Nephrotoxic and genotoxic N-acetyl-S-dichlorovinyl-L-cysteine is a urinary metabolite after occupational 1,1,2-trichloroethene exposure in humans: implications for the risk of trichloroethene exposure.

Excretion of mercapturic acids in the urine is indicative of the formation of electrophiles in the metabolism of xenobiotics. The determination of these mercapturic acids thus may be a useful method to estimate the exposure. We identified the nephrotoxic and mutagenic mercapturic acids N-acetyl-S-(1,2-dichlorovinyl)-L- cysteine and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine in the urine of workers exposed to 1,1,2-trichloroethene. A method to quantify these mercapturic acids by gas chromatography-mass spectrometry-selected ion monitoring was developed and appreciable amounts (2.8-3.8 mumole/L were found in human urine samples. Because deacetylation determines notably the amount of the excreted mercapturic acids, the formation of the resulting cysteine S-conjugates was comparably measured in subcellular fractions of rodent and human kidneys; significant species differences in acylase activity were found. The formation of mutagenic and nephrotoxic metabolites during 1,1,2-trichloroethene metabolism mandates a revision of the risk assessment of trichloroethene exposure.

Hemoglobin adducts of N-substituted aryl compounds in exposure control and risk assessment.

Arylamines, nitroarenes, and azo dyes yield a common type of metabolite, the nitroarene, which produces a hydrolyzable adduct with protein and is closely related to the critical, ultimate toxic and genotoxic metabolite. The target dose as measured by hemoglobin adducts in erythrocytes reflects not only the actual uptake from the environment but also an individual's capacity for metabolic activation and is therefore an improved dosimeter for human exposure. The usefulness of hemoglobin adducts in molecular epidemiology is now widely recognized. With regard to risk assessment, many questions need to be answered. The described experiments in rats address some of these questions. The relationship between binding to hemoglobin in erythrocytes and to proteins in plasma has been found to vary considerably for a number of diamines. The fraction of hydrolyzable adducts out of the total protein adducts formed also varies in both compartments. This indicates that the kind of circulating metabolites and their availability in different compartments is compound specific. This has to do with the complex pattern of competing metabolic pathways, and the role of N-acetylation and deacetylation is emphasized. An example of nonlinear dose dependence adds to the complexity. Analysis of hemoglobin adducts reveals interesting insights into prevailing pathways, which not only apply to the chemical, but may also be useful to assess an individual's metabolic properties. In addition, it is demonstrated that the greater part of erythrocytes and benzidine-hemoglobin adducts are eliminated randomly in rats, i.e., following first-order kinetics.

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).

Acute intoxication with trichloroethene: clinical symptoms, toxicokinetics, metabolism, and development of biochemical parameters for renal damage.

The present study reports on a 17-year-old male who ingested approximately 70 ml trichloroethene (TRI) in a suicide attempt. The patient developed fever, tremor, general motor restlessness, and sinus tachycardia and lost consciousness 5 h after poisoning. After 5 days of intubation under narcosis with forced hyperventilation and diuresis he regained consciousness. During this period blood and urine were collected and TRI and its metabolites were quantified. The highest concentration of TRI in blood was detected 13 h after ingestion. Trichloroethanol and trichloroacetic acid, metabolites of the cytochrome P450-mediated pathway, and N-acetyl-S-(1, 2-dichlorovinyl)-l-cysteine and N-acetyl-S-(2, 2-dichlorovinyl)-l-cysteine from the glutathione-dependent pathway of TRI were quantified in urine samples. Besides these known metabolites in humans, chloroacetic acid and dichloroacetic acid were identified for the first time in urine of a human exposed to TRI. Although the patient exhibited normal levels of glucose and total protein in urine, excretion of alpha1- and beta2-microglobulin as well as beta-NAG was significantly increased. In addition to these typical markers of selective tubule damage, analysis of the urinary protein pattern by SDS-PAGE revealed increased excretion of several low-molecular-mass proteins between 10,000 and 50,000 Da, clearly indicating tubular damage. Based on the elucidated glutathione-dependent mechanism for the nephrotoxicity of TRI, activation of the formed S-conjugates by beta-lyases to reactive intermediates may account for the observed renal effects after a single, high dose of TRI.

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.

Biomonitoring of aromatic amines II: Hemoglobin binding of some monocyclic aromatic amines.

Covalent binding of 13 monocyclic aromatic amines to hemoglobin was studied in female Wistar rats and hemoglobin binding indices were determined. The hemoglobin adducts were hydrolyzed under alkaline conditions. In all cases the parent amine could be identified by gas chromatography and with one exception represented the only cleavage product. The binding index varied considerably and was highest with p-chloroaniline (569) and lowest with 2,4,5-trimethylaniline (0.7). Five compounds were also studied in female B6C3F1 mice. Hemoglobin binding was lower than in rats, but to varying degrees. Hemoglobin binding correlated remarkably well with the maximum methemoglobin level achieved with the six examples studied. The results support the notion that the reaction of nitrosoarenes, as metabolites of arylamines, with hemoglobin represents a general pathway in vivo. The analysis of such hemoglobin adducts is recommended as a dosimeter in biological monitoring of humans in order to control exposure. It is too early, however, to assess the carcinogenic risk from hemoglobin binding data with these compounds.

The role of cytochrome P4503A1/2 in the sex-specific sulfoxidation of the hexachlorobutadiene metabolite, N-acetyl-S-(pentachlorobutadienyl)-L-cysteine in rats.

Hexachlorobuta-1,3-diene (HCBD) is a selective nephrotoxin and a potent nephrocarcinogen in rodents. Its toxicity and carcinogenicity is based on a multistep bioactivation pathway. Glutathione conjugation seems to be the only bioactivation pathway for HCBD leading to reactive intermediates, which are thought to be responsible for the observed nephrotoxic effects. Recent in vivo studies revealed a novel urinary metabolite in male, but not female, rats after administration of [14C]HCBD. This metabolite was identified as (E)-N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine-sulfoxide (N-Ac-PCBC-SO). The objective of this study was to elucidate the enzyme(s) involved in this particular oxidation and to find an explanation for the sex differences in the formation of N-Ac-PCBC-SO. Both cytochrome P450 and flavin-containing monooxygenases (FMO) may catalyze the oxidation of N-Ac-PCBC-SO. The use of various competitive and allosteric inhibitors of cytochrome P450 and FMO (i.e. metyrapone, N-benzylimidazole, thiobenzamide, CO, n-octylamine, and heat inactivation studies) showed that the sulfoxidation of (E)-N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)- L-cysteine is catalyzed by cytochrome P450 enzymes. In microsomes from male rats pretreated with pyridine, phenobarbital, and dexamethasone, an increase in the rates of sulfoxide formation was only seen in microsomes from dexamethasone- and phenobarbital-induced animals. Moreover, troleandomycin, a selective chemical inhibitor for enzymes of the cytochrome P4503A family, inhibited sulfoxide formation by > 80%. Correlation of sulfoxide formation with testosterone 6 beta-hydroxylation, a marker of cytochrome P4503A1/2 in the rat, underlined the finding that cytochrome P4503A is the predominant cytochrome P450 responsible for this particular oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Sulfoxidation of mercapturic acids derived from tri- and tetrachloroethene by cytochromes P450 3A: a bioactivation reaction in addition to deacetylation and cysteine conjugate beta-lyase mediated cleavage.

In the present study we investigated the formation of sulfoxides from N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine (N-Ac-TCVC), N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (N-Ac-1,2-DCVC), and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine (N-Ac-2,2-DCVC), which are formed in the glutathione dependent bioactivation of tri- and tetrachloroethene. The first aim was to elucidate the enzymes involved in these oxidation reactions. N-Ac-TCVC, N-Ac-1,2-DCVC, and N-Ac-2,2-DCVC are oxidized to the corresponding sulfoxides mainly, if not exclusively, by cytochrome P450 enzymes in liver microsomes of untreated male rats, since no role for the flavin-containing monooxygenase (FMO) could be demonstrated by heat inactivation experiments and by the use of n-octylamine. The sulfoxidation rates were increased when using liver microsomes of phenobarbital and dexamethasone pretreated male rats as well as liver microsomes of dexamethasone pretreated female rats, while no sulfoxide formation was observed in liver microsomes of untreated female rats, suggesting an involvement of cytochrome P450 3A. Also, troleandomycin, a specific chemical inhibitor for cytochrome P450 3A, drastically reduced sulfoxidation rates. The observed rates of sulfoxidation also correlated well with the rates of oxidation of testosterone at the 6-beta-position, a specific marker for P450 3A activity. The second aim of this study was to compare the cytotoxicity of the sulfoxides with the cytotoxicity of the corresponding mercapturic acids in isolated rat renal epithelial cells. Both mercapturic acids and the corresponding sulfoxides were cytotoxic. Cytotoxicity of the mercapturic acids could be blocked by (aminooxy)acetic acid (AOAA), an inhibitor of cysteine conjugate beta-lyase, while the cytotoxicity of the sulfoxides was not influenced by this treatment. Moreover, the sulfoxides were significantly more cytotoxic than the corresponding mercapturic acids at equimolar doses. The results show that mercapturic acids derived from TRI and PER are oxidized to sulfoxides by microsomal monooxygenases from rat liver. The cytotoxicity of the produced sulfoxides could not be reduced by AOAA, consistent with a role of the sulfoxides as direct acting electrophiles (i.e., Michael acceptor substrates).

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.

N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine and 2,2,2-trichloroethanol: two novel metabolites of tetrachloroethene in humans after occupational exposure.

The excretion of tetrachloroethene metabolites in urine was studied in occupationally exposed workers to identify and quantify metabolites formed by glutathione conjugation and by cytochrome P450 oxidation of tetrachloroethene in humans. The glutathione conjugation pathway has been implicated in the chronic toxicity and possible tumorigenicity of tetrachloroethene to the kidney in rats. The biosynthesis of S-(1,2,2-trichlorovinyl)glutathione and N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine in humans had not been demonstrated. In this study, we investigated the biotransformation of tetrachloroethene in humans occupationally exposed during dry cleaning. Tetrachloroethene concentrations in the air of the dry cleaning shop were 50 +/- 4 ppm; two individuals were exposed for 8 hr daily and two individuals were exposed for 4 hr daily. In urine samples collected from the individuals at the beginning and at the end of the work week, N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine and 2,2,2-trichloroethanol as tetrachloroethene metabolites in humans were identified by GC/MS. The concentrations of N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine in the urine of the individuals were not significantly different at the start and at the end of the work week; however, concentrations of both N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine and 2,2,2-trichloro compounds (trichloroacetic acid and 2,2,2-trichloroethanol) as a marker for cytochrome P450-mediated metabolism were proportional to the length of daily tetrachloroethene exposure. A remarkable difference in the excretion pattern of 2,2,2-trichloro compounds, the major tetrachloroethene metabolites, was observed. Trichloroacetic acid and 2,2,2-trichloroethanol were present in the urine of two of the exposed individuals. Only 2,2,2-trichloroethanol was identified as a major urinary tetrachloroethene metabolite in two other individuals who did not excrete detectable amounts of trichloroacetic acid. The obtained results indicate that humans also have the ability to biosynthesize nephrotoxic glutathione S-conjugates from tetrachloroethene; however, when compared with rats, the human capacity for the biosynthesis of N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine seems to be lower.

Biomonitoring of aromatic amines. III: Hemoglobin binding of benzidine and some benzidine congeners.

Covalent binding of benzidine and some congeners to hemoglobin was studied in female Wistar rats after oral administration. Hemoglobin adducts were hydrolyzed under alkaline conditions, and the arylamines extracted and analysed by HPLC with electrochemical detector. With benzidine, three cleavage products were observed, the major component being monoacetylbenzidine. This indicates that 4-nitroso-4'-N-acetylaminobiphenyl is the major reactive metabolite in erythrocytes. In addition benzidine and 4-aminobiphenyl were identified. The latter indicates a hitherto unknown metabolic pathway of benzidine. With 3,3'-dichlorobenzidine-dihydrochloride, 3,3'-dimethoxybenzidine and 3,3'-dimethylbenzidine two cleavage products were observed, the parent diamines being present in excess to or in amounts comparable to the monoacetyl derivative. With 3,3',5,5'-tetramethylbenzidine a hemoglobin adduct could not be found. When the azo dye direct red 28 was administered to the animals, the three cleavage products typical for benzidine were found, indicating that benzidine became bioavailable after reductive cleavage of the azo compound. In this case the fraction of 4-aminobiphenyl was greater than after benzidine. It is proposed to use the analysis of hemoglobin adducts in human blood to control the exposure of individuals to these carcinogenic chemicals in the course of biochemical effect monitoring.

Biotransformation of trichloroethene: dose-dependent excretion of 2,2,2-trichloro-metabolites and mercapturic acids in rats and humans after inhalation.

Chronic bioassays with trichloroethene (TRI) demonstrated carcinogenicity in mice (hepatocellular carcinomas) and rats (renal tubular cell adenomas and carcinomas). The chronic toxicity and carcinogenicity is due to bioactivation reactions. TRI is metabolized by cytochrome P450 and by conjugation with glutathione. Glutathione conjugation results in S-(dichlorovinyl) glutathione (DCVG) and is presumed to be the initial biotransformation step resulting in the formation of nephrotoxic metabolites. Enzymes of the mercapturic acid pathway cleave DCVG to the corresponding cysteine S-conjugate, which is, after translocation to the kidney, cleaved by renal cysteine S-conjugate beta -lyase to the electrophile chlorothioketene. After N-acetylation, cysteine S-conjugates are also excreted as mercapturic acids in urine. The object of this study was the dose-dependent quantification of the two isomers of N-acetyl-S-(dichlorovinyl)-L-cysteine, trichloroethanol and trichloroacetic acid, as markers for the glutathione- and cytochrome P450-mediated metabolism, respectively, in the urine of humans and rats after exposure to TRI. Three male volunteers and four rats were exposed to 40, 80 and 160 ppm TRI for 6 h. A dose-dependent increase in the excretion of trichloroacetic acid, trichloroethanol and N-acetyl-S-(dichlorovinyl)-L-cysteine after exposure to TRI was found both in humans and rats. Amounts of 3100 mumol trichloroacetic acid + trichloroethanol and 0.45 mumol mercapturic acids were excreted in urine of humans over 48 h after exposure to 160 ppm TRI. The ratio of trichloroacetic acid + trichloroethanol/mercapturic acid excretion was comparable in rats and humans. A slow rate of elimination with urine of N-acetyl-S-(dichlorovinyl)-L-cysteine was observed both in humans and in rats. However, the ratio of the two isomers of N-acetyl-S-(dichlorovinyl)-L-cysteine was different in man and rat. The results confirm the finding of the urinary excretion of mercapturic acids in humans after TRI exposure and suggest the formation of reactive intermediates in the metabolism of TRI after bioactivation by glutathione also in humans.

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.

Generation of antibodies to Di- and trichloroacetylated proteins and immunochemical detection of protein adducts in rats treated with perchloroethene.

Antibodies directed against chemical specific protein modifications are valuable tools to detect and comparatively quantify protein modifications. Both Nepsilon-(dichloroacetyl)-L-lysine and Nepsilon-(trichloroacety)l-L-lysine have been detected as modified amino acids in liver and kidneys of rats treated with perchloroethene (PER) after proteolysis. These protein modifications are formed by the interaction of reactive metabolites formed from PER with proteins. In this study we developed monospecific antibodies to dichloroacetylated and to trichloroacetylated amino acids to detect modified proteins in the target organs of PER toxicity. These antibodies were prepared by immunization of rabbits with modified keyhole limpet hemocyanin (KLH) coupled with either the dichloroacetyl or trichloroacetyl moiety. Enzyme-linked immunosorbent assays (ELISA) indicated that the polyclonal rabbit sera recognized dichloroacetylated or trichloroacetylated rabbit serum albumin (RSA), but not unmodified protein. Therefore, we further purified rabbit antisera on either Nepsilon-(dichloroacetyl)-L-lysine or Nepsilon-(trichloroacetyl)-L-lysine immobilized to immunoaffinity columns to obtain monospecific antibodies. The potential of these antibodies in the detection of di- and trichloroacetylated proteins and their selectivity for the desired dichloroacetyl or trichloroacetyl group was demonstrated in competitive enzme-linked immunosorbent assays with several structurally related compounds. Anti-dichloroacetyl (anti-DCA) antibody binding to dichloroacetylated RSA was inhibited by Nepsilon-(dichloroacetyl)-L-lysine with an IC50 value of 150 microM whereas inhibition by Nepsilon-(monochloroacetyl)-L-lysine and Nepsilon-(trichloroacetyl)-L-lysine showed an IC50 value of 100 mM. The binding of the anti-trichloroacetyl (anti-TCA) antibody to trichloroacetylated RSA was inhibited by Nepsilon-(dichloroacetyl)-L-lysine with an IC50 value of 80 mM. The inhibition by Nepsilon-(trichloroacetyl)-L-lysine was again 3 orders of magnitude stronger resulting in an IC50 value of 90 microM. Nepsilon-(acetyl)-L-lysine and unmodified RSA did not effect antibody binding to the chemically modified antigen. The antibodies were also successfully applied to detect modified proteins in subcellular fractions of liver and kidney from PER treated rats demonstrated in immunoblot. Protein adduct formation from different PER metabolism pathways was confirmed by the observation that the majority of dichloroacetylated proteins were located in kidney mitochondria and trichloroacetylated proteins were located in liver microsomes.

Glutathione-dependent biosynthesis and bioactivation of S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine, the glutathione and cysteine S-conjugates of dichloroacetylene, in rat tissues and subcellular fractions.

Dichloroacetylene is neurotoxic, nephrotoxic, and nephrocarcinogenic. The present experiments were designed to test further the hypothesis that the glutathione conjugate of dichloroacetylene, S-(1,2-dichlorovinyl)glutathione (DCVG), may be involved in dichloroacetylene-induced neurotoxicity. Hence, the biosynthesis of DCVG from dichloroacetylene and glutathione was studied in cytosolic, microsomal, and mitochondrial fractions of liver, lung, brain, and kidney. Enzymatic formation of DCVG was observed in liver microsomes and mitochondria, but only nonenzymatic formation of DCVG was seen in lung, brain, and kidney. The KM and Vmax for DCVG biosynthesis with glutathione as the variable substrate were 0.40 +/- 0.28 mM and 487 +/- 90.3 nmol/mg protein/min, respectively. No region-specific differences in the rates of DCVG biosynthesis in the brain were observed. In whole brain homogenates, DCVG was hydrolyzed to DCVC, which was biotransformed to pyruvate and S-(1,2-dichlorovinyl)-3- mercaptopyruvate, indicating cysteine conjugate beta-lyase-catalyzed beta-elimination and transamination reactions in cerebellar tissues. These findings indicate that the glutathione-dependent bioactivation of dichloroacetylene may be involved in the dichloroacetylene-induced neurotoxicity.

Sex differences in hexachlorobutadiene biotransformation and nephrotoxicity.

Hexachlorobutadiene is nephrotoxic in rats, causing damage to the proximale tubules. Renal toxicity is presumed to be due to bioactivation by glutathione S-conjugate formation. Hexachlorobutadiene is conjugated with glutathione to S-(1,2,3,4,4-pentachlorobutadienyl)glutathione and further transformed to S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBC), which is N-acetylated in the liver to form N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (N-ac-PCBC). N-ac-PCBC is accumulated in the kidney. Renal acylases cleave N-ac-PCBC to PCBC, which is a substrate for renal cysteine conjugate beta-lyase and transformed to a reactive thioketene. Binding of this intermediate to renal macromolecules is most likely responsible for the nephrotoxicity of hexachlorobutadiene. In this study, we administered [14C]HCBD (200 mg/kg, per gavage) to male and female Wistar rats and compared the distribution and biotransformation. No significant differences in the disposition and rates of excretion of [14C]hexachlorobutadiene-derived radioactivity were observed between male and female rats. A portion of the dose (15.6 +/- 4.2) was excreted in the feces and 3.1% ( +/- 0.7) in the urine of male rats, and 11.1% ( +/- 3.8) of the dose was excreted in the feces and 4.5% ( +/- 1.5) in the urine of female rats. The major metabolite excreted by female rats was N-ac-PCBC, while small amounts of PCBC were also detected. In the urine of male rats, in addition to small amounts of PCBC and N-ac-PCBC, N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide and [14C]hexachlorobutadiene were identified. Formation of the electrophile N-ac-PCBC sulfoxide must be considered as an alternative, beta-lyase-independent, bioactivation pathway for hexachlorobutadiene-derived S-conjugates. In isolated rat renal tubular cells, N-ac-PCBC sulfoxide induced a significantly more marked loss in cell viability than N-ac-PCBC. After identical doses of hexachlorobutadiene, the extent of necrosis to the pars recta of the proximal tubules was increased in male rats compared to the necrotic changes in female rats. While female animals showed a normal liver histology, male rats revealed slight toxic centrilobular liver changes in addition to the renal necroses. In vitro, only liver microsomes from male rats catalyzed the formation of N-ac-PCBC sulfoxide from N-ac-PCBC. Our results describe a new pathway of hexachlorobutadiene biotransformation in male rats, the formation of a mercapturic acid sulfoxide. The formation of this Michael acceptor may contribute to sex differences in hexachlorobutadiene nephrotoxicity.

Biotransformation, excretion and nephrotoxicity of haloalkene-derived cysteine S-conjugates.

The formation of cysteine S-conjugates is thought to play an important role in the nephrotoxicity of haloalkenes such as trichloroethene, tetrachloroethene and hexachlorobutadiene. Glutathione S-conjugates formed from these haloalkenes in the liver are processed to the corresponding cysteine S-conjugates, which may be N-acetylated to mercapturic acids and may be accumulated in the kidney. Haloalkene-derived cysteine S-conjugates are also substrates for cysteine conjugate beta-lyases and reactive intermediates are formed in this reaction. The equilibrium between cysteine S-conjugate and mercapturic acid thus influences the extent of beta-lyase dependent bioactivation and subsequently the nephrotoxicity of S-conjugates. In this study, we compared the rates of N-acetylation in vitro and the biotransformation, excretion and nephrotoxicity of S-(1,2-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC), S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) and S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBC) in rats after i.v. injection (40 micromoles/kg). Marked differences in the extent of enzymatic N-acetylation were observed; N-acetylation was most efficient with 2,2-DCVC and least efficient with 1,2-DCVC. In urine, within 48 h, most of the given 2,2-DCVC (77% of the recovered dose) and 1,2-DCVC (92%) were recovered as the corresponding mercapturic acids. In contrast, a higher percentage of cysteine S-conjugate and less of the mercapturic acid were recovered in urine after administration of PCBC and TCVC (50 and 23% of dose as mercapturic acid), respectively. Histopathological examination of the kidneys and urine clinical chemistry showed marked differences in the extent of renal damage. Necroses of the proximal tubules were found after TCVC, PCBC and 1,2-DCVC administration in male, but not in female rats. These differences in nephrotoxicity do not correlate with the balance of acetylation/deacetylation. The higher toxicity observed in male rats may indicate the involvement of other parameters such as uptake mechanisms.

Binding of hexachlorobutadiene to alpha 2u-globulin and its role in nephrotoxicity in rats.

Hexachlorobutadiene (HCBD) is nephrotoxic in rats causing damage to the proximal tubules. Renal toxicity is presumed to be due to bioactivation by glutathione S-conjugate formation and further processing by the enzymes of the mercapturic acid pathway to reactive intermediates. Recent studies revealed major sex-dependent differences in the pattern of urinary metabolites and gave evidence for the excretion of unmetabolized HCBD in the urine of male, but not female, rats. The objective of this study was to investigate the basis for the excretion of unchanged HCBD in the urine. We administered [14C]-HCBD (200 mg/kg bw, po) to male and female Sprague-Dawley (SD) and NCI Black-Reiter rats (NBR), an alpha 2u-globulin-deficient strain. No major differences in the disposition and in the rates of excretion of [14C]-derived radioactivity were observed between animals of both strains. Previously observed sex-specific differences in the formation of urinary metabolites in Wistar rats were now confirmed in SD rats and were also found in NBR rats. In contrast to male SD rats, however, NBR rats did not excrete unmetabolized HCBD with urine. [14C]-HCBD (10% of total urinary metabolites) was only present in the urine of male SD rats. Anion-exchange HPLC showed radioactivity associated with the alpha 2u-globulin fraction in urine and renal cytosol of male SD rats; the radioactive compound was identified as HCBD bound to the protein. The results indicate that the male-specific urinary excretion of HCBD is associated with its binding to alpha 2u-globulin. Light microscopic examination revealed the formation of hyaline droplets indicative of the accumulation of alpha 2u-globulin in the kidney of male SD rats after staining with Lee's methylene blue basic fuchsin. H&E staining additionally confirmed the finding of more pronounced necrotic changes in renal tubules of male SD rats than in females as previously described for Wistar rats. Binding of HCBD to alpha 2u-globulin may contribute to the pronounced nephrotoxicity in male rats.

Metabolism of tetrachloroethene in rats: identification of N epsilon-(dichloroacetyl)-L-lysine and N epsilon-(trichloroacetyl)-L-lysine as protein adducts.

Tetrachloroethene causes renal tumors in male rats after inhalation exposure. Tetrachloroethene is metabolized by cytochrome P-450 and by glutathione conjugation. Cytochrome P-450-dependent oxidation results in the formation of trichloroacetyl chloride, which may acylate cellular nucleophiles; glutathione conjugation results in the formation of S-(1,2,2-trichlorovinyl)glutathione, which is metabolized to the corresponding cysteine S-conjugate. S-(1,2,2-Trichlorovinyl)-L-cysteine is activated by renal cysteine conjugate beta-lyase to give dichlorothioketene. Covalent binding of this electrophile is presumably responsible for the renal toxicity of tetrachloroethene. In this report, we demonstrate the formation of protein adducts formed from tetrachloroethene using SDS-PAGE and immunochemical detection with rabbit anti-trifluoroacetyl serum. This serum recognizes dichloroacetylated rabbit serum albumin prepared by chemical modification of rabbit serum albumin with S-ethyl dichlorothioacetate and exhibited a high specificity for N epsilon-(dichloroacetyl)-L-lysine residues in proteins as shown by competitive ELISA. In the liver of [14C]tetrachloroethene-treated rats, the antibody recognized several modified proteins in microsomes. A protein adduct in rat liver identified by GC/MS after hydrolysis was N epsilon-(trichloroacetyl)-L-lysine. Western blots of renal fractions from rats treated with [14C]tetrachloroethene (200 mg/kg) or S-(1,2,2-trichlorovinyl)-L-cysteine (40 mumol/kg, iv) suggested the presence of modified mitochondrial and cytosolic proteins; no modified proteins were detected in microsomes. Proteins of identical molecular weight were modified by tetrachloroethene and by S-(1,2,2-trichlorovinyl)-L-cysteine in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

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.