Manfred Metzler

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.

Peroxidase-mediated oxidation, a possible pathway for metabolic activation of diethylstilbestrol

Abstract In vitro oxidation of diethylstilbestrol (DES) by peroxidase preparations from horse radish or mouse uterus in the presence of hydrogen peroxide yields β -dienestrol, which is also a major in vivo metabolite of DES in several mammalian species. The oxidation reaction appears to involve reactive intermediates, presumably the semiquinone and quinone of DES, since nonextractable binding to salmon sperm deoxyribonucleic acid and bovine serum albumin was found. The peroxidase-catalyzed oxidation of DES to reactive metabolites in estrogen target organs may be related to the organ toxicity of this synthetic estrogen.

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.

Absorption, elimination and metabolism of trichloroethylene: a quantitative comparison between rats and mice

The absorption, elimination and metabolism of 14C-trichloroethylene (Tri) was studied in adult female Wistar rats and NMRI mice after administration of 200, 20 and 2 mg/kg Tri. Dose-dependent biotransformation of Tri to metabolites was observed in both species. Induction of hepatic mono-oxygenases by phenobarbital or polychlorinated biphenyls resulted in a higher rate of biotransformation after a single oral dose of 200 mg/kg 14C-Tri to rats. An increase in radioactivity covalently bound to liver and kidney macromolecules of induced rats as compared to control rats parallels the toxic effects of Tri on these organs after induction of cytochrome P-450. The urinary metabolites were analysed by h.p.l.c. In both species, 1,1,1-trichlorocompounds (trichloroacetic acid, trichloroethanol and its glucuronide, comprising 88.9-93.5% of the radioactivity excreted in the urine) constituted the main metabolites; in addition, N-(hydroxyacetyl)-aminoethanol (4.1-7.2%), dichloroacetic acid (0.1-2.0%) and oxalic acid (0.7-1.8%) were identified. The pattern of metabolites in the 72 h urine remained constant for each species in the dose range studied and no change was induced by pretreatment. The percentage of radioactivity exhaled as 14CO2 increased with dose in mice, which may indicate dose-dependent formation of dichloroacetic acid and saturation of deactivating mechanisms for reactive intermediates in mice.

Metabolic activation of diethylstilbestrol: Indirect evidence for the formation of a stilbene oxide intermediate in hamster and rat

The synthetic estrogen diethylstilbestrol (DES) has been associated with the occurrence of vaginal and cervical tumors in young women whose mothers had received DES medication during pregnancy (1,2). Among the data available for the carcinogenic activity of DES in animals (3), the high incidence of renal tumors in male hamsters is evident (4,5). Furthermore, binding of DES to tissue macromolecules has been related to hepatic centrolobular necrosis. It has been shown by in vitro experiments that this binding requires a reactive intermediate generated from DES by a cytochrome P-450 dependent oxidation (6,7). Studies carried out in our laboratory about the biotransformation of carcinogenic trans-4-dimethylaminostilbene and its inactive cis-isomer revealed that epoxidation of the stilbene double bond represents a major pathway for the in vivo metabolism of stilbene amine derivatives (8). Since an epoxide of DES as a reactive intermediate metabolite could possibly account for the toxic properties, we are presently investigating the in vivo metabolism of DES in laboratory animals. In this communication we wish to report the identification of six new metabolites from urine and bile of hamster and rat, some of which can be taken as evidence for the metabolic epoxidation of the stilbene double bond in DES. The study was carried out using a mixture of [G-3H]-DES obtained from the Radiochemical Centre Amersham and [monoethyl-l.2-2H5] -DES prepared from perdeutero-ethanol in this laboratory by a modification of the methods of Dodds and Kuwada (9,10). Male Syrian golden hamsters (150-200 g body wt.) and female Wistar rats (200-250 g body wt.) were injected i.p. with 50 mg/kg [3H,2H]-DES dissolved in 0.5 ml of propane-l,2-diol. Urine was collected in 24-hour periods for 5 days and stored at -20°C. Bile was obtained from unanesthesized cannulated

Molecular mechanism of 1,1-dichloroethylene toxicity: excreted metabolites reveal different pathways of reactive intermediates

The excretion and biotransformation of [14C] 1,1-dichloroethylene (vinylidene chloride, VDC) after administration of a single oral dose has been investigated in female rats. Seventy-two hours after a dose of 0.5, 5.0, and 50.0 mg/kg, 1.26, 9.70, 16.47%, respectively, are exhaled as unchanged VDC, and 13.64, 11.35, 6.13% as 14CO2. The main pathway of elimination is through renal excretion with 43.55, 53.88, 42.11% of the administered radioactivity. Through the biliary system, 15.74, 14.54, 7.65% of the activity are eliminated.The isolation of the main metabolites of VDC from 24 h urine is accomplished through the combined application of solvent extraction, ion exchange chromatography and thin layer chromatography. Then gas chromatography and mass spectrometry are used for their identification. Three metabolites have been identified: thiodiglycolic acid, N-acetyl-S-(2-carboxymethyl)cysteine and methylthio-acetylaminoethanol. In addition, three smaller unidentified radioactive peaks have been found. Thiodiglycolic acid is the main metabolite in VDC metabolism. The simultaneous formation of an ethanolamine- and a cysteine-conjugation product points to different reaction pathways of the postulated intermediate reactive epoxide; ethanolamine probably originates from membrane lipids, which react with VDC-epoxide and/or its derivatives. This pathway could explain, in part, the parenchyma damaging effect of VDC.

Oxidative metabolites of diethylstilbestrol in the fetal, neonatal and adult mouse☆

Abstract Urinary metabolites of 14 C-labeled diethylstilbestrol (DES) in adult female mice were identified by radio gas chromatography and gas chromatography-mass spectrometry as β-dienestrol and ω-hydroxy-derivatives of DES, ψ-DES and dienestrol. The metabolic β-dienestrol does not appear to be formed through a DES epoxide. Most of the metabolites found in adult mice were also detected by mass fragmentography in the whole-body homogenates obtained from mice of age 1.4 and 8 days, indicating that oxidative metabolism of DES is operative in neonatal mice. Furthermore, extensive conjugation of DES and its metabolites to glucuronic acid was found in adult and neonatal mice. Part of the administered radioactivity was unextractable from tissue macromolecules in neonatal mice. The biotransformation of DES and binding of its metabolites, implies a metabolic activation of this synthetic estrogen that might be associated with its teratogenic and carcinogenic potential for neonatal and fetal mice.

Metabolism of stilbene estrogens and steroidal estrogens in relation to carcinogenicity

The oxidative metabolism of diethylstilbestrol (DES) and 17α-ethynyl estradiol, as examples of stilbene- and steroid-type estrogens, is discussed with respect to the formation of reactive intermediates. For DES, a genotoxic potential is implied by metabolic studies and positive effects in short-term tests for genetic damage. A particularly important pathway for DES carcinogenicity appears to be peroxidase-mediated oxidation. Although data for steroidal estrogens are more ambiguous, the available evidence suggests that metabolic activation by peroxidatic oxidation may also be of importance for this class of estrogens.

Excretion pattern and metabolism of hexachlorobutadiene in rats: Evidence for metabolic activation by conjugation reactions☆

Excretion, covalent binding and metabolism of hexachloro-1,3-butadiene (HCBD), a nephrotoxic and nephrocarcinogenic compound, have been studied in female rats. Seventy-two hours after administration of a single oral dose of 1 mg/kg [14C]HCBD, 5.3% of the dose were exhaled as unchanged HCBD and 76.3% were metabolized and excreted in urine and feces or exhaled as 14CO2. After a 50 mg/kg dose of [14C]HCBD, the amount of exhaled parent compound was nearly unchanged at 5.4%. At the higher dose the gastro-intestinal absorption of HCBD appeared to be saturated with the result that unchanged HCBD constituted the major portion of the 69% radioactivity eliminated. Covalent binding to proteins in kidney and liver agreed well with the organ-specific toxicity of HCBD: binding was higher in the kidney, independent of the dose. It increased significantly when the rats were pretreated with phenobarbital, an inducer of monooxygenases; it decreased when the inhibitor piperonyl butoxide was given. Urinary radioactivity in 24 hr urine was separated by column chromatography into four fractions. High performance liquid chromatography, radio gas chromatography and gas chromatography/mass spectrometry were used for further separation and identification. Two major metabolites were identified as pentachlorobutadiene methylthio ether and pentachlorobutadiene carboxymethylthio ether. Their formation is plausibly explained via glutathione conjugation, which appears to be the first step in HCBD metabolism. The mechanism of the conjugation at the olefinic double bond of HCBD is explained by an addition-elimination reaction. This pathway, which appears to lead to a destabilization of the HCBD molecule, could explain the distinct nephrotoxic effects of HCBD.

Induction of P-450 isoenzyme activities in Syrian golden hamster liver compared to rat liver as probed by the rate of 7-alkoxyresorufin-O-dealkylation

The activities of 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-deethylase (PROD), 7-ethoxycoumarin-O-deethylase (ECOD) and aromatic hydrocarbon hydroxylase (AHH) were measured in hepatic microsomes from male and female Wistar rats and Syrian golden hamsters in order to probe the basal activity and the inducibility by phenobarbital (PB) and 3-methylcholanthrene (MC) of different P-450 isoenzymes. The basal activities of EROD and ECOD, but not PROD and AHH, were higher in male hamsters than in male rats. No sex-related difference in enzyme activities was observed with hamsters, whereas male rats had a higher ECOD and AHH activity than female rats. Induction by PB led to a 450-fold and 250-fold increase in PROD activity in male and female rat liver microsomes, respectively, while MC had a more pronounced inductive effect on EROD activity in this species. In hamsters, EROD activity was induced by MC but not by PB. Unexpectedly PROD activity in male and female hamster liver microsomes was only moderately induced by PB, the extent being lower than on induction by MC. Therefore, the activity of PROD, which is useful as a specific enzymatic assay for P-450 IIB in the rat liver, cannot be used to probe PB-like inducers in the hamster liver.