Biomedical subjects

W Dekant

Publications and source records attributed to W Dekant.

At least 37 records · Page 2Linked to original sources

PubMed1999

Biotransformation and toxicokinetics of musk xylene in humans.

Musk xylene (1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene, MX) is widely used as a fragrance ingredient in detergents and toiletries and is an environmental contaminant. High concentrations of MX have been found in fish, and humans are constantly exposed to MX as a result of its stability in the environment. We investigated the biotransformation and toxicokinetics of MX in humans. A single dose of 0.3 mg/kg body wt of 15N-labeled MX (15N-MX) was given to six volunteers (three male and three female) by the oral route and to another six volunteers (three males and three females) by the dermal route. Urine was collected for 96 h after exposure. Blood samples were taken at intervals for up to 140 days after administration. The metabolite 1-tert-butyl-3,5-dimethyl-15N-4-amino-2,6-dinitrobenzene in urine and 15N-MX in plasma were quantified by gas chromatography/electron-capture mass spectrometry (GC-MS/NCI). Peak plasma concentrations of 15N-MX after oral administration were 36-262 and 1.6-5.5 ng/ml plasma after dermal administration. The toxicokinetics of 15N-MX in plasma can be described by a two-compartment kinetic model with an initial rapid decrease, due to the distribution from the blood into a second compartment (likely fat tissue) and a terminal elimination phase with an average half-life of 70 days for both routes of administration. The amount of 1-tert-butyl-3,5-dimethyl-15N-4-amino-2,6-dinitrobenzene (15N-4-A-MX) in recovered urine represented 0.1-0.5% of the oral applied dose of 15N-MX, respectively, 0.02-0.16% of dermal dose. After a short time of invasion the concentrations of 15N-4-A-MX in urine reached a maximum 18-24 h after administration. The further elimination of the metabolite occurred by first-order kinetics with an average elimination half-life of 11.8 h. After the single oral or dermal dose of 15N-MX, 15N-4-A-MX was not detected in hemoglobin. However, hemoglobin samples contained 1-tert-butyl-3, 5-dimethyl-4-amino-2,6-dinitrobenzene (4-A-MX) (11.4-18.9 fmol/mg Hb), likely derived from chronic environmental exposures.

Administration, Cutaneous↗

PubMed1999

Quantitation of alpha2u-globulin in rat kidney cytosol by capillary electrophoresis.

The renal accumulation of alpha2u-globulin has been implicated in the tumorigenicity of many nongenotoxic chemicals to the kidney of the male rat. Several chemicals inducing renal tumors in the male rat were shown to bind to alpha2u-globulin. This binding impairs the renal degradation of alpha2u-globulin, resulting in lysosomal overload, cell death, increased cell proliferation, and, presumably, renal tumor formation. To support the role of alpha2u-globulin accumulation in the renal toxicity of a chemical, a demonstration of the accumulation of this protein in the kidney of the male rat is one prerequisite. Monoclonal antibodies to alpha2u-globulin are available for quantifying alpha2u-globulin content; however, the procedure is time-consuming and complicated. We developed a method for the quantitation of alpha2u-globulin in renal cytosol using capillary electrophoresis. Renal cytosol fractions were analyzed by capillary electrophoresis as protein-SDS complexes. Using alpha2u-globulin purified from urine of male rats, the limit of detection was 10 microg/ml sample in routine analyses. Excellent run to run reproducibility in migration time (CV </= 4%) and peak areas corresponding to alpha2u-globulin (CV </= 3%) after normalization to the internal standard was observed. Significant increases in renal alpha2u-globulin content (up to 85% of total protein content) compared to controls (approx 15%) were observed in kidney cytosol of rats treated with alpha2u-globulin nephropathy-inducing agents such as trimethylpentane or the alkylphosphonates dimethyl methylphosphonate and diethyl ethylphosphonate, but not in kidney cytosol of male rats treated with tris-(2-chloroethyl)phosphate or the nephrotoxic agent hexachlorobutadiene. A good correlation of the alpha2u-globulin contents determined by capillary electrophoresis and immunoblotting with an alpha2u-globulin-specific antibody (r2 = 0.997) was obtained. Capillary electrophoresis provides a simple, rapid, and highly reproducible quantitation of alpha2u-globulin accumulation for renal tumorigens and may assist in the risk assessment process for these chemicals.

Alpha-Globulins↗

PubMed1999

Organ-specific carcinogenicity of haloalkenes mediated by glutathione conjugation.

Several halogenated alkenes are nephrotoxic in rodents. A mechanism for the organ-specific toxicity to the kidney for these compounds has been elucidated. The mechanism involves hepatic glutathione conjugation to dihaloalkenyl or 1,1-difluoroalkyl glutathione S-conjugates, which are cleaved by gamma-glutamyltransferase and dipeptidases to cysteine S-conjugates. Haloalkene-derived cysteine S-conjugates are substrates for renal cysteine conjugate beta-lyases, which cleave them to form reactive intermediates identified as thioketenes (from chloroalkene-derived S-conjugates) or thionoacyl halides (from 1,1-difluoroalkyl S-conjugates). Alternatively, cysteine S-conjugates may be N-acetylated to excretable mercapturic acids. The formation of reactive intermediates by cysteine-conjugate beta-lyase may play a role in the target-organ toxicity and in the possible renal tumorigenicity of several chlorinated olefins widely used in many chemical processes.

Animals↗

PubMed1999

Occurrence of emodin, chrysophanol and physcion in vegetables, herbs and liquors. Genotoxicity and anti-genotoxicity of the anthraquinones and of the whole plants.

1,8-Dihydroxyanthraquinones, present in laxatives, fungi imperfecti, Chinese herbs and possibly vegetables, are in debate as human carcinogens. We screened a variety of vegetables (cabbage lettuce, beans, peas), some herbs and herbal-flavoured liquors for their content of the 'free' anthraquinones emodin, chrysophanol and physcion. For qualitative and quantitative analysis, reversed-phase HPLC (RP-LC), gas chromatography-mass spectrometry (GC-MS) and RP-LC-MS were used. The vegetables showed a large batch-to-batch variability, from 0.04 to 3.6, 5.9 and 36 mg total anthraquinone per kg fresh weight in peas, cabbage lettuce, and beans, respectively. Physcion predominated in all vegetables. In the herbs grape vine leaves, couch grass root and plantain herb, anthraquinones were above the limit of detection. Contents ranged below 1 mg/kg (dry weight). All three anthraquinones were also found in seven of 11 herbal-flavoured liquors, in a range of 0.05 mg/kg to 7.6 mg/kg. The genotoxicity of the analysed anthraquinones was investigated in the comet assay, the micronucleus test and the mutation assay in mouse lymphoma L5178Y tk+/- cells. Emodin was genotoxic, whereas chrysophanol and physcion showed no effects. Complete vegetable extract on its own did not show any effect in the micronucleus test. A lettuce extract completely abolished the induction of micronuclei by the genotoxic anthraquinone danthron. Taking into consideration the measured concentrations of anthraquinones, estimated daily intakes, the genotoxic potency, as well as protective effects of the food matrix, the analysed constituents do not represent a high priority genotoxic risk in a balanced human diet.

Animals↗

PubMed1999

Biotransformation of [(12)C]- and [(13)C]-tert-amyl methyl ether and tert-amyl alcohol.

tert-Amyl methyl ether (TAME) is intended for use as a gasoline additive to increase oxygen content. Increased oxygen content in gasoline reduces tailpipe emissions of hydrocarbons and carbon monoxide from cars. Due to possible widespread use of TAME, the toxicity of TAME is under investigation. We studied the biotransformation of TAME in rats and one human volunteer after inhalation of (12)C- or (13)C-labeled TAME. In addition, the biotransformation of [(13)C]-tert-amyl alcohol was studied in rats after gavage. Urinary metabolites were identified by GC/MS and (13)C NMR. Rats (two males and two females) were individually exposed to 2000 ppm [(12)C]- or [(13)C]TAME for 6 h, and urine was collected for 48 h. Free and glucuronidated 2-methyl-2,3-butanediol and a glucuronide of tert-amyl alcohol were identified by (13)C NMR, GC/MS, and LC/MS/MS as major urinary metabolites on the basis of the relative intensities of the (13)C NMR signals. The presence of several minor metabolites was also indicated by (13)C NMR; they were identified as tert-amyl alcohol, 2-hydroxy-2-methylbutyric acid, and 3-hydroxy-3-methylbutyric acid. One human volunteer was exposed to an initial concentration of 27 000 ppm [(13)C]TAME by inhalation for 4 min from a 2 L gas sampling bag, and metabolites of TAME excreted in urine were analyzed by (13)C NMR. All TAME metabolites identified in rats were also present in the human urine samples. To study tert-amyl alcohol biotransformation, male rats (n = 3) were treated with 250 mg/kg [(13)C]-tert-amyl alcohol dissolved in corn oil by gavage, and urine was collected for 48 h. (13)C NMR of the urine samples showed the presence of metabolites identical to those in the urine of [(13)C]TAME-treated rats. Our results suggest that TAME is extensively metabolized by rats and humans to tert-amyl alcohol which may be further oxidized to diols and carboxylic acids. These reactions are likely mediated by cytochrome P450-dependent oxidations.

Administration, Inhalation↗

PubMed1999

Haemoglobin binding of a musk xylene metabolite in man.

1. Musk xylene (1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene) is used as a fragrance component in toiletries, detergents and skin care products. Musk xylene is widely distributed in the environment and has been identified as a persistent contaminant in fish and in mothers' milk. Experimental data in man indicate a slow elimination of musk xylene and a potential for accumulation. Nitroarenes may be biotransformed to the respective amines. Some aromatic amines are known to be tumorigenic in animals and in man. Quantitation of the binding of those aromatic amines to haemoglobin has been proposed as a biomarker of internal exposure. 2. To determine bioavailability, metabolic reduction and haemoglobin binding of musk xylene in man, we investigated the presence of musk xylene metabolites bound to haemoglobin in blood samples from rat and from 10 human volunteers not knowingly exposed to musk xylene. 3. Haemoglobin from the blood samples was isolated, and bound metabolites were liberated as amines by alkaline hydrolysis. In haemoglobin samples from all individuals, 1-tert-butyl-3,5-dimethyl-4-amino-2,6-dinitrobenzene and, after chemical derivatization, the corresponding N-perfluoropropyl amide were identified by GC/MS using electron-impact and electron-capture mass spectrometry. 4. The amounts of 1-tert-butyl-3,5-dimethyl-4-amino-2,6-dinitrobenzene bound to haemoglobin in the human blood samples ranged from 13 to 46 fmol/mg haemoglobin. 5. These data demonstrate that musk xylene is bioavailable in man. The use of haemoglobin binding as a biomarker for nitromusk exposure in the general population warrants further studies.

Adult↗

PubMed1999

Stereoselective formation of glutathione S-conjugates from halovinylmercapturate sulphoxides.

1. The glutathione S-transferase catalysed formation of glutathione S-conjugates from halovinylmercapturate sulphoxides was investigated in rat liver and kidney cytosol, with purified glutathione S-transferases and in rat in vivo. 2. The two diastereomers of the sulphoxides of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine show different reactivities with glutathione and glutathione S-transferases. Rat liver and kidney cytosol catalyses the formation of a 1:1 mixture of two diastereomers of (E)-N-acetyl-S-(2-glutathione-S-yl-2-chlorovinyl)-L-cysteine sulphoxide from N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine sulphoxide and of (E)-N-acetyl-S-(2-glutathione-S-yl-1,2-dichlorovinyl)-L-cysteine sulphoxide from N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine sulphoxide. In contrast, only one diastereomer of the Z-isomers was formed. 3. N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulphoxide reacted spontaneously with glutathione at high rates, a 1:1 mixture of both diastereomers of N-acetyl-S-(2-glutathione-S-yl-1-chlorovinyl)-L-cysteine sulphoxide was formed. 4. Metabolism of N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine sulfoxide and N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine sulfoxide under by alpha-class glutathione S-transferases yielded identical products as observed with the cytosolic enzymes. No reaction was observed in the presence of rat liver mu class glutathione S-transferases or human glutathione S-transferase M1. 5. Formation of these glutathione conjugates was also observed in the bile of rat after i.p. administration of the mercapturic acid sulphoxides. The results obtained show that stereochemical aspects may govern the regioselectivity and substrate specificity in glutathione S-transferase-catalysed reactions.

Acetylcysteine↗

PubMed1999

Slow oxidation of acetoxime and methylethyl ketoxime to the corresponding nitronates and hydroxy nitronates by liver microsomes from rats, mice, and humans.

Acetoxime and methylethyl ketoxime (MEKO) are tumorigenic in rodents, inducing liver tumors in male animals. The mechanisms of tumorigenicity for these compounds are not well defined. Oxidation of the oximes to nitronates of secondary-nitroalkanes, which are mutagenic and tumorigenic in rodents, has been postulated to play a role in the bioactivation of ketoximes. In these experiments, we have compared the oxidation of acetoxime and methylethyl ketoxime to corresponding nitronates in liver microsomes from different species. The oximes were incubated with liver microsomes from mice, rats, and several human liver samples. After tautomeric equilibration and extraction with n-hexane, 2-nitropropane and 2-nitrobutane were quantitated by GC/MS-NCI (limit of detection of 250 fmol/injection volume). In liver microsomes, nitronate formation from MEKO and acetoxime was dependent on time, enzymatically active proteins, and the presence of NADPH. Nitronate formation was increased in liver microsomes of rats pretreated with inducers of cytochrome P450 and reduced in the presence of inhibitors (n-octylamine and diethyldithiocarbamate). Rates of oxidation of MEKO (Vmax) were 1.1 nmol/min/mg (mice), 0.5 nmol/min/mg (humans), and 0.1 nmol/min/mg (rats). In addition to nitronates, several minor metabolites were also enzymatically formed (two diastereoisomers of 3-nitro-2-butanol, 2-hydroxy-3-butanone oxime and 2-nitro-1-butanol). Acetoxime was also metabolized to the corresponding nitronate at rates approximately 50% of those observed with MEKO oxidation in the three species examined. 2-Nitro-1-propanol was identified as a minor product formed from acetoxime. No sex differences in the capacity to oxidize acetoxime and MEKO were observed in the species examined. The observed results show that formation of sec-nitronates from ketoximes occurs slowly, but is not the only pathway involved in the oxidative biotransformation of these compounds. Due to the lack of sex-specific oxidative metabolism, other metabolic pathways or mechanisms of tumorigenicity not involving bioactivation may be involved in the sex-specific tumorigenicity of ketoximes in rodents.

Animals↗

PubMed1999

Dose-dependent protein adduct formation in kidney, liver, and blood of rats and in human blood after perchloroethene inhalation.

Perchloroethene (PER) was a widely used solvent and is an environmental contaminant. In bioassays for carcinogenicity, PER was found to increase the incidence of liver tumors in mice and of renal tumors in male rats. Toxic effects of PER after repeated administration are likely caused by bioactivation. PER bioactivation occurs by two pathways. Oxidation by cytochrome P450 results in trichloroacetyl chloride, which binds to lipids and proteins. Glutathione S-conjugate formation from PER and further processing of the formed S-(trichlorovinyl)glutathione to S-(trichlorovinyl)-L-cysteine, followed by cysteine conjugate beta-lyase catalyzed cleavage, resulted in the reactive dichlorothioketene, which binds to proteins under formation of N epsilon-(dichloroacetyl)-L-lysine in proteins. The objective of this study was to comparatively quantify the dose-dependent formation of protein adducts from PER in rats and humans using antibodies with high specificity for either N epsilon-(trichloroacetyl)-L-lysine or N epsilon-(dichloroacetyl)-L-lysine in proteins. Male and female rats (n = 2, per concentration and time point) were exposed to 400, 40, and 10 ppm PER for 6 h and killed at various time points. Formation of N epsilon-(dichloroacetyl)-L-lysine and N epsilon-(trichloroacetyl)-L-lysine in proteins was comparatively quantified in subcellular fractions from liver and kidney and in blood. In addition, three male and three female human volunteers were exposed to 10 and 40 ppm PER, and formation of protein adducts in blood was analyzed using the antibodies and GC/MS after immunoaffinity enrichment of modified proteins. In liver and kidney subcellular fractions and blood of PER-exposed rats, dose-dependent formation of N epsilon-(dichloroacetyl)-L-lysine and N epsilon-(trichloroacetyl)-L-lysine in proteins was observed. Highest concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were formed in kidney mitochondria, followed by kidney cytosol. Only low concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were present in liver proteins; blood concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were 5 to 10 fold lower than in kidney mitochondria. Highest concentrations of N epsilon-(trichloroacetyl)-L-lysine were found in microsomal and cytosolic proteins from the liver of rats exposed to PER. A higher protein adduct formation was seen in PER-exposed-male than -female rats for N epsilon-(dichloroacetyl)-L-lysine in renal mitochondrial proteins, after exposure to 400 ppm PER. In human blood samples taken 0 and 24 h after the 6 h exposures to PER, N epsilon-(trichloroacetyl)-L-lysine-containing proteins were present in low concentrations. N epsilon-(Dichloroacetyl)-L-lysine-containing proteins were not detected either by Western blotting or GC/MS after immunoaffinity chromatography. The obtained results indicate a dose-dependent covalent binding of PER metabolites to proteins in rat liver, kidney, and blood and suggest that the concentration of covalent protein adducts is much lower in blood of humans as compared to the blood of rats exposed under identical conditions.

Adult↗

PubMed1999

Biotransformation and kinetics of excretion of methyl-tert-butyl ether in rats and humans.

Methyl-tert-butyl ether (MTBE) is widely used as an additive to gasoline to increase oxygen content and reduce tail pipe emission of pollutants. Therefore, widespread human exposure may occur. To contribute to the characterization of potential adverse effects of MTBE, its biotransformation was compared in humans and rats after inhalation exposure. Human volunteers (3 males and 3 females) and rats (5 each, males and females) were exposed to 4 (4.5 +/- 0.4) and 40 (38.7 +/- 3.2) ppm MTBE for 4 h in a dynamic exposure system. Urine samples from rats and humans were collected for 72 h in 6-h intervals, and blood samples were taken in regular intervals for 48 h. In urine, MTBE and the MTBE metabolites tertiary-butanol (t-butanol), 2-methyl-1,2-propane diol, and 2-hydroxyisobutyrate were quantified; MTBE and t-butanol were determined in blood samples. After the end of the exposure period, inhalation of 40 ppm MTBE resulted in blood concentrations of MTBE 5.9 +/- 1.8 microM in rats and 6.7 +/- 1.6 microM in humans. The MTBE blood concentrations after inhalation of 4 ppm MTBE were 2.3 +/- 1.0 in rats and 1.9 +/- 0.4 microM in humans. MTBE was rapidly cleared from blood with a half-life of 2.6 +/- 0.9 h in humans and 0.5 +/- 0.2 h in rats. The blood concentrations of t-butanol were 21.8 +/- 3.7 microM in humans and 36.7 +/- 10.8 microM in rats after 40 ppm MTBE, and 2.6 +/- 0.3 in humans and 2.9 +/- 0.5 in rats after 4 ppm MTBE. In humans, t-butanol was cleared from blood with a half-life of 5.3 +/- 2.1 h. In urine samples from controls and in samples collected from the volunteers and rats before the exposure, low concentrations of t-butanol, 2-methyl-1,2-propane diol and 2-hydroxyisobutyrate were present. In urine of both humans and rats exposed to MTBE, the concentrations of these compounds were significantly increased. 2-Hydroxyisobutyrate was recovered as a major excretory product in urine; t-butanol and 2-methyl-1,2-propane diol were minor metabolites. All metabolites of MTBE excreted with urine were rapidly eliminated in both species after the end of the MTBE exposure. Elimination half-lives for the different urinary metabolites of MTBE were between 7.8 and 17.0 h in humans and 2.9 to 5.0 h in rats. The obtained data indicate that MTBE biotransformation and excretion are similar in rats and humans, and MTBE and its metabolites are rapidly excreted in both species. Between 35 and 69% of the MTBE retained after the end of the exposure was recovered as metabolites in urine of both humans and rats.

Adult↗

PubMed1998

Glutathione conjugation of perchloroethene in subcellular fractions from rodent and human liver and kidney.

Perchloroethene (Per) is a widely used industrial solvent and common environmental contaminant. In rats, long-term inhalation of Per is known to cause a small increase in the incidence of renal tubule cell tumors in males only; renal toxicity is seen in female rats and in both sexes of mice after prolonged Per exposure. The renal toxicity of Per is likely mediated by a glutathione-dependent bioactivation reaction. Glutathione S-transferase mediated formation of S-(1,2,2-trichlorovinyl)glutathione is the first step in a sequence of reactions finally resulting in the formation of reactive intermediates in the kidney. In this study, we compared the enzymatic rates of formation of S-(1,2,2-trichlorovinyl)glutathione in liver and kidney subcellular fractions from rats, mice, and from both sexes of humans (n = 11). In microsomal fractions from the liver and kidney of all three species, enzymatic formation of S-(1,2,2-trichlorovinyl)glutathione from Per could not be observed. S-(1,2,2-Trichlorovinyl)glutathione formation (the structure was confirmed by electrospray mass spectrometry) was observed in liver cytosol from both male and female rats and mice. However, the rates of S-(1,2,2-trichlorovinyl)glutathione formation in liver cytosol from male rats (84.5+/-12 pmol/mg per min) were approximately four times higher than from female rats (19.5+/-8 pmol/mg per min) and from both sexes of mice (27.9+/-6 and 26.0+/-4 pmol/mg per min). Low rates of S-(1,2,2-trichlorovinyl)glutathione formation were also seen in kidney cytosol from mice (12+/-6 pmol/mg per min), but not from rats. In human liver subcellular fractions, enzymatic formation of S-(1,2,2-trichlorovinyl)glutathione could not be detected. The human liver cytosolic fractions, however, exhibited glutathione S-transferase activity (as determined using 1-chloro-2,4-dinitrobenzene and hexachlorobutadiene as marker substrates) in the same order of magnitude as rat and mouse liver cytosol. In contrast to other marker activities for glutathione S-transferases, the ability of all human liver cytosol samples to catalyze the glutathione conjugation of 1,2-dichloro-4-nitrobenzene was three orders of magnitude lower compared to rat and mouse liver cytosol. 1,2-Dichloro-4-nitrobenzene conjugation was also four times higher in liver cytosol from male rats compared to female rats. The results suggest that the ability of the human liver to catalyze the formation of S-(1,2,2-trichlorovinyl)glutathione from Per is at least two orders of magnitude lower than that of rat liver, and that sex-specific differences in the extent of hepatic conjugation of Per with glutathione, which may be relevant for nephrotoxicity, occur in rats.

Animals↗

PubMed1998

Biotransformation of perchloroethene: dose-dependent excretion of trichloroacetic acid, dichloroacetic acid, and N-acetyl-S-(trichlorovinyl)-L-cysteine in rats and humans after inhalation.

Chronic exposure of rodents to perchloroethene (PER) increased the incidence of liver tumors in male mice and resulted in a small but significant increase in the incidence of renal tumors in male rats. The tumorigenicity of PER is mediated by metabolic activation reactions. PER is metabolized by cytochrome P450 and by conjugation with glutathione. Cytochrome P450 oxidation of PER results in trichloroacetyl chloride which reacts with water to trichloroacetic acid (TCA) which is excreted. The formation of S-(trichlorovinyl)glutathione (TCVG) from PER results in nephrotoxic metabolites. TCVG is cleaved to S-(trichlorovinyl)-L-cysteine (TCVC) and acetylated to N-acetyl-S-(trichlorovinyl)-L-cysteine (N-ac-TCVC), which is excreted with urine. TCVC is also cleaved in the kidney by cysteine conjugate beta-lyase to dichlorothioketene which may react with water to dichloroacetic acid (DCA) or with cellular macromolecules. The object of this study was to comparatively quantify the dose-dependent excretion of PER metabolites in urine of humans and rats after inhalation exposure. Three female and three male human volunteers and three female and three male rats were exposed to 10, 20, and 40 ppm PER for 6 h, and three female and three male rats to 400 ppm. A dose-dependent increase in the excretion of TCA and N-ac-TCVC after exposure to PER was found both in humans and in rats. A total of 20.4 +/- 7.77 mumol of TCA and 0.21 +/- 0.05 mumol of N-ac-TCVC were excreted in urine of human over 78 h after the start of exposure to 40 ppm PER; only traces of DCA were present. After identical exposure conditions, rats excreted 1.64 +/- 0.42 mumol of TCA, 0.006 +/- 0.002 mumol of N-ac-TCVC and 0.18 +/- 0.04 mumol of DCA. Excretion of N-ac-TCVC in male rats exposed to 400 ppm PER (103.7 nmol) was significantly higher, compared to female rats (31.5 nmol) exposed under identical conditions. N-ac-TCVC was rapidly eliminated with urine both in humans (t1/2 = 14.1 h) and in rats (t1/2 = 7.5 h). When comparing the urinary excretion of N-ac-TCVC, a potential marker for the formation of reactive intermediates in the kidney, humans received a significantly lower dose (3 nmol/kg at 40 ppm) compared to rats (23.0 nmol/kg) after identical exposure conditions. In addition, rats excreted large amounts of DCA which likely is a product of the beta-lyase-dependent metabolism of TCVC in the kidney. The obtained data suggest that glutathione conjugate formation and beta-lyase-dependent bioactivation of TCVC in PER metabolism is significantly higher in rats than in humans. Thus, using rat tumorigenicity data for human risk assessment of PER exposure may overestimate human tumor risks.

Acetylcysteine↗

PubMed1998

Renal cell cancer correlated with occupational exposure to trichloroethene.

A previous cohort-study in a cardboard factory demonstrated that high and prolonged occupational exposure to trichloroethene (C2HCl3) is associated with an increased incidence of renal cell cancer. The present hospital-based case/control study investigates occupational exposure in 58 patients with renal cell cancer with special emphasis on C2HCl3 and the structurally and toxicologically closely related compound tetrachloroethene (C2Cl4). A group of 84 patients from the accident wards of three general hospitals in the same area served as controls. Of the 58 cases, 19 had histories of occupational C2HCl3 exposure for at least 2 years and none had been exposed to C2Cl4; of the 84 controls, 5 had been occupationally exposed to C2HCl3 and 2 to C2Cl4. After adjustment for other risk factors, such as age, obesity, high blood pressure, smoking and chronic intake of diuretics, the study demonstrates an association of renal cell cancer with long-term exposure to C2HCl3 (odds ratio 10.80; 95% CI: 3.36-34.75).

Aged↗

PubMed1998

Stereo- and regioselective conjugation of S-halovinyl mercapturic acid sulfoxides by glutathione S-transferases.

Hexachloro-1,3-butadiene (HCBD) is nephrotoxic in rats. Its toxicity is due to a multistep bioactivation pathway involving glutathione conjugation. N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine resulting from further processing of the GSH conjugate of HCBD is oxidized in vitro and in vivo to the corresponding sulfoxide diastereomers by cytochromes P450 3A. N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide diastereomers represent vinyl sulfoxides which are electrophiles. They are analogous to alpha,beta-unsaturated carbonyl compounds and may be conjugated with glutathione. This study presents experimental data for the different reactivity of the two diastereomers of N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide with glutathione S-transferases in vitro. The structures of the individual diastereomers were assigned by stereoselective oxidation of N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine with sodium periodate in the presence of chloroperoxidase. The two isolated diastereomers were incubated with rat liver and kidney cytosol in the presence of glutathione. In incubations with rat liver cytosol, the formation of a glutathione conjugate, which was identified as (R)-N-acetyl-S-(4-glutathion-S-yl-1,2,3,4-tetrachlorobutadienyl )-L-cysteine sulfoxide, was observed with the (R)-sulfoxide diastereomer. The enzymatic reaction of the (S)-sulfoxide diastereomer with glutathione resulted in two GSH conjugates identified as (S)-N-acetyl-S-(4-glutathion-S-yl-1,2,3,4-tetrachlorobutadienyl )-L-cysteine sulfoxide and (S)-N-acetyl-S-(2-glutathion-S-yl-1,3,4,4-tetrachlorobutadienyl )-L-cysteine sulfoxide. In rat kidney cytosol only the S-diastereomer of N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide is transformed to (S)-N-acetyl-S-(2-glutathion-S-yl-1,3,4,4-tetrachlorobutadienyl )-L-cysteine sulfoxide, while transformation of the R-diastereomer to glutathione conjugates was not observed. In rat kidney cytosol, the rates of formation of (S)-N-acetyl-S-(2-glutathion-S-yl-1,3,4,4-tetrachlorobutadienyl )-L-cysteine sulfoxide from conjugation of the S-diastereomer were comparable to those in rat liver cytosol. Incubation of (S)-N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide with purified rat and human glutathione S-transferases indicates that both R- and S-diastereomers were conjugated to the corresponding 1,4-disubstituted compounds by mu-glutathione S-transferases. Formation of the 1,2-disubstituted conjugation product of N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide was catalyzed exclusively by alpha-glutathione S-transferases. These results are one of the first examples for differences in regio- and stereospecificity in reactions catalyzed by different glutathione S-transferase enzymes.

Acetylcysteine↗

PubMed1998

Reactivity of haloketenes and halothioketenes with nucleobases: chemical characterization of reaction products.

Halothioketenes and haloketenes are postulated as intermediates in haloolefin bioactivation. Little is known about the interactions of these reactive intermediates with macromolecules such as DNA. DNA binding, however, may be relevant in the toxicity of the parent olefins since they or their proximate metabolites are genotoxic. This prompted us to elucidate the structures and properties of potential DNA adducts formed. Adenine, cytosine, guanine, and thymine were reacted with chloro- and dichlorothioketene, chloro- and dichloroketene, and chloro- and dichloroacyl chloride. While thymine did not react, adenine and cytosine formed stable DNA base adducts with all reaction partners as demonstrated by HPLC analysis. Guanine yielded only products with chloroketene and chloroacetyl chloride. The pH-dependent UV spectra, 1H and 13C NMR, FT-IR, and elemental analysis showed (i) nucleophilic attack of the exocyclic amino groups of the DNA bases yielded haloacyl (thio)amides with all reactants as clearly demonstrated by the FT-IR spectra; (ii) the sulfur in the initial thioamides seems to be rapidly exchanged with oxygen; (iii) the acyl chlorides form identical products but in lower yields as compared to the haloketenes. Reactions of the nucleosides with haloketenes showed the formation of similar nucleoside adducts upon HPLC and MS analysis. Beside the modification of the base moieties, additional peaks in the reaction mixtures analyzed suggested acylation of the deoxyribose hydroxyl groups. In aqueous solutions at pH 7 N6-(chloroacetyl)adenine, N4-(chloroacetyl)cytosine, and N2-(chloroacetyl)guanine are not stable and cleaved to the original base or form 1,N6-acetyladenine, 3,N4-acetylcytosine, 1,N2-acetylguanine, and N2,3-acetylguanine. Under the same conditions, N6-(dichloroacetyl)adenine and N4-(dichloroacetyl)cytosine were completely hydrolyzed to adenine and cytosine, respectively. All haloacyl DNA base adducts proved to be stable at pH 5 but were rapidly degraded at neutral or alkaline pH. The compounds with an additional five-membered ring remained unchanged after 1 week at room temperature. All synthesized DNA base adducts except N2-(chloroacetyl)guanine and 1,N2-acetylguanine were fluorescent. The characterized compounds, especially the etheno (epsilon) base adduct-related derivatives, may represent potential DNA adducts formed as a consequence of haloolefin bioactivation.

Chemical Phenomena↗

PubMed1998

Reactivity of haloketenes and halothioketenes with nucleobases: reactions in vitro with DNA.

Ketenes are important and highly reactive intermediates. Thioketenes are formed by cysteine conjugate beta-lyase-dependent biotransformation of 1-halovinylcysteine S-conjugates which are metabolites of several halogenated olefins. Nucleic acid constituents react with haloketenes and halothioketenes in vitro. Thioketenes induce DNA strand breaks in incubations of 1,2-dichlorovinyl 2-nitrophenyl disulfide, a thioketene precursor, with pBr 322 plasmid DNA. After treatment of single-stranded or native calf thymus DNA with chlorothioketene generated by the hydrolysis of 1,2-dichlorovinyl 2-nitrophenyl disulfide, the formation of 3,N4-thioacetylcytosine could be demonstrated. N6-(Dichloroacetyl)adenine and N4-(dichloroacetyl)cytosine, however, adducts formed by dichloroketene in vitro, are labile to hydrolysis. Therefore, the binding of this compound to DNA constituents in intact DNA is difficult to demonstrate. Substitution of one chlorine atom by fluorine allowed us to use 19F NMR as a tool to demonstrate the formation of adducts by dihaloketenes in intact DNA. N6-(Chlorofluoroacetyl)adenine and N4-(chlorofluoroacetyl)cytosine were synthesized (yields 77%, 15%, respectively) as references and characterized by LC/MS, 1H, 13C, and 19F NMR, FT-IR, and elemental analysis. To demonstrate the ability of dihaloketenes to bind to DNA, poly-dA (1 mg) and calf thymus DNA (10 mg) were suspended in DMF and treated with different concentrations of chlorofluoroketene (50-200 micromol). Analysis of the polymeric DNA by 19F NMR showed one doublet at -137.2 ppm downfield from the reference (CFCl3). A doublet at -146.9 ppm, characteristic for chlorofluoroacetic acid, an expected product of DNA adduct hydrolysis, was not detected. These results demonstrate the formation of a stable adenine adduct by dihaloketenes in intact calf thymus DNA.

Animals↗

PubMed1998

Biotransformation of 12C- and 2-13C-labeled methyl tert-butyl ether, ethyl tert-butyl ether, and tert-butyl alcohol in rats: identification of metabolites in urine by 13C nuclear magnetic resonance and gas chromatography/mass spectrometry.

The biotransformation of the fuel oxygenates methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE) was studied in rats after inhalation exposure; the biotransformation of the initial metabolite of these ethers, tert-butyl alcohol, was studied after oral gavage. To study ether metabolism, rats were exposed for 6 h to initial concentrations of 2000 ppm of MTBE or ETBE, respectively [2-13C]MTBE and [2-13C]ETBE. Urine was collected for 48 h after the end of the exposure, and urinary metabolites were identified by 13C NMR (13C-labeled ethers) and gas chromatography/mass spectrometry (GC/MS) (12C- and 13C-labeled ethers). To study tert-butyl alcohol metabolism, rats were dosed either with tert-butyl alcohol at natural carbon isotope ratio or with 13C-enriched tert-butyl alcohol (250 mg/kg of body weight), urine was collected, and metabolites were identified by NMR and GC/MS. tert-Butyl alcohol was identified as a minor product of the biotransformation of MTBE and ETBE. In addition, small amounts of a tert-butyl alcohol conjugate, likely a glucuronide, were present in the urine of the treated animals. Moreover, the mass spectra obtained indicate the presence of small amounts of [13C]acetone in the urine of [13C]MTBE and [13C]ETBE-treated rats. 2-Methyl-1,2-propanediol, 2-hydroxyisobutyrate, and another unidentified conjugate of tert-butyl alcohol, most probably a sulfate, were major urinary metabolites of MTBE and ETBE as judged by the intensities of the NMR signals. In [13C]-tert-butyl alcohol-dosed rats, [13C]acetone, tert-butyl alcohol, and its glucuronide represented minor metabolites; as with the ethers, 2-methyl-1,2-propanediol, 2-hydroxyisobutyrate, and the presumed tert-butyl alcohol sulfate were the major metabolites present. In one human individual given 5 mg/kg [13C]-tert-butyl alcohol orally, 2-methyl-1,2-propanediol and 2-hydroxyisobutyrate were major metabolites in urine detected by 13C NMR analysis. Unconjugated tert-butyl alcohol and tert-butyl alcohol glucuronide were present as minor metabolites, and traces of the presumed tert-butyl alcohol sulfate were also present. Our results suggest that tert-butyl alcohol formed from MTBE and ETBE is intensively metabolized by further oxidation reactions. Studies to elucidate mechanisms of toxicity for these ethers to the kidney need to consider potential toxicities induced by these metabolites.

Animals↗

PubMed1998

Biotransformation, excretion, and nephrotoxicity of the hexachlorobutadiene metabolite (E)-N-acetyl-S-(1,2,3,4, 4-pentachlorobutadienyl)-L-cysteine sulfoxide.

Hexachlorobuta-1,3-diene (HCBD) is nephrotoxic in rodents. Its toxicity is based upon a multistep bioactivation pathway. Conjugation with glutathione by glutathione S-transferases to form (E)-S-(1,2,3,4,4-pentachlorobutadienyl)-L-glutathione (PCBG), further processing to the corresponding cysteine S-conjugate, and finally processing to a reactive thioketene are thought to be responsible for the observed nephrotoxic effects. A novel metabolite, identified as (E)-N-acetyl-S-(1,2,3,4, 4-pentachlorobutadienyl)-L-cysteine sulfoxide (N-AcPCBC-SO), was described after administration of [14C]HCBD to male Wistar rats. This metabolite is formed by sulfoxidation of N-acetyl-S-(1,2,3,4, 4-pentachlorobutadienyl)-L-cysteine (N-AcPCBC) mediated by cytochrome P450 3A and has been found to be cytotoxic to proximal tubular cells in vitro without activation by beta-lyase. In rats, given HCBD in vivo, only one diastereomer of the sulfoxide is excreted; however, in rat hepatic microsomes two diastereomers, (R)- and (S)-N-AcPCBC-SO, are formed. This study focuses on the mechanisms responsible for this discrepancy and on a possible contribution of N-AcPCBC-SO to the nephrotoxicity of HCBD in vivo. (R,S)-N-AcPCBC-SO (1:1 mixture of both diastereomers) and N-acetyl-alpha-methyl-S-(1,2,3,4,4-pentachlorobutadienyl)-d, L-cysteine sulfoxide (alpha-Me-N-AcPCBC-SO) were administered iv to male and female Wistar rats (20, 40, and 80 micromol/kg of body weight). alpha-Me-N-AcPCBC-SO cannot be cleaved by cysteine conjugate beta-lyase even if alpha-Me-N-AcPCBC-SO is deacetylated by acylases. Excretion of gamma-glutamyltranspeptidase, protein, and glucose in the urine, indicative for kidney damage, and histopathological examination of the kidneys showed marked differences in the renal damage in male and female rats after application of N-AcPCBC-SO and alpha-Me-N-AcPCBC-SO. Necroses of the kidney tubules were only found in male, but not female, rats. Major sex-specific differences were observed in the elimination of sulfoxides; the (R)-isomer was excreted in a 5-10-fold excess to the (S)-isomer after application of (R,S)-N-AcPCBC-SO. After purification, both isomers were administered to male rats resulting in the urinary excretion of (R)-N-AcPCBC-SO after giving the (R)-isomer; treatment with (S)-N-AcPCBC-SO, however, revealed the formation of (S)-N-acetyl-S-(2-glycinylcystein-S-yl-1,3,4, 4-tetrachlorobutadienyl)-L-cysteine. The results show major sex-specific differences in the nephrotoxic potency of N-AcPCBC-SO and alpha-Me-N-AcPCBC-SO. However, both N-AcPCBC-SO and alpha-Me-N-AcPCBC-SO are nephrotoxic in males, suggesting the formation of a vinyl sulfoxide as an additional, beta-lyase-independent mechanism in HCBD-caused nephrotoxicity.

Animals↗