Gerhard Birner

Renal cell cancer correlated with occupational exposure to trichloroethene

Green and Lash (1999) commented, in a letter to the editor, on our paper reporting an increased incidence of renal cell cancer in workers exposed to high concentrations of trichloroethene over extended periods of time (Vamvakas et al. 1998). Unfortunately, because of irregular handling of the letter by the editorial management of the journal, we were not in a position to follow common practice, that is to respond immediately and in the same issue (see footnote). We do not accept the statement at the outset of Green and Lashs letter that signi®cant methodological ̄aws make our results unreliable. Rather, we regard their criticism as unsubstantiated, for following reasons.

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

Abstract 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 β-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 μmol trichloroacetic acid+trichloroethanol and 0.45 μmol 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.

Glutathione transferase alpha as a marker for tubular damage after trichloroethylene exposure

Abstract To investigate possible persistent nephrotoxic effects of trichloroethylene (TRI), a retrospective study was carried out on 39 workers exposed to high levels of TRI from 1956 to 1975. Total protein levels in urine, as well as serum and urine creatinine and serum urea were unchanged in comparison with the control. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to differentiate between tubular and/or glomerular dysfunction. Urinary excretion of alpha-1-microglobulin and glutathione transferase (GST) alpha, as markers of proximal tubular damage, were correlated with the SDS-PAGE patterns of urinary proteins both in the TRI exposed and the control group. GST alpha was found in elevated concentrations in the urine of the TRI-exposed workers. No increase of urinary GST alpha was observed in the control group, even when alpha-1-microglobulin was elevated as a result of non-toxic damage. Both in the control and exposed groups, GST pi, a marker of distal tubular damage, was in the normal range. The results show that chronic exposure to high doses of TRI causes persistent changes to the proximal tubular system of the kidney and that GST alpha excretion into the urine is a marker well suited for quantitation of the extent of renal damage.

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 withp-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.

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

Abstract 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 β-lyases and reactive intermediates are formed in this reaction. The equilibrium between cysteine S-conjugate and mercapturic acid thus influences the extent of β-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 nephro‐ toxicity 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 μmoles/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.

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.

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.

Endogenous alkaloids in man XXVI. Determination of the dopaminergic neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) in biological samples using gas chromatography with selected ion monitoring

Highly chlorinated beta-carbolines have a potential in vivo relevance to Parkinsons disease. In this paper, a gas chromatographic method for the determination of the neurotoxic 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo), the condensation product of tryptamine and chloral hydrate, is described. The specific and sensitive assay involves purification of the biological samples by solid-phase extraction with C18 cartridges, derivatization with heptafluorobutyric anhydride, and chromatography on a non-polar fused-silica capillary column. Detection of TaClo was achieved by the registration of characteristic mass fragments of the TaClo heptafluorobutyric amide derivative using selected ion monitoring. The method was utilized to detect and quantify TaClo in blood, urine, bile, faeces, and brain tissue of rats treated with this alkaloid-type heterocycle. Four-fold deuterium-labelled TaClo was used as an internal standard.