Dehydroepiandrosterone effects on the mRNA levels of peroxisome proliferator-activated receptors and their coactivators in human hepatoma HepG2 cells.

To investigate the effect of dehydroepiandrosterone (DHEA) on intracellular mRNA levels of peroxisome proliferator-activated receptors (PPARs) and PPAR-gamma coactivators (PGCs), we conducted a quantitative real-time RT-PCR study using HepG2 cells. Treatment with 100 micromol/l DHEA for 2-20 h caused a time-dependent elevation of mRNA levels in the cells. Upon 20 h, PPAR-alpha, -gamma1, and -gamma2 mRNAs and PGC-1alpha and -1beta mRNAs increased to 157, 161, 155, 656, and 475% of control levels, respectively (p < 0.05 each). Treatment with actinomycin D for 2.5-8 h revealed a significant stabilization effect of DHEA on PPAR-gamma1 and PGC-1alpha mRNAs at both 2.5 and 8 h incubation periods and a mild but significant stabilization effect on PGC-1beta mRNA at the 8 h incubation period suggesting that DHEA can modulate turnover of these mRNA transcripts. Basal mRNA levels of PPAR-alpha and PGC-1alpha were significantly suppressed upon 20 h treatment with cycloheximide, while those of PPAR-gamma1, -gamma2, and PGC-1beta were elevated. Cycloheximide also significantly reduced DHEA-induced accumulation of PPAR-alpha, -gamma1, -gamma2, and PGC-1alpha mRNAs, demonstrating the dependence of the DHEA action on de novo protein synthesis. The findings demonstrate that a supraphysiological concentration of DHEA can substantially influence gene expression of the PPAR signalling machinery at both transcriptional and posttranscriptional levels.

Investigation of sanguinarine and chelerythrine effects on CYP1A1 expression and activity in human hepatoma cells.

Quaternary benzo[c]phenanthridine alkaloids (QBA) sanguinarine and chelerythrine exhibit a wide spectrum of biological activities whence they are used in dental care products. Recent studies indicated that cytochrome P450 CYP1A attenuates sanguinarine toxicity both in vivo [Williams, M.K., Dalvi, S., Dalvi, R.R., 2000. Influence of 3-methylcholanthrene pretreatment on sanguinarine toxicity in mice. Vet. Hum. Toxicol. 42, 196-198] and in vitro [Vrba, J., Kosina, P., Ulrichová, J., Modrianský, M., 2004. Involvement of cytochrome P450 1A in sanguinarine detoxication. Toxicol. Lett. 151, 375-387]. However, CYP1A converts sanguinarine to the products that form DNA adducts [Stiborová, M., Simánek, V., Frei, E., Hobza, P., Ulrichová, J., 2002. DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the 32P-postlabeling technique. Chem. Biol. Interact. 140, 231-242]. In our work we examined the effects of sanguinarine and chelerythrine on CYP1A1 expression and catalytic activity in human hepatoma cells-HepG2. Sanguinarine and chelerythrine did not affect basal and dioxin-inducible expression of CYP1A1 mRNA and protein in HepG2 cells. The enzymatic activity of CYP1A1 was assessed by the fluorescent measurement of 7-ethyxoresorufin-O-deethylase (EROD) activity. We observed a slight decrease of dioxin-induced EROD activity in HepG2 cells by sanguinarine and chelerythrine. This decrease was attributed to the inhibition of CYP1A1 catalytic activity, as revealed by enzyme kinetic studies on recombinant CYP1A1 protein. The IC50 values for the inhibition of CYP1A1 by sanguinarine and chelerythrine were 2.1 and 1.9muM, respectively. In conclusion, albeit the CYP1A modulates QBA cytotoxicity and genotoxicity, the QBA themselves do not affect CYP1A1 expression. The data indicate that studied alkaloids do not have specific cellular target and their biological effects are rather pleiotropic.

In vitro glycosidation potential towards olomoucine-type cyclin-dependent kinase inhibitors in rodent and primate microsomes.

Interspecies differences in glycosidation potential in mammalian tissues represent a factor contributing to ambiguity when endobiotic and/or xenobiotic metabolic pathways are extrapolated from animals to man. Using the TLC/autoradiographic technique, we conducted an in vitro investigation involving mouse, rat, monkey, as well as human liver and kidney microsomes to evaluate their glycoconjugation potential towards (3)H-labeled, purine-derived selective inhibitors of cyclin-dependent kinases such as olomoucine, bohemine, roscovitine, 6-(2-hydroxybenzyl)amino-2-(1-hydroxymethyl-2-methylpropyl)amino-9-isopropylpurine (compound A-4), and 6-(3-hydroxybenzyl)amino-2-[(1(R/S)-hydroxymethyl)propyl]amino-9-isopropylpurine (compound A-5) as aglycones. Principally, this study confirmed the aliphatic hydroxyl group of olomoucine-type inhibitors as a relatively suitable target for glucuronide, glucoside, xyloside, galactoside, and/or N-acetylaminoglucoside conjugation. Of the tissues examined, only the mouse microsomes were able to perform glucosidation and galactosidation reactions with the aglycones. On the other hand, monkey microsomes were superior to the mouse microsomes in a variety of glucuronide conjugates produced with compounds A-4 and A-5.

In vitro biotransformation of 2,6,9-trisubstituted purine-derived cyclin-dependent kinase inhibitor bohemine by mouse liver microsomes.

1. Biotransformation pathways of the cyclin-dependent kinase inhibitor 6-benzylamino-2-(3-hydroxypropylamino)-9-isopropylpurine (bohemine) by mouse liver microsomes in vitro were investigated. 2. Metabolite profiles of [8-(3)H]-labelled bohemine were established by TLC/(3)H-autoradiography and enzymatic and MS analyses were used to elucidate the chemical structures of the metabolites. The structures of the main primary metabolites were confirmed by synthesis of authentic compounds. 3. A schema of the primary NADPH-dependent pathways has been proposed involving N(2)- and N9-dealkylation, N(6)-debenzylation, aromatic hydroxylation, and C2 side chain oxidation of bohemine. Three of the primary metabolites detected, 6-(benzylamino)-2-(3-hydroxypropylamino)purine (M4), 6-amino-2-(3-hydroxypropylamino)-9-isopropylpurine (M5) and 6-(4-hydroxybenzylamino)-2-(3-hydroxypropylamino)-9-isopropylpurine (M6), all retaining their parent primary hydroxyl group, were subsequently shown to be converted, by a liver cytosolic NAD(+)-dependent system, into their corresponding carboxylic acids. M6 was subject to microsomal glycosidations requiring UDP-sugar donors. NADPH-dependent conversion of M6 into M5 by microsomes was also demonstrated. 4. Cytochrome P450 (CYP) enzymes-selective inhibitors were used to identify CYPs involved in bohemine biotransformation. The findings suggested that CYP2a and CYP3a substantially contributed to the NADPH-dependent bohemine transformation in vitro. 5. The findings will facilitate experiments designed to dissect enzymatic systems catalysing clearance of C2,C6,N9-trisubstituted purine compounds from mammalian tissues.

Fatty acid analysis of Stenotrophomonas maltophilia clinical strains showing different susceptibility to antibiotics at 30 and 37 degrees C.

Isolates of Stenotrophomonas maltophilia species display the feature "temperature-dependent susceptibility" (TDS) to antibiotics. Both 30TDS strains (at least 4 times lower value of minimum inhibitory concentration (MIC) of an antibiotic at 30 than at 37 degrees C) and 37TDS strains (at least 4 times lower value of MIC at 37 than at 30 degrees C) were described. Changes in the distribution of saturated and unsaturated fatty acids (FA) at 30 and 37 degrees C were considered as one of possible causes of the TDS phenomenon. Gas chromatography was used to determine the distribution of individual FA in five 37TDS strains of S. maltophilia (Group I); in five strains with MIC values unaffected by the cultivation temperature (Group II) and in six 30TDS (four strains) or 30/37TDS (two strains) isolates (Group III). At identical temperatures, no statistically significant differences in the distribution of major FA (iso-15:0, anteiso-15:0, 16:0 and 16:1) were registered between individual groups. Statistically significant (p < 0.05) differences between groups were found in minor FA only (iso-16:0, iso-17:0 and iso-17:1). Distribution changes of cellular FA at 30 and 37 degrees C can be considered to play only a minor role in the formation of the TDS phenomenon.

[Precision-cut tissue sections--an important system for study of metabolism in vitro]. / Tkánové rezy o definované tloust'ce--významné systémy pro studie metabolismu in vitro.

Tissue slices represent a useful in vitro method for metabolic, pharmacologic and toxicologic studies. To their major advantage belong preserved cell to cell interactions, architecture and complexity of the tissue. They enable to reduce the number of laboratory animals, that are necessary for research purposes. Tissue slices are more similar to in vivo situation than other in vitro models such as microsomes, cell suspensions or cell cultures. Slices can be cultured up to 72 hours or even more. Preparation of the slices is rapid and easy. Slices can be prepared from any organ. There is a possibility of cross-species comparison, co-culturing of slices derived from different organs. Excess of the material can be cryopreserved for later studies.

In vivo metabolism of 2,6,9-trisubstituted purine-derived cyclin-dependent kinase inhibitor bohemine in mice: glucosidation as the principal metabolic route.

Synthetic cyclin-dependent kinase inhibitors have recently been referred to as effective antiproliferative agents. This study was conducted to characterize clearance of a 3H-labeled, trisubstituted purine-type inhibitor, 8-[3H]bohemine [6-benzylamino-2-(3-hydroxypropylamino)-9-isopropylpurine], in mice. Radioactivity profiles were analyzed by liquid scintillation counting and by thin layer chromatography followed by autoradiography. Metabolite structures were elucidated by mass spectrometry, NMR, and enzymatic analyses. Bohemine was rapidly and completely metabolized in vivo and disappeared from circulation during the first 60 min following intravenous administration. The metabolites were partly eliminated by the hepatobiliary tract and partly by renal excretion. The terminal hydroxyl group located at the C2 side chain of bohemine made the compound susceptible to main metabolic attacks, i.e., distinct types of conjugation reactions with glycosyl donors as well as an oxidative reaction. Other pathways were of relatively minor significance. Bohemine O-beta-D-glucoside was the most abundant metabolite to be excreted. The enzymatic mechanism responsible for bohemine glucosidation in vitro required the presence of a UDP-glucoside donor. Additional glycosidation products were observed after inclusion of UDP-glucuronide, UDP-xylose, UDP-galactose, or UDP-N-acetylglucosamine into microsomal incubates. Glycosidations occurred faster in the kidney incubates than in hepatic ones. The second principal bohemine metabolite was a carboxylic acid, 6-benzylamino-2-(2-carboxyethylamino)-9-isopropylpurine. A cytosolic, 4-methylpyrazole-sensitive alcohol dehydrogenase class I was shown to mediate oxidation of the terminal hydroxyl group of bohemine into this acid, which was the only metabolite found in the blood in significant amounts. However, it displayed only weak cyclin-dependent kinase-1-inhibitory activity (IC(50) > 100 microM) when compared with that of bohemine (IC(50) approximately 1 microM).

Cell suspensions, cell cultures, and tissue slices--important metabolic in vitro systems.

In vitro subcellular and cellular systems have important and irreplaceable roles in the metabolic investigations that precede the development of new potential drugs. Of these model systems, tissue slices are probably the nearest to in vivo conditions. From the experimental and complexity points of view, perfused organs lie midway between tissue slices and whole organism. Preparation and working with liver slices is quick and easy, and, excess material can be cryopreserved and stored untill the next experiment. Slices can be prepared from a wide variety of organs and it is possible to co-incubate them. Another important feature is the possibility of interspecies comparison of slices. Different experiments can be run both in the short-term as well as long-term incubations. Each in vitro system has an important place for example, in the development of new medicaments. It is therefore important to compare and supplement experimental results from different in vitro systems when extrapolating to in vivo situations is done.

Tumor necrosis factor alpha in various tissues of insulin-resistant obese Koletsky rats: relations to insulin receptor characteristics.

Tumor necrosis factor alpha (TNFalpha) was found to be significantly increased in skeletal muscles and retroperitoneal fat of obese insulin-resistant Koletsky rats as compared to control Wistar rats. This increase was accompanied by a depression of insulin receptor protein tyrosine kinase (PTK) activity. Neither the insulin-binding capacity nor insulin receptor affinity were related to this TNFalpha increase in these tissues. In the liver, no significant changes of TNFalpha content and only a lowering of insulin-binding capacity were found. It is concluded that an increased TNFalpha content in muscles and fat (but not in the liver) contributes to insulin resistance by lowering insulin receptor protein tyrosine kinase activity, while other insulin receptor characteristics (insulin-binding capacity and affinity of insulin receptors to the hormone) do not seem to be influenced by this factor.

Effect of alpha-tocopherol, pyridoxine and dexpanthenol on the stress in crease of nonesterified fatty acids levels in the brain.

The supposed antistress effect of vitamins-alpha-tocopherol, pyridoxine and dexpanthenol (pantothenic acid precursor)--was followed on the model of nociceptive stress in laboratory rats. The decrease of the stress enhancement of nonesterified fatty acids (NEFA), estimated in the brain cortex, hypothalamus and the brain stem, was taken for the indicator of the antistress effect. Nonesterified fatty acids were determined with the help of gas chromatography following the separation performed by thin layer chromatographic method. Five-day application of alpha-tocopherol acetate (per os, 300 mg.kg-1) led to a decrease of the stress enhancement of arachidonic acid level in the brain stem.

Histamine of mouse lungs after single exposure to nitrogen dioxide.

Pulmonary histamine and the weight of lungs were studied in mice, exposed to a single one-hour effect of high concentrations of edemagenic gas nitrogen dioxide in a metabolic chamber. Nitrogen dioxide concentrations were chosen according to the results of nitrogen dioxide analysis of the mining atmosphere immediately after the mining blasts. The results were estimated by the method of paired comparison with the findings registered in mice, exposed to the effect of air atmosphere under identical experimental conditions. The intervals following immediately the exposure and 5 hours after were chosen for the evaluation, with regard to the dynamics of the early and late stages of hypersensitivity of the first type. i) Immediately after the exposure to the concentrations of 43, 250, 387 and 540 mg.m-3, no significant differences were observed in the amount of pulmonary histamine. In concentrations higher than 43 mg.m-3, the weight of lungs increased (the proportion of pulmonary water and the dry tissue). ii) Five hours after the exposure (nitrogen dioxide concentrations 66, 130, 137 and 270 mg.m-3), pulmonary histamine decreased, at the concentration of 137 mg.m-3 in a significant way, on the other hand, it increased significantly at the concentration of 270 mg.m-3. Both concentrations higher than 130 mg.m-3 manifested an increased weight of lungs (increased proportion of dry tissue and pulmonary water). The obtained data do not allow to establish unambiguously the part of histamine on the pulmonary changes following the effect of nitrogen dioxide. The edemagenic effect of nitrogen dioxide estimated after one-hour influence can be considered as reversible up to five hours after the exposure in concentrations lower than 130 mg.m-3. The metodical part of the study gives detailed description of exposure technique and it brings a survey of methods of histamine determination in blood and tissues.

The labilizing and stabilizing effect of physostigmine on CNS.

The authors observed biphasic changes of dopamine, norepinephrine and serotonin in the rat brain stem after a single application of physostigmine (1 mg/kg of body weight). The results confirm the hypothesis of "labilization and stabilization" of membranes.

On acute effects of some drugs on the higher nervous activity in man. Ethanol (0.5 g/kg) and its interaction with diazepam (5 mg) and meclophenoxate (100 mg). Part LVIII.

1. Acute effects of ethanol (0.5 g/kg of body weight) and its interactions with diazepam (5 mg) and meclophenoxate (100 mg) and placebo on the higher nervous activity were tested on a group of 16 healthy and unfatigued volunteers-university students (8 females, 8 males, aged 21 years). The experimental subjects were given the following combinations of the studied drugs: (a) Ethanol plus meclophenoxate, (b) Ethanol plus placebo, (c) Ethanol plus diazepam, (d) Placebo solution plus placebo. The method of artificial conditioned speech connections was made use of. Three statistically balanced trials were always carried out: before, and one and two hours after the administration of the tested drug combinations during the early and late forenoon hours. The investigation-sets consisted of two optic, two complex tactile, and two acoustic associations. The criteria for the evaluation of the results were the number of repetitions necessary for mastering the given task, then the number of correct responses and the frequencies of responses in the first eight repetitions of the sets when the active knowledge of the persons under investigation was tested. 2. One hour after the application, there appeared a statistically significant impairment in NNR and NCR, both after ethanol alone and ethanol in combinations with diazepam and meclophenoxate. The frequency of responses was impaired only after the combination of ethanol plus diazepam. 3. Two hours after the administration, the combinations of ethanol plus diazepam and ethanol plus meclophenoxate impaired significantly the number of necessary repetitions. NCR impairment was observed only after application of ethanol with diazepam. Ethanol administered separately did not impair the followed up characteristics of learning. 4. The most marked effect appeared during the formation of artificial conditioned speech connections with acoustic stimuli.

Acute effects of ethanol (0.5 mg/kg) and its interaction with diazepam (5 mg) and meclophenoxate (100 mg) on the blood pressure, the body temperature and the heart rate in man.

1. In the course of an experiment in which acute effects of perorally applied ethanol alone (0.5 g/kg of body weight) or in combination with diazepam (5 mg) and meclophenoxate (100 mg) on verbal learning were studied, the values of the blood pressure, the body temperature and the heart rate of experimental subject were simultaneously registered with the help of polyphysiograph Physiomat. 2. The method of artificial conditioned speech connections was adopted. The experimental subjects were 16 healthy and unfatigued volunteers - university students (8 females, 8 males, age 21 years). The investigation-sets consisted of two optic, two complex tactile, and two acoustic associations. Three trials were always carried out: before, and one and two hours after the administration of the studied drugs. 3. The values of the blood pressure, the body temperature and the heart rate were not significantly influenced by the applied substances. Only two hours after application of ethanol in combination with diazepam, a marked decrease of systolic and diastolic blood pressure was registered in comparison with the values observed after application of placebo.

Mechanism of psychotropic effect of toluene.

An acute 30 min exposure to toluene vapours (0.32 mmol/l of the air) provoked in the rat brain a decrease of total nonesterified fatty acids (arachidonic acid -21.7%, oleic acid -18.3%, stearic acid -7.9% in the brain cortex and arachidonic acid -6.8% in the hypothalamus). The results are in harmony with the hypothesis of the NEFA decreasing effect of the inhibitory substances in the brain.

Perspectives of physostigmine as antidote by toluene intoxication.

The administration of physostigmine (0.3 mg/kg s.c.) compensated toluene intoxication in rats together with NEFA return toward the normal values. This interaction of toluene and physostigmine supports the hypothesis of NEFA functioning as moderators of the cerebral activity.