Toxicity to isolated hepatocytes caused by the intracellular calcium indicator, Quin 2.

To determine whether incubation for several hours with intracellular Ca++ indicators caused toxicity to freshly isolated hepatocytes from rats, cells were incubated under 95% O2-5% CO2 in medium containing 2 mM Ca++ and the acetoxymethyl (AM) esters of Quin 2, Indo 1, Fluo 3, 5,5'-Dimethyl BAPTA, 4,4'-Difluoro BAPTA or Fura 2 for up to 5 hr. Quin 2-AM and Indo 1-AM (2.5 microM) induced lipid peroxidation in the cells after 1 or 3 hr of treatment, respectively. Additional experiments with Quin 2-AM (25 microM) revealed that it also caused lactate dehydrogenase leakage, cell blebbing and vitamin E loss in cells, but did not affect reduced glutathione or intracellular Ca++ content. The ability of Quin 2-AM to cause toxicity was dependent on the amount of Quin 2 which was present in the cell. Ca++ appeared to be involved in the mechanism of Quin 2-AM toxicity, for modulation of the extracellular Ca++ concentration partially inhibited lipid peroxidation, vitamin E loss, cell blebbing and lactate dehydrogenase leakage.     

Age-associated enhancement of diquat-induced lipid peroxidation and cytotoxicity in isolated rat hepatocytes.

The effect of age on the toxicity of diquat, a redox cycling compound, was investigated in hepatocytes isolated from mature (6 months) and old (24-29 months) male Fischer 344 rats. Hepatocytes of old rats were more sensitive than those of mature rats to diquat-induced cytotoxicity (lactate dehydrogenase release into the medium). Cell death was preceded by glutathione disappearance, and rates of glutathione depletion were similar in mature and old hepatocytes. In contrast, diquat-induced formation of thiobarbituric acid-reactive substances was much greater in the hepatocytes from old rats, suggesting that increased lipid peroxidation caused the enhanced cytotoxicity. Further experiments revealed that: 1) hepatocytes of mature and old rats were equally sensitive to iron-induced lipid peroxidation; 2) diquat-stimulated production of superoxide anion radical in liver microsomes did not increase with age, but decreased 43%; 3) superoxide dismutase activity was similar in hepatocytes of mature and old rats; 4) inhibition of catalase activity (which diminishes with age in male rats) did not increase diquat toxicity; and 5) malondialdehyde disappearance in intact hepatocytes decreased (33%) with age, but the toxicological significance of the decline in metabolism was uncertain. Thus, the results demonstrated that diquat-induced lipid peroxidation and cytotoxicity increase with age in male rat hepatocytes, but the enhanced sensitivity to diquat poisoning remains unexplained.     

Membrane structural changes support the involvement of mitochondria in the bile salt-induced apoptosis of rat hepatocytes

The accumulation of toxic bile salts within the hepatocyte plays a key role in organ injury during liver disease. Deoxycholate (DC) and glycochenodeoxycholate (GCDC) induce apoptosis in vitro and in vivo, perhaps through direct perturbation of mitochondrial membrane structure and function. In contrast, ursodeoxycholate (UDC) and its taurine-conjugated form (TUDC) appear to be protective. We show here that hydrophobic bile salts induced apoptosis in cultured rat hepatocytes, without modulating the expression of pro-apoptotic Bax protein, and caused cytochrome c release in isolated mitochondria. Co-incubation with UDC and TUDC prevented cell death and efflux of mitochondrial factors. Using spin-labelling techniques and EPR spectroscopy analysis of isolated rat liver mitochondria, we found significant structural changes at the membrane-water surface in mitochondria exposed to hydrophobic bile salts, including modified lipid polarity and fluidity, altered protein order and increased oxidative injury. UDC, TUDC and cyclosporin A almost completely abrogated DC- and GCDC-induced membrane perturbations. We conclude that the toxicity of hydrophobic bile salts to hepatocytes is mediated by cytochrome c release, through a mechanism associated with marked direct effects on mitochondrial membrane lipid polarity and fluidity, protein order and redox status, without modulation of pro-apoptotic Bax expression. UDC and TUDC can directly suppress disruption of mitochondrial membrane structure, which may represent an important mechanism of hepatoprotection by these bile salts.     

Mechanism of hepatotoxicity in periportal regions of the liver lobule due to allyl alcohol: studies on thiols and energy status

The possible involvement of thiols and adenine nucleotides in the selective toxicity to periportal regions by allyl alcohol was evaluated in isolated perfused rat livers. Infusion of allyl alcohol (350 microM) for 20 min depleted hepatic glutathione content by 95% in both regions of the liver lobule yet damage was undetectable as indexed by release of lactate dehydrogenase or uptake of trypan blue. Perfusion for an additional 40 min in the absence of allyl alcohol resulted in lactate dehydrogenase release (2400 U/l) and uptake of trypan blue by 75% of hepatocytes in periportal regions of the liver lobule; however, dye was not taken up by cells in pericentral areas. Because thiol content was depleted in the undamaged pericentral area, it was concluded that thiol depletion alone cannot explain local toxicity to periportal regions by allyl alcohol. Perfusion with dithioerythritol (1.5 mM) prevented damage due to allyl alcohol totally. In contrast, addition of dithioerythritol 20 min after allyl alcohol did not prevent allyl alcohol-induced damage to periportal regions indicating that irreversible changes occur during the first 20 min which ultimately lead to damage. Fasting or pretreatment of rats with diethylmaleate (0.7 g/kg; 1 hr) to deplete glutathione decreased the T1/2 required for release of lactate dehydrogenase from 45 to 35 and 22 min, respectively. When methionine was infused into livers from diethylmaleate-treated rats, the T1/2 for release of lactate dehydrogenase by allyl alcohol was increased to 45 min. Infusion of allyl alcohol for 60 min also produced a significant decrease in ATP content and in the ATP/ADP ratio in periportal but not pericentral regions of the liver lobule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of calcium and iron in Quin 2 toxicity to isolated hepatocytes

When treated with the cytosolic Ca++ indicator Quin 2-acetoxymethyl ester (Quin 2-AM), isolated hepatocytes exhibited signs of toxicity, such as extensive lipid peroxidation and vitamin E loss and release of lactate dehydrogenase. Lipid peroxidation induced by this agent was blocked completely by cotreatment of the cells with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, EDTA, ruthenium red, carbonyl cyanide m-chlorophenylhydrazone, desferal and trifluoperazine, and was partially inhibited by quinacrine and indomethacin. With the exception of carbonyl cyanide m-chlorophenylhydrazone and quinacrine, these agents also inhibited lactate dehydrogenase leakage. Although the results with ruthenium red suggested that Quin 2-AM may cause toxicity by altering handling of Ca++ by mitochondria, mitochondrial membrane potential was not altered in cells treated with Quin 2-AM until after toxicity occurred. Evidence of a direct, potentiative effect of Quin 2 on iron-induced lipid peroxidation was gained from experiments with liposomes. Treatment of cells with Quin 2-AM did not enhance nitro blue tetrazolium reduction, suggesting that Quin 2 did not stimulate O2- production by the cells. Direct chelation of Ca++ did not appear to be involved in the mechanism of Quin 2 toxicity, for an analog of Quin 2 that is virtually nonhydrolyzable, which greatly limits the binding of Ca++, also caused lipid peroxidation and cell death. These results suggest that Quin 2 causes toxicity by chelating iron or by activating some cellular process(es) that is dependent on the presence of iron or Ca++.     

Effect of quercetin on hypoxic injury in freshly isolated rat proximal tubules

The bioflavonoid quercetin, which has antioxidant properties, protects renal tubular epithelial cells from oxidant-induced injury by inhibiting lipid peroxidation. We examined the effect of quercetin on hypoxia-induced injury in freshly isolated rat renal proximal tubules. Hypoxia induced rapid loss of cellular ATP, followed by functional and structural alterations measured as a decrease in tubular potassium content and sequentially by an increase in lactate dehydrogenase release. Furthermore, hypoxia increased lipid peroxidation, measured as thiobarbituric acid-reactive substances. Quercetin significantly inhibited hypoxia-induced functional and structural tubular injury in addition to lipid peroxidation but did not alter hypoxia-induced ATP depletion. These results demonstrate the potency of the bioflavonoid quercetin in protecting proximal tubules from hypoxic injury, which is independent of tubular energy metabolism and may be related to the inhibition of lipid peroxidation.     

Effects of inorganic iron and myoglobin on in vitro proximal tubular lipid peroxidation and cytotoxicity.

Recent in vivo studies suggest that heme Fe causes proximal tubular lipid peroxidation and cytotoxicity, thereby contributing to the pathogenesis of myoglobinuric (Mgb) acute renal failure. Because hydroxyl radical (.OH) scavengers [dimethylthiourea (DMTU), benzoate, mannitol] can mitigate this injury, it is postulated that .OH is a mediator of Mgb-induced renal damage. The present study has tested these hypotheses using an isolated rat proximal tubular segment (PTS) system. An equal mixture of Fe2+/Fe3+ (4 mM total), when added to PTS, caused marked cytotoxicity [as defined by lactate dehydrogenase (LDH) release] and lipid peroxidation [assessed by malondialdehyde (MDA) increments]. Fe2+ or Fe3+ alone each induced massive MDA elevations, but only Fe2+ caused cytotoxicity. Although both DMTU and benzoate decreased LDH release during the Fe2+/Fe3+ challenge, mannitol and GSH did not, despite equivalent reductions in .OH (gauged by the salicylate trap method). GSH and catalase (but not DMTU, benzoate, or mannitol) decreased MDA concentrations, suggesting the Fe-driven lipid peroxidation was more H2O2 than .OH dependent. Deferoxamine totally blocked Fe-induced LDH release, even under conditions in which it caused an apparent increase in .OH generation. Mgb paradoxically protected against Fe-mediated PTS injury, an effect largely reproduced by albumin. In conclusion, these data suggest that: (a) Fe can cause PTS lipid peroxidation and cytotoxicity by a non-.OH-dependent mechanism; (b) Fe-mediated cytotoxicity and lipid peroxidation are not necessarily linked; and (c) Mgb paradoxically protects PTS against Fe-mediated injury, suggesting that: (i) Mgb Fe may require liberation from its porphyrin ring before exerting toxicity; and (ii) the protein residue may blunt the resulting injury.     

Iron-Mediated Cardiotoxicity Develops Independently of Extracellular Hydroxyl Radicals in Isolated Rat Hearts

Background: Myocardial iron toxicity is often attributed to free radical damage. Present studies examine the role of extracellular hydroxyl radical formation in this process. Methods: In vitro reactions examined the rate of hydroxyl radical formation using salicylate trapping with high-pressure liquid chromatography separation and electrochemical detection of 2,3- and 2,5-dihydroxybenzoic acid. Isolated rat hearts were perfused by the Langendorff technique under the same buffer conditions to determine changes in myocardial contractility, release of tissue lactate dehydrogenase activity, and formation of lipid peroxidation products when iron was added to the perfusate with or without the formation of extracellular radicals. Results: In vitro reactions, performed in Krebs buffer alone or with addition of iron (25 μM), produced levels of hydroxyl radicals that were nondetectable with salicylate trapping. Addition of iron/ascorbate (FeSO₄ = 25 μM, ascorbate = 1 mM), or iron/ascorbate/histidine (FeSO₄ = 25 μM, ascorbate = 1 mM, histidine = 15 mM) produced significant and equivalent accumulation of hydroxyl radicals. Isolated rat hearts were perfused under the same 4 conditions. Control heart contractile function was stable with little release of lactate dehydrogenase activity and low levels of thiobarbituric acid reactive substances (TBARS). There was significant and equal injury to contractile function, release of lactate dehydrogenase activity, and accumulation of TBARS in hearts in the presence (iron/ascorbate) and absence (iron alone) of extracellular hydroxyl radicals. In addition, there was significant reduction in injury with iron/ascorbate/histidine, where the formation of extracellular hydroxyl radicals was equal to those observed with iron/ascorbate alone. Additional control hearts, perfused with histidine alone, showed stable heart function. Conclusions: These findings indicate that the extracellular formation of hydroxyl radicals is not responsible for iron-mediated cardiotoxicity.     

Role for membrane fluidity in ethanol-induced oxidative stress of primary rat hepatocytes

The relationship between bulk membrane fluidizing effect of ethanol and its toxicity due to oxidative stress is still unknown. To elucidate this issue, membrane fluidity of primary rat hepatocytes was studied by measuring order parameter after inhibition of ethanol-induced oxidative stress. We showed that pretreating cells with either 4-methyl-pyrazole (to inhibit ethanol metabolism), thiourea [a reactive oxygen species (ROS) scavenger], or vitamin E (a free radical chain-breaking antioxidant) prevented the ethanol-induced increase in membrane fluidity, thus suggesting that ethanol metabolism and ROS formation were involved in this elevation. The effects of membrane stabilizing agents (ursodeoxycholic acid or ganglioside GM1), shown to prevent fluidification, next pointed to a role for this increase in membrane fluidity in the development of ethanol-induced oxidative stress. Indeed, ROS production, lipid peroxidation, and cell death were all inhibited by these agents. In contrast, the fluidizing compounds Tween 20 or 2-(2-methoxyethoxy) ethyl 8-(cis-2-n-octylcyclopropyl) octanoate, which increased the membrane fluidizing effect of ethanol, enhanced the related oxidative stress. Using electron paramagnetic resonance to determine low molecular weight iron, we finally demonstrated that membrane fluidity influence proceeded through an increase in low molecular weight iron to enhance oxidative stress. In conclusion, the present findings clearly highlight the pivotal role of membrane fluidity in ethanol-induced oxidative stress and the potential therapeutic effect of membrane stabilizing compounds.     

The critical role of mitochondrial energetic impairment in the toxicity of nimesulide to hepatocytes

We described the effects of nimesulide (N-[4-nitro-2-phenoxyphenyl]-methanesulfonamide) and its reduced metabolite in isolated rat hepatocytes. Nimesulide stimulated the succinate-supported state 4 respiration of mitochondria, indicating an uncoupling effect of the drug. Incubation of hepatocytes with nimesulide (0.1-1 mM) elicited a concentration- and time-dependent decrease in cell viability as assessed by lactate dehydrogenase leakage, a decrease of mitochondrial membrane potential as assessed by rhodamine 123 retention, and cell ATP depression. Nimesulide also decreased the levels of NAD(P)H and glutathione in hepatocytes, but the extent of the effects was less pronounced in relation to the energetic parameters; in addition, these effects did not imply the peroxidation of membrane lipids. The decrease in the viability of hepatocytes was prevented by fructose and, to a larger extent, by fructose plus oligomycin; it was stimulated by proadifen, a cytochrome P450 inhibitor. In contrast, the reduced metabolite of nimesulide did not present any of the effects observed for the parent drug. These results indicate that: 1) nimesulide causes injury to the isolated rat liver cells, 2) this effect is mainly mediated by impairment of ATP production by mitochondria due to uncoupling, and 3) on account of the activity of its nitro group, the parent drug by itself is the main factor responsible for its toxicity to the hepatocytes.     

Hepatocyte Transplantation Improves Survival in Mice with Liver Toxicity Induced by Hepatic Overexpression of Mad1 Transcription Factor

Hepatic overexpression of Mad1 with an adenoviral vector, AdMad, induced liver toxicity in immunodeficient mice. Transduction of cultured hepatocytes with AdMad inhibited cellular DNA synthesis and cell cycling, along with increased lactate dehydrogenase release, indicating cytotoxicity. When dipeptidyl peptidase IV-deficient F344 rat hepatocytes were transplanted into the liver of immunodeficient mice after treatment with AdMad, significant portions of the liver were repopulated. This was in agreement with corresponding losses of host hepatocytes, which showed increased apoptosis rates. Mortality in mice following AdMad treatment decreased significantly when animals were subjected to hepatocyte transplantation. The findings indicated that Mad1 overexpression perturbed hepatocyte survival. Investigation of pathophysiological mechanisms concerning specific cell cycle regulators in acute liver toxicity will thus be appropriate. Cell therapy has potential for treating acute liver injury under suitable circumstances.     

Carvedilol, a new vasodilator and beta adrenoceptor antagonist, is an antioxidant and free radical scavenger.

The antioxidant effect of carvedilol, a new vasodilating, beta adrenoceptor blocker was studied and compared with five other beta blockers. Carvedilol rapidly inhibited Fe(++)-initiated lipid peroxidation, measured as thiobarbituric acid reactive substance (TBARS), in rat brain homogenate with an IC50 of 8.1 microM. Under the same conditions, the IC50 values of atenolol, pindolol propranolol, celiprolol and labetalol were over 1.0 mM. Carvedilol protected against Fe(++)-induced alpha-tocopherol depletion in rat brain homogenate with an IC50 of 17.6 microM; propranolol, celiprolol and labetalol, up to 200 microM, did not show any effect. Using dihydroxyfumarate/Fe(++)-ADP as a OH.radical generating system and 5,5-dimethyl pyrroline-N-oxide (DMPO) as a trapping agent, the characteristic DMPO-OH signals were monitored by electron paramagnetic resonance. Carvedilol dose-dependently decreased the intensity of the DMPO-OH signal, with an IC50 of 25 microM, whereas propranolol, at 500 microM, and U74500A, a 21-aminosteroid, at 100 microM, had no effect. The antioxidant effect of carvedilol mainly resides in the carbazole moiety, and the substitution of a hydroxyl group at certain positions on the phenyl ring of either carbazole or the ortho-substituted phenoxylethylamine part of carvedilol resulted in an increase in antioxidant activity. Furthermore, the protective effect of carvedilol analogs against OH.-mediated neuronal death positively correlated to their antioxidant effect. We conclude that carvedilol is a far more potent antioxidant than other commonly used beta blockers. The apparent mechanism of carvedilol's inhibition of lipid peroxidation is mainly via scavenging free radicals. This novel property of carvedilol may contribute to the known cardioprotective activity of this compound.     

Evaluation of Cytotoxic Effects of Dichloromethane Extract of Guduchi (Tinospora cordifolia Miers ex Hook F & THOMS) on Cultured HeLa Cells

Extracts of Tinospora cordifolia (TCE) have been shown to possess anti-tumor properties, but the mechanism of the anti-tumor function of TCE is poorly understood. This investigation elucidates the possible mechanism underlying the cytotoxic effects of dichlormethane extracts of TCE, after selecting optimal duration and concentration for treatment. HeLa cells were exposed to various concentrations of TCE, which has resulted in a concentration-dependent decline in the clonogenicity, glutathione-S-transferase (GST) activity and a concentration-dependent increase in lipid peroxidation (TBARS) with a peak at 4 h and lactate dehydrogenase (LDH) release with a peak at 2 h. Our results suggest that the cytotoxic effect of TCE may be due to lipid peroxidation and release of LDH and decline in GST.     

Mechanism of oxmetidine (SK&F 92994) cytotoxicity in isolated rat hepatocytes

Oxmetidine is an H2-receptor antagonist that has efficacy in the treatment of peptic ulcers. Isolated rat hepatocytes exposed to oxmetidine (0.5 mM) rapidly lost viability as estimated by increased leakage of lactate dehydrogenase, increased formation of plasma membrane surface blebs and decreased intracellular potassium concentration [K+]. Oxmetidine caused a reduction in hepatocyte reduced glutathione concentration that paralleled cell death; malondialdehyde formation was not observed. Hepatocyte respiration (O2 consumption) and intracellular ATP concentration were decreased markedly by oxmetidine in a concentration-related fashion. Oxmetidine (50 microM) blocked pyruvate/malate-supported state 3 (ADP-stimulated) respiration, caused a decrease in the ADP:0 ratio and a loss of respiratory control in isolated rat liver mitochondria. In contrast, oxmetidine did not block succinate-supported ADP-stimulated O2 consumption in isolated rat liver mitochondria. These data demonstrate that: 1) oxmetidine was cytotoxic to isolated rat hepatocytes in suspension and 2) the mechanism of oxmetidine-induced hepatocyte injury may be related to sustained inhibition of mitochondrial oxidative phosphorylation leading to decreased cellular ATP content and cell death. Although the exact site of action of oxmetidine within the mitochondrion has not been completely elucidated, it appears to reside in the inner mitochondrial membrane electron transport chain before ubiquinone oxidoreductase.     

Potentiation of adriamycin toxicity by ethanol in perfused rat liver.

Adriamycin, which has a quinone nucleus, damages periportal regions of the lobule in perfused rat liver in an oxygen-dependent manner, presumably by redox cycling. Because redox cycling requires reducing equivalents, we investigated whether ethanol, which generates NADH via alcohol dehydrogenase, would increase hepatotoxicity due to concentrations of adriamycin which by themselves were not toxic in perfused rat liver. Perfusion with adriamycin (100 microM) alone did not significantly alter oxygen uptake or cell death evaluated by release of lactate dehydrogenase or uptake of trypan blue. In contrast, oxygen uptake due to adriamycin was increased about 35 mumol/g/hr and lactate dehydrogenase release was elevated to values around 240 U/g/hr in the presence of ethanol (10 mM). As expected, ethanol increased NADH fluorescence detected from the liver surface due to reduction of NAD+ in a concentration-dependent manner (half-maximal effect = ca. 1 mM). The increase in NADH fluorescence due to ethanol and the stimulation of oxygen uptake due to adriamycin had similar dependencies on ethanol concentration. Upon infusion of adriamycin, oxygen uptake increased concomitantly with a decrease in NADH fluorescence, most likely due to utilization of NADH. The half-maximal change in both processes also occurred with concentrations of ethanol around 1 mM. Furthermore, methylpyrazole (4 mM), an alcohol dehydrogenase inhibitor, prevented the increase in NADH fluorescence due to ethanol as well as the stimulation of oxygen uptake due to adriamycin in the presence of ethanol. Ethylhexanol, another agent which increased NADH, also potentiated oxygen uptake due to adriamycin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysophosphatidic acid induces rapid and sustained decreases in epidermal growth factor receptor binding via different signaling pathways in BEAS-2B airway epithelial cells

L-methionine (Met) has been implicated in parenteral nutrition-associated cholestasis in infants and, at high levels, it causes liver toxicity by mechanisms that are not clear. In this study, Met toxicity was characterized in freshly isolated male and female mouse hepatocytes incubated with 5 to 30 mM Met for 0 to 5 h. In male hepatocytes, 20 mM Met was cytotoxic at 4 h as indicated by trypan blue exclusion and lactate dehydrogenase leakage assays. Cytotoxicity was preceded by reduced glutathione (GSH) depletion at 3 h without glutathione disulfide formation. Exposure to 30 mM Met resulted in increased cytotoxicity and GSH depletion. It is interesting to note that female hepatocytes were resistant to Met-induced cytotoxicity at these concentrations and showed increased cellular GSH levels compared with hepatocytes exposed to medium alone. The effects of amino-oxyacetic acid (AOAA), an inhibitor of Met transamination, and 3-deazaadenosine (3-DA), an inhibitor of the Met transmethylation pathway enzyme S-adenosylhomocysteine hydrolase, on Met toxicity in male hepatocytes were then examined. Addition of 0.2 mM AOAA partially blocked Met-induced GSH depletion and cytotoxicity, whereas 0.1 mM 3-DA potentiated Met-induced toxicity. Exposure of male hepatocytes to 0.3 mM 3-methylthiopropionic acid (3-MTP), a known Met transamination metabolite, resulted in cytotoxicity and cellular GSH depletion similar to that observed with 30 mM Met, whereas incubations with D-methionine resulted in no toxicity. Female hepatocytes were less sensitive to 3-MTP toxicity than males, which may partially explain their resistance to Met toxicity. Taken together, these results suggest that Met transamination and not transmethylation plays a major role in Met toxicity in male mouse hepatocytes.     

The effect of simvastatin on erythrocyte membrane fluidity during oxidative stress induced by cardiopulmonary bypass: a randomized controlled study

BACKGROUND: Abnormal erythrocyte deformability can cause severe complications during cardiopulmonary bypass (CPB) surgery, including both hemolysis and perfusion abnormalities. OBJECTIVES: The goals of this study were to evaluate changes in erythrocyte membrane fluidity and lipid peroxidation during CPB and to examine the effect of simvastatin treatment on these parameters. METHODS: Patients undergoing cardiac surgery involving CPB were selected and randomized to receive either simvastatin 40 mg/d or placebo for 3 weeks before surgery. Three blood samples were obtained at different times during surgery for analysis of erythrocyte membrane fluidity, anion permeability, and lipid peroxidation. Erythrocyte ghosts were prepared and incubated with a lipophilic fluorescent probe (diphenyl-hexatriene), and fluorescence anisotropy was evaluated by spectrophotofluorimetric assay as a measure of membrane fluidity. Anion permeability was evaluated by the specific absorption of methemoglobin (CM) at 590 and 635 nm after treatment of heparinized blood with NaNO2. The formation of thiobarbituric acid-reactive substances was evaluated as an index of lipid peroxidation. Aspartate transaminase and lactate dehydrogenase were also measured as indices of hemolysis. RESULTS: Forty patients met the inclusion criteria (20 simvastatin, 20 placebo). Their characteristics differed significantly at baseline only in terms of the lipid profile; the statin group had higher levels of high-density lipoprotein cholesterol (P = 0.01) and lower levels of low-density lipoprotein cholesterol (P = 0.001) than the placebo group. CPB was found to significantly modify characteristics of the erythrocyte membrane. Compared with preoperative values, CPB induced decreases in both mean (SD) erythrocyte membrane fluidity and anion permeability (preoperative CM: 0.69 [0.02]; 24-hour postoperative CM: 0.18 [0.02]; P < 0.001) and an increase in mean (SD) membrane lipid peroxidation (preoperative malonyl dialdehyde [MDA]: 0.21 [0.01] nmol/mL; postoperative MDA: 0.10 [0.02] nmol/mL; P < 0.001). Treatment with simvastatin was associated with a significant reduction in mean (SD) membrane lipid peroxidation both preoperatively and at 24 hours postoperatively compared with placebo (preoperative MDA: 0.07 [0.01] vs 0.10 [0.02] nmol/mL, respectively; P < 0.05; postoperative MDA: 0.10 [0.04] vs 0.21 [0.01] nmol/mL; P < 0.05). In addition, statin treatment was associated with significant increases in anion permeability preoperatively and postoperatively compared with placebo (preoperative CM: 0.79 [0.01] vs 0.69 [0.02]; P < 0.01; 24-hour postoperative CM: 0.30 [0.01] vs 0.18 [0.02]; P < 0.01). CONCLUSION: The results of this study suggest that among these patients undergoing CPB surgery, use of simvastatin for 3 weeks before the surgery had significant beneficial effects on erythrocyte membrane fluidity, lipid peroxidation, and anion permeability.     

Metabolism and toxicity of coumarin on cultured human, rat, mouse and rabbit hepatocytes

Summary— We compared the cytotoxic effect of coumarin and its derivatives, 7-hydroxycoumarin (7-OHC), 4-hydroxycoumarin (4-OHC), o -hydroxyphenyl acetic acid (OHPAA) and o -coumaric acid (CA), on cultured hepatocytes from human, rat, mouse and rabbit liver. At 10⁻⁵ and 5 × 10⁻⁵ M, coumarin and its derivatives did not give rise to any signs of toxicity on cultured hepatocytes of the four species. At 10⁻⁴ M, coumarin, but not its derivatives, induced release of lactate dehydrogenase (LDH) into the medium, especially in rat hepatocyte cultures. Intracellular LDH activities were correspondingly reduced. The cytotoxic effect of coumarin in cultured rat hepatocytes was evidenced on morphological examination and from the results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction test. At higher concentrations (5 × 10⁻⁴ M), 7-OHC and CA were also found to be cytotoxic in cultured rat hepatocytes. The cytotoxic effect of coumarin (5 × 10⁻⁴ M) was decreased in the presence of SKF 525-A, a cytochrome P450 inhibitor. Interspecies comparisons showed that rat hepatocytes were the most sensitive to the toxicity of coumarin and its derivatives, whereas human hepatocytes were the most resistant. Our results suggest that the cytotoxicity of coumarin is metabolism and species-dependent. Thus, the rat may not be a suitable model for evaluating the pharmacological hazards of coumarin in humans.     

Cytotoxicity and Oxidative Stress Alterations Induced by Aldrin in BALB/c 3T3 Fibroblast Cells

The intensive use of pesticides in agro-vet practices resulted in their persistence in environmental medium responsible for adverse effect on living system. The present study was planned to evaluate the cytotoxicity and oxidative stress potential of aldrin on BALB/c 3T3 mouse fibroblast cells. Short exposure of 3 h was given to the BALB/c 3T3 fibroblast cells under standard in vitro conditions. Inhibitory concentration-50 was estimated at the end of exposure period by neutral red uptake assay and was found to be 49.7 μg/ml. Further, cells were exposed with three concentrations of aldrin (12.4, 24.8 and 49.7 μg/ml) and 0.1 % dimethyl sulfoxide was used as negative control. Lipid peroxidation, antioxidant enzymes and non-enzymes were determined along with glucose-6-phosphate dehydrogenase, lactate dehydrogenase and alkaline phosphatase enzymes in BALB/c 3T3 fibroblast cells. Cells were also monitored for cell morphology during experiment. Exposure of aldrin to BALB/c 3T3 fibroblast cells resulted in increase in lipid peroxidation and decrease in antioxidant enzyme/non-enzyme system. Further, it caused decrease in glucose-6-phosphate dehydrogenase enzyme activity and excess leakage of lactate dehydrogenase and alkaline phosphatase enzymes into the medium. Aldrin-treated cells showed high degree of alteration in cell morphology indicating cell damage and death. These changes were noticed in dose dependent manner. In conclusion, the result of present study suggests that increase in lipid peroxidation and decrease in activity of antioxidant defence via oxidative stress by aldrin in BALB/c 3T3 cells were responsible for cytotoxicity.     

Production of chemotactic activity for polymorphonuclear leukocytes by cultured rat hepatocytes exposed to ethanol

Acute alcoholic hepatitis is characterized by infiltration of the liver parenchyma with polymorphonuclear leukocytes. As a possible explanation for this phenomenon, we have found that ethanol stimulates cultured rat hepatocytes to generate potent chemotactic activity. Hepatocytes (greater than 99% pure), isolated from the livers of Sprague-Dawley rats, responded to incubation with ethanol (2.0-10 mM) by releasing chemotactic activity for human polymorphonuclear leukocytes into culture supernatants in a time- and concentration-dependent fashion. Chemotactic activity was maximal after incubation of hepatocytes with 10 mM ethanol for 6 h. It was undetectable in the absence of ethanol and was reduced in the presence of either the alcohol dehydrogenase inhibitor, 4-methylpyrazole, or the acetaldehyde dehydrogenase inhibitor, cyanamide. Ethanol failed to stimulate generation of chemotactic activity by either rat dermal fibroblasts, hepatic sinusoidal endothelial cells, or Kupffer cells. The chemotactic activity generated by ethanol-treated rat hepatocytes was recovered from culture supernatants in the lipid phase after extraction with chloroform/methanol. Thin-layer chromatography and high performance liquid chromatography of chloroform/methanol extracts demonstrated that the chemotactic factor probably is a polar lipid. This chemotactic lipid may account, in part, for the leukocytic infiltration of the liver parenchyma that is observed during the course of acute alcoholic hepatitis.