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Archives of Toxicology (v.69, #5)

The role of CYP enzymes in cocaine-induced liver damage by Markku Pasanen; Pertti Pellinen; Frej Stenbäck; Risto O. Juvonen; Hannu Raunio; Olavi Pelkonen (pp. 287-290).

Cocaine is hepatotoxic in several species, including man. A high dose of cocaine produces metabolismdependent, mainly pericentral, liver damage. At 24h after a single dose of cocaine, mouse hepatic P450 content decreases but CYP2A activities; coumarin 7-hydroxylase and testosterone 15α-hydroxylase increase concomitant with prominent diffuse cell necrosis. Repeated adminision of cocaine for up to 5 days decreases CYP1A1/2, 2A4/5, 2Cx, and 2E1 related enzymatic activities. However, after five doses of cocaine, CYP2B10 increases in conjunction with the healing process. In the acute phase, the increased CYP2A activities do not participate in cocaine bioactivation. CYP3A enzymes are principally responsible for the cocaine N-demethylation in human and mouse liver microsomes. The hepatic metabolic CYP enzyme profile will change during prolonged cocaine intake, this being accompanied by altered cell morphology. Possible connections to cocaine toxicity in man are discussed.

Keywords: Cytochrome P450; Cocaine; Hepatotoxicity Hepatotoxin; Coumarin 7-hydroxylase

Increased incidence of renal cell tumors in a cohort of cardboard workers exposed to trichloroethene by D. Henschler; S. Vamvakas; M. Lammert; W. Dekant; B. Kraus; B. Thomas; K. Ulm (pp. 291-299).

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.

Keywords: Trichloroethene; Renal cell cancer; Occupational exposure; Epidemiological study

A note on individual differences in the urinary excretion of optical enantiomers of styrene metabolities and of styrene-derived mercapturic acids in humans by E. Hallier; H. W. Goergens; H. Karels; K. Golka (pp. 300-305).

Urine samples from 20 male workers in the polyester industry exposed by inhalation to styrene concentrations ranging from 29 to 41 ppm were investigated. Excretion products of styrene metabolism, mandelic acid and mercapturic acids, were purified from the urine over an extraction column packed with Porapak Q, with subsequent ether elution. The optical enantiomers R- and S-mandelic acid were then determined by thin layer chromatography (TLC) using chiral plate material and selective staining with vanadium pentoxide. Quantitative analysis of these compounds was performed using ocmmercial reference substances. Styrene-specific mercapturic acids were analyzed by a modified TLC method, using synthesized reference substances. The concentration of racemic mandelic acid in the individual urine samples ranged from 80 to 1610 mg/l, and the ratio of the R- and S-enantiomers ranged from 0.7 to 2.2. These individual variations are not explained by differences in individual styrene exposure levels, or by differences in the concentration of the urine samples (in relation to creatinine excretion). Styrene-specific mercapturic acids were detected in the urine of only 1 of the 20 workers, at a concentration much lower than expected from previous investigations by others in humans and laboratory animals, in which less specific analytical methods had been used. The results point to marked interindividual differences in metabolism of styrene, probably related to enzyme polymorphisms.

Keywords: Styrene; Mandelic acid enantiomers; Styrene-specific mercapturic acids; Individual metabolic variation; Enzyme polymorphism

Gas chromatography-mass spectrometry determination of ethylenethiourea hemoglobin adducts: a possible indicator of exposure to ethylene bis dithiocarbamate pesticides by Roberta Pastorelli; Riccardo Allevi; Stefano Romagnano; Giovanna Meli; Roberto Fanelli; Luisa Airoldi (pp. 306-311).

Ethylenebisdithiocarbamates (EBDC) are an important class of fungicides used to control crop diseases and prevent mold. Ethylenethiourea (ETU), reported to be their main degradation and metabolic product in animals and man, may have teratogenic and carcinogenic properties. The feasibility of monitoring exposure to ETU on the basis of the formation of adducts to hemoglobin (Hb) was investigated. Rats given a single oral dose of ETU (from 62.5 to 500 mg/kg body wt) formed stable covalent ETU-Hb adducts. Mild acid hydrolysis of the protein regenerated ETU, allowing its detection by isotope dilution gas chromatography-mass spectrometry (GC-MS). The amount of released ETU increased with the dose. The dose-response curve fitted a linear model only between 62.5 mg/kg and 250 mg/kg. Acid-releasable ETU was also positively identified in the hemoglobin of workers exposed to Mancozeb, an EBDC formulation. In the exposed group, 40% had ETU-Hb adducts levels ranging from 0.5 to 1.42 pmol ETU/mg Hb. Such adducts might be useful for measuring EBDC exposure in humans.

Keywords: Adducts; Ethylenethiourea; Ethylenebisdithiocarbamate fungicides; Hemoglobin; Dosimetry

Chemical form of selenium-containing metabolite in small intestine and liver of mice following orally administered selenocystine by Tatsuya Hasegawa; Makoto Mihara; Tomofumi Okuno; Katsuhiko Nakamuro; Yasuyoshi Sayato (pp. 312-317).

The chemical form of a selenium-containing metabolite in the small intestine following a single oral administration of selenocystine was investigated with ICR male mice. Selenium content in the small intestine of animals treated with 50 mg/kg selenocystine significantly increased 15 min, 1 h and 6 h after treatment. In contrast, selenocystine significantly depressed the intestinal reduced glutathione (GSH) level at 1 h after administration. A significant negative correlation between the selenium level and the level of GSH in the small intestine was observed (r=−0.83, p<0.001). Analysis of the intestinal metabolite of selenocystine showed that selenium-containing metabolites elute in two fractions from a Sephadex G-25 column: the low-molecular fraction (peak I) contained the selenocystine, while the high-molecular fraction (peak II) contained selenocysteine-containing metabolite. An in vitro experiment was performed to gain insight into the mechanism for selenocysteine-containing metabolite production in the intestinal cytosol. When selenocystine or selenocysteine reacted with excess GSH in the presence of intestinal homogenate, the peak II fraction which involved the selenocysteine-containing metabolite was recognized in the Sephadex G-25 chromatogram. From an examination of the distribution of the selenocysteine-containing metabolite, it was recognized that this metabolite exists in plasma and liver cytosol of mice after oral administration of selenocystine. These results suggested that the mice treated with selenocystine produce selenocysteine-containing metabolite by reaction of selenocystine with excess GSH in the small intestine, and the metabolite is then transported to the liver through blood plasma.

Keywords: Selenium; Selenocystine; Chemical form; Selenium metabolite; Intestine

Diquat increases cysteine proteinase inhibitors greatly in rat plasma and tissues by Kayoko Minakata; Osamu Suzuki; Sachiko Oh-ishi; Izumi Hayashi; Shinichi Saito; Naoko Harada (pp. 318-321).

Biochemical and gross pathological effects of diquat were studied with special attention to cysteine proteinase inhibitor level which was often increased in acute and chronic disorder. Diquat was fed continuously to rats at the dose of 1000 ppm in the diet. After 10 days, anorexia and severe diarrhea were observed but epistaxis and hypokinesia were not apparent. The rats were killed after feeding the diet for 13.5 days and plasma components such as acute phase reactant proteins and some vitamins which act as antioxidants were examined. The results showed that α-cysteine proteinase inhibitor (α-CPI) increased to 9-fold and vitamin C radical increased to 1.6-fold, whereas α1 proteinase inhibitor (α1-PI) decreased to 0.9-fold and vitamins C and E were the same as the control. Among three components of α-CPI, the T kininogen level in intoxicated rat plasma was about 20-fold, whereas the high molecular weight kininogen level was about 2-fold of the control. Diquat also enhanced the cysteine proteinase inhibitor (CPI) level to 20-fold in kidney and to 7- to 10-fold in the other organs. The large increment of T kininogen in these organs was also confirmed immunologically. The kidney showed a granular degeneration and its weight increased to 1.2-fold of control. The other organs showed neither gross pathological alteration nor weight change, compared with the control. The diquat distribution was highest in spleen and next highest in kidney among several organs. These results were compared with those caused by paraquat.

Keywords: Diquat; Cysteine proteinase inhibitor T kininogen; Rats; Acute phase reactant protein Vitamin C radical

Cardiorespiratory function in nerve agent poisoned and oxime+atropine treated guinea-pigs: effect of pyridostigmine pretreatment by Franz Worek; Ladislaus Szinicz (pp. 322-329).

The effect of pyridostigmine pretreatment on the efficacy of oxime+atropine treatment in sarin and VX poisoning was investigated in a guinea-pig model with on-line monitoring of respiratory and circulatory parameters. The carotid artery, jugular vein and trachea were cannulated in female urethane-anesthetized Pirbright-white guinea-pigs. After baseline measurements the animals received pyridostigmine (PYR, 0.05 μmol/kg, i.v.), 30 min later sarin (100 or 200 μg/kg=5 or 10xLD₅₀, i.v.) or VX (45 or 90 μg/kg=10 or 20xLD₅₀, i.v.), followed 2 min later by atropine (10 mg/kg, i.v.) plus HI 6 or HLö 7 (30 μmol/kg each, i.v.). Sixty-minute survival time and rate and respiratory and circulatory parameters were recorded. Diaphragm acetylcholinesterase (AChE) activity was determined spectrophotometrically. Identical groups without PYR were included for comparison. With regard to survival time and rate, PYR pretreatment slightly improved the efficacy of HI 6 plus atropine in sarin 5xLD₅₀ poisoned animals, reduced the efficacy of oxime+atropine treatment in the other sarin groups and had no effect in VX poisoning. Compared to nonpretreated oxime+atropine groups, PYR slightly improved respiratory function in sarin and in VX poisoning (only HI 6). PYR did not affect circulatory function in VX poisoning but reduced circulatory parameters in sarin poisoning to varying extent’s. The oxime efficacy in reactivating diaphragm AChE decreased in the order sarin > VX without significant differences between pretreated and non-pretreated groups. The data suggest that pyridostigmine pretreatment does not enhance the efficacy of oxime+atropine in sarin or VX poisoning.

Keywords: Organophosphate; Pyridostigmine; HI 6; HLö 7; Respiration; Circulation

Organophosphate polyneuropathy and neuropathy target esterase: Studies with methamidophos and its resolved optical isomers by M. Lotti; A. Moretto; M. Bertolazzi; M. Peraica; F. Fioroni (pp. 330-336).

Methamidophos (O, S-dimethyl phosphorothioamidate) causes polyneuropathy in man and hens. However, experiments in the hen show that lower doses of methamidophos either protect from or promote the neuropathy caused by certain organophosphates. The initiation of neuropathy as well as protection from neuropathy are thought to be related to neuropathy target esterase (NTE), whereas promotion is likely to be due to interactions with another unknown target. Methamidophos is a racemate and we report studies with its resolved optical isomers, aimed at elucidating which isomer is responsible for the described effects. The time-course of acetylcholinesterase (AChE) and NTE activity in nervous tissues of hens after inhibition by single doses of either isomer showed that after d-(+) methamidophos (25 mg/kg PO) peak inhibition of both enzymes was achieved within 24 h (80–90%). However, after l-(-) methamidophos (15 mg/kg PO), peak inhibition (80–90%) was obtained within 24 h for AChE, whereas similar NTE inhibition (120 mg/kg PO) was observed only 4 days after dosing. The minimal neuropathic doses of d-(+) and l-(-) methamidophos were 60 and 120 mg/kg PO, respectively, and correlated with > 80% NTE inhibition in nervous tissues. OPIDP initiation by either isomer was slightly promoted by phenylmethanesulfonyl fluoride (120 mg/kg SC). d-(+) Methamidophos (25 mg/kg PO) partially protected from dibutyl dichlorovinylphosphate (DBDCVP) neuropathy (up to 0.8 mg/kg SC). This effect correlated with about 70% NTE inhibition. l-(-) Methamidophos (15 or 60 mg/kg PO) did not protect from DBDCVP neuropathy (0.2–0.8 mg/kg SC). d-(+) and l-(-) methamidophos (25 mg/kg PO) promoted DBDCVP neuropathy (0.4 mg/kg SC), and d-(+) methamidophos (24 mg/kg PO) also promoted DFP neuropathy (0.3 mg/kg SC). These effects were unrelated to the degree of NTE inhibition they caused: about 70% by d-(+) methamidophos and extrapolated to about 10–15% by l-(-) methamidophos. We conclude that when racemic methamidophos is given to hens, initiation and protection from OPIDP is due to the interaction of d-(+) methamidophos with NTE. Promotion of OPIDP is due to both isomers as the result of their interaction with unknown site(s). It is possible that the neuropathy due to racemic methamidophos or isomers is a self promoted neuropathy because the promoting doses of both isomers are much lower than the neuropathic ones, and because the neuropathy they initiate is only slightly promoted by phenylmethanesulfonyl fluoride.

Keywords: Organophosphates; Methamidophos; Isomers; NTE; Polyneuropathy; Protection; Promotion

Visual evoked potentials in individuals exposed to long-term low concentrations of toluene by Andelko Vrca; Dubravko Božičević; Višnja Karačić; Radovan Fuchs; Danica Prpić-Majić; Marta Malinar (pp. 337-340).

An investigation of visual evoked potentials was carried out in two groups of subjects; 49 workers employed in a printing-press where toluene has been used exclusively as an organic solvent for the last 30 years, and 59 workers not occupationally exposed to any known neurotoxic substances. The average length of work service in the printing-press was 21.4 years. The level of exposure was assessed by determination of the concentration of toluene in peripheral blood, the concentration of hippuric acid and ortho-cresol in urine in subgroups of subjects chosen at random from both groups. N75, P100 and N145 waves of the pattern reversal visual evoked potentials were analyzed. In the group of exposed subjects, significantly greater amplitudes were found in all waves, with significantly longer latency of the P100 wave.

Keywords: Neurotoxicity; Organic solvents; Toluene; Chronic exposure; Visual evoked potentials

Effect of cadmium on lung lysosomal enzymes in vitro by Shri N. Giri; Mannfred A. Hollinger (pp. 341-345).

Labilization of lysosomal enzymes is often associated with the general process of inflammation. The present study investigated the effect of the pneumotoxin cadmium on the release and activity of two lung lysosomal enzymes. Incubation of rat lung lysosomes with cadmium resulted in the release of β-glucuronidase but not acid phosphatase. The failure to “release” acid phosphatase appears to be the result of a direct inhibitory effect of cadmium on this enzyme. The K₁ for cadmium was determined to be 26.3 μM. The differential effect of cadmium on these two lysosomal enzymes suggests that caution should be exercised in selecting the appropriate enzyme marker for assessing lysosomal fragility in the presence of this toxicant. Furthermore, the differential basal release rate of the two enzymes from lung lysosomes may reflect the cellular heterogeneity of the lung.

Keywords: Cadmium; Lung; Lysosomes; β-Glucuronidase; Acid phosphatase

In vitro metabolism of cyclosporin A with rabbit renal or hepatic microsomes: analysis by HPLC-FPIA and HPLC-MS by C. Pham-Huy; N. Sadeg; T. Becue; C. Martin; G. Mahuzier; J. -M. Warnet; M. Hamon; J. R. Claude (pp. 346-349).

Cyclosporin A (CsA) is in vivo mainly metabolized by hepatic cytochrome P450 IIIA to more than 21 metabolites, the major ones known as: M1, M17 and M21. The aim of this work is to explore the in vitro metabolism of CsA after incubation, in the presence of NADPH, with renal or hepatic microsomes obtained from rabbits pretreated with rifampycin (enzyme inducer) or erythromycin (enzyme inhibitor). The presumed metabolites were separated by semi-preparative high-performance liquid chromatography (HPLC) and identified in each collected fraction by fluorescence polarization immunoassay (FPIA) (HPLC-FPIA) using a non-specific polyclonal antibody. They were also analyzed by HPLC-mass spectrometry (MS) using fast atom bombardment (HPLC-MS-FAB). Five collected fractions gave positive results with FPIA. The major metabolites found were M1, M17 and M21 after identification by HPLC-MS-FAB and comparison with three corresponding standard metabolites. The CsA biotransformation rates were calculated by the amount of unmetabolized CsA and were linear with time. These mean rates (V_m) for 12-min incubation by renal microsomes of rabbits treated with rifampicin or erythromycin or untreated (control) were 0.11, 0.02 and 0.04 nmol/min × mg microsomal protein, respectively. These rates were 15-, 37-, and 30-fold lower than those obtained with hepatic microsomes of rabbits treated identically. As CsA metabolites are less cytotoxic than the parent drug, this weak renal biotransformation of CsA after in vitro incubation should be one of the mechanisms of its in vivo nephrotoxicity.

Keywords: In vitro metabolism; Cyclosporin A; Renal and hepatic microsomes; HPLC-mass spectrometry; FPIA

2-Acetylaminofluorene inhibits the activation of immune responses by blocking cell cycle progression at G₁ phase by W. S. Koh; K. -H. Yang; T. C. Jeong; B. Delany; N. E. Kaminski (pp. 350-356).

2-Acetylaminofluorene (AAF) inhibited in a dose dependent manner mouse spleen cell blastogenesis in response to phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Io) activation, the T-cell lectin, concanavalin A (Con A), and following stimulation by alloantigens as measured by the mixed lymphocyte response (MRL). AAF also markedly suppressed the T-cell dependent antibody forming cell (AFC) response to sRBC. AAF was most inhibitory on both the sRBC IgM AFC response and Con A stimulated proliferation when added during the first 24 h following initiation of culture. Direct addition of high concentrations of AAF (100 μM) to spleen cell cultures at 48 h following Con A stimulation produced a very modes inhibition (<20%) of T-cell proliferation as compared to 90% when added at the time cultures were initiated. Similarly, AAF (75 and 100 μM) produced a greater than 80% inhibition of the in vitro AFC response when spleen cells were sensitized with antigen in presence of AAF. In contrast, no inhibition of the IgM AFC response was produced when AAF (75μM) was added to spleen cell cultures 48 or 72 h after antigen sensitization. Con A-triggered cell-cycle progression was attenuated at the G₁ stage by the addition of AAF (50 and 100 μM) with no inhibition of S to G₂/M phase transition. These results suggest that the mechanism of AAF-mediated immune suppression is through a blockade of cell cycle progression from G₁ to S phase.

Keywords: AAF; Immunosuppression; Cell cycle

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Archives of Toxicology (v.69, #5)