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Archives of Toxicology (v.71, #5)
Observation of hepatotoxic effects of 2-n-pentylaminoacetamide (Milacemide) in rat liver by a combined in vivo/ in vitro approach by V. Rogiers; Yves Vandenberghe; Tamara Vanhaecke; Albert Geerts; André Callaerts; Jacqueline Carleer; Jose Roba; A. Vercruysse (pp. 271-282).
Milacemide or 2-n-pentylaminoacetamide hydrochloride, a new glycine derivative, was found to cause elevations of plasma transaminases in patients suffering from severe depression and Alzheimer's disease. However, no signs of liver toxicity were observed during the course of earlier conducted subchronic and chronic in vivo studies in rodents and cynomolgus monkeys. In this study an in vivo/in vitro approach has been proposed to detect early alterations in key metabolic and functional liver capacities. Milacemide was administered by continuous i.v. infusion for 7 days to male Sprague-Dawley rats using subcutaneously implanted osmotic pumps. Doses were given of 0, 250 and 500 mg/kg per day. Body weight and food intake were recorded and at day 7 of exposure, Milacemide concentration, glucose, urea, triglycerides and cholesterol levels and alanine (ALT) and aspartate aminotransferase (AST) activities were measured in plasma. Non-esterified fatty acids were determined in serum. On day 8, after overnight fasting, hepatocytes were isolated. A portion of the cells derived from untreated animals (no osmotic pumps) were cultured in a primary monolayer and exposed in vitro to different Milacemide concentrations.The xenobiotic biotransformation capacity of the isolated hepatocytes was studied by measuring the cytochrome P450 content, ethoxycoumarin-O-deethylase (ECOD), pentoxyresorufin-O-deethylase (PROD), ethoxyresorufin-O-deethylase (EROD), aldrin epoxidase (AE), epoxide hydrolase (EH) and glutathione S-transferase (GST) enzyme activities. Triglycerides, cholesterol and phospholipid contents were measured on the isolated cells. At plasma concentrations of 43 and 130 μM Milacemide, the ALT activity was unchanged or significantly decreased, whereas the AST activity was increased in both cases. Other clinical chemistry parameters remained unchanged. Weight gain was significantly lower in rats treated with the high Milacemide dose. In addition, decreased food consumption was observed in all treated animals leading to significantly lower food efficiency factors for the rats treated with the high dose. Milacemide had a specific inhibitory effect on xenobiotic biotransformation: ECOD activity decreased to 60% of the control value for both Milacemide doses, PROD activity remained unaffected whereas EROD activity decreased to 65% of the control value. A decrease was also observed at the highest drug concentration for AE (to 41%), EH␣(to 65%), cytochrome P450 content (to 80%) and GST (to␣85%). At 500 mg Milacemide kg/day, hepatocyte triglycerides levels increased 3.1-fold while cholesterol and phospholipid levels remained unaffected. Electron and light microscopy on total liver and isolated hepatocytes indicated a concentration-dependent accumulation of lipid droplets, the occurrence of numerous vacuoles in the cytoplasm and other structural abnormalities. When the cultured hepatocytes of control animals (without osmotic pumps) were exposed to Milacemide, the appearance of vacuoles and myeloid bodies could be confirmed in vitro. The results of this study using an in vivo/in vitro approach clearly show potential hepatotoxic properties of Milacemide, an effect not observed in conventional toxicity studies.
Keywords: Key words Milacemide ; 2-n-Pentylaminoacetamide hydrochloride ; Hepatotoxicity ; Hepatocytes ; Liver cells
Enhancement of SDZ ICT 322-induced cataracts and skin changes in rats following vitamin E- and selenium-deficient diet by U. W. Längle; A. Wolf; A. Cordier (pp. 283-289).
The indole-3-carboxylic acid scopoine ester, SDZ ICT 322, is a selective hydroxytryptamine (5-HT3) antagonist, which after chronic treatment causes posterior subcapsular cataracts and skin changes such as hair loss, hyperaemia, desquamation and hyperkeratosis in rats. The detailed mechanisms underlying these changes are not yet known. In order to evaluate a possible oxidative stress-induced pathomechanism of SDZ ICT 322, the antioxidative defence capacity in rats was modulated by feeding a special vitamin E- and selenium-deficient (VE/SeD) diet. For this purpose 32 male Wistar rats, age 4 weeks, were pretreated for 8 weeks with either a VE/SeD diet or a normal standard diet. Each dietary group was divided into 8 control and 8 SDZ ICT 322-treated animals. SDZ ICT 322 was administered in feed to rats at an adjusted daily dose level of 125 mg/kg for 14 weeks. Plasma levels of SDZ ICT 322 as well as of the N-desmethyl metabolite were similar in rats fed the different diets in weeks 3, 6 and 14. In SDZ ICT 322 treated VE/SeD rats cataracts were observed by week 7, whereas in rats fed normal diet cataracts were first seen in week 14. In the normal dietary group no corneal opacity was found after SDZ ICT 322-treatment; however, a corneal opacity was seen in the deficiency group in parallel with one of the cataract animals in week 7. The incidence and severity of clinical skin signs were greater and their onset was earlier in the deficiency dietary group: onset occurred after 6␣weeks compared to 9 weeks on the normal diet. Thiobarbituric acid reactive substances, an indicator mainly of oxidized lipids, were statistically significantly increased in the urine of rats administered SDZ ICT 322 and the VE/SeD diet. Uric acid, which is an endogenous antioxidant was statistically significantly decreased in the urine and plasma of SDZ ICT 322 VE/SeD treated rats. The clinical eye and skin changes occurring early after VE/SeD feeding, the unchanged drug plasma exposure and unchanged drug metabolite formation in combination with the general pro-oxidative activity of the drug suggest an oxidative stress-mediated pathomechanism of SDZ ICT 322 at high dose levels in rats.
Keywords: Key words Selective 5-HT3 antagonist ; Cataracts ; Adverse skin changes ; Vitamin E-selenium deficiency ; Oxidative stress
Reduction of the ochratoxin A-induced cytotoxicity in Vero cells by aspartame by I. Baudrimont; A. M. Betbeder; E. E. Creppy (pp. 290-298).
Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus as well as other moulds. This mycotoxin contaminates animal feed and human food and is nephrotoxic for all animal species studied so far. OTA is immunosuppressive, genotoxic, teratogenic and carcinogenic. Recently lipid peroxidation induced by OTA has been reported. OTA, a structural analogue of phenylalanine, inhibits protein synthesis by competition with phenylalanine in the phenylalanine-tRNA aminoacylation reaction, constituting the main mechanism of OTA-induced cytotoxicity. Since it seems impossible to avoid contamination of foodstuffs by toxigenic fungi, investigation is required for preventing the toxicity of OTA. An attempt to prevent its toxic effect, mainly the inhibition of protein synthesis, has been made using aspartame (l-aspartyl-l-phenylalanine methyl ester) a structural analogue of both OTA and phenylalanine. Protein synthesis was assayed in monkey kidney cells (Vero cells) treated by increasing concentrations of OTA (10–100 μM). After 24 h incubation, protein synthesis was inhibited by OTA in a concentration dependent manner (the 50% inhibitory concentration, IC50, was c.␣14.5 μM). Aspartame (A19), at tenfold higher concentrations than OTA (100–1000 μM), was found to partially protect against the OTA-induced inhibition of protein synthesis in Vero cells, and more efficiently when added 24 h prior to the toxin (IC50 34 μM) than together (IC50 22 μM). As expected A19(250 μM) prevented the OTA-induced leakage of certain enzymes, including lactate dehydrogenase, γ-glutamyl transferase, alkaline phosphatase, into the culture medium, and the concomitant decrease of their intracellular activity in OTA (25 μM)-treated cells. In order to investigate the effect of aspartame (A19) on OTA-protein binding as explanation of the above results, the mycotoxin time- and concentration-dependent binding to human samples was studied in static diffusion cells with two compartments separated by a dialysis membrane. When A19 (34 μM) was added to the upper compartment containing plasma before installing OTA (50, 250, 1240 μM) in the lower one, OTA binding was largely prevented (95–98%). When A19 (34 μM) was added to the lower compartment simultaneously with the toxin (50, 250, 1240 μM), for the lowest concentration of OTA, the same efficiency was shown in preventing OTA binding, but at the two high concentrations A19 seemed less efficient.
Keywords: Key words Ochratoxin A ; Aspartame (A19) ; Vero cells ; Cytotoxicity ; Binding to plasma proteins
In vitro haematotoxic effects of three methylated hydroxylamines by Anita A. M. G. Spooren; Chris T. A. Evelo (pp. 299-305).
Hydroxylamine (HYAM, HONH2) and some of its derivatives are known to cause erythrotoxic effects both in vitro and in vivo. Previous studies have shown that the primary in vitro effect of HYAM and O-ethyl hydroxylamine (OEH) is methaemoglobin formation, leading to liberation of free radicals which cause lipid peroxidation, enzyme inhibitions and glutathione depletion. By contrast, N-substituted N,O-dimethyl hydroxylamine (NODMH), primarily induces impairment of glucose 6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR). The oxidative potency of HYAM and the O-derivative was larger than the potency of the N,O-derivative. This seemed to indicate that attachment of an alkyl group to the nitrogen atom of hydroxylamine leads to decreased reactivity. To achieve a better understanding of the structure activity relationship for hydroxylamines three methylated derivatives were tested: N-methyl hydroxylamine (NMH), N-dimethyl hydroxylamine (NDMH) and O-methyl hydroxylamine (OMH). We were also interested in the erythrotoxic potency of OMH which recently entered industrial production. Methaemoglobin formation, high release of lipid peroxidation products, inhibition of NADPH methaemoglobin reductase and glutathione S-transferase (GST) and depletion of total glutathione (GT) were seen for OMH. The reducing enzymes G6PDH and GR were not impaired by OMH. These findings for OMH are consistent with the proposed mechanism for O-derivatives. Since both the effects caused by OMH and its potency are comparable to those of HYAM and OEH this indicates that possible occupational exposure to this compound may be approached similarly to HYAM and OEH. NMH only inhibited G6PDH and GR activity, which is fully in accord with the proposed mechanism for N-substituted derivatives of HYAM. However, NDMH a double N-substituted compound, caused a strikingly different scheme of reactivity: inhibition of G6PDH but not of GR, severe methaemoglobin formation, only little lipid peroxidation and some impairment of NADPH methaemoglobin reductase. This study confirms that O-derivatives of HYAM are potent haemoglobin oxidators, leading to other oxidative effects. The main effect was confirmed for single N-derivatives as inhibition of the two protective enzymes G6PDH and GR. However, the results for NDMH indicate that this simple classification of O-derivatives and N-derivatives has to be extended for double N-substituted compounds which give a mixture of effects.
Keywords: Key words Hydroxylamines ; Human erythrocytes ; Oxidizing effects ; Potency ranking ; Structure activity relations
Recombinant expression and catalytic analysis of rapid and slow acetylator Syrian hamster chimeric NAT2 alleles by David W. Hein; Ronald J. Ferguson; Mark A. Doll; Anne C. Deitz (pp. 306-313).
Polymorphic aromatic amine N-acetyltransferase (NAT2) catalyzes the N-acetylation of aromatic amines and the metabolic activation of N-hydroxyarylamines (via O-acetylation) and N-hydroxy-N-acetylarylamines (via N,O-acetylation) to electrophilic intermediates that mutate DNA. Acetylation capacity in humans and other mammalian species such as Syrian hamsters is subject to a genetic polymorphism. NAT2 is regulated by a single gene (NAT2) containing a single coding exon of 870 bp. Syrian hamster slow acetylator differs from the rapid acetylator NAT2 coding region by three nucleotide substitutions at T36C, A633G, and C727T. We measured expression of immunoreactive NAT2 protein and aromatic amine N-acetylation, N-hydroxyarylamine O-acetylation and N-hydroxy-N-acetylarylamine N,O-acetylation by recombinant NAT2 proteins expressed from alleles containing all combinations of the T36C, A633G, and C727T substitutions. The C727T substitution, which creates an opal stop codon in slow acetylator NAT2, was the sole mutation responsible for substantial reduction in expression of a truncated NAT2 protein with reduced capacity for the deactivation of aromatic amines (N-acetylation) and the metabolic activation of N-hydroxyarylamines (O-acetylation) and N-hydroxy-N-acetylarylamines (N,O-acetylation). The reductions in aromatic amine N-acetylation correlated very highly with the reductions in metabolic activation of the corresponding N-hydroxyarylamines and N-hydroxy-N-acetylarylamines.
Keywords: Key words N-acetyltransferase ; Acetylation polymorphism ; NAT2 ; Syrian hamster
Mutagenicity testing of organic extracts of diesel exhaust particles after fractionation and recombination by L. Østby; Solveig Engen; A. Melbye; I. Eide (pp. 314-319).
A new strategy for the evaluation of mixtures is presented. The mixture used was the organic extract of diesel exhaust particles (DEP). After extraction with dichloromethane (DCM), the crude extract was fractionated according to polarity into five fractions: aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, dinitro-PAHs, and polar compounds. After dissolving in dimethylsulphoxide␣(DMSO), the three fractions containing the primary mutagens (fractions 3–5) were recombined in different combinations to create new extracts. The blend matrix was obtained using a mixture design at three dose levels to support an empirical model with linear, interaction, and quadratic terms (Taylor polynome). The recombined extracts were tested in the Ames Salmonella assay using strain TA100. Multivariate data analysis was performed with projections to latent structures (PLS). The best model describing the relation between the mutagenicity (response) and the three fractions (variables) contained two interaction terms. The model showed high correlation (r 2) and prediction properties (Q 2), the latter obtained after cross validation. Interaction terms are only indications of possible synergism or antagonism and have to be evaluated with respect to dose-additivity and response-additivity. The incorporation of dose in the design reduced the number of samples (recombined extracts) significantly, compared to determining dose-response curves on each sample (i.e. the recombined extracts in different dilutions). Furthermore, instead of running two independent experiments as required in the standard procedure for the Ames test, predictions and verifications of a few new samples were used. The principle of fractionation and recombination, and the use of mixture design may in principle be extended to an unlimited number of variables. An adaptation of mixture design to the isobole method is discussed.
Keywords: Key words Combination toxicology; Complex mixtures; Diesel exhaust particles; Experimental design; Multivariate data analysis; Projections to latent structures
Development of a physiologically based model for the toxicokinetics of C(±)P(±)-soman in the atropinized guinea pig by Jan P. Langenberg; C. Van Dijk; Richard E. Sweeney; Donald M. Maxwell; Leo P. A. De Jong; Hendrik P. Benschop (pp. 320-331).
A physiologically based model was developed which describes the in vivo toxicokinetics of the highly reactive nerve agent C(±)P(±)-soman at doses corresponding to 0.8–6 LD50 in the atropinized guinea pig. The model differentiates between the summated highly toxic C(±)P(−)-soman stereoisomers at supralethal doses and the individual nontoxic C(±)P(+)-isomers. Several toxicant-specific parameters for the soman stereoisomers were measured in guinea pig tissue homogenates. Cardiac output and blood flow distribution were measured in the atropinized, anesthetized, and artificially ventilated guinea pig. The model was validated by comparison of the time courses for the blood concentrations of the two pairs of stereoisomers in the guinea pig after i.v. bolus administration with the blood concentrations predicted by the model. The predictions put forward for the summated C(±)P(−)-isomers are in reasonable agreement with the experimental data obtained after doses corresponding to 2 and 6 LD50. In view of large differences in the rates of hydrolysis of the C(±)P(+)-isomers, these two isomers had to be differentiated for satisfactory modeling of both isomers. In order to model the toxicokinetics of C(±)P(−)-soman at a dose of 0.8 LD50, the almost instantaneous elimination of the C(+)P(−)-isomer at that dose had to be taken into account. The sensitivity of the predictions of the model to variations in the parameters has been studied with incremental sensitivity analysis. The results of this analysis indicate that extension to a model involving four individual stereoisomers is desirable in view of large differences in the biochemical characteristics of the two C(±)P(−)- and C(±)P(+)-isomers.
Keywords: Key words Physiologically based model ; C(±)P(±)-soman ; Stereoisomers ; Toxicokinetics ; Guinea pig
Renal cell carcinomas in trichloroethene(TRI) exposed persons are associated with somatic mutations in the von Hippel-Lindau (VHL) tumour suppressor gene by Thomas Brüning; Gregor Weirich; M. A. Hornauer; Heinz Höfler; Hiltrud Brauch (pp. 332-335).
Renal cell carcinomas (RCC) develop as a consequence of somatic mutations of the von Hippel-Lindau (VHL) tumour suppressor gene. Recent epidemiological studies show that high and prolonged occupational exposures to trichloroethene (TRI) are associated with an increased incidence of RCC. Tumour tissues from 23 RCC patients with occupational histories of very high TRI exposure were analysed for somatic mutations within the VHL gene. DNA was isolated from microdissected tumour cells, amplified by polymerase chain reaction (PCR), and analysed in single strand conformation polymorphism (SSCP) and sequencing. RCC tissues of all 23 TRI exposed persons analysed thus far showed aberrations of the VHL gene, with 30% having aberrations in exon 1, 44% in exon 2, and 26% in exon 3. By comparison to much lower reported VHL mutation frequencies of 33–55% in TRI-unexposed RCC patients, these results indicate a specifically high mutation frequency at the VHL gene in TRI-exposed RCC patients; four of these aberrations have thus far been confirmed as VHL mutations by sequence analysis. This finding indicates the VHL gene being a susceptible and specific target in TRI induced renal carcinogenesis. Furthermore, the frequent involvement of exon 2 identifies potential `hot spots' for this carcinogen. In addition to the available epidemiological studies the results are now further proof for human renal carcinogenicity induced by high occupational exposures to TRI.
Keywords: Key words Trichloroethene ; Renal cell cancer ; VHL ; Tumour suppressor genes
Lack of biliary excretion of Cd linked to an inherent defect of the canalicular isoform of multidrug resistance protein (cMrp) does not abnormally stimulate accumulation of Cd in the Eisai hyperbilirubinemic (EHB) rat liver by Naoki Sugawara; Koji Arizono; Toshiichi Kitajima; Hideaki Inoue; Yu-Rong Lai (pp. 336-339).
A new mutant, the Eisai hyperbilirubinemic (EHB) rat, shows no inherent expression of the canalicular isoform of the multidrug resistance protein (cMrp) in the liver. It has defective biliary secretion of organic anions such as bilirubin glucuronides, bromosulfophthalein (BSP), cysteinyl leukotrienes, glutathione (GSH) and bile acid sulfate and glucuronides. When rats were injected intravenously with CdCl2, biliary excretion of Cd over 30 min was 0.28% and 0.004% of the total dose in Sprague-Dawley (SD) and EHB rats, respectively. Six SD rats and five EHB rats were fed a diet containing Cd. Bile Cd was detected at the level of 2 ng/20 min in SD rats, but not in EHB rats. There was no significant difference of hepatic Cd concentration between SD and EHB rats. Furthermore, there were no significant differences of renal and intestinal Cd, and hepatic and renal metallothionein (MT) concentrations between the SD and EHB groups. Biliary excretion of reduced-GSH for 20 min was 1.3 ± 0.3 mg and 3.6 ± 0.9 μg in SD and EHB rats, respectively. Our results suggest that hepatobiliary excretion of exogenous Cd is mediated mainly via carrier transport, including a cMrp or GSH carrier, but that the lack of the transport pathway does not contribute to abnormal accumulation of Cd in the liver.
Keywords: Key words EHB rat ; cMrp ; ATP-driven anion transport ; Biliary excretion of Cd and glutathione ; Hepatic accumulation of Cd
Binding of Cu to metallothionein in tissues of the LEC rat with inherited abnormal copper accumulation by D. Klein; S. Michaelsen; S. Sato; A. Luz; A. Stampfl; K. H. Summer (pp. 340-343).
Long-Evans Cinnamon (LEC) rats aged 16 ± 4 weeks with histopathological alterations of liver and kidney, exhibited elevated Cu levels in liver, kidney and spleen which were 52, 27 and 5 times higher than those of the respective tissues of age-matched Wistar rats. About 61% of hepatic and about 38% of renal Cu was recovered in the cytosolic fraction. Metallothionein (MT) levels were found to correlate with the cytosolic Cu concentrations in liver and kidney. According to differential MT analysis, about 68 and 82% of hepatic and renal MT was loaded with Cu. The portion of MT which binds Cu was negatively correlated with the ratio of cytosolic Zn/Cu in all organs investigated. Despite high MT levels and the high percentage of Cu binding to MT, particularly in liver and kidney, considerable amounts of Cu remained unbound to MT. This non-MT bound Cu showed good correlation with the total cytosolic Cu content, and might play a crucial role in the pathogenesis of Cu toxicosis.
Keywords: Key words LEC rat ; Copper ; Metallothionein ; Hepatitis ; Liver