E. Chiesara

FURTHER STUDIES ON THE INHIBITION AND STIMULATION OF MICROSOMAL DRUG METABOLIZING ENZYMES OF RAT LIVER BY VARIOUS COMPOUNDS.

Abstract A variety of drugs produce an inhibition of the in vitro and in vivo metabolism of pentobarbital, hexobarbital, meprobamate, carisoprodol and strychnine and most of these drugs produce after 36–48 hr a stimulation of the in vitro and the in vivo metabolisms of pentobarbital et al. On the other hand, a number of drugs, known as the stimulator of the microsomal drug-metabolizing enzymes, produce an inhibition of these enzymes, if they are added directly to the incubation mixture or given to the rats shortly before the administration of pentobarbital et al. It would therefore appear that many drugs cause the biphasic effects on the microsomal drug-metabolizing enzymes, chlorcyclizine, phenaglycodol, thiopental, hydroxyzine, benadryl, zoxazolamine, phenylbutazone, SKF 525A, DPEA, MG 3062. There is no direct relationship between these effects and glutethimide, phenaglycodol and thiopental stimulate the drug metabolism more markedly than SKF 525A, DPEA and MG 3062, and the latter compounds inhibit the drug metabolisms more markedly than the former. Phenobarbital, meprobamate and urethan have strong stimulatory action, but they have no or only very weak inhibitory action, while JB 516, ipronizaide, imipramine and azacyclonol have strong inhibitory action, but they have no stimulating action. The results of the present studies are of practical importance in the evaluation of the action of two compounds administered simultaneously or after short time intervals.

Influence of sex difference on the pharmacological action and metabolism of some drugs

Abstract A sex difference in the pharmacological effects of pentobarbital, carisoprodol, strychnine, OMPA in adult rats has been demonstrated. These sex differences almost disappeared after the administration of SKF 525 A. The differences were also demonstrated in vitro using liver slices and microsomal fractions, and by in vivo metabolic studied. They were modified by castration and by sex hormone treatments. The anabolic action of male sex hormones appears to account for the increased enzyme activities in male rats.

Factors influencing induction of hepatic microsomal drug-metabolizing enzymes.

Abstract The influence of some factors on the induction of the microsomal drug-metabolizing enzymes by phenobarbital was studied to obtain further information on the mechanism of induction. Only the administration of ethionine 30 min before the administration of phenobarbital completely inhibits the effect of phenobarbital. On the contrary, norleucine, p -fluorphenylalanine and methioninesulphoxide did not modify the effect of phenobarbital. An activator or an inhibitor were not found in the liver of the phenobarbital or the phenobarbital + ethionine pretreated rats. Treatment with phenobarbital can increase the enzyme activity also in rats which have low enzyme activities after administration of carbon tetrachloride and low dietary protein.

Early oxidative damage in primary cultured trout hepatocytes: a time course study

The aim of this study was to evaluate the influence of the two-step hepatocyte isolation procedure on primary cultured trout (Oncorhynchus mykiss) hepatocytes over time. We characterised the possible changes of a variety of some cellular parameters within the first 24-48 h after seeding. We followed the time dependent changes of these parameters during subsequent culture times in order to see if the cells maintained a differentiated status. Scanning electron microscopy revealed bleb formation and 20% cell damage in freshly isolated hepatocytes. During subsequent culture times the bleb dimension appear to be reduced. Heat shock proteins 70 and 50 (HSP70, HSP50) were induced by hepatocyte isolation. During the first 4 h of culture, the hepatocytes showed a variation in mitochondrial activity, an increase in free radical species (ROS), and a decrease in both glutathione (GSH) content and catalase (CAT) activity; the generation of free radicals led to an increase in the formation of 8-hydroxydeoxyguanosine (8-OHdG) in the DNA. The cells showed detectable ethoxyresorufin-O-deethylase activity after 4 h of culture, which had rapidly increased by the 24th hour. After 24 h, mitochondrial and CAT activity, free radical production, and the content of GSH and 8-OHdG returned to their original levels. P450 activity was retained for at least 48 h after seeding. Our data show that trout hepatocytes suffer significant cell injury as a result of the isolation procedure, but primary cultured cells metabolically recover from this stress after a few hours: they are capable of repairing their damaged surfaces, recovering their antioxidant defences and retaining their ability to repair DNA. Our results also confirm that trout hepatocytes in a primary culture maintain their in vivo-like metabolic activities for 3-8 days.

Mechanism of potentiation of barbiturates and mepbrobamate actions by imipramine.

Abstract Imipramine (Tofranil) markedly potentiates the actions of hexobarbital, pentobarbital, meprobamate and carisoprodol, but it does not potentiate the action of barbital and chloralhydrate. On the other hand, unlike SKF 525 A, imipramine can reinduce sleep in rats awakening from pentobarbital hypnosis. Studies of the effect of imipramine on the in vitro metabolism of hexobarbital, pentobarbital, meprobamate and carisoprodol indicate that imipramine is an inhibitor of the microsomal drug-metabolizing enzymes. It inhibits especially the enzyme responsible for the metabolism of pentobarbital. Imipramine potentiates the inhibitory actions of SKF 525 A and phenylisopropylhydrazine on the metabolism of hexobarbital and meprobamate. An inhibition of in vivo metabolism of pentobarbital and meprobamate was also demonstrated. In the imipramine treated rats, carisoprodol, meprobamate hexobarbital and pentobarbital concentrations in the brain, determined on recovery from the loss of the righting reflex, are a little lower than in controls. The results indicate that imipramine would have to be classified both as a prolonging agent and a potentiating agent by virtue of its inhibitory action on drug metabolism and of its powers of increasing the sensitivity of the central nervous system towards the drugs.

Adaptation to oxidative stress: effects of vinclozolin and iprodione on the HepG2 cell line

It is well known that the dicarboximide fungicides, vinclozolin and iprodione, induce lipid peroxidation by means of oxygen activation in fungi, but their action on mammalian cells is not yet clear. We therefore investigated the effect of 1- and 24-h treatments with vinclozolin at concentrations of 25, 50, 100 microg/ml and iprodione at concentration of 62.5, 125, 250 microg/ml on malonaldehyde and free radical production and on reduced glutathione levels in the human HepG2 hepatoma cell line. The concentrations were chosen on the basis of neutral red cytotoxicity assays. One-hour treatment with the different concentrations of either vinclozolin or iprodione increased both malonaldehyde and free radical content, and decreased reduced glutathione levels, whereas 24-h treatment decreased malonaldehyde content and free radical production, and increased reduced glutathione concentration. These results suggest that the mammalian cells respond to the initial oxidative damage caused by the two dicarboximide fungicides by means of a characteristic adaptative phenomenon within 24 h. This hypothesis is supported by the antagonized effects caused by treatment with the two dicarboximide fungicides and buthionine sulfoximine 0.5 mM, a specific and irreversible inhibitor of reduced glutathione synthesis. The data confirm that the two dicarboximide fungicides maintain their specific action in mammalian cells, although this action is masked by adaptation.

Increased activity of microsomal strychnine-metabolizing enzyme induced by phenobarbital and other drugs

Abstract This report deals with induction of increased activity of microsomal strych-nine-metabolizing enzyme and of decreased toxicity of strychnine by phenobarbital, phenaglycodol, glutethimide, thiopental, nikethamide, primidone, diphenylhydantoine, urethane, meprobamate and carisoprodol (inducing drugs). The increased activity of microsomal strychnine metabolizing enzyme and the decreased toxicity developed 12 hr after the injection of the inducing drugs, the maximal increase occurred after about 36–48 hr. The development of these two effects was inhibited by ethionine injected shortly before the inducers. In the case of intravenous injection of strychnine in rats pretreated 48 hr before with the inducing drugs, the decreased toxicity was not observed. SKF 525 A injected 30 min before strychnine also overshadowed the decreased toxicity in the pretreated rats. The increased activity of strychnine-metabolizing enzyme accounted for the decreased strychnine toxicity. A decreased picrotoxin toxicity similar to that observed with strychnine was also demonstrated.

Different effects of PCB101, PCB118, PCB138 and PCB153 alone or mixed in MCF-7 breast cancer cells

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that can be a potential health hazard. In the present study we analyzed the potential estrogenic effect in MCF-7 cells of four biologically relevant PCB congeners, alone or in mixtures, present in dairy products, vegetable oil and fish: PCB101, PCB118, PCB138 and PCB153. The mixture of four PCB was tested at seven different concentrations. We investigated the ability of these PCBs, alone or mixed, to induce cell proliferation, and the level of estrogen-regulated protein pS2, in human MCF-7 breast cancer cells. PCB153 (35 microM) stimulated cell proliferation from 48 h up to day 6, PCB118 (40 microM) only at 48 h, but PCB101 (45 microM) and PCB138 (15 microM) applied to the cells for 6 days had no effect. In contrast, the various concentrations of mixtures significantly reduced cell proliferation at different times. No change in pS2 levels was seen after treatment with the PCBs alone or mixed. In exploring the mechanism of these events, we found that PCB153 induced mitogen-activated protein kinase (MAPK) ERK1/2 at 4, 8 and 12 h, while the antiproliferative effect seemed to be related to an apoptotic action beginning at 12 h and ending at 48 h. These findings indicate that these PCBs alone or mixed have no estrogenic effect in MCF-7 cells, although PCB153 induce an ERK1/2-mediated mitogenic effect. On the contrary the mixture of PCBs induces an antiproliferative effect, ascribable to an apoptotic action.

Drinking water quality: An in vitro approach for the assessment of cytotoxic and genotoxic load in water sampled along distribution system

An in vitro approach was performed to assess the quality of drinking water collected at two treatment/distribution networks located near the source (Plant #1) and the mouth of River Po (Plant #2). The water was sampled at different points of each distribution network, before (raw water) and after the chlorine dioxide disinfection, and in two points of the pipeline system to evaluate the influence of the distribution system on the amount and quality of the disinfection by-product. Cytotoxicity and genotoxicity of water extracts were evaluated in human peripheral lymphocytes and Hep-G2 cells by the use of the micronucleus (MN) test and Comet assay. Raw water samples of both plants induced cytotoxic effects, but not the increases of MN frequency in Hep-G2 cells and in human lymphocytes. Increases of DNA damage in human leukocytes was detected by Comet assay for raw water of Plant #2 at concentration > or = 0.25 Leq/mL. The disinfection process generally has reduced the toxicity of water samples, even if potential direct DNA-damaging compounds have been detectable in drinking water samples. The proposal approach, if currently used together with chemical analysis, can contribute to improve the monitoring drinking water.

Benomyl affects the microtubule cytoskeleton and the glutathione level of mammalian primary cultured hepatocytes.

Rat primary hepatocyte cultures have been used to study the effect of Benomyl alone or in combination with Pirimiphos-methyl. The results presented demonstrate that Benomyl alone is responsible for the microtubular disorganization in both a time- and dose-dependent manner, that the effect is reversible after the agent is removed, and that Benomyl is a potent glutathione-depleting agent. Pirimiphos-methyl, alone or combined with Benomyl had no effect on microtubule organization, but reinforced the decrease in glutathione.