Relative cytotoxicity of psychotropic drugs in cultured rat hepatocytes

The relative cytotoxic effects of ten psychotropic drugs were assessed in rat hepatocyte monolayer cultures. Clear concentration-related toxicity was seen in the narrow range of 10^–5M to S × 10^–5M. The four cytotoxicity endpoints chosen were: release of the cytosolic enzyme, lactate dehydrogenase, and impairment of biosynthesis and secretion of proteins, bile acids and glycerolipids. LDH leakage and inhibition of protein secretion into the culture medium proved to be the parameters which allowed the best differentiation between the test compounds. The inhibition of glycerolipid secretion was the most sensitive test in relation to concentration and time of exposure. Based on the effects of these endpoints, the following ranking of relative in vitro toxicity, using equimolar drug concentrations, could be established: clomipramine > imipramine = thioridazine > chlorpromazine > amitriptyline = fluperlapine > haloperidol > promazine > clozapine sulpiride. This ranking order of in vitro cytotoxicity correlated well with the potential of the drugs to impair liver function in man. Only clozapine had to be classified as a false negative. There was, however, no correlation between the cytotoxicity and the intracellular accumulation of the test drugs. Furthermore, the comparison of the data obtained with psychotropics with the data from five other amphiphilic cationic drugs was consistent with the widely accepted concept of a direct toxic interaction of the drugs with cytomembranes. This nonspecific toxicity of the membrane-active drugs was further corroborated by a positive correlation between their potential to induce LDH leakage in hepatocytes and their ability to induce hemolysis in red cells. In conclusion, the results obtained in our study strongly suggest that it is possible to assess the relative cytotoxicity of psychotropic drugs in rat hepatocyte cultures. It is proposed that this in vitro system provides a useful tool to evaluate new drugs at an early stage of their development, and to identify the most promising candidates within a class of structurally related compounds. In addition, it allows information to be obtained on possible mechanisms of cytotoxicity.Abbreviations AIB aminoisobutyric acid - AMT amitriptyline - BSA bovine serum albumin - CLP clomipramine - CLZ clozapine - CPZ chlorpromazine - FLU fluperlapine - HAL haloperidol - HC₅₀ dose causing 50% hemolysis - IMP imipramine - LDH lactate dehydrogenase - PZ promazine - SUL sulpiride - TCA trichloroacetic acid - TRZ thioridazine     

Oltipraz-induced phase 2 enzyme response conserved in cells lacking mitochondrial DNA

Oltipraz, a member of a class of 1,2-dithiolethiones, is a potent phase 2 enzyme inducing agent used as a cancer chemopreventive. In this study, we investigated regulation of the phase 2 enzyme response and protection against endogenous oxidative stress in lymphoblastic leukemic parental CEM cells and cells lacking mitochondrial DNA (mtDNA) (rho0) by oltipraz. Glutathione (GSH) levels (total and mitochondrial) and glutathione S-transferase (GST) activity were significantly increased after pretreatment with oltipraz in both parental (rho+) and rho0 cells, and both cell lines were resistant to mitochondrial oxidation, loss of mitochondrial membrane potential, and cell death in response to the GSH depleting agent diethylmaleate. These results show that the phase 2 enzyme response, by enhancing GSH-dependent systems involved in xenobiotic metabolism, blocks endogenous oxidative stress and cell death, and that this response is intact in cells lacking mtDNA.     

Nimesulide-induced hepatic mitochondrial injury in heterozygous Sod2(+/-) mice

Nimesulide, a preferential COX-2 inhibitor, has been associated with rare idiosyncratic hepatotoxicity. The underlying mechanisms of liver injury are unknown, but experimental evidence has identified oxidative stress as a potential hazard and mitochondria as a target. The aim of this study was to explore whether genetic mitochondrial abnormalities, resulting in impaired mitochondrial function and mildly increased oxidative stress, might sensitize mice to the hepatic adverse effects of nimesulide. We used heterozygous superoxide dismutase 2 (Sod2(+/-)) mice as a model, as these mice develop clinically silent mitochondrial stress but otherwise appear normal. Nimesulide was administered for 4 weeks (10 mg/kg, ip, bid), at a dose equivalent to human therapeutic dosage. We found that the drug potentiated hepatic mitochondrial oxidative injury (decreased aconitase activity, increased protein carbonyls) in Sod2(+/-), but not wild-type, mice. Furthermore, the nimesulide-treated mutant mice exhibited increased hepatic cytosolic levels of cytochrome c and caspase-3 activity, as well as increased numbers of apoptotic hepatocytes. Finally, nimesulide in vitro caused a concentration-dependent net increase in superoxide anion in mitochondria from Sod2(+/-), but not Sod2(+/+) mice. In conclusion, repeated administration of nimesulide can superimpose an oxidant stress, potentiate mitochondrial damage, and activate proapoptotic factors in mice with genetically compromised mitochondrial function.     

ANTIPYRETIC ACTION OF DEXAMETHASONE ON EGTAZIC ACIDINDUCED FEVER IN RABBITS

本文用脑室灌注和Ｆｕｒａ２测定细胞内游离钙技术观察了地塞米松（ｄｅｘａｍｅｔｈａｓｏｎｅ,ＤＥＸ）对家兔乙二醇双（２氨基乙醚）四乙酸性发热效应和下丘脑细胞内游离钙浓度（［Ｃａ２＋］ｉ）的影响,借此深入探讨地塞米松解热作用的中枢机制。结果发现：脑室灌注乙二醇双（２氨基乙醚）四乙酸（０６ｎｍｏｌ）引起家兔结肠温度明显升高,静脉注射地塞米松（５ｍｇ／ｋｇ）显著抑制家兔乙二醇双（２氨基乙醚）四乙酸性发热,地塞米松（６０～１２０μｍｏｌ／Ｌ）并不影响下丘脑细胞内［Ｃａ２＋］ｉ,而事先脑室灌注抑制基因转录的放线菌素Ｄ（３ｎｍｏｌ）则完全取消了地塞米松对乙二醇双（２氨基乙醚）四乙酸性发热的解热作用。这些结果提示：地塞米松显著抑制家兔乙二醇双（２氨基乙醚）四乙酸性发热,其机制与地塞米松激活脑内某些基因的表达有关,而与下丘脑神经细胞跨膜钙离子流无关。     

Mitochondrial permeability transition as a source of superoxide anion induced by the nitroaromatic drug nimesulide in vitro

Nimesulide, a widely used nonsteroidal anti-inflammatory drug containing a nitroaromatic moiety, has been associated with rare but serious hepatic adverse effects. The mechanisms underlying this idiosyncratic hepatotoxicity are unknown; however, both mitochondrial injury and oxidative stress have been implicated in contributing to liver injury in susceptible patients. The aim of this study was, first, to explore whether membrane permeability transition (MPT) could contribute to nimesulide's mitochondrial toxicity and, second, whether metabolism-derived reactive oxygen species (ROS) were responsible for MPT. We found that isolated mouse liver mitochondria readily underwent Ca2+-dependent, cyclosporin A-sensitive MPT upon exposure to nimesulide (at >or=3 microM). Net increases in mitochondrial superoxide anion levels, determined with the fluorescent probe dihydroethidium, were induced by nimesulide only in the presence of Ca2+ and were cyclosporin A-sensitive, indicating that superoxide production was a consequence, rather than the cause, of MPT. In addition, nimesulide caused a rapid dissipation of the inner mitochondrial transmembrane potential (at >or=3 microM), followed by a concentration-dependent decrease in ATP biosynthesis. Because nimesulide, unlike the related nitroaromatic drug nilutamide, did not produce any detectable ROS during incubation with mouse hepatic microsomes, we conclude that mitochondrial uncoupling causes MPT and that ROS production is a secondary effect.     

Differential toxicity of cocaine and its isomers, (+)-cocaine and (-)-psi-cocaine, is associated with stereoselective hydrolysis by hepatic carboxylesterases in cultured rat hepatocytes.

Cocaine induces acute lethal cell injury in rat hepatocytes following N-oxidative metabolic activation by cytochrome P450-dependent and flavin-dependent monooxygenases. Beside this oxidative bioactivation pathway, hepatic carboxylesterases may cleave the carboxymethylester or the benzoylester linkage which leads to molecules found to be non-toxic in vivo. To elucidate the structural requirements of the cocaine molecule for its bioactivation and inactivation, the cytotoxic potential of the natural (-)-cocaine relative to two isomeric forms, (+)-cocaine* (the unnatural enantiomer) and (-)-psi-cocaine (the C2 epimer of the unnatural cocaine) were investigated. Primary short-term cultures of rat hepatocytes obtained from phenobarbital (PB)-pretreated rats were exposed to the drugs for up to 24 h. (-)-Cocaine produced marked time- and concentration-dependent release of lactate dehydrogenase (LDH) into the extracellular medium, whereas the other forms were not cytotoxic (0-1 mM). Furthermore, depletion of cellular glutathione (GSH) with diethylmaleate enhanced LDH release in (-)-cocaine-treated cells and caused marginal cytotoxicity in hepatocytes exposed to the other isomers. To investigate the mechanisms that could be responsible for these isomer-specific effects, the time-dependent metabolic degradation was determined both in cultured hepatocytes and in hepatic microsomes in the presence or absence of the serine carboxylesterase inhibitors, phenylmethylsulfonylfluoride (PMSF) or NaF. All three cocaine analogs were enzymatically degraded, but the rates of ester cleavage greatly varied among the stereoisomers. (-)-Cocaine was primarily N-oxidized via SKF-525A-sensitive pathways, whereas (+)-cocaine was predominantly hydrolyzed by PMSF-sensitive carboxylesterases. In contrast, (-)-psi-cocaine, which is very stable in the absence of cells at 37 degrees C and pH 7.4, was subject to extremely fast enzymatic ester cleavage. In conclusion, these results indicate that the isomer-specific differential cytotoxicity of (-)-cocaine, (+)-cocaine and (-)-psi-cocaine in hepatocytes may be related to stereoselective differences in the rates of hydrolytic inactivation by hepatic carboxylesterases and that the N-oxidative pathway, resulting in hepatocyte injury, may thus be relevant only for (-)-cocaine.