The kinetics of cannabinoid distribution and storage with special reference to the brain and testis.

Male and female rats were given either single or repeated intramuscular injections of 2 microCi of 14C-delta 8-tetrahydrocannabinol. They were sacrificed by groups of three at regular intervals 2, 3, 4, 6, or 24 hours after the last injection. Samples of blood lung, brain and pituitary, spleen, liver, fat, testis, and ovary were removed. Some samples were pyrolysed in an automatic oxygen train system to measure 14C-CO2, which reflects total cannabinoid concentration; other samples were kept for measurements of individual cannabinoids after extraction. After a single administration of 14C-delta 8-THC, maximal concentration of total radioactivity was reached in the brain between 2 and 4 hours amounting to 6 ng/gm tissue, or 0.06 per cent of the administered dose. After two weeks of chronic administration, concentrations of radioactivity progressively increased in liver and neutral fat, while cannabinoid levels in brain and testis remained unchanged. These data illustrate the efficiency of the blood-brain and blood-testicular barrier in limiting the access and accumulation of cannabinoids in these tissues.

In vivo covalent binding of cismethrin and bioresmethrin to hepatic proteins.

The covalent binding level of [14C]alcohol-labelled cismethrin, bioresmethrin and/or their metabolites to hepatic proteins was measured after a single i.v. injection of pyrethroids in rats pretreated with malathion. In the first control group, the radiolabel binding level was different for cismethrin (35.3 pmol X mg-1 protein) and bioresmethrin (22.6 pmol X mg-1 protein) and in the treated rats, the amount of binding was reduced similarly for the two pyrethroids (13 pmol X mg-1 protein). After 13 days of chronic i.p. treatment, the covalent binding level on microsomal protein was 3 times higher for cismethrin than for bioresmethrin and the ratio of cismethrin to bioresmethrin covalent binding level was similar to that after a single i.v. injection. It is suggested that the difference in covalent binding distribution and concentration in the various subcellular fractions of liver was due to the different routes of metabolism of the two resmethrin isomers.

A multicentre study of acute in vitro cytotoxicity in rat liver cells.

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 IC(50) 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 IC(50) 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.

Fate of isopropyl-iodoamphetamine (IMP) in rat liver microsomes.

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.

Effects of a diphenyl-ether herbicide, oxyfluorfen, on human BFU-E/CFU-E development and haemoglobin synthesis.

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-Erythroid) were exposed to oxyfluorfen, a diphenyl-ether herbicide in the presence of erythropoietin, and the haematoxicity evaluated in vitro by scoring the development 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 differentiation 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.

Comparison of toxicity induced by T-2 toxin on human and rat granulo-monocytic progenitors with an in vitro model.

T-2 toxin is a trichothecene mycotoxin produced by various 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 alimentary intoxication and, among others, haematological symptoms. Granulo-monocytic progenitors from human umbilical cord blood on the one hand and granulo-monocytic 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 relationships showed a strong and rapid effect of T-2 toxin on rat colony forming unit-granulocyte and macrophage (CFU-GM) between 5 x 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. IC50 were determined on day 7, 10 and 14. They were, respectively, 1.6 x 10(-9) M; 3.6 x 10(-9) M; 1.4 x 10(-9) M for human cells, and 2.2 x 10(-9) M; 3.3 x 10(-9) M; 2.6 x 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.

Comparison of toxicity induced by HT-2 toxin on human and rat granulo-monocytic progenitors with an in vitro model.

HT-2 toxin is a trichothecene mycotoxin occurring naturally in various agricultural products. Many in vitro studies have shown that HT-2 toxin is a major metabolite of the parent compound T-2 toxin. In man as well as in animals both toxins have been shown to cause alimentary intoxications and haematological disorders. Granulomonocytic progenitors (CFU-GM) from human umbilical cord blood and from rat bone marrow were cultured in the presence of HT-2 toxin (10(-7) M to 10(-10) M) for 14 days. The concentration-effect relationship was studied and the IC50 were determined on Days 7, 10 and 14. The IC50 was 1.8 x 10(-9) M, 3.5 x 10(-9) M and 1.8 x 10(-9) M for human cells, and 2 x 10(-9) M, 2.3 x 10(-9) M and 2.2 x 10(-9) M for rat cells. The results have been compared with previous findings for T-2 toxin. Although T-2 and HT-2 toxins had a similar IC50 on human and rat CFU-GM, the expression of their cytotoxicities was different. These findings are relevant to investigation of the cellular targets of T-2 and HT-2 in elucidating the mechanism of trichothecene haematotoxicity.

[Neurotoxicity of pyrethrins in warm-blooded animals]. / Neurotoxicité des pyréthrinoïdes chez les animaux à sang chaud.

The synthetic pyrethroids, a new class of insecticides, have excellent insecticide properties with good biodegradability, therefore a low remanence. Their mammalian toxicity is low in considering amounts used in agriculture. On the other hand, experimental animals exposed to mean so high concentrations of pyrethroids display neurological symptoms. This paper is a review relating the electrophysiological, biochemical and pharmacological aspect of the patent neurotoxicity of pyrethroids. Benign cases human intoxication and acute animal intoxication are reported.

[Allergenic character of tetrahydrocannabinol (delta 9 THC), active principle of Indian hemp (Cannabis saliva var. indica)]. / Caractère allergisant du tétrahydrocannabinol (delta9 THC) principe actif du chanvre indien (Cannabis saliva var. indica).

The maximization test applied on the Hartley Guinea Pig and the mast cell degranulation test performed on the sensibilised Guinea Pig serum resulted in: 1, extreme allergenicity; 2, the formation of circulating antibodies anti delta9 THC.

Cytotoxicity, cytogenotoxicity and allergenicity tests on certain pyrethroids.

Pyrethroids are potent synthetic insecticides which have been increasingly employed in recent years. Such compounds have been shown to bind covalently to hepatic proteins. Covalent binding is often associated with toxic effects. Possible cytotoxic, cytogenotoxic and allergenic effects could be due to covalent binding of these compounds and/or their metabolites to endogenous macromolecules. In the present paper we examine possible cytotoxic effects of certain pyrethroids on human lymphocytes and L 1210 lymphoblastoid mouse cells, cytogenotoxic effects with micronuclei test and allergenic effects with Magnusson and mast cell degranulation tests. Under our experimental conditions, the tested compounds showed neither acute cytotoxic nor cytogenotoxic effects, though, Cismethrin presented slight antimitotic effects statistically different to those with the control. Slight allergenic character of Cismethrin, Bioresmethrin and Deltamethrin was revealed by Magnusson and mast cell degranulation tests.

In vitro covalent binding of the pyrethroids cismethrin, cypermethrin and deltamethrin to rat liver homogenate and microsomes.

Phenobarbital-induced rat liver homogenate and microsomes were used to study covalent binding of 14C-labelled (at the alcohol moiety) cismethrin, 14C-labelled (at the alcohol and acid moieties) cypermethrin, and 14C-labelled (at the alcohol and acid moieties) deltamethrin. Covalent binding was dependent on pyrethroid concentration. With liver homogenate, inhibition of esterases by tetraethylpyrophosphate and of mitochondrial respiration by rotenone or potassium cyanide only slightly altered the covalent binding level. With microsomes, inhibition of cytochrome P-450 and mixed function oxidases by carbon monoxide and piperonyl butoxide reduced the covalent binding so far as to be nearly absent. Eighty percent inhibition of epoxide hydrolase decreased the covalent binding by 50%. The comparison of data between alcohol and acid labelling of the same pyrethroid suggested that, in vitro, the whole molecule is bound to proteins and that hydrolysis can occur afterwards. The experiments stress the role of cytochrome P-450-dependent monoxygenases in the covalent binding process.

In vitro toxicity induced by deoxynivalenol (DON) on human and rat granulomonocytic progenitors.

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 x 10(-7) mol/L. IC50 values on days 7, 10, and 14 were, respectively, 3 x 10(-8), 2.9 x 10(-8), 3.9 x 10(-8) mol/L for human CFU-GM and 2.6 x 10(-7), 1.5 x 10(-7), and 1.6 x 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.

Detection and quantitation of cannabinoids in biological fluids: specificity and kinetics after smoking.

A procedure for the quantitation and detection of cannabinoids in biological fluids (plasma and urine) is described. This method is based on isotopic derivatization and double labeling. delta 9-Tetrahydrocannabinol and two of its metabolites (11-hydroxy-delta 9-THC and 8-beta-hydroxy-delta 9-THC) can be detected and measured in plasma or urine. The use of TLC enables specific separation of cannabinoids. The sensitivity of detection (4 ng/mL) is compatible with cannabinoid kinetics. The procedure can be applied to routine measurements and pharmacokinetic studies.

Isolation and toxicological study of tetrahydrocannabivarol, an homolog of tetrahydrocannabinol (delta 1--THC).

Selection and culture in a Phytotron of plants of Cannabis sativa L. with high content of propyl-THC are obtained. Then it was possible to isolate a large quantity of propyl-THC by column chromatography. Spectroscopic analysis is done. Pharmacological tests are being run; first results revealed the allergenicity of this substance by comparison with the properties of tetrahydrocannabinol, the active principle of Cannabis.

[Binding of delta-8-THC to human serum proteins: binding as a function of lipoprotein composition]. / Liaison du delta8-THC aux protéines sériques humaines: étude de sa fixation en fonction de la composition lipoprotéinique

In accordance with the function of the human lipoprotein composition, using serum of different lipid profiles, the distribution of delta8tetrahydrocannabinol, as isomer of delta9THC, was studied through the use of ultracentrifugation and fraction isolation, followed by biochemical analysis of the VLDL, LDL, and HDL fractions, as well as that of the non-lipoprotein protein fraction. The results indicate the existence of a linear correlation between the distribution of delta8THC and the phospholipids of the lipoprotein cenapse. No correlation between the quantity of bound delta8THC and the other lipoprotein constituents cholesterol, triglycerides, total lipids or protein could be observed.

Cytoprotective effects of Gypsophila saponins towards isolated rat hepatocytes.

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.

In vitro covalent binding of new brain tracer, para-125I-amphetamine, to rat liver and lung microsomes.

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.