Conditioning to ethanol in the fruit fly-a study using an inhibitor of ADH.

To identify processes involved in the choice of ethanol by adult Drosophila, flies homozygous Adh(F), reared in the absence of alcohol were placed in contact with: a) an ethanol-free medium, b) a medium containing ethanol, c) a medium supplemented with 4-methylpyrazole (4-MP, an inhibitor of the ADH pathway), d) a medium containing ethanol and 4-MP. The choice of ethanol over a medium without ethanol was evaluated by measuring the duration of extension of the proboscis of the flies in each of the media. A slight preference for the ethanol-supplemented medium was observed in the naive flies, which was enhanced by previous exposure to ethanol. Exposure to ethanol and 4-MP, however, led to an avoidance of ethanol. There was a reduction in ADH activity on treatment of the flies with 4-MP, and signs of malaise (reduced locomotor activity, loss of balance) were observed in the flies who ingested both ethanol and inhibitor. We concluded that the preference for ethanol stems from an associative learning related to ethanol utilization. Inhibition of enzymes of ADH pathway led to a conditioned aversion due to disturbance of ethanol metabolism giving rise to malaise.

IL-13 increases the cPLA2 gene and protein expression and the mobilization of arachidonic acid during an inflammatory process in mouse peritoneal macrophages.

Pretreatment of mouse peritoneal macrophages with interleukin-13 (IL-13) potentiates the mobilization of arachidonic acid (AA) and the production of HETEs but does not affect the production of cyclooxygenase metabolites triggered by the suboptimal concentration of an inflammatory agonist (opsonized-zymosan). Cycloheximide suppresses these effects of IL-13 suggesting that de novo protein synthesis is involved. Indeed, IL-13 induces a time-dependent increase in the levels of cytosolic PLA2 (cPLA2) protein and mRNA. This study demonstrates a new pathway for IL-13 to modulate the inflammatory process in macrophages via modifications of cPLA2 expression and subsequent AA mobilization.

Metabolism and cytotoxicity of chlorpropham (CIPC) and its essential metabolites in isolated rat hepatocytes during a partial inhibition of sulphation and glucuronidation reactions: a comparative study.

The changes in metabolism and cytotoxicity of chlorpropham (CIPC) and its major metabolites, 4-hydroxychlorpropham (4-OH CIPC), 3-chloroaniline, and 3-chloroacetanilide were investigated in isolated rat hepatocyte suspensions after a partial inhibition of sulphation and glucuronidation and the two reactions combined in an attempt to assess the part of each of them in the enhanced CIPC toxicity observed in vivo after D-galactosamine treatment. With sulphation and glucuronidation effective, CIPC has a cytolytic effect and reduces intracellular ATP and K+ level while 4-OH CIPC has a weak cytolytic effect but modifies ATP and K+ level in a greater extent than CIPC. Inhibition of sulphation does not affect the cytotoxicity of CIPC or 4-OH CIPC because there is a compensatory increase in the amount of 4-OH CIPC glucuronide formed and the level of free 4-OH CIPC always remain low. In contrast, when incubations are carried out with either CIPC or 4-OH CIPC, the presence of D-galactosamine leads to a decrease of glucuronide and sulphate conjugates accompanied, respectively, by a 3.6-fold and 6. 9-fold increase of the free 4-OH CIPC level in the culture medium. This alteration of the metabolism is followed by a marked reduction of ATP synthesis with a concomitant modification of cell permeability. The cytolytic effect is due to CIPC itself, whereas the effect on energy supply was attributed to free 4-OH CIPC. The results demonstrate a combined effect of free 4-OH CIPC and D-galactosamine on intracellular ATP level that could account for the partial inhibition of sulphation. This change in the CIPC metabolism could explain the increased CIPC toxicity observed in vivo after D-galactosamine pretreatment.

Effect of interleukin-4 on allergen-induced arachidonic acid metabolism of rat peritoneal macrophages during immediate hypersensitivity reactions.

The aim of this study was to investigate the [3H]arachidonic acid metabolism of rat peritoneal macrophages, induced by allergen (ovalbumin) and the impact of interleukin-4 on this process. We established that ovalbumin induces an increase of [3H]arachidonic acid mobilisation from membrane lipids and of [3H]arachidonic acid catabolism, principally by the 5-lipoxygenase pathway, when the macrophages are sensitized and when serum is present. The allergen effect is not modified by the presence of interleukin-4 in the culture medium of macrophages 15 h before the allergen challenge. We also showed that, whereas the basal [3H]arachidonic acid metabolism of macrophages from control and actively sensitized rats is not different, interleukin-4 increases the [3H]arachidonic acid mobilisation and catabolism by cyclooxygenase and 5-lipoxygenase pathways in macrophages from control rats although it does not in macrophages from actively sensitized rats. In macrophages from control rats, the interleukin-4 effect is diminished by the addition of IgEs to their culture medium. In summary, interleukin-4 has an enhancer effect on the macrophage arachidonic acid catabolism that depends on the sensitization condition of the cell but that has no consequences on the further increased arachidonic acid metabolism induced by the allergen.

Effects of modulation of sulphation and glucuronidation on chlorpropham metabolism and cytotoxicity in isolated rat hepatocytes.

After modulation of sulphation and glucuronidation, the relationship between the changes in metabolism and cytotoxicity of chloropropham (CIPC), a widely used herbicide, was investigated in isolated rat hepatocyte suspensions. Under physiological conditions, CIPC had a cytolytic effect, modified membrane permeability and reduced intracellular ATP level. CIPC was metabolized by hepatocytes mainly into 4-OH chlorpropham sulphate (37%) and glucuronide conjugates (18%). Inhibition of sulphation, by omitting sulphate from the isolation and incubation media, did not affect the cytotoxicity of CIPC, since there was a 2.5-fold compensatory increase in 4-OH CIPC glucuronide. Inhibition of glucuronidation by adding 4 mM D-galactosamine in the incubation medium led to a 66% decrease of glucuronide conjugate and simultaneously to a 32% decrease of sulphate conjugate. In that case, concentrations of free 4-OH CIPC in both hepatocytes and incubation medium were markedly increased, while those of 3-chloroaniline and 3-chloroacetanilide were slightly modified and remained low. This alteration of metabolism was accompanied by modification of cell permeability and reduction in ATP synthesis. The cytolytic effect was due to CIPC itself, whereas the effect on energetic metabolism was attributed to a metabolite. Results demonstrated for the first time a partial inhibition of sulphation by D-galactosamine (4 mM), probably due to the effect of D-galactosamine on intracellular ATP levels.

Cadmium accumulation and cytotoxicity in rat hepatocytes co-cultured with a liver epithelial cell line.

Cadmium accumulation and cytotoxicity were compared in hepatocytes in primary culture (HPC) or co-cultured (COC) with a rat liver epithelial cell line (RLEC). Cells were exposed for a 15-day period at 0, 0.045, 0.45 and 0.9 mum-Cd in the incubation medium. Cadmium uptake in COC was always linearly Cd dose and time dependent. In contrast, at the two highest doses for RLEC and only at the highest dose for HPC, a plateau in Cd uptake was observed. In viable cells 95% of the intracellular Cd was found in the cytosol, entirely protein bound. In the three types of culture the amount of Cd-metallothionein (MT) complex was proportional to Cd uptake and was in the order: COC > HPC > RLEC. Lactate dehydrogenase release in the medium and the DNA content of the plated cells at the end of exposure accounted for a marked Cd-induced cytotoxic effect in RLEC and a lesser effect in HPC. In contrast, no significant cytolysis was observed in COC. This suggests that although greater Cd accumulation was observed in COC than in HPC and RLEC, the detoxification procedure, dependent on Cd-MT complex formation, was more efficient and longer preserved in COC owing to a greater maintenance of hepatocyte specific functions, as evidenced by albumin secretion. This COC model offers a powerful tool for studying long-term accumulation and cytotoxicity of Cd in vitro at low exposure levels.

Ethanol-inducible cytochrome P-450 activity and increase in acetaldehyde bound to microsomes after chronic administration of acetaldehyde or ethanol.

Chronic ethanol consumption results in acetaldehyde adduct formation with proteins such as haemoglobin and liver proteins in vivo. Our purpose was to study the binding of acetaldehyde to liver microsomal proteins, a site of ethanol oxidation via cytochrome P-450 (especially P-450 II E1), after chronic administration of ethanol or acetaldehyde for 21 days to rats. The liver microsomal oxidation of 1-butanol by the ethanol-inducible P-450 also was examined. Acetaldehyde bound to liver microsomal proteins was higher in ethanol-fed rats compared with acetaldehyde-treated rats (0.735 vs 0.413 nmol/mg of protein respectively). The biotransformation of n-butanol to butyraldehyde by liver microsomes was increased (by 136%) in ethanol-fed rats vs controls, whereas in acetaldehyde-treated rats this increase was much lower (only 27%). However, in this last group, a significant negative relationship between the quantity of acetaldehyde bound to microsomal proteins and the monooxygenase-catalyzed transformation of butanol by liver microsomes was demonstrated (r = -0.79, P less than 0.01). These results suggest that proteins of liver microsomes are a target for acetaldehyde binding during ethanol oxidation and such adduct formation could impair the oxidative properties of the alcohol-inducible cytochrome P-450.

Cadmium-induced alterations of chlorpropham metabolism in isolated rat hepatocytes.

In order to investigate the various steps of chlorpropham (CIPC) metabolism which could be influenced by cadmium, isolated rat hepatocytes were incubated in the presence of CIPC (0.1 mM) and of increasing Cd concentrations (0-180 microM). The results showed that Cd accumulation in hepatocytes was in good correlation to its concentration in the incubation medium. At 90 microM Cd, hydroxylation of CIPC was only slightly decreased by 30%, while CIPC hydrolysis into 3-chloraniline was unaffected by the presence of Cd. Accordingly, unchanged CIPC increased in hepatocytes. At 27 microM Cd, free 4-hydroxychlorpropham (4-OHCIPC) increased in the intracellular medium as a consequence of a strong suppression of both sulfation and glucuronidation which was related to the strong depletion of the intracellular ATP level under the combined influences of both cadmium and free 4-OHCIPC. Acetylation of 3-chloroaniline, which represents a minor pathway of CIPC metabolism, was already markedly suppressed (43%) with the lowest Cd concentration (27 microM). These in vitro results suggest that Phase II reactions are more sensitive to Cd than Phase I processes and that Cd enhanced the CIPC cytotoxicity as shown by alterations of the membrane integrity.

Effect of chronic acetaldehyde intoxication on ethanol tolerance and membrane fatty acids.

Recent studies have suggested that acetaldehyde participates directly in the pathogenesis of alcoholism. Its action has been attributed mainly to its physico-chemical properties. Results of direct intoxication of laboratory animals with acetaldehyde have been reported, but only for short periods of exposure and at high doses. These are probably not representative of the conditions found during alcohol intoxication. The pulmonary route of administration described here enables long term intoxication with acetaldehyde, at levels corresponding to values measured during chronic ethanol intoxication. Chronic administration of acetaldehyde during 3 weeks induced a metabolic tolerance to ethanol as tested by the sleeping time after a challenge dose of ethanol; behavioural tolerance (measured by blood alcohol levels on waking) was not observed. At the end of the intoxication, phospholipid fatty acids of erythrocyte and synaptosome membranes were also analysed. Small changes in levels of the shorter fatty acids were observed in the phosphatidyl-choline fraction. By comparison with the effects of ethanol on the same membrane preparations, only a small part of this effect can be attributed to acetaldehyde. The first metabolite of ethanol has, however, a sure effect on the pattern of fatty acid phospholipids.

Ethanol tolerance in the rat after inhalation of acetaldehyde for a period of 21 days.

Ethanol tolerance is related to alterations in fatty acid content and physical properties of membranes. Studies have suggested a specific role for acetaldehyde in the pathogenesis of alcoholism. We measured tolerance to EtOH and erythrocyte membrane fatty acid composition after intoxication by continuous inhalation of AcH vapor for a period of 21 days. Pathophysiological and nutritional parameters were compared between treated and pair weight controls. We showed that: intoxication with AcH is technically possible via the pulmonary route, producing plasma AcH levels comparable to those seen after ethanol intoxication. The dose of AcH needs to be increased progressively to maintain constant plasma levels this indicates metabolic tolerance. the AcH-intoxicated animals had a metabolic tolerance to EtOH. AcH intoxication led to alterations in fatty acid composition similar to those seen after EtOH intoxication, especially in the saturated/unsaturated ratio of the phosphatidyl-choline and phosphatidyl-inositol fractions. AcH probably plays a part in the phenomenon of tolerance to EtOH.

Sensitivity of individual erythrocyte membrane phospholipids to changes in fatty acid composition in chronic alcoholic patients.

The erythrocyte membrane levels of total phospholipids and cholesterol and the fatty acid composition of individual groups of phospholipids (phosphatidylethanolamine, phosphatidylserine plus phosphatidylinositol, and phosphatidylcholine) were studied in 10 patients admitted for ethanol detoxification and in 14 control subjects. The fatty acid composition of the patient phospholipids was modified but the level of cholesterol and the level of phospholipids remained unchanged. The fatty acid changes were mainly confined to phosphatidylcholine. The modifications concerned the levels of the octadecenoic acids (18:1) which rose (p less than 0.01), and linoleic acid (18:2) which fell (p less than 0.05). These results suggest that chronic ethanol ingestion may perturb the cell membrane organization with, in consequence, possible effects on cell morphology and functions.

Membrane fatty acid changes and ethanol tolerance in rat and mouse.

Ethanol tolerance and erythrocyte membrane lipids were studied in Sprague-Dawley rats at various times during and after chronic administration, by inhalation, of ethanol vapor. Tolerance increased during the three weeks treatment period and reverted to base line ten days after the treatment was stopped. Chronic ethanol treatment led to changes in the composition of membrane phospholipid fatty acids. These changes partially reverted after treatment ceased. At all times the changes in 16:0, 18:0, 18:1 and 18:2 were correlated with the degree of ethanol tolerance. Analysis of the effect of ethanol treatment (ip injections over a one week period) in three strains of mice showed that the changes of phospholipid fatty acids in erythrocyte membranes were related to whether the strain developed a tolerance to the hypnotic effect of ethanol (DBA, C 57), or not (Swiss). These results show that membrane phospholipid fatty acid composition and ethanol tolerance co-vary during chronic treatment. During the withdrawal period, ethanol sensitivity reverts to control values while the return of the fatty acids to the normal state is incomplete.

Lipid composition of the synaptosome and erythrocyte membranes during chronic ethanol-treatment and withdrawal in the rat.

Male Sprague-Dawley rats were intoxicated by inhalation of ethanol vapor for 21 days. This allowed high tolerance to the hypnotic effect of ethanol and withdrawal syndrome to be developed. The chronic intoxication brought about modifications of the synaptosome and erythrocyte membrane lipid composition which were not due to the reduction in food intake that parallels intoxication. The fatty acid composition of the phospholipids was modified but the level of cholesterol and the level of phospholipid remained unchanged. The modifications concerned the levels of linoleic (18:2) and arachidonic (20:4) acids which decreased in the synaptosomes. In the red blood cell membranes, ethanol affected the levels of the octadecenoic acids (18:1) which rose, and linoleic acid (18:2) which fell. These disturbances were present when the withdrawal syndrome was at its highest and also 3 days after withdrawal when the signs of hyperexcitability were no longer visible in the animal. Modifications in the brain membrane lipid composition parallel the behavioral tolerance to ethanol; however the present results show that the apparent readaptation of the central nervous system to withdrawal of alcohol occurs earlier than the return to normal of the membrane lipid modifications.

Ethanol sensitivity and membrane lipid composition in three strains of mouse.

Strains of mouse (Swiss OF 1, DBA/2J Ico and C57 BL/6J Ico) with different sensitivities to the hypnotic effects of acute ethanol administration were also found to have differences in fatty acid composition in synaptosomal and erythrocyte membrane phospholipids. Chronic ethanol treatment altered membrane fatty acid composition in quantitatively different ways depending on whether the mice developed tolerance (DBA, C57) or not (Swiss). These results support the hypothesis that membrane lipid composition plays a role in the sensitivity to the effects of ethanol.

Comparison of rat blood preparation methods for acetaldehyde assay.

A comparison is made of four previously described methods for the preparation of blood for acetaldehyde (AcH) assay in the rat. The spontaneous formation of AcH which occurs during the treatment of blood containing ethanol and the recovery of known amounts of AcH added to the blood were studied. The methods using sodium nitrite-sulfosalicylic acid or perchloric acid (PCA) in saline gave low levels of spontaneous formation (1 to 2 microM AcH for 48 mM ethanol). In the recovery studies it was seen that semicarbazide does not allow displacement of all the AcH; treatment of the blood with the reactant sodium nitrite-sulfosalycilic acid and use of the hemolysis method gave levels of recovery lower than 50%. Only treatment of the blood with perchloric acid in NaCl allowed all the AcH added to the blood to be recovered. In vivo, PCA in saline releases the AcH which was seen to remain bound in the red blood cells with the semicarbazide method. So the recommended procedure for accurate assay of blood AcH in the rat is cold deproteinization in PCA/saline before head-space gas chromatography. The levels of in vivo blood AcH (4.1 +/- 0.33 microM) obtained in the rat using this method for a blood alcohol concentration of 52 mM are lower than those previously described in the literature.

The gastro-intestinal metabolism of ethanol in the rat. Effect of chronic alcoholic intoxication.

By collecting the blood coming exclusively from the stomach or intestine of rats, it has been shown that ethanol can be oxidized in the digestive mucosa on the basis of the appearance, during its absorption, of acetaldehyde and acetate. This has been shown to be the result of the action of alcohol- and aldehyde-dehydrogenases present in these tissues. The metabolic ability of these enzymes was tested after chronic alcoholic intoxication in association with various treatments with "anti-alcoholic" drugs. The modifications observed correspond to the effects of the treatments on the levels of the enzyme activities but are less clearcut in the in vivo conditions.

[Validity of the method of alcohol administration by the pulmonary route in studies of the alcoholic syndrome in the rat]. / Validite de la méthode d'alcoolisation par voie pulmonaire dans les études du syndrome alcoolique chez le rat.

Alcohol intoxication by the pulmonary route was studied in the rat. This method of continuous alcoholization quickly leads to deep intoxication. After 3 weeks the animals showed metabolic alterations and a strong withdrawal syndrome. Nutritional control was obtained by adjusting the growth of a group of animals (pair-weight animals) with that of the alcoholic rats. It was shown that prolonged periods in an atmosphere rich in ethanol did not cause any alteration of the lungs: neither the constituents nor the histological structure of the lungs were modified. Measurement of the various blood parameters (pH, arterial O2 and CO2 partial pressures, level of haemoglobin, etc.) showed the absence of adverse respiratory effects. This inhalation procedure would therefore seem to be a model which is well adapted to the study of various aspects of alcoholism.