The effects of cyclopentane and cyclopentene analogues of GABA at recombinant GABA(C) receptors.

The pharmacological effects of the enantiomers of cis-3-aminocyclopentanecarboxylic acids ((+)- and (−)-CACP), the enantiomers of trans-3-aminocyclopentanecarboxylic acids ((+)- and (−)-TACP), and the enantiomers of 4-aminocyclopent-1-ene-1-carboxylic acids ((+)- and (−)-4-ACPCA) were studied on human homomeric ρ₁ and ρ₂ GABA_C receptors expressed in Xenopus oocytes using two-electrode voltage clamp methods. These compounds are conformationally restricted analogues of γ-aminobutyric acid (GABA) held in a five-membered ring. (+)-TACP (EC₅₀ (ρ₁)=2.7±0.2 μM; EC₅₀ (ρ₂)=1.45±0.22 μM), (+)-CACP (EC₅₀ (ρ₁)=26.1±1.1 μM; EC₅₀ (ρ₂)=20.1±2.1 μM) and (−)-CACP (EC₅₀ (ρ₁)=78.5±3.5 μM; EC₅₀ (ρ₂)=63.8±23.3 μM) were moderately potent partial agonists at ρ₁ and ρ₂ GABA_C receptors, while (−)-TACP (100 μM inhibited 56% and 62% of the current produced by 1 μM GABA at ρ₁ and ρ₂ receptors, respectively) was a weak partial agonist with low intrinsic activity at these receptors. In contrast, (+)-4-ACPCA (K_i (ρ₁)=6.0±0.1 μM; K_i (ρ₂)=4.7±0.3 μM) did not activate GABA_C ρ₁ and ρ₂ receptors but potently inhibited the action of GABA at these receptors, while (−)-4-ACPCA had little effect as either an agonist or an antagonist. The affinity order at both GABA_C ρ₁ and ρ₂ receptors was (+)-TACP>(+)-4-ACPCA(+)-CACP>(−)-CACP(−)-TACP(−)-4-ACPCA. This study shows that the cyclopentane and cyclopentene analogues of GABA affect GABA_C receptors in a unique manner, defining a preferred stereochemical orientation of the amine and carboxylic acid groups when binding to GABA_C receptors. This is exemplified by the partial agonist, (+)-TACP, and the antagonist, (+)-4-ACPCA.     

Neuroprotective and cardioprotective actions of an association of pyruvate, vitamin E and fatty acids

Oxidative stress may involve overproduction of hydrogen peroxide which can generate highly cytotoxic hydroxyl radicals in the presence of ferrous ions. This work demonstrates that TCAT (Tricomponent Antioxidant Therapy), an association of pyruvate, vitamin E and fatty acids, provides neuronal and cardiac protection in oxidative stress, ex vivo. Mouse P19 neuron cultures were exposed for 30 min to low millimolar H2O2 concentrations in the absence or presence of Fe2+. Concentrations 1X (10 mmol/L pyruvate, 0.1 U/mL vitamin E and 0.1% fatty acids) and 3X of TCAT, respectively, prevented neuronal death caused by these treatments. Analysis of the contribution of TCAT components to neuroprotection showed that vitamin E and fatty acids enhanced pyruvate action whereas they displayed no neuroprotection by themselves. In contrast, vitamin E and fatty acids were as potent antioxidants as pyruvate in an in vitro cell-free assay, indicating that TCAT protection is modulated by the existence of the cellular membrane barriers. Isolated rat hearts were perfused under electrolysis or subjected to regional ischemia-reperfusion. TCAT 1X prevented the electrolysis-induced decrease in left ventricular pressure, heart rate and coronary flow. At 0.25X concentration, TCAT abolished the incidence of irreversible ventricular fibrillation in ischemia-reperfusion. The results indicate that TCAT could have a broad therapeutic utility in neurological and cardiac injuries associated with oxidative stress. The protective action of TCAT can surpass that of its components, revealing a benefit of the association.     

Aflatoxin deposition and clearance in the eggs of laying hens

Hens fed a diet containing 3310 μg of AFB₁ and 1680 μg of AFB₂ per kg feed for 28 days showed a significant decrease in egg production and egg weights by wk 3 and 4 of feeding, respectively. Transfer of aflatoxins to the eggs occurred rapidly, reaching maximum levels after 4–5 days, and remained relatively constant throughout aflatoxin feeding. The mean values for combined residue levels in eggs were less than 0.5 μg/kg. Levels of AFB₂, AFM₁ and AFM₂ were similar in yolk and albumen while levels of B₁ and B_2a were higher in the yolk. Upon removal of the aflatoxin-containing diet, residues in eggs decreased rapidly. Clearance of aflatoxin residues from the albumen occurred faster than from the yolk. Thus, no residues were detected in the albumen and in the yolk after 5 and 7 days of withdrawal, respectively. No aflatoxin residues could be recovered from whole eggs after feeding the aflatoxin-free diet for 4 days.     

Effects of carbon tetrachloride on embryonic development studied in the post-implantation rat embryo culture system and in chick embryos in ovo

The effects of carbon tetrachloride on embryonic development were investigated in a mammalian and a non-mammalian system. In the former, whole-rat embryos, taken at 9.5 days of gestation, were exposed in vitro to different concentrations of CCl₄ (10, 100, 300, 600 and 1000 μg/ml) in rat serum with or without a rat liver microsomal activating system (S-9 mix). In the latter system, chick embryos in ovo were exposed to different concentrations of CCl₄ vapour (25, 35 and 75 ppm). When studied in the whole-rat embryo culture system without metabolic activation, concentrations of up to 300 μg CCl₄/ml had no effect on the overall development. Concentrations -600 μg CCl₄/ml affected the somite number, growth and morphology of the embryos, which can be interpreted as general toxicity. In the presence of S-9 mix, toxicity occurred at concentrations -300 μg/ml. In ovo exposure to CCl₄ showed that 25 ppm caused a decrease in the number of somites. At 35 ppm, CCl₄ induced further toxicity, as indicated by reduced somite number and growth and increased malformation rates. The results indicate that effects on morphogenic events appeared in both systems at concentration levels that also affected the overall development and that, independently of the choice of species or route of administration, CCl₄ has no potential to induce specific malformation patterns. The presence of a metabolic system in the rat embryo cultures approximately doubled the toxicity of CCl₄.     

Suicidal inactivation of haemoproteins by reductive metabolites of halomethanes: a structure-activity relationship study

Human haemoglobin (Hb), methaemalbumin (MHA) or rat liver microsomal cytochrome P-450 (P-450) were incubated anaerobically at μM concentrations with 1 mM carbon tetrachloride (CCl₄), trichlorobromomethane (CCl₃Br), chloroform (CHCl₃) or methylene chloride (CH₂Cl₂) in presence of 1 mM sodium dithionite as the reducing agent. At the end of a 5-min incubation, haem was measured by various methods i.e. binding spectrum with CO, pyridine-haemochromogen haem assay and porphyrin fluorescence, and compared for the four analogues. Statistically significant losses were observed, with all three haemoprotein systems, for CCl₃Br, CCl₄ and CHCl₃, but not CH₂Cl₂. For Hb, the loss was greater with CCl₃Br (haem assay, 63%; porphyrin fluorescence, 48%; CO binding, 24%) than with CCl₄ (haem assay, 31%) or CHCl₃ (haem assay, 13%). On the other hand, with MHA, CCl₄ gave a dramatic loss (haem assay, 88%; porphyrin fluorescence, 83%; CO binding, 67%), which was greater than that observed with CCl₃Br (haem assay, 49%; porphyrin fluorescence, 38%; CO binding, 25%). No loss was found with CHCl₃. Finally, with microsomes, the inactivation was larger with CCl₄ (CO binding, 58%; haem assay, 50%; porphyrin fluorescence, 33%) than with CCl₃Br (CO binding, 33%; haem assay, 10%) or CHCl₃ (haem assay, 9%; CO binding, 6%). In a separate set of similar experiments, an ion-pairing reverse phase HPLC method showed the formation of substrate-dependent haemderived products during incubation of CCl₃Br with Hb or microsomes, and of CCl₃ with Hb. A correlation between potential for free radical formation (CCl₃Br > CCl₄ > CHCl₃ > CH₂Cl₂) and extent of haem inactivation was observed with all methods for Hb, but not for microsomal P-450 or MHA. The results indicate that these halomethanes may be activated differently by different haemoproteins and suggest that their potential ability to undergo reductive metabolism may not be the only critical factor involved in P-450 haem inactivation by these chemicals.     

Effects of the CCl 4 -cycloheximide interaction on protein synthesis and lipid metabolism in rat liver

A protective effect against triglyceride accumulation in liver was observed in CCl₄-poisoned rats pretreated with cycloheximide. This protection is not related to the action of cycloheximide on protein synthesis and secretion of triglyceride from liver into plasma in CCl₄-treated rats. The metabolism of CCl₄ is not affected by cycloheximide. Reduced levels of plasma free fatty acids in cycloheximide treated rats could explain the phenomenon, in spite of the behaviour of protein synthesis and triglyceride secretion.     

5-HT3 receptors augment neuronal, cholinergic contractions in guinea pig trachea European Journal of Pharmacology, 234 (1993) 109–112

Serotonin (5-HT) and the selective 5-HT₃ receptor agonist, 2-methyl-5-hydroxytryptamine enhanced electrical field stimulated contractions of the isolated guinea pig trachea. 5-HT (EC₅₀ = 3.5 μM) was twice as potent as 2-methyl-5-hydroxytryptamine (EC₅₀ = 7.4 μM). The effects of 5-HT and 2-methyl-5-hydroxytryptamine were antagonized by the selective 5-HT₃ receptor antagonist, zacopride (apparent pA₂ = 7.60 against 2-methyl-5-hydroxytryptamine). 2-Methyl-5-hydroxytryptamine (10 μM) had no effect on contractile responses to exogenous acetylcholine. Furthermore, the increase in electrical field stimulated contraction by 2-methyl-5-hydroxytryptamine was unchanged by hexamethonium (100 μM) but contractions were blocked by atropine (1 μM). These results suggest that excitatory 5-HT₃ receptors exist on postganglionic cholinergic nerves in the isolated guinea pig trachea.     

Effects of culture duration on hydrogen peroxide-induced hepatocyte toxicity

The effects of culture duration on primary cultured mouse hepatocyte antioxidant levels (superoxide dismutase, catalase, glutathione peroxidase, vitamin E, and glutathione) and susceptibility to glucose oxidase (GO)- and hydrogen peroxide (H2O2)-induced cell killing and lipid peroxidation were examined. Membrane fatty acid composition was also evaluated. Adult male B6C3F1/CrlBR mouse hepatocytes were isolated by collagenase perfusion of the liver and cultured on 60-mm plastic dishes in Leibovitz's L-15 medium supplemented with glucose (1 mg/ml), dexamethasone (1 microM), fetal bovine serum (10%, v/v), and gentamicin sulfate (50 micrograms/ml) for 0 hr (freshly isolated cells) to 96 hr. Hepatocyte toxicity (determined by lactate dehydrogenase release and lipid peroxidation) after a 2-hr exposure to GO (0.8-80 micrograms/ml) or H2O2 (1-5 mM) decreased with increased time in culture. This decreased hepatocyte sensitivity to GO and H2O2 toxicity was not related to antioxidant enzyme activity since superoxide dismutase, catalase, and glutathione peroxidase declined during the 96-hr culture period. In contrast, glutathione and vitamin E levels in the cultured hepatocytes rose to 274.9 +/- 8.3% and 220.6 +/- 18.6% of the levels in freshly isolated cells (129.6 +/- 11.5 nmol and 0.10 +/- 0.01 nmol per 10(6) hepatocytes, respectively). The percentage of polyunsaturated fatty acids in hepatocyte phospholipids and triglycerides decreased with culture duration while the percentage of oleic acid increased in esterified and free fatty acid pools after 2 hr in culture. Total fatty acids were not affected by time in culture. These results suggest that the decreased hepatocyte susceptibility to the toxic effects of hydrogen peroxide may have been due to elevations in cellular GSH and vitamin E levels and decreases in membrane polyunsaturated fatty acids. The data also indicate that hepatocytes in primary culture undergo changes in antioxidant levels and fatty acid composition that may affect free radical toxicity at different times in culture.     

Acetylcholine receptor agonists stimulate [H]taurine release from rat sympathetic ganglia

The endogenous taurine content, and the uptake and release of [³H]taurine were examined using the rat superior cervical ganglion. Taurine was found to be one of the most abundant amino acids in the superior cervical ganglion, and the superior cervical ganglion took up [³H]taurine from the incubation medium. Carbachol stimulated the release of [³H]taurine in a concentration-dependent manner with an EC₅₀ of 26 μM and maximal stimulation at 100 μM. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium stimulated release with the same potency but with greater efficacy than carbachol. The nicotinic receptor antagonist hexamethonium (1 mM) inhibited carbachol-stimulated release by 74% (±)-Muscarine stimulated release with an EC₅₀ of 8 μM but with a maximal effect of only 32% of that produced by 100 μM carbachol. Oxotremorine another muscarinic receptor agonist, was ineffective, even at 1 mM. The muscarinic receptor antagonist atropine inhibited carbachol-stimulated release by 30% at 10μM. These results show that ³H taurine release from rat superior cervical ganglion can be stimulated by cholinergic receptor agonists. Release is mediated predominantly by a nicotinic receptor and partially by a muscarinic receptor.     

Interspecies differences in acetaminophen sensitivity of human, rat, and mouse primary hepatocytes

Most of the experiments studying acetaminophen (APAP) induced hepatotoxicity were performed using moue as model specie, right because its high sensitivity. While the toxic responses can be called forth easily in mice, the human relevancy of these results is questionable. In this study human, rat, and mouse primary hepatocytes were treated with increasing concentrations of APAP, and cell viability was measured by MTT cytotoxicity assay. Pronounced interspecies differences were obtained in cell viability following 24 h of APAP treatment starting at 24 h after seeding (EC₅₀: 3.8 mM, 7.6 mM, and 28.2 mM, in mouse, rat, and human hepatocyte culture, respectively). The longer time of culturing highly increased the resistance of hepatocytes of all species investigated. In rat hepatocyte culture EC₅₀ values were 6.0 mM, 12.5 mM, and 18.8 mM, when starting APAP treatment after 24, 48, and 72 h of seeding. Although N-acetylbenzoquinoneimine, a minor metabolite of APAP, which is mainly formed by CYP2E1 at high APAP concentration in every species studied, is thought to initiate the toxic processes, no correlation was found between CYP2E1 activities and hepatocyte sensitivity of different species. We conclude that the toxicity induced by APAP overdose highly depends on the animal model applied.     

Protective effect of caffeic acid phenethyl ester against carbon tetrachloride-induced hepatotoxicity in mice

This study was designed to investigate the protective effects of the phenethyl ester of caffeic acid (CAPE) against carbon tetrachoride (CCl₄)-induced hepatotoxicities in mice. Pretreatment with CAPE prior to administration of CCl₄ significantly prevented the increases in serum alanine, aspartate aminotransferase and alkaline phosphatase activities, hepatic lipid peroxidation formation, and depletion of glutathione content. In addition, CAPE prevented CCl₄-induced apoptosis and necrosis, as indicated by liver histopathology and DNA laddering studies. To determine whether the Fas/Fas ligand (FasL) pathway is involved in CCl₄-induced acute liver injury, Fas and FasL proteins and caspase-3 and -8 activities were tested by western blotting and ELISA. CAPE markedly decreased CCl₄-induced Fas/FasL protein expression levels and, in turn, attenuated CCl₄-induced caspase-3 and -8 activities in mouse liver. Moreover, the effect of CAPE on CYP2E1, the major isozyme involved in CCl₄ bioactivation, was investigated. Treatment with CAPE significantly decreased the CYP2E1-dependent hydroxylation of aniline. In addition, CAPE attenuated the CCl₄-mediated depletion of antioxidant enzyme (catalase, superoxide dismutase and glutathione-S-transferase) activities. These findings suggest that the protective effects of CAPE against CCl₄-induced acute liver injury may involve its ability to block CYP2El-mediated CCl₄ bioactivation and to protect against Fas/FasL-mediated apoptosis.     

5-Fluorotryptamine is a partial agonist at 5-HT3 receptors, and reveals that size and electronegativity at the 5 position of tryptamine are critical for efficient receptor function

Antagonists, but not agonists, of the 5-HT₃ receptor are useful therapeutic agents, and it is possible that partial agonists may also be potentially useful in the clinic. Here we show that 5-fluorotryptamine (5-FT) is a partial agonist at both 5-HT_3A and 5-HT_3AB receptors with an R_max (I_max / I_max5-HT) of 0.64 and 0.45 respectively. It is about 10 fold less potent than 5-HT: EC₅₀ = 16 and 27 μM, and K_i for displacement of [³H]granisetron binding = 0.8 and 1.8 μM for 5-HT_3A and 5-HT_3AB receptors respectively. We have also explored the potencies and efficacies of tryptamine and a range of 5-substituted tryptamine derivatives. At 5-HT_3A receptors tryptamine is a weak (R_max = 0.15), low affinity (EC₅₀ = 113 μM; K_i = 4.8 μM) partial agonist, while 5-chlorotryptamine has a similar affinity to 5-FT (EC_50 = 8.1 μM; K_i = 2.7 μM) but is a very weak partial agonist (R_max = 0. 0037). These, and data from 5-methyltryptamine and 5-methoxytryptamine, reveal the importance of size and electronegativity at this location for efficient channel opening.     

Fucoidan partly prevents CCl4-induced liver fibrosis

Fucoidan, a sulfated polysaccharide extracted from brown algae, has a wide range of biological activities, including anti-inflammatory, anti-viral, and anti-tumor activities. In the present study, we investigated the effects of fucoidan on CCl₄-induced liver fibrosis. Administration of fucoidan reduced CCl₄-induced acute and chronic liver failure. Hepatic fibrosis induced by CCl₄ was also attenuated by injection of fucoidan. Damage to hepatocytes and activation of hepatic stellate cells are key events in liver fibrosis, and, interestingly, treatment of hepatocytes with fucoidan prevented CCl₄-induced cell death and inhibited the proliferation hepatic stellate cells. These results indicate that fucoidan might be a promising anti-fibrotic agent possessing dual functions, namely, protection of hepatocytes and inhibition of hepatic stellate cell proliferation.     

Toxicity, metabolism and absorption of selenite by isolated rat hepatocytes

The uptake of various levels of selenite by isolated rat hepatocytes was investigated. The LD₅₀ value of selenite was about 500 μM. The activity of lactic dehydrogenase in the medium was correlated with cell viability as determined by trypan blue exclusion. After incubation of selenite with hepatocytes, protein-bound Se was the predominant form (80–90% of the cellular Se) present. Subcellular fractionation indicated that most of the radioactivity was present in the cytosol when hepatocytes were incubated with ⁷⁵Se-selenite. The uptake of ⁷⁵Se by isolated rat hepatocytes was linear with selenite concentration up to the highest amount tested, 200 μM. Sulfite inhibited the uptake of selenite by hepatocytes.     

Effect of carbon tetrachloride treatment on ethanol metabolism

Administration of a single dose (2.5 ml/kg body weight, p.o.) of carbon tetrachloride to rats was found to cause a marked decrease in activity of the hepatic microsomal ethanol oxidizing system (MEOS). As early as 6 hr after CCl₄ administration 50 per cent decrease of MEOS activity was observed; this decrease amounted to 58 and 63 per cent at 10 and 20 hr respectively. With identical CCl₄ treatment, there was no change in hepatic alcohol dehydrogenase activity. At 20 hr, when the reduction of MEOS was greatest, there was no significant effect of the CCl₄ on the rate of ethanol uptake by liver slices or on the rate of ethanol metabolism in vivo as measured in the whole body or as estimated from the rate of decrease of blood ethanol concentration. It is, therefore, suggested that MEOS does not play a significant role in ethanol metabolism in vivo in the rat.     

Protection of extract from leaves of Ardisia compressa against benomyl-induced cytotoxicity and genotoxicity in cultured rat hepatocytes

The potential of the Ardisia compressa extract (EA) was examined regarding its capacity to reduce the cytotoxic effect of benomyl on rat hepatocytes. The protective effect was evaluated by Janus Green dye exclusion method. An approximate 50% cytotoxic effect of benomyl on hepatocytes was observed at 35 μg/ml after 2 hr of incubation. (−)Epigallocatechin 3-gallato (EGCG) and EA decreased the viability of hepatocytes at concentrations above 3 μg/ml and 2.52 μg, equivalent to (+)catechin/ml, respectively. A protective effect against benomyl was observed when hepatocytes were previously exposed to EGCG (3 μg/ml) or EA (2.52 μg, equivalent to (+)catechin/ml) followed by incubation with benomyl (35 μg/ml) alone. When EGCG or EA were in contact with cells, either simultaneously or after pretreatment with benomyl, did not protect hepatocytes. EGCG (1.3×10⁻² μg/ml) or EA (9.8×10⁻² μg, equivalent to (+)catechin/ml) inhibited 57% and 34%, respectively, the unscheduled DNA synthesis (UDS) induced by benomyl at a concentration of 23×10⁻² μ , when both were incubated with hepatocytes prior to benomyl. The simultaneous incubation of benomyl with EGCG or EA did not protect the cell against the genotoxic effect of benomyl. These results indicate that the dried leaves extract of Ardisia compressa protect rat hepatocytes from benomyl-induced cytotoxicity and genotoxicity.     

Antioxidant activity of the oyster mushroom, Pleurotus ostreatus, on CCl4-induced liver injury in rats

This study was undertaken to investigate the putative antioxidant activity of the oyster mushroom Pleurotus ostreatus on CCl₄-induced liver damage in male Wistar rats. Intraperitoneal administration of CCl₄ (2 ml/kg) to rats for 4 days resulted in significantly elevated (p < 0.05) serum levels of glutamic oxaloacetic transaminase (SGOT), glutamic pyruvate transaminase (SGPT) and alkaline phosphatase (SALP) compared to controls. In the liver, significantly elevated levels (p < 0.05) of malondialdehyde (MDA) and lowered levels (p < 0.05) of reduced glutathione (GSH) were observed following CCl₄ administration. Quantitative and qualitative analysis of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (Gpx) revealed lower activities of these antioxidant enzymes in the liver of CCl₄-administered rats. An analysis of the isozyme pattern of these enzymes revealed variations in relative concentration presumably due to hepatotoxicity. When rats with CCl₄-induced hepatotoxicity were treated with the extract of P. ostreatus, the serum SGOT, SGPT and SALP levels reverted to near normal, while the hepatic concentration of GSH, CAT, SOD and Gpx were significantly increased (p < 0.05) and that of MDA significantly (p < 0.05) lowered, when compared to CCl₄-exposed untreated rats. Histopathological studies confirmed the hepatoprotective effect conferred by the extract of P. ostreatus. These results suggest that an extract of P. ostreatus is able to significantly alleviate the hepatotoxicity induced by CCl₄ in the rat.     

Glibenclamde blocks the relaxant action of pinacidil and cromakalim in airway smooth muscle

Concentration-relaxation curves for pinacidil and cromakalim were obtained in isolated guinea-pig tracheas contracted by histamine. Pinacidil produced complete relaxation with an EC₅₀ value of 2.8 μM. The antidiabetic concentration-relaxation curve for pinacidil without changing the maximal relaxant response. Cromakalim produced 85% relaxation with an EC₅₀ value of 1.1 μM. Glibenclamide (0.1 μM) displaced the concentration-relaxation curve to the right and at higher concentrations (1–10 μM) caused nearly complete suppression of the maximal relaxant response to cromakalim. Glibenclamide not only prevented the effects of pinacidil and cromakalim but also produced a concentration-dependent and complete reversal of submaximal relaxations produced by these drugs. Glibenclamide was a selective antagonist of the relaxation of airway smooth muscle induced by pinacidil and cromakalim. Concentration-relaxation curves for theophylline, terbutaline and verapamil were unaffected by glibenclamide.