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.     

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.     

Metabolism of reverse triiodothyronine by isolated rat hepatocytes.

Reverse triiodothyronine (rT3) is metabolized predominantly by outer ring deiodination to 3,3'-diiodothyronine (3,3'-T2) in the liver. Metabolism of rT3 and 3,3'-T2 by isolated rat hepatocytes was analyzed by Sephadex LH-20 chromatography, high performance liquid chromatography, and radioimmunoassay, with closely agreeing results. Deiodinase activity was inhibited with propylthiouracil (PTU) and sulfotransferase activity by sulfate depletion or addition of salicylamide or dichloronitrophenol. Normally, little 3,3'-T2 production from rT3 was observed, and 125I- was the main product of both 3,[3'-125I]T2 and [3',5'-125I]rT3. PTU inhibited rT3 metabolism but did not affect 3,3'-T2 clearance as explained by accumulation of 3,3'-T2 sulfate. Inhibition of sulfation did not affect rT3 clearance but 3,3'-T2 metabolism was greatly diminished. The decrease in I- formation from rT3 was compensated by an increased recovery of 3,3'-T2 up to 70% of rT3 metabolized. In conclusion, significant production of 3,3'-T2 from rT3 by rat hepatocytes is only observed if further sulfation is inhibited.     

Carrier-mediated efflux of ketone bodies in isolated rat hepatocytes

The rate of efflux of ketone bodies has been studied in isolated hepatocytes prepared from starved rats and preloaded with D-3-[14C]hydroxybutyrate. Efflux of ketone bodies was temperature-dependent, saturable and inhibited by alpha-cyano-3-hydroxycinnamate and phloretin. The rate of efflux was also reduced by 6 mmol/l lactate and pyruvate added to the external medium. Under conditions of simulated metabolic acidosis in the hepatocyte suspension medium, ketone body efflux rate was reduced. The experimental data suggest that hepatic plasma membrane ketone body transit is carrier-mediated.     

Ethanol inhibition of codeine and morphine metabolism in isolated rat hepatocytes

Suspensions of isolated hepatocytes from male Wistar rats were prepared according to a two step Ca++-free collagenase perfusion method. Codeine, morphine or norcodeine were incubated with hepatocytes at 37 degrees C for up to 90 min in the absence and presence of ethanol. The elimination rate constant (Kel) of codeine and morphine was reduced with approximately one-third and one-fourth, respectively, in the presence of 60 mM ethanol, whereas the presence of ethanol did not alter the Kel of norcodeine significantly. The inhibition of codeine metabolism was dose-dependent, extending from approximately 15% at 10 mM ethanol to 40 to 50% at 100 mM. A 3-fold increase in the ratio of morphine concentration (formed from codeine) to the amount of codeine metabolized was observed in the presence of ethanol as compared to control cells. The mean morphine concentration was 170% higher in the ethanol-treated suspensions than in the controls. The ratio of norcodeine concentration to codeine metabolized was unchanged. The inhibition of morphine metabolism was accompanied by a similar reduction of morphine-3-glucuronide formation. The accumulation of morphine observed in the cell medium in the presence of ethanol might be due to inhibition of other metabolic pathways from codeine, thus shunting to morphine formation, combined with the inhibitory effect of ethanol on morphine metabolism per se.     

Influence of Cl- on organic anion transport in short-term cultured rat hepatocytes and isolated perfused rat liver.

Transport of 35S-labeled sulfobromophthalein [35S]BSP was studied in short-term cultured rat hepatocytes incubated in bovine serum albumin. At 37 degrees C, initial uptake of [35S]BSP was 5-10-fold that at 4 degrees C, linear for at least 15 min, saturable, inhibited by bilirubin, and reduced by greater than 70% after ATP depletion or isosmotic substitution of sucrose for NaCl in medium. Replacement of Na+ by K+ or Li+ did not alter uptake, whereas replacement of Cl- by HCO-3 or gluconate- reduced uptake by approximately 40%. Substitution of Cl- by the more permeant NO-3 enhanced initial BSP uptake by 30%. Efflux of [35S]BSP from cells to media was inhibited by 40% after ATP depletion or sucrose substitution. To confirm these results in a more physiologic system, transport of [3H]bilirubin was studied in isolated livers perfused with control medium or medium in which Cl- was replaced by gluconate-. Perfusion data analyzed by the model of Goresky, revealed 40-50% reductions in influx and efflux with gluconate- substitution. These results are consistent with existence of a Cl-/organic anion-exchange mechanism similar to that described by others in renal tubules.     

Evidence for lipid peroxidation by paraquat in the perfused rat lung

In order to evaluate the effect of paraquat on oxidative radical reactions in the lung, we studied MDA production and chemiluminescence (spontaneous and tBuOOH-induced) in the isolated rat lung. After 2 hr of perfusion with 3.0 mM paraquat, MDA content in lung homogenates was 16 +/- 7 nmol/gm dry weight higher than in control lungs (mean +/- S.E., n = 7, p less than 0.05 by paired test); during 30 min of perfusion, malondialdehyde efflux was 33 +/- 15 nmol/gm dry weight higher than in control perfusates (n = 6, p less than 0.05). Spontaneous chemiluminescence was not augmented by 2 hr of perfusion with concentrations of paraquat ranging from 0.75 to 6.0 mM. On the other hand, tBuOOH-induced chemiluminescence was 17% +/- 3 higher immediately after the addition of hydroperoxide and reached a 16% +/- 6 higher plateau for the paraquat-perfused lungs than for control lungs (n = 10, p less than 0.05). Spectral analysis of the light emitted during induced chemiluminescence demonstrated peak intensity between 630 and 730 nm for both control and paraquat-treated lungs. Increased MDA production and increased induced chemiluminescence indicate that perfusion with paraquat enhances lipid peroxidation in the isolated rat lung.     

Zinc supplementation ameliorates electromagnetic field-induced lipid peroxidation in the rat brain

Extremely low-frequency (0-300 Hz) electromagnetic fields (EMFs) generated by power lines, wiring and home appliances are ubiquitous in our environment. All populations are now exposed to EMF, and exposure to EMF may pose health risks. Some of the adverse health effects of EMF exposure are lipid peroxidation and cell damage in various tissues. This study has investigated the effects of EMF exposure and zinc administration on lipid peroxidation in the rat brain. Twenty-four male Sprague-Dawley rats were randomly allocated to three groups; they were maintained untreated for 6 months (control, n = 8), exposed to low-frequency (50 Hz) EMF for 5 minutes every other day for 6 months (n = 8), or exposed to EMF and received zinc sulfate daily (3 mg/kg/day) intraperitoneally (n = 8). We measured plasma levels of zinc and thiobarbituric acid reactive substances (TBARS), and levels of reduced glutathione (GSH) in erythrocytes. TBARS and GSH levels were also determined in the brain tissues. TBARS levels in the plasma and brain tissues were higher in EMF-exposed rats with or without zinc supplementation, than those in controls (p < 0.001). In addition, TBARS levels were significantly lower in the zinc-supplemented rats than those in the EMF-exposed rats (p < 0.001). GSH levels were significantly decreased in the brain and erythrocytes of the EMF-exposed rats (p < 0.01), and were highest in the zinc-supplemented rats (p < 0.001). Plasma zinc was significantly lower in the EMF-exposed rats than those in controls (p < 0.001), while it was highest in the zinc-supplemented rats (p < 0.001). The present study suggests that long-term exposure to low-frequency EMF increases lipid peroxidation in the brain, which may be ameliorated by zinc supplementation.     

Effects of diphenyl-phenylenediamine on gentamicin-induced lipid peroxidation and toxicity in rat renal cortex

The hypothesis that lipid peroxidation is linked causally to the pathogenesis of aminoglycoside nephrotoxicity was tested by determining whether administration of the antioxidant, diphenyl-phenylenediamine (DPPD) would inhibit lipid peroxidation and ameliorate gentamicin-induced proximal tubular cell injury. Rats were injected with saline, gentamicin or gentamicin plus DPPD for 4 days and were sacrificed 48 hr later. Gentamicin increased malondialdehyde in renal cortex from a control level of 0.65 +/- 0.04 to 1.01 +/- 0.03 nmol/mg of protein, P less than .01; it was reduced to 0.20 +/- 0.03 by DPPD, P less than .01 compared to control. Arachidonic acid comprised 27.6 +/- 0.5% of the fatty acid in renal cortical phospholipid of control rats. Gentamicin lowered arachidonic acid to 16.7 +/- 0.9%, P less than .01, and promoted a shift toward saturated fatty acids. DPPD reversed these changes. Gentamicin depressed catalase activity from a control value of 0.211 k/min to 0.154 +/- 0.008 k/min, P less than .01. DPPD depressed catalase further to 0.095 +/- 0.066 k/min, P less than .01. Total glutathione and reduced glutathione were depressed whereas the fraction of total glutathione in the oxidized state was augmented by gentamicin. These changes were prevented by DPPD. The renal cortical phospholipidosis induced by gentamicin was not altered by DPPD. The increased urinary excretions of alanine aminopeptidase and N-acetyl-beta-glucosaminidase induced by gentamicin were augmented further by DPPD. In DPPD rats serum creatinine (0.45 +/- 0.04 mg/dl) was higher (P less than .01) than that of gentamicin rats (0.35 +/- 0.01 mg/dl), which was higher (P less than .01) than that of control rats (0.26 +/- 0.01 gm/dl).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonlinear elimination kinetics of 5-fluoro-2'-deoxyuridine in isolated perfused rat liver and isolated hepatocytes

The kinetic parameters of the cytostatic agent 5-fluoro-2'-deoxyluridine (FUDR) were studied in isolated rat hepatocytes and in the isolated perfused rat liver. In both experimental setups a dose dependency of the elimination parameters, half-life and clearance, was observed with a calculated turning point around 250 microM. In the medium of rat hepatocytes incubated at low (0.1 microM) to high (2000 microM) FUDR, the majority of the metabolites consisted of the catabolite alpha-fluoro-beta-alanine. The nucleobase metabolites, 5-fluorouracil and its primary product 5,6-dihydro-5-fluorouracil, approached apparent steady-state levels comprising 10 to 15% of the initial concentration. In the intracellular phase of hepatocytes incubated at 300 microM FUDR almost 90% of the FUDR-derived material was alpha-fluoro-beta-alanine, whereas essentially no unchanged FUDR could be detected. Similar results were obtained at extracellular FUDR concentrations exceeding 300 microM. In the isolated perfused rat liver, the clearance decreased to 15 to 20% of the corresponding values when the initial concentration was raised from 24 to 2400 microM. At the end of perfusion alpha-fluoro-beta-alanine comprised 90 to 95% of FUDR-derived total radioactivity in the tissue even at initially 2400 microM FUDR, although at this FUDR dosage 20% of the substrate remained unmetabolized in the medium. These results suggest that the limitation of hepatic FUDR elimination is not due to saturable hepatic metabolism but must be due to saturable uptake of these pyrimidine derivatives across the cellular membrane of parenchymal liver cells.     

The effect of arginine vasopressin on ureagenesis in isolated rat hepatocytes

The effect of arginine vasopressin (AVP) on ureagenesis was measured in isolated rat hepatocytes with ammonium chloride and L(+)-lactate as substrates. AVP was found to stimulate urea synthesis and the dose-response curve suggests that such an effect is present at concentrations of the hormone as low as 25-50 pmol/l. Both the dose-response curve and the concentrations of NH+4 employed suggest that the effect observed could be of physiological significance.     

Oxygen-dependent lipid peroxidation during lung ischemia.

The effect of alveolar oxygen tension on lung lipid peroxidation during lung ischemia was evaluated by using isolated rat lungs perfused with synthetic medium. After a 5-min equilibration period, global ischemia was produced by discontinuing perfusion while ventilation continued with gas mixtures containing 5% CO2 and a fixed oxygen concentration between 0 and 95%. Lipid peroxidation was assessed by measurement of tissue thiobarbituric acid-reactive products and conjugated dienes. Control studies (no ischemia) showed no change in parameters of lipid peroxidation during 1 h of perfusion and ventilation with 20% or 95% O2. With 60 min of ischemia, there was increased lipid peroxidation which varied with oxygen content of the ventilating gas and was markedly inhibited by ventilation with N2. Perfusion with 5-, 8-, 11-, 14-eicosatetraynoic acid indicated that generation of eicosanoids during ischemia accounted for approximately 40-50% of lung lipid peroxide production. Changes of CO2 content of the ventilating gas (to alter tissue pH) or of perfusate glucose concentration had no effect on lipid peroxidation during ischemia, but perfusion at 8% of the normal flow rate prevented lipid peroxidation. Lung dry/wet weight measured after 3 min of reperfusion showed good correlation between lung fluid accumulation and lipid peroxidation. These results indicate that reperfusion is not necessary for lipid peroxidation with ischemic insult of the lung and provide evidence that elevated PO2 during ischemia accelerates the rate of tissue injury.     

Mitochondrial function of isolated rat hepatocytes from normal and cirrhotic liver

Mitochondrial fractions were obtained from purely isolated hepatocytes of the normal and cirrhotic livers. Mitochondrial function of isolated hepatocytes was evaluated to compare between those from the normal and those from the cirrhotic livers in addition to the evaluation of the mitochondrial function of the normal and cirrhotic liver tissues. Respiratory control, ADP/O ratio and ATP synthesis were significantly lower in the cirrhotic liver tissue than those in the normal liver tissue. However, the mitochondrial function of isolated hepatocytes showed no difference between normal and cirrhotic groups. By electron microscopic examination, debris was more observed in the mitochondrial fractions from the normal and cirrhotic tissues compared to those from hepatocytes. However, no difference of the shape and size of the mitochondria was seen between the fraction from hepatocytes and that from the tissue in each group. These results suggest that as far as hepatocyte itself is concerned, the cirrhotic liver preserves the sufficient function as well as the normal liver.     

Chemopreventive efficacy of pronyl-lysine on lipid peroxidation and antioxidant status in rat colon carcinogenesis

Colon cancer is a serious health problem in most of the countries and is the leading cause of cancer mortality throughout the world. The major objective of this study was to examine the chemopreventive effect of dietary pronyl-lysine (2 mg/kg body weight), a bread crust antioxidant, on intestinal and colonic tissue lipid peroxidation (LPO) and antioxidant status in rat colon carcinogenesis. Male Wistar rats were divided into seven groups and were fed a modified pellet diet for 34 weeks. Rats were given a weekly subcutaneous injection of 1,2-dimethyl hydrazine (DMH) (20 mg/kg body weight) for the first 15 weeks. Pronyl-lysine was supplemented to rats during the pre-initiation, initiation, post-initiation and also throughout the study period. All the rats were sacrificed at the end of 34 weeks and their colons were evaluated histologically. The activity of lipid peroxidation (LPO) and antioxidant status in the tissues such as the intestines, colon and cecum were estimated. Our results showed diminished levels of colonic, and cecal LPO products such as conjugated dienes, lipid hydroperoxides and thiobarbituric acid reactive substances, and also reduced activities of the antioxidants superoxide dismutase, catalase and glutathione dependent enzymes (glutathione peroxidase, glutathione-S-transferase, glutathione reductase) in DMH-treated rats, while on supplementing dietary pronyl-lysine the levels of LPO products and antioxidants were significantly reversed ( P  < 0.05). Thus, our results strongly suggest that the administration of pronyl-lysine throughout the study period (group 7) and the post-initiation (group 6) stages of colon carcinogenesis significantly inhibits colon cancer incidence and prevents DMH induced histopathological lesions.     

Insulin resistance in uremia. Characterization of lipid metabolism in freshly isolated and primary cultures of hepatocytes from chronic uremic rats.

We have studied the mechanism(s) of hyperlipidemia and liver insulin sensitivity in a rat model of severe chronic uremia (U). Basal lipid synthesis was decreased in freshly isolated hepatocytes from U when compared with sham-operated ad lib.-fed controls (alfC). Basal lipid synthesis in pair-fed controls (pfC) was in between U and alfC. Similarly, the activity of liver acetyl CoA carboxylase, fatty acid synthetase, citrate cleavage enzyme, malate dehydrogenase, and glucose-6-phosphate dehydrogenase was diminished in U. Muscle and adipose tissue lipoprotein lipase was also decreased. Insulin stimulated lipid synthesis in freshly isolated hepatocytes from alfC. Hepatocytes from U and pfC were resistant to this effect of insulin. To ascertain if the insulin resistance in U was due to starvation (chow intake 50% of alfC) or to uremia itself, the U and pfC were intragastrically fed an isocaloric diet via a Holter pump the last week of the experimental period. Hepatocytes from orally fed U and pfC were also cultured for 24 h in serum-free medium. While freshly isolated and cultured U hepatocytes remained insulin resistant, those from pfC normalized, in vivo and in vitro, when they were provided with enough nutrients. Conclusions: (a) Hyperlipidemia in uremia is not due to increased synthesis, but to defect(s) in clearance. (b) Insulin does not stimulate lipid synthesis in uremia. This finding, along with our recent demonstration that insulin binding and internalization are not decreased in the uremic liver, suggests that a post-binding defect(s) in the liver plays an important role in the mechanism(s) of insulin resistance in uremia. (c) Cultured hepatocytes from uremic rats remain insulin resistant. This quality renders these cells useful in studying the postinsulin binding events responsible for the insulin-resistant state in the absence of complicating hormonal and substrate changes that occur in vivo.     

Stereoselective glutathione conjugation and amidase-catalyzed hydrolysis of alpha-bromoisovalerylurea enantiomers in isolated rat hepatocytes

alpha-Bromoisovalerylurea (BIU) is used as model substrate for studies on the pharmacokinetics of glutathione conjugation in vivo. Its metabolism in isolated rat hepatocytes is presently studied. A major part of the substrate was conjugated with glutathione, but also amidase-catalyzed hydrolysis occurred, resulting in the products urea and alpha-bromoisovaleric acid (BI). The amidase activity was located in the microsomal fraction of the rat liver. The product of hydrolysis, BI, also was conjugated efficiently with glutathione. In glutathione-depleted hepatocytes, no glutathione conjugates but only urea and BI were formed. A pronounced stereoselectivity in the metabolism of the BIU enantiomers was observed: (R)-BIU was conjugated with glutathione much faster than (S)-BIU. (S)-BIU was hydrolyzed substantially in the cells and the glutathione conjugate of the hydrolytic product, (S)-BI, could be detected. At high BIU concentrations (500 microM of the racemate) intracellular glutathione was seriously depleted; then, the cosubstrate availability most likely was the rate-limiting factor in the conjugation of BIU with glutathione. More urea was formed from (racemic) BIU in isolated rat hepatocytes in the present study than in the perfused liver and the intact rat in previous studies. This in vivo-in vitro difference is tentatively assigned to differences in glutathione availability in these systems. The results suggest that BI may also be a useful model substrate to study the kinetics of glutathione conjugation in vivo and in vitro.     

Age-associated changes in beta-adrenergic modulation on rat cardiac excitation-contraction coupling.

Previous studies have demonstrated that the ability of beta-adrenergic receptor (beta AR) stimulation to increase cardiac contractility declines with aging. In the present study, the control mechanisms of excitation-contraction (EC) coupling, including calcium current (ICa), cytosolic Ca2+ (Cai2+) transient and contraction in response to beta AR stimulation were investigated in ventricular myocytes isolated from rat hearts of a broad age range (2, 6-8, and 24 mo). While the baseline contractile performance and the Cai2+ transient did not differ markedly among cells from hearts of all age groups, the responses of the Cai2+ transient and contraction to beta-adrenergic stimulation by norepinephrine (NE) diminished with aging: the threshold concentration and the ED50 increased in rank order with aging; the maximum responses of contraction and Cai2+ transient decreased with aging. Furthermore, the efficacy of beta AR stimulation to increase ICa was significantly reduced with aging, and the diminished responses of the contraction and Cai2+ transient amplitudes to NE were proportional to the reductions in the ICa response. These findings suggest that the observed age-associated reduction in beta AR modulation of the cardiac contraction is, in part at least, due to a deficit in modulation of Cai2+, particularly the activity of L-type calcium channels.