Hepatic ATP Research Articles

Protection by albumin against ischaemia- and hypoxia-induced hepatic injury.

In previous studies using isolated perfused rat livers, we have shown that reactive oxygen species are involved in hypoxic and ischaemic liver damage. Since albumin was shown to possess strong antioxidant properties we now investigated the capacity of albumin to prevent ischaemic and hypoxic damage in isolated perfused rat livers. Both, partial ischaemia and hypoxia/reoxygenation, resulted in marked hepatic injury as evidenced by an increased release of hepatic enzymes (GPT, LDH), by a strong decline of bile flow and by a decrease in hepatic GSH levels. With partial ischaemia, hepatic ATP depletion and calcium accumulation were also observed. Bovine serum albumin, added to the perfusate at concentrations of 0.1 or 1%, provided nearly complete protection against both types of liver injury. The same level of protection was also afforded by sulfhydryl-blocked and fatty acid-free bovine albumin preparations and by human albumin. In conclusion, the protective effect of albumin in our models of oxidative liver injury is neither due to the thiol moiety nor to the presence of oxidizable fatty acids in the albumin fraction. More likely, albumin provides protection by an unspecific binding of redox-active transition metal ions capable of catalyzing reactions which yield hydroxyl or hydroxyl-like radicals. Besides, unspecific sacrifice reactions of albumin with highly reactive oxygen species or other endogenous compounds may also be implicated.

Influence of inducers and inhibitors of cytochrome P450 on the hepatotoxicity of hydrazine in vivo.

Hydrazine hepatotoxicity in vivo, as manifested by triglyceride accumulation, depletion of ATP and reduced glutathione (GSH) was shown to be dose related. The effect of pretreatment of rats with various inhibitors and inducers of cytochrome P450 on these dose-response relationships was investigated. Pretreatment with the inhibitor piperonyl butoxide increased triglyceride accumulation whereas pretreatment with the inducers phenobarbital and beta-naphthoflavone (BNF) resulted in reduced triglyceride accumulation. Pretreatment with the inducers acetone and isoniazid also enhanced triglyceride accumulation. Only phenobarbital pretreatment also significantly reduced GSH and ATP depletion. A linear correlation was found between hepatic glutathione and ATP levels in non-pretreated animals given various doses of hydrazine. However, exponential relationships were found between hepatic triglycerides and both hepatic ATP and glutathione. The results suggest that i) the hepatotoxicity of hydrazine can be modulated by inducing or inhibiting particular isoenzymes of cytochrome P450, ii) ATP and GSH depletion may not be directly involved in the development of fatty liver.

Phosphate loading prevents the decrease in ATP and increase in food intake produced by 2,5-anhydro-D-mannitol.

The fructose analogue 2,5-anhydro-D-mannitol (2,5-AM) triggers feeding in rats apparently by its action in the liver. In vivo phosphorylation of this analogue decreases hepatic inorganic phosphate and ATP by trapping of phosphate in the mono- and diphosphorylated forms of 2,5-AM. To determine whether hepatic phosphate depletion and decreased ATP are involved in the eating response to 2,5-AM, rats were treated with excess sodium phosphate before injection of 2,5-AM. Phosphate loading prevented both the increase in food intake and the decrease in liver ATP, without affecting the changes seen in plasma fuels produced by 2,5-AM treatment. Phosphate loading did not influence water intake or eating elicited by insulin or 2-deoxy-D-glucose, indicating that the effect on 2,5-AM-induced eating was behaviorally specific and not due to malaise. These data suggest that 2,5-AM elicits eating by trapping phosphate and reducing ATP in liver.

Glycogenesis from glucose and ureagenesis in isolated perfused rat livers. Influence of ammonium ion, norvaline, and ethoxyzolamide.

The probable involvement of hepatic carbamyl-P in the reciprocal relationship between hepatic ureagenesis and glycogenesis from glucose was explored. Isolated perfused liver preparations from 48-h fasted rats were employed. Moderate (9.2 mM) and relatively high levels of glucose (34 mM) were perfused. Hepatic glycogenesis, glucose-6-P, carbamyl-P, and citrulline levels, hepatic urea formation, and ureagenesis based upon perfusate urea levels were measured. Experimental probes selected to modify hepatic ureagenesis and carbamyl-P production and utilization included: (a) NH4Cl, maintained at 5 mM by continuous infusion (NH4+ is a substrate for carbamyl-P synthase I and glutamate dehydrogenase); (b) norvaline, an inhibitor of ornithine transcarbamylase which catalyzes the first committed step in the urea cycle; and (c) ethoxyzolamide, an inhibitor of carbonic anhydrase which produces HCO3-, an essential substrate for carbamyl-P synthase I. NH4+ increased ureagenesis and decreased glycogenesis. The inclusion of norvaline with NH4+ decreased ureagenesis and increased glycogenesis. Ethoxyzolamide with or without NH4+ inhibited both ureagenesis and glycogenesis, and decreased the hepatic glucose-6-P level. Glycogenesis was greater at 34 mM than 9.2 mM glucose, increased in norvaline-containing preparations correlative with increased availability of carbamyl-P, and decreased when carbamyl-P formation was inhibited by ethoxyzolamide. Kinetic analysis indicated a Km, Glc of 31 mM for glucose phosphorylation preliminary to glycogenesis. Glycogen formation via the "indirect pathway" (i.e. involving extrahepatic glycolysis, transport of lactate to the liver, and glyconeogenesis therefrom) was quantitatively insufficient to account for the observed glycogenesis. Glucokinase is contraindicated by the inverse relationship between hepatic glycogenesis and ATP availability in the ethoxyzolamide-treated preparations. In contrast, carbamyl-P:glucose phosphotransferase activity of the glucose-6-phosphatase system has the characteristics to bridge hepatic ureagenesis and glycogenesis.

Effects of inhalation anesthetics on myocardial and hepatic energy metabolism in normotensive and spontaneously hypertensive rats subjected to hemorrhage

Forty spontaneously hypertensive rats (SHRs) and forty normotensive Wistar-ST rats (NRs) were used to assess the influence of anesthetics on myocardial and hepatic energy metabolism after hemorrhage. They were divided into five pairs of groups: a control group (pentobarbital 6 mg.100 g BW-1 ip), and four others which received 1.2% halothane, 2.2% enflurane, 1.4% isoflurane, and 3.3% sevoflurane, respectively. Following a 10 min stabilization period, blood (2 ml.100 g BW-1) was gradually withdrawn over a 5 min period from a femoral artery. Thirty min after the induction of hemorrhage, the heart and liver were removed and myocardial and hepatic metabolites (ATP, lactate, pyruvate and glycogen) were measured by enzymatic methods. There were no significant differences in myocardial metabolites among either the anesthetic groups or between SHRs and NRs. However, hepatic ATP levels in all SHR groups were significantly lower than those in NR groups. Moreover, ATP levels in the inhalation anesthetic groups of SHRs were significantly higher than that in the control group of SHRs. All inhalation anesthetics, especially isoflurane, may reduce metabolic deterioration of the liver during hemorrhage when compared to barbiturate anesthesia.

Hepatic phosphate trapping, decreased ATP, and increased feeding after 2,5-anhydro-D-mannitol

The mechanism by which the fructose analogue 2,5-anhydro-D-mannitol (2,5-AM) elicits feeding behavior was investigated by studying its metabolism and biochemical effects in liver. Thin-layer chromatography of liver extracts from rats given 2,5-AM containing 14C-labeled 2,5-AM showed that the analogue is phosphorylated in vivo with a time course that parallels the eating response. In vivo 31P nuclear magnetic resonance spectroscopy of rat liver during intravenous infusion of 2,5-AM and high-resolution nuclear magnetic resonance analyses of liver extracts showed that 2,5-AM is rapidly phosphorylated in liver, trapping hepatic phosphate and decreasing ATP, inorganic phosphate, and phosphate diesters. These changes occurred in a time frame in which the feeding response is elicited in conscious animals given the same dose of 2,5-AM by the same route. During an interval in which 2,5-AM increased eating, it also increased urinary uric acid excretion, implicating enhanced adenosine degradation in the reduction in hepatic ATP. These results provide the first direct evidence that changes in a high-energy phosphate-carrying compound in liver may provide a signal to initiate eating behavior.

Adenosine triphosphate protection of chlordecone-amplified CCl4 hepatotoxicity and lethality

Dietary exposure to a nontoxic level of chlordecone (10 ppm for 15 days) followed by a single exposure to a subtoxic dose of CCl4 (100 microliters/kg, ip) is known to result in a 67-fold amplification of CCl4 toxicity. The hypothesis that the underlying mechanism is due to incapacitation of hepatocytes leading to an ablation of the early-phase hormetic response of tissue repair as a consequence of precipitous decline in hepatic glycogen and ATP, received experimental support from Mehendale in 1990. The present study was designed to investigate if direct administration of ATP to rats maintained on the chlordecone diet would result in protection from the hepatotoxic and lethal effects of the chlordecone+CCl4 combination. Male Sprague-Dawley rats (125-150 g) were maintained either on a diet containing no added contaminants (control) or on a diet containing 10 ppm chlordecone for 15 days, and were challenged with CCl4 (100 microliters/kg, ip) on day 16. Without ATP administration all rats died within 72 h, while administration of ATP (100 mg/rat, sc) to chlordecone-pretreated rats at -1, +1, 3, 5, 12, 24 and 36 h of CCl4 injection resulted in 100% survival. Injection of ATP, at -1, +1, 3 and 5 h of CCl4 administration to chlordecone pretreated rats decreased plasma enzyme elevations (alanine and aspartate aminotransferase, sorbitol dehydrogenase) as well as substantially preventing elevation of plasma bilirubin levels at 6, 12 and 24 h. Hepatic ATP levels were also elevated at 6 and 12 h, but not at 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration of atracurium during reperfusion of rat livers after 21 h of cold ischaemic storage in different solutions

The pharmacokinetics of atracurium are not altered by impaired hepatic function. The drug is therefore used widely in liver transplant patients. In previous work on the hepatotoxic effects of atracurium in an isolated, perfused rat liver model, we could not detect biochemical (release of lactate dehydrogenase or aspartate aminotransferase) or histological evidence of liver cell damage, except a reduction in hepatic tissue ATP content. In the present study, rat livers were reperfused with Krebs-Henseleit bicarbonate buffer with or without atracurium after 21 h of cold ischaemic storage in University of Wisconsin (UW), Bretschneider's HTK or Euro-Collins solution. UW-protected livers showed a complete restoration of ATP, total adenine nucleotides and energy charge during reperfusion, but the addition of atracurium diminished the regeneration capacity to about 50%. The energy charge (an index for determination of liver viability) was also reduced markedly.

Hepatic Failure Leads to Lethality of Chlordecone-Amplified Hepatotoxicity of Carbon Tetrachloride

Hepatic Failure Leads to Lethality of Chlordecone-Amplified Hepatotoxicity of Carbon Tetrachloride. Soni, M. G., and Mehendale, H. M. (1993). Fundam. Appl. Toxicol. 21, 442-450.Chlordecone (Kepone) amplification of CCI4 toxicity occurs at small, nontoxic levels of chlordecone and CCI4 and results in highly increased irreversible hepatotoxicity culminating in lethality. Although it is generally assumed that CCI4 lethality is due to hepatic failure, no definitive studies are available in the literature bridging massive liver failure and death. The present studies were designed to evaluate whether hepatic failure is the cause of the lethality during chlordecone-amplified CCI4 toxicity. Male Sprague-Dawley rats were maintained on control or a chlordecone (10 ppm) diet for 15 days and injected with CCI4 (100 μl/kg, ip) on Day 16. Rats were killed at 0, 6, 12, 24, 36, and 48 hr after CCI4 challenge. Hepatic failure was evaluated by measuring plasma glucose, ammonia, bilirubin, aspartate transaminase (AST), alanine transaminase (ALT), sorbitol dehydrogenase (SHD), hepatic ATP, glycogen, and by histological and histomorphometric analyses. Plasma creatinine, urea, and kidney histopathology were also assessed for possible renal injury. As expected CCI4 administration to chlordecone-pretreated rats resulted in 20% lethality by 36 hr. which progressed with time, and all rats died within 72 hr. A significant and progressive hypoglycemia was observed with a 60% reduction in plasma glucose at 48 hr. Hepatic glycogen content dropped precipitously. Similarly, hepatic ATP levels remained suppressed (80% of control) at all the time points studied. Plasma ammonia levels were significantly elevated, and by 48 hr, a threefold increase was observed. Plasma ALT, AST, SDH, and bilirubin increased progressively until the death of rats receiving the chlordecone + CCI4 combination. Histopathologically, the liver sections revealed a progressive and irreversible damage evidenced by vacuolation, accumulation of fat, and increase in hepatocellular necrosis resulting in disrupted hepatolobular structure. Administration of the same dose of CCI4 to rats maintained on a normal diet resulted in only marginal changes in plasma enzymes, no increase in serum bilirubin, and no significant injury in lever sections at 24 hr. CCI4 administration to chlordecone-pretreated rats resulted in a marginal increase in plasma creatinine and urea only at later time points. Histopathological examination of sections of kidneys from rats receiving either chlordecone + CCI4 combination or the same dose of CCI4 alone did not reveal any evidence of significant renal injury. The clinical end points of hepatic function measured in the present study are consistent with the sequel of hepatic failure and hepatic encephalopathy leading to animal death in chlordecone-amplified CCI4 toxicity.

Impaired hepatic energy metabolism during taurocholate-induced acute pancreatitis in rats

Abstract The effect of acute pancreatitis on hepatic functions was evaluated. At 6 h after the induction of acute pancreatitis by injecting 5% sodium taurocholate into the rat pancreatico-biliary duct, malate dehydrogenase leakage from hepatic mitochondria was significantly increased in in-vitro incubation as compared with the control group. Hepatic ATP levels were significantly decreased and hepatic AMP levels were, on the contrary, significantly increased as compared with the control group. Reflecting these data, Hepatic adenylate energy charge levels were also significantly decreased as compared with the control group. These results indicate the impaired hepatic energy metabolism in the early stage of acute pancreatitis, and also indicate the intimate relationship between liver and exocrine pancreas in the pathological condition, such as in acute pancreatitis.

Platelet-activating factor in hepatic ischemia/reperfusion injury. The effects of a PAF antagonist combined with a prostaglandin I2 analogue.

The effects of TCV-309, a specific platelet-activating factor (PAF) antagonist, and OP-41483, a prostaglandin I2 analogue, on warm ischemia/reperfusion injury of the rat liver were studied. Rats were divided into five groups by the duration of warm ischemia and the treatment used. The NS1 group (normal saline pretreatment) had 60 min of warm ischemia, while the NS2 group (normal saline pretreatment), the PGI2 group (OP-41483, 500 ng/kg/min pretreatment), the TCV group (TCV-309, 3 micrograms/kg), and the PGI2+TCV group (both the above dosages) underwent 120 min of warm ischemia. Postoperative survival after 30 days, bile secretion, serum endotoxin levels, and tissue glutathione levels after 60 min of reperfusion were compared between the groups. The survival rates for the NS1, NS2, PGI2, TCV, and PGI2+TCV groups were 80%, 0%, 50%, 80%, and 86.7%, respectively. Bile secretion, which has a strong correlationship with hepatic cellular ATP level, was strongly correlated with survival. The NS2 group had a high serum endotoxin level--however, the PGI2 and PGI2+TCV groups had normal levels. Although there were some discrepancies between survival and the tissue glutathione level, combined treatment with the PGI2 analogue and TCV-309 was most effective in inhibited oxidative stress. In conclusion, TCV-309 increased the survival rate after 120 min of warm hepatic ischemia without endotoxemia by the PGI2 analogue. This finding suggest that warm ischemia/reperfusion injury is related to the generation of PAF. Combined pretreatment with TCV-309 and a PGI2 analogue may be useful in liver transplantation.

Glutathione Depletion Alters Hepatocellular High-Energy Phosphate Metabolism

Oxygen free radicals have recently been implicated as a major cause of tissue injury in critically ill patients. Glutathione (GSH) is a potent endogenous antioxidant that may be important in minimizing oxidant-induced organ damage. However, this tripeptide is depleted during severe illness. In order to determine the effect of GSH depletion on hepatic high-energy phosphate metabolism, in vivo31P magnetic resonance spectroscopy was used to measure phosphate ratios in male Wistar rats given 1 ml/kg of diethylmaleate (DEM), an agent that binds and thus depletes tissue GSH, or corn oil vehicle intraperitoneally. Spectra of the liver were obtained in noninjected animals (baseline, n = 15) and in rats 2 and 24 hr after the intraperitoneal injection of DEM (n = 20) or corn oil (control, n = 20). These spectra were used to measure hepatocellular pH, phosphomonoester to ATP (PME/ATP), and phosphodiester to ATP ratios, measures of hepatocellu]ar damage; and the inorganic phosphate (Pi)/ATP ratio, a measure of energy status. In addition, tissue GSH, phosphofructokinase, citrate synthase, and β-OH-acyl-Co-A dehydrogenase activities as well as hepatocellular ATP were measured in vitro in representative liver samples. Hepatic GSH levels were maximally depressed by 85% 2 hr after the injection of DEM (6.94 ± 0.34 vs 0.94 ± 0.22 μM/g wet wt, baseline vs 2° DEM). This was associated with a marked increase in the PME/ATP and Pi/ ATP ratios by 25 and 33%, respectively, and both ratios were significantly correlated with the severity of hepatic GSH depletion (r = 0.63, P < 0.001 and r = 0.42, P < 0.01, respectively). In addition, the intracellular pH decreased from 7.41 ± 0.02 to 7.33 ± 0.02 (P < 0.05) 2 hr after the injection of DEM. At this time citrate synthase and β-OH-acyl-Co-A dehydrogenase activities were significantly elevated in the GSH-depieted animals. In contrast, ATP levels did not change significantly (2.92 ± 0.07 vs 3.20 ± 0.28 μM/g wet wt, baseline vs 2° DEM) and PFK activity was similar in both groups. We conclude that (1) GSH depletion in rats alters the hepatocellular energy status primarily due to increased ATP turnover; (2) under these conditions, hepatic ATP synthesis via the oxidation of fatty acids accelerates to meet an increased energy demand; and (3) these changes in hepatocellular energetics and PME/ATP ratios may indicate early hepatocyte injury and are likely due to increased oxidative stress unveiled by GSH depletion. The GSH depletion associated with critical illness may contribute to the development of liver dysfunction in severely injured patients.

Serum complement mediates endotoxin-induced cysteinyl leukotriene formation in rats in vivo.

To investigate potential mediators responsible for cysteinyl leukotriene formation during endotoxemia, male Fischer rats received an intravenous bolus injection of 5 mg/kg Salmonella enteritidis endotoxin and cysteinyl leukotrienes were measured by gas chromatography-mass spectrometry. The biliary excretion of leukotriene (LT) C4 (0.20 +/- 0.02 pmol.min-1.g liver-1) and N-acetyl-LTE4 (0.37 +/- 0.07 pmol.min-1.g-1) was increased by 190 and 1,000%, respectively, during the first 30 min after endotoxin injection. Endotoxin also caused a temporary reduction of hepatic ATP levels by 84%. Depletion of serum complement almost completely abolished the endotoxin-induced increase of cysteinyl leukotrienes in bile without affecting the biliary excretion mechanism. Intravenous injection of activated complement factors caused cysteinyl leukotriene formation and reduced the hepatic ATP content similar to endotoxin. Depletion of glutathione in the liver prevented cysteinyl leukotriene formation and the complement-induced ATP depletion. It is concluded that endotoxin-induced cysteinyl leukotriene generation in vivo is mediated predominantly through activation of complement. The vasoconstrictive cysteinyl leukotrienes are then responsible for ATP depletion in the liver.

Absence of mitochondrial enhancement in the remnant liver after partial hepatectomy in cirrhotic rats

Changes in energy metabolism after 70% partial hepatectomy were investigated in normal and carbon-tetrachloride-(CCl4)-induced-cirrhotic rats by evaluating hepatic mitochondrial ATP synthesizing activity as well as liver tissue levels of adenine nucleotides and lipid peroxide. Preoperative concentrations of ATP and total adenine nucleotides (TAN: ATP + ADP + AMP) in mumol/g dry weight (dw) and the energy charge potential (ECP) in the cirrhotic livers were 8.53 SEM 0.25, 14.73 SEM 0.54, and 0.74 SEM 0.01, respectively, and were significantly lower than those of normal livers (12.04 SEM 0.34, 15.75 SEM 0.12, and 0.86 SEM 0.01, P less than 0.01 in TAN). There was no difference in the preoperative mitochondrial phosphorylation rate (PR = x 10(-10) mol ATP/sec per mg mitochondrial protein) between normal and cirrhotic livers (21.01 SEM 0.95 and 21.55 SEM 1.03, respectively). After hepatectomy, in the normal livers these values decreased slightly after 12 h, remained low until 48 h, and returned to the preoperative value at 72 h. PR was remarkably enhanced and reached the maximum level of 32.54 SEM 2.07 at 24 h (P less than 0.001, compared to the sham-operated rats) and gradually returned to the preoperative value at 72 h. In the cirrhotic livers, ATP and ECP levels were drastically decreased at 12 h and recovered to the preoperative levels within 24 h, while TAN level remained unchanged. Enhancement of PR was not observed in any of the cirrhotic livers during the experiment. Lipid peroxidation was transiently increased postoperatively with no difference between normal and cirrhotic livers both in the sham-operated and the hepatectomized rats. These findings suggest that the energy status was more depressed in the cirrhotic livers than in normal livers both before and after hepatectomy. This depressed energy status might be attributed to the low preoperative tissue levels of adenine nucleotides and ECP level in the cirrhotic livers as well as to the absence of the enhancement of PR in the remnant livers.

A possible mechanism for the increase in brain tyrosine levels induced by cyanide in mice

Subcutaneous administration of cyanide significantly increased blood tyrosine levels of mice in a dose dependent manner. Tyrosine aminotransferase activity in liver of mice was significantly decreased in the presence of cyanide (8, 10, 20, 40, 50, 65, 80 and 100 μ M), also in a concentration-dependent manner, with a positive correlation between the percentage increase of blood tyrosine levels and the percentage decrease of hepatic tyrosine aminotransferase activity. These results suggest that the increased tyrosine levels induced in blood by cyanide may be related to its inhibition of tyrosine aminotransferase activity in the liver. Cyanide decreased hepatic ATP content and increased blood ammonia levels and brain tyrosine in a dose-dependent manner. As it is known that hyperammonaemia increases the uptake of neutral amino acids such as tyrosine into the brain from blood, the mechanism by which tyrosine levels increase in the brain may be based on increases of both tyrosine and ammonia levels in blood.

Platelet-activating factor causes hypoketonaemia in starved rats.

1. The aim of this work was to examine whether platelet-activating factor could mimic the hypoketonaemia seen in septic and endotoxic experimental animals. Platelet-activating factor was administered either by the intraperitoneal (high dose) or intravenous (jugular vein, low dose) routes. 2. Intraperitoneal injection of platelet-activating factor (25 micrograms/kg body weight) decreased the blood ketone body concentration (acetoacetate plus 3-hydroxybutyrate) transiently (30 min after injection) in starved rats. Continuous intravenous infusion of platelet-activating factor (40 ng min-1 kg-1 for 5 h) caused comparable hypoketonaemia. 3. The hepatic acetoacetate concentration also decreased transiently after injection of platelet-activating factor and there was an increase in the 3-hydroxybutyrate/acetoacetate ratio. The hepatic ATP concentration decreased at 15 and 30 min after injection of platelet-activating factor. Infusion of platelet-activating factor caused a similar decrease in hepatic ketone body concentration, but no significant change in the 3-hydroxybutyrate/acetoacetate ratio or adenine nucleotide concentrations. 4. Platelet-activating factor administered by infusion, but not by injection, decreased the plasma non-esterified fatty acid concentration. The plasma glycerol concentration also decreased after infusion of platelet-activating factor, suggesting decreased lipolysis in adipose tissue. 5. Changes in plasma insulin concentration or white adipose tissue blood flow did not appear to contribute to the decrease in the plasma non-esterified fatty acid concentration after infusion of platelet-activating factor. However, there was a significant decrease in blood flow to interscapular brown adipose tissue in both the infused and injected groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in hepatic lipogenic enzyme activities in voles and mice treated with monosodium aspartate

Changes in activities of hepatic lipogenic enzymes, atp citrate lyase and acetyl-CoA carboxylase, were measured in voles and C57BL mice following neonatal administration of monosodium aspartate ( msa). Hepatic lipogenic enzyme activities in voles were considerably lower than those in mice; these low activities were considered to be one of the characteristics of voles as a herbivore. In the msa-treated voles and mice, the plasma insulin concentrations increased significantly. The msa-treated mice showed remarkable obesity and increased lipogenic enzyme activities. In the msa-treated voles, signs of obesity were not observed and hepatic atp citrate lyase activity increased significantly; acetyl-CoA carboxylase activity did not increase.

The toxicological relevance of paracetamol-induced inhibition of hepatic respiration and ATP depletion

In order to elucidate the role of mitochondrial dysfunction in paracetamol-induced hepatotoxicity, the effects of paracetamol on the oxygen consumption and ATP content of the isolated perfused rat liver were correlated with parameters of hepatic viability and hepatotoxicity. Paracetamol at 5 g/L reduced the oxygen consumption of the livers by about 80% and hepatic ATP content by 96%. Hepatotoxicity was evident from the nearly complete interruption of bile secretion, a marked release of enzymes [glutamate-pyruvate transaminase (GPT), lactate dehydrogenase (LDH)] in the perfusate, a depletion of hepatic glutathione and an accumulation of calcium in the liver. Paracetamol-induced hepatotoxicity could be prevented completely by using livers from non-fasted rats as well as by addition of fructose to the perfusate of livers from fasted animals. Both treatments resulted in an increased energy supply from anaerobic glycolysis as evidenced by a large release of lactate and pyruvate into the perfusate, but did not inhibit paracetamol-induced decline of oxygen consumption. The decrease in hepatic oxygen consumption depended on the dose of paracetamol and occurred first at a concentration of 0.2 g/L (−10%). LDH and GPT release, on the other hand, was elevated at 2 and 5 g/L and calcium accumulation occurred at 5 g/L paracetamol only. Inhibition of mixed-function oxidases by dithiocarb did not prevent the decrease in oxygen consumption and the resulting hepatic injury induced by paracetamol. The oral administration of the high dose of 5 g/kg paracetamol in vivo to rats exerted strong hepatotoxicity but produced maximal serum levels of 800 mg/L paracetamol only and did not decrease hepatic oxygen consumption as measured in vitro. Our results show that in the isolated perfused rat liver in vitro, only high concentrations of paracetamol can produce “chemical hypoxia” by attacking mitochondria so as to cause hepatic injury. Such high concentrations of paracetamol are not attained in vivo, however. “Chemical hypoxia”, thus, seems not to be relevant to the well-known hepatotoxic action of paracetamol.

Cardiac and hepatic metabolism in spontaneously hypertensive rats following acute blood loss.

Seven spontaneously hypertensive rats (SHRs) and eight Wistar-St rats were used to assess the influence of hemorrhage on myocardial and hepatic energy metabolism. They received 2% halothane and pancuronium, 0.3 mg.kg(-1), during preparation. After discontinuation of halothane, blood (2 ml.100 g body weight(-1)) was gradually withdrawn over a 5 min period from a femoral artery. Thirty min after induction of hemorrhage, the heart and liver were removed and myocardial and hepatic metabolites (ATP, lactate, pyruvate and glycogen) were measured by the enzymatic methods. Acidosis and decreased hematocrit were noted in the both groups after hemorrhage. Mean arterial pressure (MAP) in SHR was significantly higher than that in Wistar rat before hemorrhage. However, there were no significant differences in MAP and heart rate between the two groups after hemorrhage. Although there were no significant differences in cardiac metabolites, a significant decrease of hepatic ATP and an increase of hepatic lactate/pyruvate ratio were found in SHR when compared with Wistar rat. These results suggest that human hypertensive disease may run a high risk in connection with acute hemorrhage.

Breakdown of hepatic tight junctions during reoxygenation injury.

We investigated whether reoxygenation following anoxia increased biliary permeability and whether or not allopurinol had a protective effect. Isolated rat livers were perfused for 30 min in a one-pass system with buffer equilibrated with 100% nitrogen after stabilization, and then for 60 min with the oxygenated buffer. Hepatic tight junction permeability was assessed by quantifying the early appearance in the bile of horseradish peroxidase (HRP) injected with the perfusate. This early peak represents paracellular passage of HRP, whereas a later second peak results from transcellular passage. In the control livers, 7% of the total HRP passage (93 +/- 50 pg/g liver) was paracellular and 93% was transcellular. After 30 min of reoxygenation following anoxia, however, 516 +/- 20 pg/g liver of HRP passed paracellularly. Addition of allopurinol (5 micrograms/ml) to the perfusate from the start of perfusion reduced paracellular passage of HRP to 219 +/- 49 pg/g liver after anoxia and reperfusion (P less than 0.01). Allopurinol also reduced the cumulative lactate dehydrogenase (LDH) release during the first 30 min of reoxygenation from 2.1 +/- 0.3 x 10(4) to 1.4 +/- 0.4 x 10(4) units/g liver (P less than 0.01). Reduction of the anoxic period from 30 min to 25 min significantly reduced the change in tight junction permeability and the extent of cellular injury: Paracellular passage of HRP was 336 +/- 20 pg/g and LDH release was 0.7 +/- 0.1 x 10(4) units/g liver, both significantly lower than those at 30 min (P less than 0.01). No significant difference in hepatic ATP levels after 60 min of reoxygenation was noted among the experimental groups, but all had lower levels than the control group. The protective effect of allopurinol suggests that the mechanism of biliary reoxygenation injury involves free radical generation. Susceptibility of tight junctions suggests a pattern of injury similar to that involved in anoxic damage of the vascular endothelium.