Ethoxidine (N-methyl-12-ethoxy-2,3,8,9-tetramethoxybenzo[c]phenanthridinium methylsulfonate salt) is a synthetic 2-methoxy-12-ethoxy derivative of the natural alkaloid fagaronine. This new inhibitor of DNA-topoisomerase I is considered as a potential antitumor agent with higher in vitro activity than fagaronine. In order to further improve the efficiency of ethoxidine, its in vitro biotransformation by hepatic monooxygenases and the structures of its metabolites were investigated by high-performance liquid chromatography (HPLC) combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) and accurate mass measurement by time-of-flight mass spectrometry (TOFMS). When ethoxidine was incubated with BNF-treated rat liver microsomes or with cells expressing different recombinant human cytochrome P450, the same four ethoxidine metabolites (m(1)-m(4)) were detected and were formed exclusively by CYP1A1. The structures of these metabolites were assigned from ESI-MS/MS mass spectra and compared with those of ethoxidine derivatives. Accurate mass measurements of in-source ESI-TOFMS fragment ions exhibited successive neutral losses of C(2)H(4) and CO for ethoxidine and its metabolites. Whereas a 15 Da loss (methyl radical) was observed for the metabolites m(1)-m(4) containing a quaternary ammonium group, a 16 Da loss (methane) was observed for ethoxidine and could have resulted from the presence of two methoxy groups at adjacent positions (C-2 and C-3). The proposed oxidative modifications of ethoxidine were further confirmed by determination of the number of exchangeable hydrogen atoms and by the proposed elemental compositions of the metabolites based on accurate mass measurements by TOFMS. Two major metabolites resulted from O-demethylation of ethoxidine; one was tentatively identified as 12-ethoxyfagaronine (m(3)) and the second as an O-demethylated ethoxidine isomer (m(4)). Two polar metabolites were shown to be O-demethylated (m(1)) and hydroxylated (m(2)) derivatives of 12-ethoxyfagaronine. When 12-ethoxyfagaronine was incubated under the same conditions as ethoxidine, m(2) was formed, thus supporting the proposal that 12-ethoxyfagaronine is the primary oxidative product of ethoxidine.

The diphenyl-ether herbicides exert their phytotoxic activity by preventing chlorophyll formation in plants as a result of inhibition of protoporphyrinogen oxidase. This enzyme is the last step of the common pathway for chlorophyll and haem biosynthesis. The aim of this work is to determine whether herbicide inhibitors of plant protoporphyrinogen oxidase could act on the human protoporphyrinogen oxidase involved in haemoglobin synthesis and cause heamatologic diseases. Human erythroblastic progenitors (BFU-E/CFU-E: Burst Forming Unit-Erythroid and Colony Forming Unit-Ery throid) were exposed to oxyfluorfen, a diphenyl-ether herbicide in the presence of erythropoietin, and the haematoxicity evaluated in vitro by scoring the develop ment of BFU-E/CFU-E colonies after 7 and 14 days of culture. The toxic effect on differentiation has been evaluated using four criteria: morphology, total protein, total porphyrin, and haemoglobin content. The study of BFU-E/CFU-E proliferation and differentia tion showed a cytotoxic effect of oxyfluorfen only at very high concentrations. In contrast, haemoglobin synthesis can be inhibited by concentration of oxyfluorfen (10-4 M) that have no adverse effect on cellular proliferation.

Deoxynivalenol (DON) is a trichothecene mycotoxin produced by various species of fungi. Trichothecenes are known as major contaminants of cereals and cereal-containing foods. DON has been detected in agricultural products worldwide and persists in products after processing. In humans as well as in animals, DON has been shown to induce both alimentary and hematological toxicities. Granulo-monocytic progenitors (CFU-GM) from human umbilical cord blood from rat bone marrow were cultured in the presence of DON (from 10-6 to 10-8 mol/L) for 14 days. DON rapidly inhibits human and rat CFU-GM in a concentration-dependent manner between 10-6 and 2.5 × 10-7 mol/L. IC50 values on days 7, 10, and 14 were, respectively, 3 × 10-8, 2.9 × 10-8, 3.9 × 10-8 mol/L for human CFU-GM and 2.6 × 10-7, 1.5 × 10-7, and 1.6 × 10-7 mol/L for rat CFU-GM. The present study defines the cytotoxic and inhibitory DON concentrations for rat and human CFU-GM and provides a system for further investigation of cellular DON targets and elucidation of the mechanism of trichothecene hematotoxicity. Moreover, we propose one of the trichothecenes tested in our studies as a reference molecule for in vitro studies, since one mycotoxin seems to be the most potent myelotoxic inhibitor of CFU-GM detected to date.

T-2 toxin is a trichothecene mycotoxin produced by vari ous species of fungi. Trichothecenes are known as major contaminants of cereals and their derivatives. In man as well as in animals, T-2 toxin has been shown to induce ali mentary intoxication and, among others, haematological symptoms. Granulo-monocytic progenitors from human umbilical cord blood on the one hand and granulo-mono cytic progenitors from rat bone marrow on the other, were cultured in the presence of T-2 toxin (from 10 -7 to 10 -10 M) for 14 days. A study of concentration and effect relation ships showed a strong and rapid effect of T-2 toxin on rat colony forming unit-granulocyte and macrophage (CFU-GM) between 5.10 -9 M and 10 -9 M. On the other hand, human CFU-GM were able to grow in the presence of the same T-2 toxin concentrations. IC 50 were determined on day 7, 10 and 14. They were, respectively, 1.6.10-9 M; 3.6.10 -9 M; 1.4.10 -9 M for human cells, and 2.2.10 -9 M; 3.3.10 -9 M; 2.6.10 -9 M for rat cells. The present study was prompted by the need to define precisely the cytotoxic and inhibitory T-2 toxin concentrations for rat and human CFU-GM. It is particularly relevant for the investigation of cellular T-2 toxin targets and in order to elucidate the mechanism of trichothecene haematotoxicity.

Changes in different microsomal membrane functions were measured in the liver of rats 3, 6, or 9 weeks following an oral infection with 20 metacercariae of Fasciola hepatica. The parasitic pathology noted at autopsy was accompanied by increased levels in both plasma aspartate aminotransferase (EC 2.6.1.1) and microsomal γ-glutamyltransferase (EC 2.3.2.2). Heme oxygenase activity of microsomes was significantly decreased by Weeks 3 and 6 postinfection and this decrease correlates with those of total microsomal cytochrome P450 and certain P450-dependent monooxygenase activities, namely, benzphetamine demethylation, ethoxycoumarin deethylation, and benzopyrene hydroxylation. Microsomal epoxide hydrolase (EC 3.3.2.3) was only altered 6 weeks after the infection. During the early stages of the parasitism, there were decreases in both microsomal calcium uptake and calcium ATPphosphohydrolase activity (EC 2.6.1.1), whereas membrane fluidity, estimated by the order parameter S, was lower in the infected rats than that in the controls. These alterations could be related to the already described increase in liver cytosolic calcium or lipid peroxidation which occurs in experimental fascioliasis.

A multicentre validation study of the acute in vitro cytotoxicities of 31 liquid or solid chemicals was carried out by six laboratories, using primary rat hepatocyte cultures as a model system. We report here a comparison of neutral red uptake IC50 and LD50 values. Oral, i.p. and i.v. LD50 values were available for 27, 24 and 18 chemicals, respectively, and an IC50 value was obtained for 15, 14 and 11 of these compounds, respectively. A significant correlation was found only between IC50 and i.v. LD50 values.

A multicentre validation study of the acute in vitro cytotoxicities of 31 liquid or solid chemicals was carried out by six laboratories, using primary rat hepatocyte cultures as a model system. We report here a comparison of neutral red uptake IC50 and LD50 values. Oral, i.p. and i.v. LD50 values were available for 27, 24 and 18 chemicals, respectively, and an IC50 value was obtained for 15, 14 and 11 of these compounds, respectively. A significant correlation was found only between IC50 and i.v. LD50 values.

Para-iodoamphetamines are currently used in nuclear medicine to detect brain perfusion abnormalities with tomoscintigraphy. Little is known about their metabolism pathways in rat and humans. N-isopropyl-125I-iodoamphetamine (IMP) interactions were studied with rat liver microsomes. The first dealkylated metabolite (IAMP) at low concentration gave a type I binding complex then, with a high concentration, a very stable type II complex with oxidized cytochrome P-450 FeIII. In contrast, IMP only gave a type I binding complex in the absence of NADPH. In the presence of NADPH, IAMP and IMP produced 455 nm absorbing complexes, which were enhanced when phenobarbital-treated rat liver microsomes were used. During in vitro metabolic activation, covalent binding of IMP and IAMP on rat liver microsomal proteins was observed. This process was mixed function oxidase (MFO) dependent. The covalent binding level was higher with IAMP and was not affected by flavine oxidase inhibitors. These results confirm the interaction of IMP and IAMP with microsome proteins and cytochrome P-450 and suggest that an N-oxidation of IMP occurs after N-dealkylation. As cytochrome P-450 and dealkylated IMP (IAMP) were found in brain, cerebral metabolism in brain and evolution of activity biodistribution with the course of time can be suggested.

A multicentre validation study of the acute in vitro cytotoxicity of drugs involving six French laboratories from INSERM or pharmaceutical companies has been carried out. Thirty liquid or solid chemicals such as antibiotics, anticancer drugs and solvents were selected and incubated for 20 hr with normal rat hepatocytes and FaO hepatoma cells. Miniaturized and automated methods were defined for the evaluation of cytotoxic effects. Four endpoints were evaluated: the ratio of extracellular lactate dehydrogenase to total lactate dehydrogenase, total cellular protein content, reduction of a tetrazolium salt, and neutral red uptake. For each test IC50 values were calculated. A good interlaboratory reproducibility was demonstrated. The neutral red assay was found to be the most sensitive and the least reproducible endpoint. More compounds were shown to be cytotoxic to hepatocytes than to hepatoma cells (18 v. 12). On the basis of the IC50 values a few compounds were found to be much less cytotoxic than predicted from in vivo data, suggesting that a simple experimental protocol and non-specific cytotoxicity parameters are not sufficient to test certain drug families. However, such methods appear to provide a useful means of defining the concentration range of the drug that will be selected for further analysis using more specific tests.

On utilise souvent le DMSO afin de solubiliser des produits dont on veut etudier le metabolisme hepatique a l'aide de cultures foetales hepatocytes de rat. Se pose donc la question de la toxicite intrinseque de ces solvants

p-125I-amphetamine (I-Amp) is retained significantly in liver and lung during brain tomoscintigraphy. To attempt to explain this clinical observation, we have investigated the interaction of I-Amp with rat liver and lung microsomal proteins. Studies using spectral shift technique indicate that low concentration of I-Amp gives a type I complex and high concentration appears very stable type II complex with cytochrome P-450 Fe III. In the presence of NADPH, I-Amp gives rise to a 455 nm absorbing complex with similar properties to the Fe-RNO complexes. This complex formation was greatly enhanced with phenobarbital treated liver microsomes. The in vitro binding study shows that I-Amp and/or its metabolites was covalently bound to macromolecules in the presence of the molecular oxygen and NADPH-generating system. Incubation in the presence of glutathione, cystein and radical scavengers decreases binding. Mixed function oxydase (MFO) inhibitors diminish the amount of covalent binding and alter the extent of metabolite formation. The total covalent binding level increased with liver microsomes from PB pretreated rats as it was observed with the 455nm complex formation. The radioactivity distribution on microsomal proteins was examinated with SDS polyacrylamide gel electrophoresis and autoradiography. This experiment proves that the radiolabelled compounds are bound on the cytochrome P-450. The radioactivity bound increased when the PB induced rat liver microsomes were used. All these results indicate that I-Amp was activated by an oxydative process dependent on the MFO system which suggests a N-oxydation of I-Amp and the formation of reactive entities which covalently bind to proteins.

Saponins are glycosides widely distributed in the plant kingdom and are found in many foods. The hepatoprotective potential of glucuronogypsogenin (GG) and gypsoside (GY) towards isolated rat hepatocytes treated by three toxic models used at sub-lethal doses: galactosamine (5 x 10(-3) M), CCl4 (5 x 10(-4) M) and erythromycin (5 x 10(-4) M) was investigated. Two schedules were carried out corresponding to curative or preventive treatment. No protection was observed on hepatocytes treated with GY before or after addition of the toxicants. In contrast, a protective action was detected when hepatocytes were pretreated with GG (5 x 10(-5) M) as probe, by the normalisation of LDH leakage and ATP content. It depends on the toxicant: the cytoprotective spectrum is 5 x 10(-5) to 5 x 10(-7) M with galactosamine; 5 x 10(-5) to 5 x 10(-6) M with CCl4; and around 5 x 10(-5) M with erythromycin. Taking into account the importance of LDH as an indicator of membrane damages, GG was assumed to interact with membrane hepatocyte.

AbstractThe catalytic reduction of (+)‐cismethrin 1, a synthetic pyrethroid, with tritium gas in various solvents and in presence of different catalysts was studied. The best result was obtained with 5% Pd‐ BaSO4 catalyst in methanol giving rise to 3H‐5‐benzyl‐3‐furylmethyl (+)‐cis‐3‐isobutyl‐2,2‐dimethylcylopropane carboxylate 2 with a specific activity of 98 Ci. mmol−1 (3626 GBq. mmol−1). The incorporation of tritium was verified by H‐NMR and MS analyses.