Sulphuric Acid Conjugates Research Articles

Human sulphotransferases are involved in the activation of aristolochic acids and are expressed in renal target tissue

Use of herbal preparations containing Aristolochia species has led to progressive nephropathy and urothelial cancer in humans. Analysis of DNA adducts formed in human target tissues and studies in animal models have pointed out a major role of the secondary plant metabolites, aristolochic acids, in these effects. Only a minority of the users of Aristolochia-containing products developed nephropathy and cancer, suggesting differences in individual susceptibility. Differences in metabolic activation and inactivation frequently affect the susceptibility towards chemicals. Others have shown that the activation of aristolochic acids to DNA-reactive and mutagenic metabolites requires reduction of their aryl nitro group. The biological activity of numerous nitro- and aminoarenes, after appropriate phase I metabolism, is strongly enhanced in the presence of acetyltransferases or sulphotransferases (SULTs). In the present study, we demonstrate that expression of human SULTs in bacterial and mammalian target cells reinforces the mutagenic activity of aristolochic acids. Using Salmonella typhimurium TA1538 as the recipient organism, we identified the expression of all 12 human SULT forms. SULT1A1 led to the strongest increase in the mutagenicity of aristolochic acids. Some activation was also observed with SULT1B1, but not with the remaining forms. The role of SULT1A1 in the activation of aristolochic acids was corroborated using S. typhimurium TA100- and Chinese hamster V79-derived target cells engineered for expression of human SULT1A1 when compared with control cells. Furthermore, pentachlorophenol, an inhibitor of SULT1A1, strongly reduced the mutagenic effect of aristolochic acids in V79-hCYP2E1-hSULT1A1 cells. Moreover, we demonstrate that SULT1A1 and SULT1B1 are expressed in human kidney using immunoblot analysis, but their levels are substantially lower than in liver. Finally, we discuss the possibility that reactive sulphuric acid conjugates produced in other tissues are transferred to kidney and ureter.

Excretion of metabolites in urine and faeces from rats dosed with the heterocyclic amine, 2-amino-9 H-pyrido[2,3- b]indole (AαC)

2-amino-9 H-pyrido[2,3- b]indole (AαC) is a mutagenic and carcinogenic heterocyclic amine formed during ordinary cooking. In model systems AαC can be formed by pyrolysing either tryptophan or proteins of animal or vegetable origin. In the present study, the in vivo metabolism of AαC in rats was investigated. Rats were dosed with tritium labelled AαC. Urine and faeces were collected over three days. The metabolites of AαC were characterised by HPLC-MS and quantified by liquid scintillation counting. Conjugated metabolites were characterised by enzymatic hydrolyses with β-Glucuronidase or arylsulfatase. The data showed that the metabolic pattern of AαC was similar in all rats. About 55% of the dose was excreted in urine and faeces during 72 h and the major amount of AαC metabolites (31%) was excreted during the first 24 h. In addition to a small amount of unmetabolised AαC seven conjugated metabolites were characterised. Three minor metabolites were characterised as AαC- N 2-glucuronide and glucuronic acid conjugates of 3-OH-AαC and 6-OH-AαC. Four metabolites were all characterised as sulphuric acid conjugates and accounted for the largest amount of metabolites excreted in urine. The two major sulphuric acid conjugates were identified as AαC-3- O-sulfate and AαC-6- O-sulfate, while the minor sulphuric acid conjugates were proposed to be other O-sulfonated metabolites. In faeces only AαC was excreted and accounted for about 12% of dose during the first 24 hours. Any activated metabolites of AαC were not detected in rat urine or faeces. In future accumulation or binding of AαC to macromolecules such as DNA and proteins has to be studied.

Stable heterologous expression of hydroxysteroid sulphotransferase in Chinese hamster V79 cells and their use for toxicological investigations

Various benzylic alcohols are metabolically activated to electrophilic, potentially mutagenic and carcinogenic sulphuric acid esters. The involved sulphotransferases are not expressed in the cell lines in culture which are commonly used for mutagenicity testing. The liver of adult female rats is very efficient in the bioactivation of 1-hydroxymethylpyrene. The major enzyme involved was purified and identified as hydroxysteroid sulphotransferase a. Its cDNA was stably expressed in Chinese hamster V79 cells, which are particularly suited for the quantitative detection of various types of mutations and other genotoxic and cytotoxic effects. The mRNA, protein and enzyme activity levels in the constructed cell lines (V79rSTa-1 and V79rSTa-2) were measured, and the cells were also used in mutagenicity and cytotoxicity investigations with benzylic alcohols. 1-Hydroxymethylpyrene, 9-hydroxymethylanthracene and 6-hydroxymethylbenzo[ a]pyrene showed enhanced cytotoxicity in V79rSTa-1 and V79rSTa-2 cells, as compared with sulphotransferase-deficient control cells. In addition, 1-hydroxymethylpyrene induced sister chromatid exchanges, and 6-hydroxymethylbenzo[ a]pyrene induced gene mutations in V79rSTa-1 cells. We intend carrying out more investigations with other chemicals on these cell lines. Their advantages, as compared with systems with external metabolising systems, include the formation of the active metabolites within the target cell, as in ST-proficient cells in vivo, eliminating the problems which may result from restricted intercellular transport of reactive and ionized sulphuric acid conjugates. Furthermore, cells expressing other sulphotransferases, including human enzymes, may be constructed and used for comparative investigations.

Hydrolysis, absorption and metabolism of di(2-ethylhexyl) terephthalate in the rat.

1. The hydrolysis of di(2-ethylhexyl) terephthalate (DEHT) and di(2-ethylhexyl) phthalate (DEHP) were studied using rat gut homogenate fractions in vitro. Both isomers were hydrolysed by the intestinal fraction; however, DEHP was hydrolysed to 2-ethylhexanol (2-EH) and mono(2-ethylhexyl) phthalate (MEHP) in about equal proportions, whereas DEHT was hydrolysed to 2-EH and terephthalic acid (TPA). The half-lives for disappearance of the diesters were determined to be 12.6 min for DEHP and 53.3 min for DEHT. 2. The absorption and metabolism of DEHT were studied by administering [hexyl-2-14C]DEHT (in corn oil) by oral gavage at a dose level of 100 mg/kg to 10 adult male Sprague-Dawley rats. Urine, faeces and expired air were collected for 144 h and analysed for the presence of radioactivity, and faeces and urine were analysed for unlabelled metabolites. 3. Radioactivity was eliminated in faeces (56.5 +/- 12.1% of dose) primarily as unchanged DEHT, small amounts of MEHT and polar metabolites; excreted in urine (31.9 +/- 10.9% of dose) principally as MEHT and metabolic products of 2-EH; and expired as 14CO2 (3.6 +/- 0.9% of dose). Less than 2% of the administered radioactivity was found in the carcass. Small amounts of 14C were found in the tissues with the highest amounts found in liver and fat. 4. Metabolites identified in urine included terephthalic acid (equivalent to 51% of dose), oxidized metabolites of 2-EH and MEHT, and glucuronic and sulphuric acid conjugates (equivalent to about 10% of dose).(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme immunoassay for 4-hydroxy-2-(4-methylphenyl)benzothiazole

A specific enzyme immunoassay (EIA) has been developed for 4-hydroxy-2-(4-methylphenyl)benzothiazole (KB-2714) (1), an active metabolite of 4-acetoxy-2-(4-methylphenyl)benzothiazole (KB-2683) (2) which is a promising anti-rheumatic agent. The EIA was based upon antiserum elicited against 5-(2-carboxyphenylazo)-4-hydroxy-2-(4-methylphenyl)benzothiazole (3)-bovine serum albumin conjugate and beta-galactosidase-labelled 5-amino-4-hydroxy-2-(4-methylphenyl)benzothiazole (5). The sensitivity of the EIA was significantly improved by the utilization of a bridge heterologous combination system. An appropriate dose-response curve of EIA for 4-hydroxy-2-(4-methylphenyl)benzothiazole was obtained in the range of 10 pg tube-1-20 ng tube-1. The specificity of EIA proved to be satisfactory in terms of cross-reactivities to 14 benzothiazole-related compounds including glucuronic acid and sulphuric acid conjugates. The proposed method was evaluated to be useful for the determination of 1 in urine and plasma with acceptable recovery and inter- and intra-assay precision.

The disposition and metabolism of (+)-cyanidanol-3 in patients with alcoholic cirrhosis.

The effect of alcoholic cirrhosis of the liver on the disposition and metabolism of (+)-cyanidanol-3 has been studied in three patients. Following oral intake of U-14C-(+)-cyanidanol-3 2 g, the unchanged drug was detected in plasma between 0.5 and 24 h after dosing and the metabolites, measured only as 14C-levels, were still detectable at 120 h. A mean of 55% of the dose was excreted in the urine, in which the major metabolites were the glucuronic and sulphuric acid conjugates of (+)-cyanidanol-3 and 3'-O-methyl-(+)-cyanidanol-3, although one patient excreted a larger proportion as non-conjugated metabolites. The pattern of metabolism in the patients did not differ significantly from that in control subjects, suggesting that the drug is handled by the cirrhotic liver in a similar manner to the normal liver.

Determination of trimethoprim metabolites including conjugates in urine using high-performance liquid chromatography with combined ultraviolet and electrochemical detection

A high-performance liquid chromatographic method for the determination of trimethoprim metabolites in pig urine was developed. The metabolites — glucuronic acid and sulphuric acid conjugates of phenolic metabolites formed by demethylation of trimethoprim — were quantitated after treatment of urine with β-glucuronidase ( Escherichia coli). The sulphuric acid conjugate was not susceptible to enzymatic hydrolysis and was therefore assayed as the conjugate by use of ion-pair chromatography on the reversed-phase column. In order to find suitable conditions for enzymatic hydrolysis of the glucoronides, the conjugates were obtained by gel chromatography of urine from a [ 14C]trimethoprim-treated pig.

The metabolism of benzo(a)pyrene in isolated perfused lungs from variously-treated rats.

Abstract Rats were pretreated either by the injection of 3‐methylcholanthrene (MC) 40 mg/kg intraperitoneally for 3 days or by giving phenobarbitone (PB) 0.5 g/1 in the drinking water for 7 days. Benzo(a)pyrene (BP) and its metabolites were measured by thin‐layer chromatography (TLC) and radiometry in samples of perfusion medium up to two hours. In perfusion medium the half‐life of BP was about 15 min. in the perfusions of lungs from MC‐pretreated rats and 60‐90 min. in the perfusions of lungs from PB‐pretreated and control rats. The appearance of water‐soluble metabolites was most marked in the perfusions of lungs from MC‐pretreated rats being at fifteen minutes 2.5‐fold and at two hours 2‐fold in comparison to the controls. In the perfusions of lungs from PB‐pretreated rats the accumulation was retarded perhaps indicating an inhibition of lung enzymes by PB. Water‐soluble metabolites included glucuronic and sulphuric acid conjugates as well as those conjugates resistant to acid hydrolysis. Phenols were the most pronounced organic‐soluble metabolites at the beginning of perfusion of lungs from MC‐pretreated rats. At fifteen minutes the amount of phenols in the perfusions of lungs from PB‐pretreated rats was 1/6 and in controls 1/3 of that in the perfusions of lungs from MC‐pretreated rats. Towards the end of perfusion phenols decreased in the perfusions of lungs from MC‐pretreated rats. In other perfusions all metabolite fractions increased slowly during the perfusion. Covalently bound radioactivity and BP‐hydroxylase in lung homogenates were studied after perfusions. The covalent binding of radioactivity in the lungs from MC‐pretreated rats was 3.5 times higher than in control lungs and the lungs from PB‐pretreated rats. There was a correlation between covalent binding and the activity of BP‐hydroxylase in lung homogenates. Exposure of rats to cigarette smoke resulted in effects on the BP metabolism resembling those of MC pretreatment.

Ion-pair chromatography of acidic drug metabolites and endogenic compounds

Liquid-liquid chromatographic systems based on ion-pair partition with silica microparticles as the support for the stationary phase have been used for the separation of anionic compounds of biochemical and pharmacological interest. A high separating efficiency can be obtained with both aqueous and organic mobile phases and the retention is easily regulated by the nature and the concentration of the quaternary ammonium counter ion, present in the aqueous phase. The influence of the composition of the liquid phases on the selectivity and separating efficiency has been studied, as well as equilibration methods and the stability of the systems. Examples are given of separations of sulphonamides, barbiturates, glucuronic and sulphuric acid conjugates of steroidal compounds and phenols, glycine conjugates of carboxylic acids (hippuric, nicotinuric and salicyluric acid) and anionic metabolites of biogenic amines (indoleacetic, benzoic, mandelic and phenylacetic acid derivatives).

The comparative metabolism of mesitylene, pseudocumene, and hemimellitene in rats

The principal routes of metabolism of the three isomers of trimethylbenzene, mesitylene, pseudocumene, and hemimellitene, have been quantitatively investigated in rats and rate constants measured for the urinary excretion of conjugates with glycine, glucuronic acid, and sulphuric acid. Approximately 78% of an oral dose of mesitylene is excreted as 3,5-dimethylhippuric acid; an additional 7.6 and 8.2% were excreted as glucuronic and sulphuric acid conjugates. The corresponding values for the glycine, glucuronic and sulphuric acid conjugates of pseudocumene and hemimellitene were 43.2, 6.6, and 12.9% and 17.3, 19.4, and 19.9%, respectively. It is considered that 3,4-dimethylbenzoic acid is the principal metabolite of pseudocumene. The relatively greater toxicity of hemimellitene may in part be attributed to the fact that it is more slowly metabolized and excreted than either mesitylene or pseudocumene. It has been established that the effect of phenobarbital administration on the of metabolism of all three compounds is due primarily to an increase in the rate of aromatic hydroxylation rather than in the rate of formation of the corresponding carboxylic acid. By comparing the metabolism of the isomeric di- and trimethylbenzenes, it has been possible to evaluate the influence of the position of the methylsubstituents on the routes of metabolism.

Metabolism of the phenolic antioxidant 3,5-di- tert-butyl-4-hydroxyanisole (Topanol 354). III. The metabolism in rats of the major autoxidation product, 2,6-di- tert-butyl- p-benzoquinone

When 2,6- di-tert- butyl-p-[ 14 C] benzoquinone (DBQ), the major autoxidation product of Topanol 354, was administered orally to rats, an average of 36% of the label was excreted in the urine and 63% in the faeces in 7 days. In studies with biliary-cannulated rats, 35% of an oral dose was recovered in the bile in 48 hr. The major urinary metabolites have been identified as glucuronic and/or sulphuric acid conjugates of 2,6-di- tert-butylhydroquinone (DBHQ; 11·3% of dose), 3,3-dimethyl-5-hydroxy-7- tert-butylcoumaran (7·2%) and 2- tert-butyl-6-[(2-hydroxy-1,1-di-methyl)ethyl]- p-benzoquinone (6·1%). The principal biliary metabolite was a glucuronic acid conjugate of DBHQ (26·7% of the dose). As the distribution and biotransformation of DBQ were similar in almost all respects to the fate of the corresponding metabolite formed from the parent antioxidant in vivo, it may be concluded that ingestion of the autoxidation product in the small amounts likely to be present in foods will not be associated with biological effects other than those identified after the feeding of Topanol 354.

Metabolism of the phenolic antioxidant 3,5-Di- tert-butyl-4-hydroxyanisole (topanol 354). III. The metabolism in rats of the major autoxidation product, 2,6-Di- tert-butyl- p-benzoquinone

When 2,6-di- tert-butyl- p-[ 14C]benzoquinone (DBQ), the major autoxidation product of Topanol 354, was administered orally to rats, an average of 36 % of the label was excreted in the urine and 63 % in the faeces in 7 days. In studies with biliary-cannulated rats, 35 % of an oral dose was recovered in the bile in 48 hr. The major urinary metabolites have been identified as glucuronic and/or sulphuric acid conjugates of 2,6-di- tert-butylhydroquinone (DBHQ; 11·3% of dose), 3,3-dimethyl-5-hydroxy-7- tert-butylcoumaran (7·2%) and 2- tertbutyl-6-[(2-hydroxy-1,1-di-methyl)ethyl]- p-benzoquinone (6·1%). The principal biliary metabolite was a glucuronic acid conjugate of DBHQ (26·7 % of the dose). As the distribution and biotransformation of DBQ were similar in almost all respects to the fate of the corresponding metabolite formed from the parent antioxidant in vivo, it may be concluded that ingestion of the autoxidation product in the small amounts likely to be present in foods will not be associated with biological effects other than those identified after the feeding of Topanol 354.