Hepatic ATP Levels Research Articles

Cocaine-induced liver injury in mice elicits specific changes in DNA ploidy and induces programmed death of hepatocytes.

Liver injury was induced by a single dose (60 mg/kg) of cocaine in male albino Swiss mice untreated or pretreated with phenobarbital (in drinking water 1 gm/L), for 5 days before cocaine administration. One parameter of liver injury, serum isocitrate dehydrogenase activity, showed sharp increases at 24 hr of cocaine treatment; we also noted decrease hepatic levels of ATP, GSH, cytochrome P-450 and NADPH/NADP+ ratio and increases in malondialdehyde concentration. Histopathological study of liver slices showed perivenous and periportal necrosis induced by cocaine in untreated mice and mice pretreated with phenobarbital, respectively. A regenerative postnecrotic response, which peaked at 48 hr, was demonstrated by the appearance of mitotic cells. Mitotic index analysis showed that proliferative cells appear to be unevenly distributed in the hepatic acinus and were mainly located in the vicinity of the damaged acinar region. Genomic DNA ploidy and the distribution of DNA in the phases of the cell cycle were studied in nuclei of isolated hepatocytes. At 12 hr of cocaine administration, both in untreated and phenobarbital-pretreated mice, the following changes were observed: a sharp decrease in tetraploid (4N) cells (40% to 17% and 25% to 6%, respectively) and octoploid (8N) cells (5% to 2% and 2% to 1%, respectively), together with the appearance of a hypodiploid population (13% and 31%, respectively). Hypodiploid population was characterized as apoptotic cells by detection of DNA fragmentation in agarose gel. These results suggest that a significant percentage of cell death induced by cocaine occurs by means of the apoptosis death program. Comparison of the initial values of DNA ploidy with those obtained at 7 days of cocaine administration showed remarkable increases in polyploid populations (4N and 8N) and a decrease in diploid cells (2N), indicating that the process of differentiation occurs when liver restores its functionality.

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.

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.

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)

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.

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.

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.

Does oxygen supply improve graft viability in liver preservation?

While the clinical results of orthotopic liver transplantation have greatly improved, the viability of liver grafts and extension of the safe time for preservation are necessary factors in need of improvement. The liver is one of the organs most sensitive to anoxia. The addition of an oxygen carrying agent to the preservation solution was evaluated. Pyridoxalated hemoglobin-polyoxyethylene conjugate (PHP) is used as an oxygen carrier. Viaspan (UW) served as a control solution. Test solution (PHP+UW) composition was composed of a 1:1 mixture of PHP and UW solutions with hemoglobin 4.0g%, hydroxyethyl starch 2.5g%, osmolality 320 mOsm/kg H2O, and colloidal osmotic pressure 33 mmHg. The oxygen carrying capacity of PHP+UW solution is about 10 times higher than UW solution at 4 degrees C. Male Lewis rats (BW: 250-300 g) were divided into five groups. After flushing the solution via the portal vein, rat livers were harvested. Two preservation methods, simple storage and perfusion (0.1 ml/min/g liver), were studied at 4 degrees C for 24 or 48 hours. OxyHb, MetHb, pO2, pH, Na, K, GOT, and GPT of perfusate, hepatic mitochondrial functions after preservation, and tissue adenine nucleotides by HPLC were measured. Light microscopy on the tissue was also performed. No significant differences were noted in perfusate biochemical parameters. Oxygen consumption during the perfusion was significantly higher in the PHP+UW than in the UW group. Hepatic mitochondrial functions and tissue ATP levels were better preserved in perfusion than in simple storage, and in PHP+UW than in UW at 48 hours. The oxygen carrying agent, PHP, can provide significantly higher levels of oxygen to liver grafts and improve graft viability.

The response of the rat liver in situ to bromobenzene— In vivo proton magnetic resonance imaging and 31P magnetic resonance spectroscopy studies

Proton magnetic resonance imaging (MRI) and 31P magnetic resonance spectroscopy (MRS) have been used to study the response of the rat liver in situ to bromobenzene, a classic hepatotoxicant. A localized region of high proton signal intensity was seen in the perihilar region of the liver 24 hr after injection of a sublethal dose of bromobenzene. The signal intensity of the entire liver was increased at 48 hr with a gradual return approaching control values by 120 hr. These results are consistent with acute hepatic edema followed by repair of the damaged tissue. In vivo 31P MRS studies of the same rat livers were performed under conditions whereby localized, quantitative spectra could be obtained without surgical intervention. Initial concentrations of the major endogenous phosphorus-containing metabolites within the livers of control rats were 2.97 ± 0.43 m m for the phosphomonoesters (PME), 2.92 ± 0.56 m m for inorganic phosphate, 11.3 ± 1.0 m m for phosphodiesters (PDE), 4.09 ± 0.54 m m for ATP, and 0.56 ± 0.50 m m for ADP and the intracellular pH was 7.39 ± 0.14 (mean ± SD, n = 10). Bromobenzene was found to cause statistically significant ( p < 0.05) changes in several of these metabolites: a decrease in hepatic ATP levels (20% at 24 hr; 27% at 48 hr), a decrease in PDE levels (15% at 24 hr; 18% at 48 hr), and an increase in the PME (63% at 24 hr; 84% at 48 hr). Both the proton MRI and the 31P MRS changes have an onset of 15–20 hr and maximum effect at 25–60 hr, but the MRS changes returned to normal well before the MRI changes. The decreased ATP levels indicate deleterious effects of bromobenzene on the bioenergetic status of the liver in situ, while the increase PME, due to a selective increase in phosphocholine, suggests the activation of a phosphatidylcholine-specific phospholipase C in response to tissue damage. Trolox C, a potent inhibitor of lipid peroxidation, prevented the bromobenzene-induced hepatic edema (i.e., the increase in proton MRI signal intensity) and the bioenergetic deterioration (i.e., the decrease in ATP levels). However, the bromobenzene-induced increase in PME levels was not prevented by Trolox C. These results indicate that the process of lipid peroxidation plays a significant role in the hepatotoxicity of bromobenzene within the intact animal.

Association between decreased splenic ATP levels and immunodepression: amelioration with ATP-MgCl2

Although it is known that decreased ATP levels in liver and kidney contribute to organ dysfunction after shock, it remains unknown whether there is any association between decreased splenocyte ATP levels and splenic immune functions. Moreover, although studies have shown that ATP-MgCl2 treatment after shock improves hepatic and renal ATP levels and organ function, it remains unknown whether splenocyte ATP levels and immune functions are similarly affected by this agent. To study this, C3H/HeN mice were bled to a mean blood pressure of 35 mmHg, maintained at that pressure for 60 min, resuscitated with shed blood and Ringer lactate, and treated with ATP-MgCl2 (80 mumol/kg) or vehicle (saline). Splenocytes (SPL) were harvested at various intervals after hemorrhage, ATP levels were assessed by 31P nuclear magnetic resonance spectrometry, and functions were determined by measuring proliferative capacity and interleukin (IL)-2, and IL-3 synthesis. Hemorrhage depleted SPL ATP levels to 1.7 +/- 0.8% of control levels. However, there was a significant increase in ATP levels of ATP-MgCl2-treated mice (77 +/- 11%, P less than 0.05) compared with vehicle-treated animals (13 +/- 4.1%) at 1 h after resuscitation. SPL ATP levels returned to control by 2 h after resuscitation in the ATP-MgCl2 group, whereas ATP levels of the vehicle-treated mice remained significantly depressed (P less than 0.05) for up to 12 h after resuscitation. At 1 h after resuscitation, SPL proliferative capacity and IL-2 and IL-3 synthesis were all profoundly depressed in the vehicle-treated group (P less than 0.05 vs. control).(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphocreatine protects ATP from a fructose load in transgenic mouse liver expressing creatine kinase

The effects of an intraperitoneal dose of fructose on hepatic metabolism in transgenic mice expressing creatine kinase in liver were investigated using phosphorus-31 nuclear magnetic resonance (31P-NMR). Transgenic mice were fed diets containing varying amounts of creatine (Cr; 0-12%). It has previously been shown that 31P-NMR spectra of transgenic mice have a peak due to phosphocreatine (PCr), the intensity of which was proportional to the amount of Cr in the diet. No PCr peak was detected in control mice or transgenic mice not fed Cr. In the present study NMR spectra were collected before and for a 1-h recovery period after infusion of 0.15 mmol/10 g body wt fructose. In all mice infusion of fructose resulted in a two- to threefold elevation of phosphomonoesters. In control and non-Cr-fed transgenic mice this was accompanied by a 60% reduction of the inorganic phosphate (Pi) and a 50% fall in ATP. In transgenic mice fed Cr, the extent of reduction of Pi was dependent on the level of PCr and was markedly reduced compared with controls. Falls in Pi of 46, 24, and 6% were detected 12.5 min after fructose infusion in low, intermediate, and high PCr-containing livers, respectively. The presence of PCr also protected hepatic ATP levels from a fructose load. Transgenic mice fed on high or intermediate Cr diets showed no significant loss of ATP. However, livers with low levels of PCr lost ATP during a fructose challenge. From the equilibrium established by creatine kinase, free ADP levels were calculated throughout the fructose dose. Fructose caused a 2.5-fold increase in free ADP. This rise in ADP was independent of the total Cr or whether Pi and ATP were reduced by fructose infusion. These results indicate that an increase in ADP is not sufficient to cause depletion of ATP during a fructose challenge.

A 31P-NMR study on the energy state of rat liver in an experimental model of chronic dietary iron overload

31P-NMR spectroscopy of rat liver perchloric acid extracts was utilized to assess the hepatic energy state in an experimental model of chronic dietary iron over load. Oral administration of iron for a period of 65 days that induces a steady ten-fold increase in hepatic iron concentration causes a significant decrease in the hepatic ATP level not associated with appreciable modifications of ADP and Pi levels. The phosphorylation ratio appears on the average decreased. The values of the energy state parameters revert to the normal if the concentration of iron in the liver is reversed below the critical level upon withdrawal of iron treatment after 45 days for a period of 20 days. The implication of these energy modifications for the pathogenesis of cell damage in the siderosis is discussed.

Protection from chlordecone-amplified carbon tetrachloride toxicity by cyanidanol: Biochemical and histological studies

Chlordecone (CD) pretreatment is well known to greatly potentiate CCl 4 toxicity. Previous work has shown that suppression of hepatocellular regeneration permits an ordinarily limited liver injury to progress in an irreversible manner. Insufficient hepatocellular energy has been proposed as a mechanism for suppressed hepatocellular regeneration. Since cyanidanol reportedly increases cellular ATP, this compound was employed to test the above hypothesis. The present study was designed to investigate the sequential biochemical and histological changes over a time course of 120 hr after CCl 4 administration. Male Sprague-Dawley rats (125–150 g) were maintained on 10 ppm CD diet for 15 days and were challenged with either a standard protocol dose (100 μl/kg) or a low (50 μ/kg, L) dose of CCl 4. Cyanidanol pretreatment at 48, 24, and 2 hr before CCl 4 administration to rats maintained on CD diet resulted in 100 or 70% animal survival, for CCl 4 (L) or the standard dose of CCl 4, respectively. Preliminary studies indicated that neither simultaneous nor subsequent administration of cyanidanol with CCl 4 challenge affords such protection. Prior treatment with cyanidanol and a latency period were found necessary for protection. Without cyanidanol, CD + CCl 4 combination caused 50 and 100% lethality after CCl 4 (L) and the standard dose, respectively, while the same doses of CCl 4 alone did not cause lethal effects. Plasma enzymes (alanine aminotransferase, asparatate aminotransferase, sorbitol dehydrogenase) in control rats showed only moderate and transient increases after CCl 4 challenge. The combination of CD + standard dose of CCl 4 resulted in progressive and marked elevations of all three serum enzymes at all time intervals until the death of animals. Cyanidanol pretreatment resulted in significant decline in the plasma enzyme elevations at later time points. Cyanidanol pretreatment increased hepatic ATP synthesis in control or CD rats. CCl 4 administration to control rats did not alter hepatic ATP levels, while in CD-fed rats hepatic ATP levels were significantly decreased. Cyanidanol pretreatment to CD + CCl 4 combination-treated rats did not significantly prevent the decline in hepatic ATP and glycogen levels. However, in the surviving rats a recovery in these parameters was observed. Light microscopic examination of livers from animals that received CCl 4 alone revealed only marginal cellular injury, at early time points only. However, CCl 4 challenge to rats maintained on CD resulted in progressive injury, characterized by the appearance of ballooned cells, necrotic cells, and cells with lipid droplets in the liver. Cyanidanol pretreatment to these rats caused decreased vacuolation and significantly reduced the progression of liver necrosis. These results show that cyanidanol protects against an irreversibly progressive phase of liver injury and the interactive lethality of CD + CCl 4. In conclusion, the cyanidanol protection of CD-amplified CCl 4 toxicity requires a latency period and appears to be through mechanisms other than interference with the injurious events such as free radical injury, bioactivation, or covalent binding of CCl 4. These findings are consistent with the possibility that protection might be through a facilitation of cellular supply of energy.

Altered hepatic energy status in chlordecone (kepone®)-potentiated ccl 4 hepatotoxicity

Previous studies have demonstrated that increased intracellular calcium, depletion of glycogen, and suppressed hepatocellular division resulting in progression of hepatic lesion without recovery are associated with chlordecone (CD)-potentiated CCl 4 hepatotoxicity. Since these phenomena are indicative of compromised hepatic energy status, the present studies were designed to investigate this possibility. Neither hepatic ATP content nor mitochondrial Mg 2+-ATPase was altered significantly in rats maintained on diets contaminated with either CD (10 ppm), mirex (10 ppm) or phenobarbital (PB; 225 ppm) alone for 15 days. Similarly, CCl 4 (100 μL/kg) administration alone did not alter hepatic ATP levels or mitochondrial Mg 2+-ATPase activity in rats maintained on a normal diet. However, CCl 4 administration to CD pretreated rats resulted in significantly decreased hepatic ATP content as early as 1 hr (36%), and this decrease was irreversibly progressive with time (81% at 6 hr). Oligomycin-sensitive Mg 2+-ATPase was decreased significantly only starting at 6 hr (21%) after CCl 4 administration, indicating that depletion of ATP at early time points was most likely due to rapid utilization consequent to toxic events. CCl 4 administration to mirex or PB pretreated rats resulted in a smaller decrease in ATP levels (18–24%) only at 24 hr, returning to normal levels by 36–48 hr, in accord with rapid recovery from limited liver injury. These findings indicate that CCl 4 administration to CD but not to PB or mirex pretreated rats results in a severely compromised energy status of the liver. The progressive and early depletion of liver ATP and the inhibition of Mg 2+-ATPase in CD + CCl 4 treated rats indicate the association of compromised energy status with altered Ca 2+ homeostasis, depletion of glycogen, and suppressed cell division in CD-potentiated CCl 4 toxicity.

Protective Effects of Verapamil on Ischemia-Induced Hepatic Damage in the Rat

The preventive effects of the calcium channel blocker, verapamil, on ischemic liver damage were studied using a rat total hepatic ischemic model. A marked improvement in the survival was obtained by verapamil administration. Following 90 min of hepatic ischemia, 8 of 9 rats (89%) survived in the verapamil-treated group compared to only a 50% survival rate in the saline-treated control group. Furthermore, 56% of the rats still survived after 120 min of ischemia, while there was no survivor in the control group. The recovery of hepatic ATP level following ischemia was significant in the verapamil-treated group, showing the well-preserved mitochondrial function afforded by verapamil administration.

Effect of oral glutathione on hepatic glutathione levels in rats and mice.

Administration of oral glutathione (GSH) increases hepatic GSH levels in fasted rats, in mice treated with GSH depletors such as diethyl maleate and in mice treated with high doses of paracetamol. An increase in hepatic GSH levels after administration of oral GSH does not occur in animals treated with buthionine sulphoximine, an inhibitor of GSH synthesis. Administration of oral GSH leads to an increase in the concentration of L-cysteine, a precursor of GSH, in portal blood plasma. Oral administration of L-methionine produced a significant decrease of hepatic ATP in fasted rats, but not in fed rats. Administration of N-acetylcysteine or GSH did not affect the hepatic ATP levels. The results show that the oral intake of GSH is a safe and efficient form of administration of its constituent amino acids in cases when GSH synthesis is required to replete hepatic GSH levels.

Phase-related changes in tissue energy reserves during hemorrhagic shock

In view of the well-known fact that the liver is more sensitive to ischemia than skeletal muscle, it was the purpose of the present study to determine the relationship between the hemorrhage-induced changes in plasma glucose and lactate concentrations and the status of the energy reserves of these two tissues. Sprague-Dawley rats were bled to a constant mean arterial blood pressure of 40 mm Hg and held there by removal or reinfusion of blood. The stages of shock defined on the basis of the net blood loss were early compensatory, maximal compensatory, early decompensatory, and late decompensatory phases. The results showed a depletion of hepatic ATP levels which occurred between the early compensatory and maximal compensatory phases of shock, coincident with the most dramatic increases in plasma glucose and lactate seen during the shock protocol. Hepatic ATP levels fell no further through the decompensatory phases of shock while plasma glucose declined to hypoglycemic levels and plasma lactate was maintained at the same high level attained at the maximal compensatory phase. Since hepatic sources of glucose were exhausted by the maximal compensatory phase and hepatic energy stores were depleted to a point which precludes significant gluconeogenesis, the large increase in plasma lactate was probably largely due to loss of the hepatic sink for lactate during this phase of shock. In contrast to the liver, soleus muscle showed no change in the levels of glycogen, ATP, CrP, free creatine, or total creatine compared to time-matched controls in any phase of hemorrhagic shock suggesting the absence of significant muscle ischemia. The possibility that red skeletal muscle may act as a sink for lactate is considered.

Rapid changes in glucose metabolism following the administration of ethionine: its role in regulating hepatic protein synthesis.

The relationship between the changes in portal glucagon, insulin, glucose, and hepatic protein synthesis were investigated during ethionine intoxication. There was a 50% decrease in blood glucose, a seven-fold increase in portal glucagon and a 90% increase in portal insulin, all of which coincided temporally with the inhibition of hepatic protein synthesis. When reversal of ethionine intoxication was initiated with adenine it simultaneously restored blood glucose, insulin, glucagon, and protein synthesis. Protein synthesis could not be adequately restored by glucose, but in this case hepatic ATP levels did not increase. In addition, glucose given by stomach tube prior to ethionine did not prevent the action of ethionine, though it did maintain plasma glucose levels and prevented the decrease in plasma insulin and increase in plasma glucagon. These results show that in vivo regulation of hepatic protein synthesis during ethionine intoxication is not likely to be mediated by portal insulin, glucose, and glucagon.