetic data showed that p-NP inhibited the sulfation of DCNP competitively. Likewise the sulfation of p-NP and dopamine was competitively inhibited by DCNP. However, non-competitive inhibition was observed in the sulfation of p-NP by DCNP, measured at varying concentrations of PAP 35S. The above kinetic data suggest that DCNP is an alternate-substrate inhibitor of rat liver PST.

Sulfotransferase-mediated DNA binding of N-hydroxyarylamines(amide) in liver cytosols from human and experimental animals

Characteristics of cytosolic sulfotransferase-mediated binding of carcinogenic N-hydroxyarylamines(amide) have been investigated and compared among experimental animal species and humans in vitro. Human cytosols exhibited significant sulfating activities towards 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-hydroxy-Glu-P-1), N-hydroxy-2-aminofluorene (N-hydroxy-AAF) and N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF), but had no detectable activity toward 2-hydroxyamino-3-methyl-imidazo[4,5-f]quinoline (N-hydroxy-IQ). Although the extent of the covalent binding of these N-hydroxyarylamines(amide) differed significantly among individuals, clear correlations were observed among the sulfation of N-hydroxyarylamines (amide) and also with p-nitrophenol sulfation. Hepatic cytosols from mouse, rat, guinea-pig, hamster, rabbit, dog and monkey also mediated the binding of N-hydroxy-Glu-P-1, N-hydroxy-AF and N-hydroxy-AAF, while only rat cytosols showed detectable DNA binding of N-hydroxy-IQ. Among the species examined, rat showed the highest capability for activating these N-hydroxyarylamines(amides). Significant sex-related differences were detected in rat, dog and monkey for all substrates examined, except N-hydroxy-IQ. Clear correlations were observed in the animal species between N-hydroxyarylamines(amide), but not with p-nitrophenol. Using an ion-exchange chromatographic system, sulfating activity of p-nitrophenol in human livers was separated into two fractions and the PAPS-dependent DNA binding of N-hydroxy-AF was supported mainly by the later fraction. On Western blots, an immunoreactive protein was detected in these fractions using an antibody raised against rat hepatic N-hydroxy-AAF sulfotransferase. The band was also detected in human hepatic cytosols with considerable individual variation in their amounts. These results indicate the involvement of a closely related form(s) of sulfotransferase in the PAPS-mediated activation of N-hydroxyarylamines(amide) in human as well as in the experimental animal species.

Substrate specificity and some properties of phenol sulfotransferase from human intestinal Caco-2 cells

The phase II metabolic reactions, sulfation and glucuronidation, were studied in a human colon carcinoma cell line (Caco-2), which has been developed as a model of intestinal enterocytes. Phenol sulfotransferase (PST, EC 2.4.2.1) was isolated from Caco-2 cells cultured for 7, 14 and 21 days. The enzyme catalyzed the sulfation of both p-nitrophenol and catecholamines ( e.g., dopamine) as well as most catecholamine metabolites. The affinity (K m) of PST for dopamine was much higher than for p-nitrophenol, and the specific activity of PST with both substrates increased with the age of the cells. The thermal stability of Caco-2 PST increased with cell age and was not dependent on the acceptor substrate used. The thermolabile PST from 7-day old cells was more sensitive to NEM than was the thermostable enzyme from 21-day old cells. No UDP-glucoronyltransferase (EC 2.4.1.17) activity was detected in 7-, 14- and 21-day old Caco-2 cells with any of the methods used.

Enzymatic acetylation and sulfation of N-hydroxyarylamines in bacteria and rat livers.

In mammalian hepatic cytosol both acetyltransferase and sulfotransferase are involved in the activation of N-hydroxy derivatives of arylamines and arylamides. The role of acetyltransferase is also shown in Salmonella, whereas no rigid evidence is provided on the role of sulfotransferase in Salmonella. In Ames mutagenesis test without S9-mix, the number of revertants of Salmonella typhimurium TA98 induced was 10-fold higher with 2-hydroxyamino-3-methylimidazo[4,5-f] quinoline (N-hydroxy-IQ) than with 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-hydroxy-Glu-P-1). The extents of the binding to calf thymus DNA of N-hydroxy-Glu-P-1 were, however, 3.9 to 8.6-fold higher than that of N-hydroxy-IQ in both acetyl CoA- and PAPS-fortified rat hepatic cytosol systems. To understand the mechanism causing the apparent discrepancy between the results of the mutation and DNA binding, the activating capacities of cytosols of S. typhimurium TA98 and TA98/1,8-DNP6 strains on the binding of N-hydroxy-Glu-P-1 and N-hydroxy-IQ have been examined in comparison with those of rat livers. Although both N-hydroxyarylamines were activated by hepatic cytosols in the presence of PAPS, no significant DNA binding of these N-hydroxyarylamines was detected in the presence of PAPS and either one of the two strains of bacterial cytosols. In addition, both cytosols of TA98 and TA98/1,8-DNP6 strains showed no measurable activity on the sulfation of p-nitrophenol, suggesting no capacity for sulfotransferase-mediated activation of N-hydroxyarylamines in Salmonella. On the contrary, the extents of the acetyl CoA-dependent binding of N-hydroxy-IQ in cytosols of TA98, but not of TA98/1,8-DNP6, were respectively 6- and 9-fold higher than those in hepatic cytosols of male and female rats, although the extents of the binding of N-hydroxy-Glu-P-1 were rather higher in hepatic than in bacterial cytosols. In addition, the covalent binding of N-hydroxy-2-acetylaminofluorene to DNA was detected in hepatic, but not in bacterial cytosols, although the binding of N-hydroxy-2-aminofluorene was detectable in both hepatic and bacterial cytosols in the presence of acetyl CoA. These results indicate that the metabolic activating capacities of Salmonella and rat liver cytosols differ qualitatively, and the difference in the substrate specificity of acetyltransferase between Salmonella and rat livers may be involved, in part, in the difference of their DNA damage in bacteria and mammals.

Aryl sulfotransferase in rat liver: multiplicity and substrate specificity.

Rat liver aryl sulfotransferase was purified by chromatography on diethylaminoethyl-cellulose or chromatofocusing and three fractions, referred to by Sekura and Jakoby as I, II and IV, were obtained in the order of their elution, each containing sulfation activity. p-Nitrophenol (PNP) at mM order and beta-naphthol were substrates common to all three fractions, but PNP at microM order and tyramine were substrates only for IV. IV corresponded to the enzyme designated M by Rein et al. and was active with monoamine, as predicted from our previous results with rat liver cytosol. However, the effectiveness of IV in bringing about the sulfation of PNP at mM order was not evident from our previous results. The characteristics of aryl sulfotransferase multiplicity on the basis of thermostability of sulfation activity could not be determined since essentially the characteristics were the same for all three purified fractions. The multiplicity of aryl sulfotransferase purified from rat liver was different from that of human platelets, indicating possible species and/or tissue differences in this enzyme.

Effect of aging on oxidative metabolism and conjugation in perfused livers of an inbred strain of mice: A pilot study

A study was carried out to examine the possibility that altered mitochondrial function with aging might cause changes in hepatic rates of oxygen uptake and β-oxidation of fatty acids. Livers of 2–4 month and 20–25 month old CBA mice were perfused, and rates of respiration and ketogenesis were measured. Livers of old mice had significantly decreased rates of oxygen utilization (190 ± 16 vs 151 ± 9 μmol/gm/hr, p.05) and significantly lower rates of ketogenesis as indicated by rates of acetoacetate and β-hydroxybutyrate production (84 ± 8 vs 63 ± 7, p <.05). To determine whether NADPH supply, which is largely derived from β-oxidation of fatty acids in the fasted state, might be rate limiting for mixed-function oxidation in aged mice, rates of p-nitroanisole O-demethylation were measured in livers of young and old mice. Age had no effect on mixed-function oxidation of p-nitroanisole. Rates of glucuronidation of p-nitrophenol were also not affected by age; however, rates of sulfation of p-nitrophenol were significantly higher in livers of old (7.2 ±.6) than in young (3.5 ± 0.8) mice. This pilot study raises the possibility that the disposition of ingested fatty acids and xenobiotic compounds requiring sulfation may be significantly altered with aging. Moreover, it demonstrates that the perfused mouse livers can be used as a model in aging studies.

Glucuronidation and sulfation of p-nitrophenol in isolated rat hepatocyte subpopulations. Effects of phenobarbital and 3-methylcholanthrene pretreatment

Parenchymal cells, isolated from untreated (control), phenobarbital(PB)- or 3-methylcholanthrene(3-MC)-treated rats, were separated into four subpopulations according to cell density, and glucuronidation and sulfation of p-nitrophenol (PNP) in the hepatocyte subpopulations were investigated. PB enhanced the glucuronidation almost 2-fold but not the sulfation, while 3-MC enhanced both glucuronidation (3-fold) and sulfation (2-fold) in the original cell suspensions. Some gradation trends were found in the conjugation activities among the hepatocyte subpopulations: In the control experiment, the extent of glucuronidation in four subpopulations was virtually the same but sulfation in high-density hepatocytes was slightly higher than in low-density ones. Both glucuronidation and sulfation were higher in low-density hepatocytes from PB-treated rats, though the gradation was very modest. Glucuronidation and sulfation tended to be slightly higher in middle-density hepatocytes in the 3-MC experiment. However, no definite correlation in conjugation activities vs. cell density, like those seen in cytochrome P-450s vs. cell density in the hepatocytes isolated from PB-treated rats, were found in the subpopulations from control or inducer-treated rats. Simultaneous studies on acetylation of p-aminobenzoic acid (PABA) revealed that the activities in the subpopulations were virtually the same and the inducers had little influence on the activity.