Acute interaction of halothane and enflurane with the metabolism of ethanol in isolated hepatocytes and liver cytosol preparations from the rat

The effects of halothane and enflurane on ethanol (40 mM) oxidation were studied in isolated rat hepatocytes. Anaesthetic (halothane, enflurane and diethyl ether) effect on the activity of alcohol dehydrogenase (ADH) was studied in incubations of cytosol preparations from rat liver. Mean rates of ethanol metabolism ranged from 0.44 to 0.49 mumol ethanol metabolized/mg cell protein/hour in control hepatocytes from fasted and fed animals. These rates were enhanced by 2- and 3-fold in hepatocytes from fed and fasted animals, respectively, when pyruvate (5 mM) was added. Halothane and enflurane both caused dose dependent inhibition of ethanol metabolism (15-40%) in all hepatocytes without exogenous addition of pyruvate. The inhibitory effect was present also after pyruvate stimulation in hepatocytes from fasted animals, but disappeared in hepatocytes from fed animals when pyruvate was added. The rate of ethanol oxidation by cells from fed rats was enhanced by approximately 40% when the concentration of ethanol was increased from 20 mM to 80 mM. The anaesthetic inhibition of ethanol metabolism was about 20% more pronounced at the higher ethanol concentration compared to the lower concentration when no pyruvate was added. In the presence of pyruvate the effect of anaesthetics was again reversed regardless of ethanol concentration. Halothane (2 mM) and enflurane (2 mM) both caused about 25% inhibition of the ADH-activity in cytosol preparations while ether (30 mM) caused more than 50% inhibition. No inhibition of hepatocyte uptake of ethanol was caused by any of the three anaesthetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibody-directed characterization of cytochrome P450 isozymes responsible for toluene metabolism in rat liver

Monoclonal antibodies (MAbs) were used to study the contribution of cytochromes P450IA1/IA2, P450IIB1/IIB2, P450IIC11/IIC6 and P450IIE1 to toluene side-chain (benzyl alcohol, BA, formation) and ring (o- and p-cresol formation) oxidation in liver microsomes from fed, one-day fasted, and phenobarbital (PB)-, 3-methylcholanthrene (MC)- and ethanol-treated rats. All rats were fed synthetic liquid diets. MAb 1-7-1 against P450IA1/IA2 inhibited markedly o-cresol formation and slightly p-cresol formation but not BA formation only in microsomes from MC-treated rats. MAbs 2-66-3, 4-7-1 and 4-29-5 against P450IIB1/IIB2 strongly inhibited BA, o-cresol and p-cresol formation only in PB-induced microsomes. MAb 1-68-11 against P450IIC11/IIC6 inhibited BA formation at high toluene concentration in the following order: fed greater than fasted greater than ethanol = MC greater than PB, and ethanol greater than or equal to fed = fasted greater than MC greater than PB on the basis of the percentage and net amount inhibition, respectively. MAb 1-91-3 against P450IIE1 inhibited BA formation at low toluene concentration, but not at high concentration, in the following order: ethanol greater than fasted = fed greater than MC, and ethanol greater than fasted greater than fed greater than MC on the basis of percentage and net inhibition, respectively. MAbs 1-68-11 and 1-91-3 also inhibited p-cresol formation at high and low toluene concentrations, respectively. These results indicate that (i) both P450IIE1 and P450IIC11/IIC6 are constitutive isozymes mainly responsible for the formation of BA and p-cresol from toluene as low- and high-Km isozymes, respectively; (ii) P450IIE1, but not P450IIC11/IIC6, is induced by one-day fasting and ethanol treatment; (iii) both P450IIE1 and P450IIC11/IIC6 are decreased by PB and MC treatments; (iv) P450IIE1 is inhibited by high concentration of toluene; (v) P450IIB1/IIB2 can contribute to the formation of BA, o- and p-cresol from toluene, while P450IAI/IA2 preferentially contributes to the formation of o-cresol.     

Oxidation of pyrazole to 4-hydroxypyrazole by intact rat hepatocytes

4-Hydroxypyrazole has been identified as a major metabolite found in the urine of rats and mice after in vivo administration of pyrazole, a potent inhibitor of alcohol dehydrogenase and of ethanol metabolism. The locus and the enzyme systems responsible for the oxidation of pyrazole have not been identified. In the current report, isolated hepatocytes from fed rats were shown to oxidize pyrazole to 4-hydroxypyrazole. An HPLC procedure employing UV and electrochemical detection was utilized to separate and quantify the 4-hydroxypyrazole. The apparent Km for pyrazole by intact hepatocytes was about 2 mM, whereas the apparent Vmax was about 0.06 nmol 4-hydroxypyrazole per min per mg liver cell protein. The production of 4-hydroxypyrazole was inhibited by carbon monoxide and metyrapone, as well as by competitive drug substrates such as aniline or aminopyrine. These results implicate a role for cytochrome P-450 in the oxidation of pyrazole by the hepatocytes. Ethanol was an effective inhibitor of pyrazole oxidation. Hepatocytes were also isolated from rats treated with acetone and 4-methylpyrazole, to attempt to evaluate whether pyrazole oxidation is induced. The rate of 4-hydroxypyrazole production by hepatocytes after acetone and 4-methylpyrazole treatment was actually lower than that of controls. Kinetic assays suggested the presence of an endogenous inhibitor (perhaps the inducer itself) in the induced hepatocytes. In contrast, hepatocytes isolated from rats fasted for 48 hr showed a 2-fold increase in the oxidation of pyrazole to 4-hydroxypyrazole. The Km for pyrazole was the same in hepatocytes from fasted and fed rats, whereas Vmax was increased after fasting. The locus and enzyme system responsible for the oxidation of pyrazole to 4-hydroxypyrazole, and the site of sensitivity to ethanol, appears to be the cytochrome P-450 system of the hepatocyte.     

Inhibition of Paranitroanisole and Antipyrine Monooxygenation in Isolated Rat Hepatocytes by Compounds Interacting with Mitochondrially Related Carbohydrate Metabolism

Abstract: The monooxygenation of paranitroanisole (PNA) and antipyrine (AP) were measured in isolated rat hepatocytes incubated with compounds interacting with mitochondrially related carbohydrate metabolism. Phenylpyruvate, an inhibitor of pyruvate carboxylase, reduced the rate of PNA and AP metabolism to about 60 and 20%, respectively, in hepatocytes both from fasted and fed rats. Inhibition of amino acid transaminase with aminooxyacetate, decreased the metabolism of both PNA and AP to 60–70% of control values in hepatocytes from fasted rats, whereas this effect was not seen in fed rats. n-Butylmalonate, an inhibitor of mitochondrial malate/phosphate exchange, had only minimal effects on PNA and AP monooxygenation in both the nutritional states. The simultaneous presence of glyoxylate and pyruvate, known to inhibit the NADPH specific isocitrate dehydrogenase, reduced the metabolism of both PNA and AP in hepatocytes from fasted rats to about 60 and 35% of control values respectively, while the effect was not so marked in hepatocytes from fed rats. The metabolism both of PNA and of AP in hepatocytes from fasted rats was reduced to 50–60% of control values with the addition of NH₄Cl. This effect could be blocked either by incubating the hepatocytes with pyruvate or by using hepatocytes isolated from fed rats. The addition of various carbon intermediates generally reduced the effect of the inhibitors used. Phenobarbital-treatment did not change the effects observed with cells from uninduced animals. The inhibitors did not alter PNA or AP metabolism in microsomal incubations, and therefore most likely reduced the monooxygenation in intact cells by affecting NADPH generation pathways.     

Ethanol oxidation by isolated hepatocytes from fed and starved rats and from rats exposed to ethanol,phenobarbitone or 3-amino-triazole: No evidence for a physiological role of a microsomal ethanol oxidation system

In isolated hepatocytes from fed and starved rats, basal rates of ethanol oxidation were 1.15 and 0.71 μmoles/g wet wt, respectively, and were unchanged over the ethanol concentration range 8–96 mM. The addition of 4-methyl pyrazole (4 mM), a competitive inhibitor of alcohol dehydrogenase, largely abolished ethanol oxidation from 8mM ethanol, while at an ethanol concentration of 96 mM, the oxidation rate was inhibited by 87 per cent. Pyrazole was a less effective inhibitor of alcohol oxidation than 4-methyl pyrazole. In hepatocytes isolated from rats treated with ethanol, phenoharbitone or 3-amino-triazole, basal rates of ethanol oxidation were the same at ethanol concentrations of 8–96 mM and the rates were similar to, and never exceeded, the rate found in hepatocytes from normal fed rats. 4-Methyl pyrazole inhibited ethanol oxidation to the same extent in all liver cell preparations. regardless of the treatment the donor animal had received. Pyruvate stimulated cellular ethanol oxidation irrespective of the prior treatment of the donor animal. This stimulation, together with the ethanol-induced accumulation of lactate, was abolished by 4-methyl pyrazole. This suggests that the capacity for alcohol oxidation in isolated liver cells is generally limited by the lack of suitable acceptors for the hydrogen generated in the cytoplasm by the alcohol dehydrogenase-catalysed oxidation of ethanol to acetaldehyde. Methylene blue, phenazine methosulphate and menadione stimulated both ethanol oxidation and respiration, irrespective of the prior treatment of the donor animal. This enhancement of ethanol oxidation and respiration was prevented by 4-methyl pyrazole. These artificial electron acceptors appear to act by circumventing normal pathways for the oxidation of cytoplasmic NADH generated in the conversion of ethanol to acetaldehyde. In cells from each treatment group, antimycin was more effective than rotenone as an inhibitor of ethanol oxidation; inhibition of ATP formation by oligomycin had least effect on alcohol oxidation. Ethanol oxidation by cells from alcohol-treated rats was most affected by these inhibitors of mitochondrial respiration. These results indicate that under a wide variety of experimental conditions the contribution of the postulated microsomal ethanol oxidizing system to ethanol oxidation in isolated, intact liver cells appears minimal. Thus they cast doubt on a physiological role for this system in vivo.     

Combined Effects of Ethanol and pH-Change on Protein Synthesis in Isolated Rat Hepatocytes

Abstract: Rat liver parenchymal cells were isolated and incubated for 80 min. in buffered salt solutions. ¹⁴C-valine incorporation in the presence of a high concentration of unlabelled valine (4.2 mM) was taken as a measure of protein synthesis. The pH dependence of the synthesis of cell and medium proteins was studied at pH values varying from 6.6 to 7.9. The results showed that maximum protein synthesis occurred close to the physiological pH value. Protein synthesis was reduced at both lower and higher pH-values. Protein synthesis was inhibited by the addition of ethanol (30 mM). The relative inhibition caused by ethanol got more pronounced as the pH of the hepatocyte suspensions was lowered. The metabolism of ethanol resulted in lowering of the pH in cell suspensions. It is suggested that the depression of protein synthesis by ethanol could be mediated by the fall in pH due to ethanol metabolism.     

Toluene metabolism in isolated rat hepatocytes: effects of in vivo pretreatment with acetone and phenobarbital

Hepatocytes isolated from control, acetone- and phenobarbital-pretreated rats were used to study the metabolic conversion of toluene to benzyl alcohol, benzaldehyde, benzoic acid and hippuric acid at low (<100 M) and high (100–500 M) toluene concentrations. The baseline formation rates of toluene metabolites (benzyl alcohol, benzoic acid and hippuric acid) were 2.9±01.7 and 10.0±2.3 nmol/mg cell protein/60 min at low and high toluene concentrations, respectively. In vivo pretreatment of rats with acetone and phenobarbital increased the formation of metabolites: at low toluene concentrations 3- and 5-fold, respectively; at high toluene concentrations no significant increase (acetone) and 8-fold increase (phenobarbital). Apparent inhibition by ethanol, 7 and 60 mM, was most prominent at low toluene concentrations: 63% and 69%, respectively, in control cells; 84% and 91% in acetone-pretreated cells, and 32% (not significant) and 51% in phenobarbital-pretreated cells. Ethanol also caused accumulation of benzyl alcohol. The apparent inhibition by isoniazid was similar to that of ethanol at low toluene concentrations. Control and acetone-pretreated cells were apparently resistant towards metyrapone; the decrease was 49% and 64% in phenobarbital-pretreated cells at low and high toluene concentrations, respectively. In these cells, the decrease in presence of combined ethanol and metyrapone was 95% (low toluene concentrations). 4-Methylpyrazole decreased metabolite formation extensively in all groups. Benzaldehyde was only found in the presence of an aldehyde dehydrogenase inhibitor. Increased ratio benzoic/hippuric acid was observed at high toluene concentrations. These results demonstrate that toluene oxidation may be studied by product formation in isolated hepatocytes. However, the influence of various enzymes in the overall metabolism could not be ascertained due to lack of inhibitor specificity.     

Effects of anaesthetics on protein synthesis in isolated rat hepatocytes: inhibition by diethyl ether in contrast to no influence by pentobarbital and fentanyl

Hepatocytes were isolated from livers of fasted rats by a two-step Ca++-free/collagenase perfusion method. Suspensions of parenchymal liver cells were incubated in the absence and presence of three different anaesthetics, diethyl ether, pentobarbital and fentanyl at various concentrations. Their influence on the hepatocytes was monitored by measuring protein synthesis as the incorporation of L-(U-14C) valine (50 mCi/mol, 4.2 mM) into liver proteins. Diethyl ether representing anaesthetics mainly affecting cellular membranes unspecifically, inhibited protein synthesis markedly, concentrations of approximately 10, 20 and 30 mM caused 27, 50 and 74 per cent inhibition respectively, of cellular protein synthesis. The rate of synthesis process of these proteins or that ether also inhibited protein secretion from cells to media. The effect of diethyl ether was completely reversible when the anaesthetic was removed by changing the medium. Pentobarbital representing barbiturate anaesthetics, reduced the synthesis of cell and medium proteins very little, while the opiate anaesthetic fentanyl had no inhibitory effect. These results demonstrate a potential hepatotoxic mechanism for membrane active drugs like diethyl ether. They also indicate that special precautions should be taken when this type of anaesthesia is used during the study of hepatic protein synthesis.     

Effect of toluene on ethanol preference in rats

The effect of toluene on the preference of ethanol was studied in rats. Toluene was administered orally by stomac tube in doses of 200, 400 or 800 mg/kg daily for 5 weeks or by inhalation at a concentration of 2000 p.p.m. 6 or 8 hr daily for 5 or 6 days per week for 2 weeks in rats of different age. During toluene inhalation exposure the rats had access to either tap water or ethanol-containing water (6 or 10%). After the exposure and during oral administration the rats had access to both ethanol-free and ethanol-containing water. Toluene inhibited the body weight gain in the highest oral dose group and in rats exposed to toluene and forced to drink ethanol in the inhalation experiments. In these experiments, the intake of fluid was reduced in the exposure period in rats forced to drink ethanol-containing water and further reduced in rats exposed to both ethanol and toluene. Exposure to toluene alone increased the fluid consumption. The preference of ethanol defined as consumed ethanol-containing water in per cent of the total water intake was not influenced by oral administration of toluene. It was, however, reduced by toluene given by inhalation to rats forced to drink ethanol-containing water during the exposure period. Toluene exposure alone or forced ethanol intake alone caused in these experiments a short-lasting reduction of the ethanol preference. It is concluded that toluene decreases the preference of ethanol in rats forced to drink ethanol during exposure to toluene.     

Interaction of ethanol oxidation with glucuronidation in isolated hepatocytes.

1. Glucuronidation of harmol, 2-naphthol, 4-methylumbelliferone and phenolphthalein in isolated hepatocytcs was inhibited up to 50 per cent in the presence of low concentrations of ethanol (10 mM). Sulphate conjugation was unaffected. The inhibitory effect of ethanol was reversed by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase dependent ethanol oxidation. 2. The oxidation of harmine to harmol was not affected by 10 mM ethanol, but in hepatocytes isolated from phenobarbital-treated rats glucuronidation of the formed harmol was inhibited about 30 per cent in the presence of this amount of ethanol. 3. Ethanol increased the intracellular NADH/NAD⁺ ratio as did lactate and sorbitol. The latter two substances were also inhibitory to glucuronidation having no effect on the sulphate conjugation. 4. The synthesis of UDPglucuronic acid was inhibited by ethanol both in the presence and absence of a substrate undergoing glucuronidation. It is suggested that the inhibitory effect of ethanol on glucuronidation is due to a decreased UDPglucuronic acid synthesis caused by the increased NADH/NAD⁺ ratio resulting from the alcohol dehydrogenase dependent oxidation of ethanol.     

Ethanol Interference with Morphine Metabolism in Isolated Guinea Pig Hepatocytes

Abstract: It has previously been shown that guinea pig hepatocytes metabolise morphine in a fashion similar to humans. The metabolism of morphine (5 μM) and the formation of metabolites morphine-3-glucuronide, morphine-6-glucuronide and normorphine was studied in the absence and presence of ethanol (5, 10, 25, 60 and 100 mM) in freshly isolated guinea pig hepatocytes. In order to gain more detailed information, a mathematical model was estimated on experimental data and used to analyse the effects of ehtanol on the reaction rates of the different morphine metabolites. Ethanol inhibited the rate of morphine elimination in a dose-related manner, at the high ethanol concentrations the elimination rate was 40 per cent of the control rate. The formation of morhine-glucuronides was influenced in a biphasic manner. Five and 10 mM ethanol increased both the morphine-3-glucuronide and morphine-6-glucuronide levels after 60 min incubation compared to the control, whereas at the higher ethanol concentrations (25-100 mM) the levels of morphine-glucuronides were reduced. Data from the mathematical model, however, demonstrated that the reaction rates for morphine-glucuronide formation were decreased at all ethanol concentrations and in a dose-dependent manner, the interpretation of this being that at the lower (5 and 10 mM) ethanol concentraions employed in this study, other metabolic pathways of morphine are more heavily inhibited than the glucuronidations, resulting in a shunting towards morphine-3-glucuronide and morphine-6-glucuronide. The pharmacodynamic consequences of these pharmacokinetic effects are thus somewhat diffucult to predict since morphine-6-glucuronide has a higher agonist potency than morphine. At high concentrations ethanol inhibition of morphine metabolism will increase the concentration of morphine and subsequently the euphoric and the toxic effects. The lower quantities of morphine-6-glucuronide formed in the presence of high ethanol concentrations on the other hand most probably imply reduction of such effects and the net pharmacodynamic effect would be uncertain. At low ethanol concentrations, however, morphine-6-glucuronide concentrations increased and morphine metabolism was less inhibited leading to a possible potentiation of the effects of morphine. Thus, a low ehtanol concentration might exert a more pronounced ethanol-drug effect interaction than a higher ethanol concentration.     

Effect of volatile anesthetics on protein synthesis and secretion by rat hepatocyte suspensions

The effect of volatile anesthetics on protein synthesis and secretion by isolated rat hepatocytes in suspension was investigated. Halothane and enflurane inhibited protein synthesis in a dose-dependent manner. Diethyl ether had little effect on protein synthesis while isoflurane caused a mild inhibition. This effect was more pronounced in hepatocytes from phenobarbital treated male rats when compared to hepatocytes from control rats. Protein synthesis in hepatocytes from phenobarbital treated female rats was inhibited similar to that seen with control male rat hepatocytes. Isoflurane, enflurane, and halothane also caused a dose-dependent inhibition of protein secretion, while diethyl ether was only mildly inhibitory. From these studies it appears that inhibition of protein synthesis and secretion might be an early and sensitive indicator of cellular injury by volatile anesthetics.     

An experimental study on ethanol elimination at subphysiological temperatures

Hepatocytes isolated from fed and fasted rats have been used to study the rate of ethanol elimination at different incubation temperatures. In the presence of exogenous pyruvate, hepatocytes from fed and 42 hr fasted rats, eliminated ethanol at 37 degrees by a rate of 11.6 +/- 3.4 and 6.4 +/- 0.8 nmol/min./mg of cell protein (+/- S.D.; n = 5), respectively, which are comparable to the rates obtained in vivo. The ethanol oxidation rate in cells from rats of both nutritional states correlated linearily to the incubation temperature (t = 24-37 degrees) with a temperature coefficient (Q10) of 1.8-2.3. (Q10, (= temperature coefficient) is the factor by which the enzyme activity is increased on raising the temperature 10 degrees). These findings indicate that the oxidation is not controlled by processes which involve membrane transitions in the temperature range 24-37 degrees. Our results indicate that a hypothermic individual with a body temperature of 27 degrees would have a 40-50 per cent inhibition of the ethanol elimination rate. Thus, the observed dependency of the ethanol oxidation on the body temperature has to be regarded in back-calculations of blood ethanol concentrations in forensic toxicology.     

Effect of Volatile Anesthetics on Protein Synthesis and Secretion by Rat Hepatocyte Suspensions

ABSTRACT

The effect of volatile anesthetics on protein synthesis and secretion by isolated rat hepatocytes in suspension was investigated. Halothane and enflurane inhibited protein synthesis in a dose-dependent manner. Diethyl ether had little effect on protein synthesis while isoflurane caused a mild inhibition. This effect was more pronounced in hepatocytes from phenobarbital treated male rats when compared to hepatocytes from control rats. Protein synthesis in hepatocytes from phenobarbital treated female rats was inhibited similar to that seen with control male rat hepatocytes. Isoflurane, enflurane, and halothane also caused a dose-dependent inhibition of protein secretion, while diethyl ether was only mildy inhibitory. From these studies it appears that inhibition of protein synthesis and secretion might be an early and sensitive indicator of cellular injury by volatile anesthetics.     

Glucuronidation in rat intestinal epithelial cells

Glucuronidation of 1-naphthol was studied in mucosal cells isolated from the rat intestine. Glucuronidation was directly dependent on the supply of extracellular carbohydrates. Basal glucuronidation (ca. 0.3 nmoles/min · mg cell protein) was increased 2- to 3-fold by adding glucose or fructose to the incubation medium. Saturation of glucuroniation was achieved by adding 0.3 mM glucose, while saturation by fructose was not reached at concentrations below 2 mM. No carbohydrate reserves able to support glucuronidation appear to be present in intestinal cells, since no difference in glucuronidation was observed between cells prepared from fasted (18 or 42 h) and control rats. Glucuronidation was decreased by adding d-galactosamine to the incubation medium, but only when extracellular glucose was present. Various chemicals which are known to inhibit glucuronidation in hepatocytes (ethanol, diethyl ether, sorbitol) did not influence the glucuronidation of 1-naphthol in isolated intestinal cells. Only when ethanol was added to mucosal cells in the absence of extracellular glucose was a small decrease in glucuronidation observed.     

Effect of ethanol metabolism on initiation of protein synthesis in rat hepatocytes

Rat liver parenchymal cells were incubated in the presence and absence of ethanol (80 mM). Polysomes were isolated and analysed on sucrose gradients. Ethanol was shown to (1) inhibit the incorporation of ¹⁴C-valine into proteins, (2) result in a shift in the distribution of polysomes towards smaller sizes, (3) inhibit the formation of 40S initiation complexes, and (4) diminish the concentration of glucose-6-phosphate in the hepatocytes. Addition of 4-methylpyrazole (0.5 mM) partially prevented the inhibition of protein synthesis and completely restored the polysomal distribution. It is concluded that ethanol inhibits protein synthesis partly by a mechanism linked to ethanol metabolism. This effect takes place at the level of initiation and may be mediated by a reduced gluconeogenesis.     

The Effects of Metformin Compared to the Effects of Phenformin on the Lactate Production and the Metabolism of Isolated Parenchymal Rat Liver Cell

Abstract: The metabolic effects of metformin were compared to the effects of phenformin on isolated parenchymal liver cells from fed and fasted rats with ethanol or glycerol as the only substrate. Both biguanides caused a fall in the hepatic oxygen-consumption, in the cellular ATP-content and in the ATP/ADP-ratio. The lactate production and the concentration-ratios of lactate/pyruvate and of P-hydroxybutyrate/acetoacet-ate rose. The production rate of ketone bodies remained unchanged. This response was the same whether the hepatocytes were from fed or fasted rats and whether glycerol or ethanol was substrate. Only quantitative differences in the response on the biguanides were detected. The effects of the biguanides were dose dependent. Phenformin was ten times more potent than metformin. The same holds for their therapeutical potency. The findings indicate inhibition of the oxidative phosphorylation by both biguanides resulting in reduction of the cytoplasmic and the mitochondrial redox potentials causing enhanced lactate production. It is concluded that metformin excerts the same effects as phenformin on the hepatic metabolism, when the concentration ratio is about ten to one. Both biguanides give rise to elevated lactate production. This effect is highly increased when ethanol is present.     

L-asparaginase effects on inhibition of protein synthesis and lowering of the glutamine content in cultured rat hepatocytes

Primary cultures of rat hepatocytes were exposed to various concentrations of L-asparaginase derived from Escherichia Coli. Protein synthesis was inhibited by about 33% and cellular glutamine was reduced proportionally to the enzyme concentration. However, protein synthesis was inhibited only by amounts of enzyme able to reduce glutamine to critical levels below 10 nmol/mg cell protein. These data suggest that the glutaminase activity which probably contaminates E. coli asparaginase may be responsible for reduced liver protein synthesis.     

The effects of fructose on adenosine triphosphate depletion following mitochondrial dysfunction and lethal cell injury in isolated rat hepatocytes

Mitochondrial injury in aerobic mammalian cells is associated with a rapid depletion of adenosine triphosphate (ATP) which occurs prior to the onset of lethal cell injury. In this report, the relationships between ATP depletion and lethal cell injury were examined in rat hepatocytes using oligomycin as a model mitochondrial toxicant and fructose as an alternative carbohydrate source for glycolysis. Oligomycin was more potent in causing lethal cell injury in hepatocytes isolated from fasted animals than cells from fed animals. The onset of cell injury (leakage of lactate dehydrogenase) in cells from fed animals correlated with the depletion of stored glycogen and ATP. The degree and time course profile of oligomycin-induced ATP depletion could be duplicated with 50 mM fructose alone in hepatocytes from fasted animals; however, fructose did not cause lethal cell injury. Oligomycin caused marked accumulation of adenosine monophosphate (AMP) and inorganic phosphate (Pi) and a conservation of adenine nucleotides. In contrast, fructose (50 mM) caused a decrease in Pi, no persistent change in AMP, and a depletion of the adenine nucleotide pool. Fructose, at concentrations greater than 1.0 mM, protected hepatocytes from oligomycin-induced toxicity. Blockade of mitochondrial ATP synthesis with oligomycin resulted in massive ATP depletion. In the presence of oligomycin, 5.0 mM fructose maintained cellular ATP content similar to that of control cells, whereas 50 mM fructose did not, demonstrating the biphasic effect of increasing fructose concentrations on cellular ATP content. Fructose-induced protection of hepatocytes from oligomycin toxicity was due to glycolytic fructose metabolism as hepatocytes incubated with iodoacetate (30 microM), fructose, and oligomycin had reduced viability and ATP content. In conclusion, interruption of mitochondrial ATP synthesis leads to marked ATP depletion and lethal cell injury. Cell injury is clearly not due to ATP depletion alone since increased glycolytic ATP production from either glycogen or fructose can maintain cell integrity in the absence of mitochondrial ATP synthesis and at low cellular ATP levels.     

Fasting increases the susceptibility of rat hepatocytes to the cytotoxic effects of N-hydroxy-acetylaminofluorene. Effects on mitochondrial respiration and membrane potential.

Isolated rat hepatocytes were incubated with the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Cells from fasted rats were much more susceptible to the cytotoxic effects of 1 mM N-OH-AAF than cells from fed rats: after approximately 90 min exposure the former were all dead but the latter still viable. Even after 240 min 25% of the "fed" cells were still viable. The loss of viability was preceded by a decrease in mitochondrial membrane potential (MMP) and inhibition of respiration; the mitochondrial respiration as measured in permeabilized cells appeared uncoupled. Addition of 15 mM fructose prevented cell death and the loss of MMP in cells both from fed and fasted rats to a large extent; however, uncoupling was not prevented. After incubation of hepatocytes from fasted rats with 1 mM [3H]N-OH-AAF for 120 min, 12 nmol [3H]N-OH-AAF became bound per mg cell protein. Addition of fructose decreased this to 7 nmol. In cells from fed animals 4 nmol [3H]N-OH-AAF became bound after 120 min, in this case fructose had no effect. Part of the protective effect of fructose might be explained by a decrease in intracellular ATP, which prevents the formation of reactive intermediates of N-OH-AAF resulting in a decrease of covalent binding, in addition, fructose protects via a yet to be determined mechanism.