Dietrich Henschler

Increased incidence of renal cell tumors in a cohort of cardboard workers exposed to trichloroethene

A retrospective cohort study was carried out in a cardboard factory in Germany to investigate the association between exposure to trichloroethene (TRI) and renal cell cancer. The study group consisted of 169 men who had been exposed to TRI for at least 1 year between 1956 and 1975. The average observation period was 34 years. By the closing day of the study (December 31, 1992) 50 members of the cohort had died, 16 from malignant neoplasms. In 2 out of these 16 cases, kidney cancer was the cause of death, which leads to a standard mortality ratio of 3.28 compared with the local population. Five workers had been diagnosed with kidney cancer: four with renal cell cancers and one with a urothelial cancer of the renal pelvis. The standardized incidence ratio compared with the data of the Danish cancer registry was 7.97 (95% Cl: 2.59–18.59). After the end of the observation period, two additional kidney tumors (one renal cell and one urothelial cancer) were diagnosed in the study group. The control group consisted of 190 unexposed workers in the same plant. By the closing day of the study 52 members of this cohort had died, 16 from malignant neoplasms, but none from kidney cancer. No case of kidney cancer was diagnosed in the control group. The direct comparison of the incidence on renal cell cancer shows a statistically significant increased risk in the cohort of exposed workers. Hence, in all types of analysis the incidence of kidney cancer is statistically elevated among workers exposed to TRI. Our data suggest that exposure to high concentrations of TRI over prolonged periods of time may cause renal tumors in humans. A causal relationship is supported by the identity of tumors produced in rats and a valid mechanistic explanation on the molecular level.

Bacterial β-lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene

The metabolism of beta-lyase and the mutagenicity of the synthetic cysteine conjugates S-1,2-dichlorovinylcysteine (DCVC), S-1,2,2-trichlorovinylcysteine (TCVC), S-1,2,3,4,4-pentachlorobuta-1,3-dienylcysteine (PCBC) and S-3-chloropropenylcysteine (CPC) were investigated in Salmonella typhimurium strains TA100, TA2638 and TA98. The bacteria contained significantly higher concentrations of beta-lyase than mammalian subcellular fractions. Bacterial 100,000 X g supernatants cleaved benzthiazolylcysteine to equimolar amounts of mercaptobenzthiazole and pyruvate. DCVC, TCVC and PCBC produced a linear time-dependent increase in pyruvate formation when incubated with bacterial 100,000 X g supernatants; pyruvate formation was inhibited by the beta-lyase inhibitor aminooxyacetic acid (AOAA). CPC was not cleaved by bacterial enzymes to pyruvate. DCVC, TCVC and PCBC were mutagenic in three strains of S. typhimurium (TA100, TA2638 and TA98) in the Ames-test without addition of mammalian subcellular fractions; their mutagenicity was decreased by the addition of AOAA to the preincubation mixture. CPC was not mutagenic in any of the strains of bacteria tested. These results indicate that beta-lyase plays a key role in the metabolism and mutagenicity of haloalkenylcysteines when tested in S. typhimurium systems. The demonstrated formation in mammals of the mutagens DCVC, TCVC and PCBC during biotransformation of trichloroethylene (Tri), tetrachloroethylene (Tetra) and hexachlorobutadiene (HCBD) may provide a molecular explanation for the nephrocarcinogenicity of these compounds.

Carcinogenicity study of trichloroethylene by longterm inhalation in three animal species

Pure trichloroethylene (tri), stabilized by an amine base, was administered by inhalation at 0, 100, and 500 ppm for 6 h/day, 5 days/week, for 18 months to mice, rats and Syrian hamsters of both sexes. No significant increase in tumor formation was observed in any species or dosing group, except in malignant lymphomas, which were increased in female mice in the following incidence rates: 9/29 (controls), 17/30 (100 ppm), and 18/28 (500 ppm). Whether or not this high occurrence of lymphomas, which is peculiar to this strain of mice (NMRI) has any relationship to tri-exposure, cannot be decided upon by the present experiment. It is concluded that from these findings no indication for a carcinogenic potential of pure trichloroethylene can be deduced.

Novel metabolites of trichloroethylene through dechlorination reactios in rats, mice and humans☆

The excretion and biotransformation of [14C]trichloroethylene (Tri) has been studied in female rats and mice. Seventy-two hours after a single oral dose of 200 mg/kg, rats exhaled 52% and mice 11% of the recovered radioactivity as unchanged Tri, and 1.9% and 6%, respectively, as 14CO2. Rats excreted 41.2% of the recovered radioactivity in the urine, in contrast to mice where urinary activity amounted to 76%. The isolation of urinary metabolites was accomplished by reversed-phase HPLC, using a water-methanol gradient. After chemical derivatization, a combination of radio-GC and GC/MS was used for identification. The metabolites identified in rat urine were: trichloroacetic acid (15.3%); trichloroethanol, free (11.7%) and as the glucuronide (61.9%); dichloroacetic acid (2.0%); oxalic acid (1.3%) and N-(hydroxyacetyl)-aminoethanol (HAAE) (7.2%). In mice, trichloroethanol (free and in several conjugated forms) is the main metabolite of Tri (94.3%), but small amounts of HAAE (4.1%) and oxalic acid (0.7%) are also excreted. Only traces of dichloro- and trichloroacetic acids were found in this species. In human male subjects, HAAE was also identified as a urinary metabolite of Tri after exposure of two volunteers to 200 ppm Tri for 6 hr. The identification of HAAE and oxalic acid as metabolites indicates hydrolytic dechlorination reactions in the metabolism of Tri.

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.

Analysis of ras mutations in human melanocytic lesions : activation of the ras gene seems to be associated with the nodular type of human malignant melanoma

We have analyzed the Ha-ras, Ki-ras and N-ras gene for point mutations at codons 12, 13 and 61 via restriction fragment length polymorphism/polymerase chain reaction analysis and subsequent direct sequencing in non-cultured fresh-frozen tissues of 16 superficial spreading melanomas (SSM), 13 nodular malignant melanomas (NMM), 2 lentigo malignant melanomas (LMM), 1 dysplastic nevus, 1 congenital nevus and 5 normal nevi from 38 patients. Mutations were found in 4 melanoma samples, all belonging to the nodular malignant type. Three of them were mutated in N-ras and one in the Ha-ras gene. Mutation in N-ras was also detected in the congenital nevus. All mutations were exclusively located at the first two base pairs of codon 61. No Ki-ras mutation was detected in any lesion. No mutation could be found in SSM and LMM in addition to dysplastic and normal nevi. The frequency ofras mutation in NMM was 31%, whereas in SSM it was 0%. Our study suggests (a) an association betweenras mutations (mainly N-ras) and the NMM as a subgroup of human melanoma; (b) that activation of Ki-ras is not involved in the pathogenesis of melanoma. The role of UV radiation in point mutations ofras genes in human melanoma is discussed.

Carcinogenicity of trichloroethylene: fact or artifact?

Technical trichloroethylene has been found carcinogenic in mice after high daily doses per os. A GC-MS analysis of this technical sample revealed the presence of considerable amounts of epichlorohydrin and 1.2-epoxibutane as stabilizers. These epoxides are highly mutagenic in the Ames test and are, most probably, responsible for the carcinogenic effect found in mice. The question whether trichloroethylene is carcinogenic or not remains open.ZusammenfassungTechnisches Trichloräthylen erwies sich nach hohen, täglichen oralen Dosen an Mäusen als carcinogen. Eine GC-MS-Analyse des benutzten technischen Präparates ergab die Gegenwart beträchtlicher Gehalte an Epichlorhydrin und 1.2-Epoxibutan, die im Ames-Test stark mutagen sind; diese Epoxide tragen höchstwahrscheinlich die carcinogene Wirkung in dem für den Carcinogeneseversuch verwendeten technischen Produkt, wo sie als Stabilisatoren zugesetzt werden. Die Frage, ob Trichloräthylen carcinogen ist oder nicht, bleibt offen.

What is there that is not poison? A study of the Third Defense by Paracelsus

University of Miami School of Medicine, 1931 South Bayshore Drive, Coconut Grove, Florida 33133, USA 2 Institute of Toxicology and Pharmacology, University of W/irzburg, Verbacher Strage 9, D-8700 W/irzburg, Federal Republic of Germany 3 Department of Toxicology, Karolinska Institute, Box 60208, S-10401 Stockholm 60, Sweden 4 Institute of History of Medicine, University of W/irzburg, K611ikerstral3e 6, D-8700 Wiirzburg, Federal Republic of Germany

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.

Correlation of alkylating and mutagenic activities of allyl and allylic compounds: standard alkylation test vs. kinetic investigation.

Thirty-nine allylic and non-allylic compounds have been tested in the standard 4-(p-nitrobenzyl)pyridine (NBP) alkylating procedure and the Salmonella typhimurium mutagenicity assay. Fourteen of these were found directly mutagenic (without addition of S-9 mix activating enzyme system). With twelve of these compounds, a good correlation of alkylating and mutagenic potencies was established; the remaining two do not meet the chemical conditions of the NBP procedure on account of HCl elimination with these two compounds. The other 25 substances were inactive in both systems. The quantitative correlation proved to be almost linear in the lower activity ranges (E approximately 2; revertants/muml approximately 600). The reasons for some deviations from the linear relationship have been analyzed and discussed on the basis of structural features. In addition to the standard alkylation test, a modified NBP-test was performed in order to obtain kinetic data and activation energy values. The results with 6 representative allylic compounds show that the overall correlation is not substantially improved above that of the standard procedure: nonetheless, additional information on reaction characteristics is obtained with some substances.