Hepatic expression of hepatocyte growth factor gene mRNA in acute liver failure.

Hepatocyte growth factor plays a key role in liver regeneration but the role of liver in its synthesis in acute liver failure is unclear. We therefore measured hepatic expression of hepatocyte growth factor mRNA in this condition in comparison to H3 histone mRNA, a marker of cellular proliferation. Hepatocyte growth factor mRNA levels were quantified by specific RNase protection assay in nine patients with acute liver failure and found to be similar to those in six normal controls. Hepatocyte proliferation, as assessed by H3 histone mRNA expression, was not detected in normal liver but was present in six of nine patients with acute liver failure (P < 0.05) and was not correlated with expression of hepatocyte growth factor mRNA (rs = -0.28). Liver is unlikely to be the source of the high serum hepatocyte growth factor levels observed in acute liver failure.

The human and mouse GATA-6 genes utilize two promoters and two initiation codons.

GATA-6 has been implicated in the regulation of myocardial differentiation during cardiogenesis. To determine how its expression is controlled, we have characterized the human and mouse genes. We have mapped their transcriptional start sites and demonstrate that two alternative promoters and 5' noncoding exons are utilized. Both transcript isoforms are expressed in the same tissue-specific and developmental stage-specific pattern, and their ratio appears similar wherever examined. The more upstream noncoding exon showed a substantial degree of homology between the two mammalian species, suggesting a conserved regulatory function. Moreover, in transfection assays we show that elements within this exon act to promote its transcription. Positive regulatory elements that effect transcription from the more downstream exon were not apparent in this assay, revealing a regulatory distinction between the two promoters. We also demonstrate alternative initiator codon usage in both the human and mouse GATA-6 genes. Both isoforms of the protein are synthesized in vitro regardless of which 5' noncoding exon is present in the RNA, although the larger protein has greater transcriptional activation potential in transfection assays. Thus, GATA-6 function in the cell is controlled by a complex interplay of transcriptional and translational regulation.

Analysis of N-ras gene mutation and p53 gene expression in human hepatocellular carcinomas.

AIM:To study the relationship between N-ras gene mutation and p53 gene expression in the carcinogenesis and the development of human hepatocellular carcinomas (HCC).METHODS:The N-ras gene mutation and the p53 gene expression were analyzed in 29 cases of HCC by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and immunohistochemistry.RESULTS:Thirteen cases of HCCs were p53 positive (44.8%), which showed a rather high Cpercen-tage of p53 gene mutation in Guangxi. The aberrations at N-ras codon 2-37 were found in 79.31% of HCCs and 80.77% of adjacent non-tumorous liver tissues. More than 2 point mutations of N-ras gene were observed in 22 cases (75.86%). Twelve cases (41.37%) of HCCs showed both N-ras gene mutation and p53 gene expression.CONCLUSION:N-ras gene and p53 gene may be involved in the carcinogenesis and the development of HCC.That 38% of HCCs with N-ras gene mutation did not express p53 protein indicates that some other genes or factors may participate in the carcinogenesis and the development of HCC.

Molecular cloning of human GATA-6 DNA binding protein: high levels of expression in heart and gut.

Human GATA-6 has been cloned from foetal heart by a combination of PCR-based methods and cDNA library screening. The 3.8 kbp cDNA has a coding sequence of 1347 bp the 449 aa protein is virtually identical in the two zinc-finger binding domains to other human GATA sequences, but varies considerably in the amino and carboxy terminal regions. The sequence shows greatest similarity to GATA-6-like sequences from rat, mouse, chicken and Xenopus. Northern analysis and in situ hybridisation show that GATA-6 is expressed at high levels in human adult and foetal heart as well as in gut derivatives. It is postulated that GATA-6, in concert with GATA-4, plays a crucial role in the regulation of cardiac differentiation.

Regional cerebral edema and chloride space in galactosamine-induced liver failure in rats.

The pathogenesis of cerebral edema, which is a major complication of fulminant hepatic failure, is poorly understood. In previous studies, increased regional brain water content was observed in rats at an early stage of acute liver failure caused by galactosamine. At a later stage when the animals had developed deep coma, brain water content was reduced, possibly as a result of generalized dehydration. In the present investigation, we have determined brain water content at a late stage of liver failure, 48 hours after galactosamine, in animals that had been maintained in fluid balance by continuous intraperitoneal infusion of glucose solution. In these animals, brain water content, determined from the ratio of wet to dry weight, showed a greater increase than that observed previously at the early stage (hindbrain region [cerebellum, pons, and brain stem] increased by 4.2%; forebrain region increased by 1.4% compared with controls). Regional analysis of brain water, using a tissue-specific gravity method, showed a significant increase in cerebellar gray matter water content. Analysis of chloride space showed the extra fluid to be mainly extracellular in the hindbrain region, but not in the forebrain region. Ultrastructural examination of capillaries in gray matter from cerebellum and cerebrum showed no evidence of gross disruption of the tight junctions. Swelling of the astroglial foot processes was observed in the cerebellar gray matter. These results suggest that both vasogenic and cytotoxic mechanisms of edema formation occur in the brain during liver failure.

Over-expression of GATA-6 in Xenopus embryos blocks differentiation of heart precursors.

Xenopus GATA-6 transcripts are first detected at the beginning of gastrulation in the mesoderm, and subsequent domains of expression include the field of cells shown to have heart-forming potential. In this region, GATA-6 expression continues only in those cells that go on to form the heart; however, a decrease occurs prior to terminal differentiation. Artificial elevation of GATA-6, but not GATA-1, prevents expression of both cardiac actin and heart-specific myosin light chain. This effect is heart-specific because cardiac actin expression is unaffected in somites. Expression of the earlier marker XNkx-2.5 was unaffected and morphological development of the heart was initiated independently of the establishment of the contractile machinery. We conclude that a reduction in the level of GATA-6 is important for the progression of the cardiomyogenic differentiation programme and that GATA-6 may act to maintain heart cells in the precursor state. At later stages, when the elevated GATA-6 levels had decayed, differentiation ensued but the number of cells contributing to the myocardium had increased, suggesting either that the blocked cells had proliferated or that additional cells had been recruited.

Protective effects of Osbeckia octandra against galactosamine and tert-butyl hydroperoxide induced hepatocyte damage.

Ayurvedic and other 'traditional' medical practitioners in Sri Lanka use the mature leaves of the plant Osbeckia octandra for its hepatoprotective properties. In this study the effects of an aqueous extract of Osbeckia octandra against injury induced by D-galactosamine and tert-butyl hydroperoxide (TBH) were investigated in freshly isolated rat hepatocytes. The plant extract (500 micrograms/ml) significantly reduced the inhibition of protein synthesis (as assessed by the incorporation of 14C-leucine into protein) in hepatocytes incubated for 1 h with 10 mM galactosamine by a mean of 25.6 +/- 3.6% and decreased the release of cellular lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) enzyme activities into the medium by 55.3% and 32.8%, respectively. With TBH, the plant extract decreased lipid peroxidation (estimated from malondialdehyde formation) by a mean of 29.9 +/- 1.1% together with a 46.8% and 54.7% decrease in the release of LDH and AST, respectively into the incubation medium. Significant protection was also obtained when the Osbeckia extract was added to the incubation medium up to 30 min after pre-exposure of the hepatocytes to either galactosamine or, to a lesser extent, TBH. The results support the use of Osbeckia as a hepatoprotective agent.

Protective effects of Osbeckia octandra against paracetamol-induced liver injury.

1. Osbeckia octandra is a plant used in traditional medicine to treat jaundice and other liver disorders. In this study, the effects of Osbeckia leaf extract on paracetamol-induced liver injury were investigated both in vivo in mice and in rat hepatocytes in vitro. 2. Oral administration of Osbeckia extract (330 mg/kg) at the same time as paracetamol (450 mg/kg) to mice, resulted in a significant protection (p < 0.05) against liver damage, as assessed by improvements in the blood Normotest (39.1 +/- 1.9 versus 46.3 +/- 2.0 s), total liver glutathione (730 +/- 39 versus 574 +/- 27 micrograms/250 mg liver), plasma aspartate aminotransferase level (916 +/- 225 versus 1965 +/- 291 iu/l), and liver histopathology at 24 h after paracetamol administration. 3. In experiments to assess the direct effects of Osbeckia extract, significant protection was also found in freshly isolated rat hepatocytes against damage induced by 185 microM 2,6-dimethyl N-acetyl p-quinoneimine (2,6-diMeNAPQI, an analogue of NAPQI, the toxic metabolite of paracetamol) in vitro. When Osbeckia extract (500 micrograms/ml) was added to the incubation medium at the same time as 2,6-diMeNAPQI significant changes in cell viability (78.4 +/- 3.3 versus 47.2 +/- 5.8% of control, p < 0.001), cell reduced glutathione (GSH) level (35.0 +/- 3.1 versus 23.8 +/- 1.5%, p = 0.009), and reduced release of lactate dehydrogenase (129.9 +/- 6.6 versus 224.6 +/- 12.1%, p < 0.001) were demonstrated after 1 h incubation as compared with 2,6-diMeNAPQI alone. 4. Significant protection was still obtained against 2,6-diMeNAPQI in vitro when addition of Osbeckia extract was delayed by 20 min. These results indicate that Osbeckia extract can protect against paracetamol-induced liver injury.

Skeletal muscle protease activities are unaltered in cirrhotic rats but altered in response to ethanol and acetaldehyde in vitro.

This study was carried out in an attempt to differentiate between the contribution of liver impairment and direct actions of alcohol in myopathy of alcoholic liver disease. Using an animal model of cirrhosis we have previously shown that protein synthetic potential in muscle was not significantly altered. We therefore investigated the possibility that muscle degradation is increased. Cirrhosis was induced by carbon tetrachloride gavage in male rats receiving phenobarbitone in their drinking water. Controls were given phenobarbitone alone. After 135 days the free, latent and total activities of the lysosomal enzymes cathepsin B and cathepsin D in gastrocnemius muscle were unaffected by the induction of experimental cirrhosis when expressed relative to tissue wet weight, protein or DNA. The non-lysosomal enzyme neutral protease was also measured in gastrocnemius muscle from control and cirrhotic rats. There was no difference between the two groups in the free, latent or total activities. Addition of ethanol and acetaldehyde to the assay mixtures in some cases significantly altered the relative activities of the proteases in latent and free compartments of the cirrhotic tissues. In control tissues a different pattern of response emerged. It is concluded that in cirrhosis, at least in the carbon tetrachloride-induced rat model, there is no change of the activity of cathepsin B and D and the neutral protease activity in gastrocnemius. Small but significant effects of ethanol and its metabolite acetaldehyde on latent and free muscle protease activity were demonstrated.

Effect of supernatants from Kupffer cells stimulated with galactosamine and endotoxin on the function of isolated rat hepatocytes.

Activated Kupffer cells may release substances that are involved in liver injury induced by galactosamine and endotoxin. In the present study Kupffer cells were isolated from rat livers, cultured for 24 hours and incubated with galactosamine, endotoxin (LPS) or tumor necrosis factor-alpha for 4 or 24 hours. The Kupffer cell-conditioned media were than added in separate experiments to freshly prepared isolated rat hepatocytes to determine their cytotoxic effect. No significant effects on the rate of protein synthesis, as assessed by the incorporation of 14C-leucine on lactate dehydrogenase enzyme release from hepatocytes during 1 h incubation was found as compared with conditioned media from control Kupffer cells. In further experiments, Kupffer cells incubated for 4 hours with LPS and galactosamine were shown to produce thromboxane B2 and also the potentially cytoprotective prostaglandins PGE2 and small amounts of prostacyclin measured as 6-keto-PGF1 alpha. It is concluded that under the conditions of the present experiments, factors secreted by cultured Kupffer cells have no cytotoxic effects on isolated rat hepatocytes during short-term incubation.

Metabolic effects of beta-adrenergic receptor blockade in advanced alcoholic cirrhosis.

It has been postulated that the adverse metabolic effects of beta-adrenergic blockade with propranolol in cirrhosis may be related to altered delivery and utilisation of oxygen, particularly in patients with advanced alcoholic liver disease (ALD). Consequently, in 10 patients with decompensated ALD, we assessed (a) systemic and hepatic oxygen delivery (DO2), extraction ratio (%O2E) and consumption (VO2), (b) myocardial VO2 (assessed by the rate-pressure product [RPP], together with full systemic and splanchnic haemodynamics) and (c) hepatic redox state (HRS), measured indirectly by the arterial ketone body ratio (KBR i.e. ratio of acetoacetate/beta-hydroxybutyrate), prior to and following intravenous propranolol (0.1-2 mg/kg). Results are expressed as mean +/- S.E.M. Propranolol reduced DO2 (700 +/- 33 vs. 583 +/- 32 ml/min/m2, p < 0.05) and myocardial VO2 (RPP 72 vs. 58, p < 0.05). The %O2E increased however, (18.5 +/- 1.3 vs. 22.6 +/- 1.6%, p < 0.05), resulting in unaltered systemic VO2 (127 +/- 7.3 vs. 131 +/- 6.9 ml/min/m2, p > 0.10). Similarly hepatic VO2 did not change. KBR was not altered (0.44 +/- 0.08 vs. 0.48 +/- 0.07), and in fact improved in two patients (Child C12 and C13) from 0.17 to 0.34 and 0.12 to 0.27, respectively. In conclusion, the results of this study suggest that an underlying O2 debt exists in patients with advanced alcoholic cirrhosis and that beta-adrenergic blockade with propranolol 'normalises' the O2 supply-consumption relationship resulting in more efficient O2 utilisation without adversely affecting HRS. The mechanism of this action may be related to the antagonism of beta 2-mediated arteriovenous shunting resulting in appropriate blood redistribution.

Synthesis and metabolism of cysteinyl leukotrienes by the isolated pig kidney.

The metabolism and synthesis of cysteinyl leukotrienes by the isolated perfused pig kidney has been investigated. Kidneys were maintained for up to six hours in a recirculating perfusion system by using an oxygenated Krebs-Henseleit buffer containing albumin and the perfluorinated oxygen carrier, FC-43. Perfusion pressure was maintained at 12-13.5 kPa, with perfusion flow rates of 150-250 ml/min resulting in a urine output of between 20-180 ml/hr. Infusion of 3H-LTC4 into the renal artery resulted in rapid and complete metabolism, with the major urinary metabolites comprising LTE4, omega-hydroxy-LTE4, omega-carboxy-LTE4 and N-acetyl-omega-hydroxy-LTE4. The capacity of the isolated kidney to synthesize cysteinyl leukotrienes was monitored by measuring urinary LTE4 excretion; there was a basal urinary excretion of LTE4 (median 43 pg/min, range 8-470 pg/min). Neither lipopolysaccharide or human recombinant tumor necrosis factor alpha had any effect on basal excretion. Treatment with the calcium ionophore A23187, however, resulted in a 38.1 +/- 9.6-fold increase in urinary LTE4 excretion. We conclude that the isolated pig kidney, in the absence of circulating cells, can synthesize cysteinyl leukotrienes in the absence of circulating cells, which can then undergo extensive oxidative metabolism.

Intravenous acetylcysteine in paracetamol induced fulminant hepatic failure: a prospective controlled trial.

OBJECTIVE: To see whether intravenous acetylcysteine would improve outcome in patients with fulminant hepatic failure after paracetamol overdose. DESIGN: A prospective randomised controlled study. SETTING: The Institute of Liver Studies, King's College Hospital, London. PATIENTS: 50 consecutive patients (21 male) aged 16-60 with fulminant hepatic failure after paracetamol overdose who had not previously received acetylcysteine. INTERVENTIONS: Conventional intensive liver care plus either acetylcysteine (25 patients) in the same dose regimen as used early after a paracetamol overdose, except that the infusion was continued until recovery from encephalopathy or death, or an equivalent volume of 5% dextrose (25 patients). MAIN OUTCOME MEASURES: Survival; incidence of cerebral oedema, renal failure, and hypotension requiring inotropic support; liver function as assessed by prolongation of the prothrombin time; and degree of encephalopathy. RESULTS: The rate of survival was significantly higher in the acetylcysteine treated group than in the controls (48% (12/25 patients) v 20% (5/25); p = 0.037, 95% confidence interval for difference in proportions surviving 3% to 53%). Acetylcysteine treated patients had a lower incidence of cerebral oedema (40% (10/25) v 68% (17/25); p = 0.047, 95% confidence interval for difference in incidence 2% to 54%), and fewer developed hypotension requiring inotropic support (48% (12/25) v 80% (20/25); p = 0.018, 95% confidence interval 7% to 57%). Rates of deterioration and recovery of liver function, however, were similar in the two groups. No adverse reactions to acetylcysteine were seen. CONCLUSIONS: Acetylcysteine is safe and effective in fulminant hepatic failure after paracetamol overdose.

Inhibitors of hepatic DNA synthesis in fulminant hepatic failure.

In certain etiological groups of patients with fulminant hepatic failure, poor survival may be due to lack of liver regeneration. In vitro experiments have shown that fulminant hepatic failure serum is cytotoxic to rabbit hepatocytes and inhibits DNA synthesis on short-term incubation with isolated regenerating rat hepatocytes. When fulminant hepatic failure serum is injected into partially hepatectomized rats at the time of maximal DNA synthesis, [3H]thymidine incorporation into hepatic DNA is reduced significantly. The effect is greater with sera obtained from patients with fulminant hepatic failure due to non-A, non-B hepatitis or an adverse drug reaction and is associated with a less than 10,000-dalton fraction. No stimulation of DNA synthesis is observed with injection of the greater than 10,000-dalton serum fraction into normal rats. In preliminary experiments, no increase in epidermal growth factor production has been found in liver failure. Overall, the substances present in fulminant hepatic failure serum appear to be inhibitory rather than stimulatory for liver cell regeneration.

Substantial quantities of the high energy derivative oligophosphoglyceroyl-ATP are located in mitochondria in rat liver.

Perfusion with [8-14C]adenosine demonstrated the likely existence in rat liver of oligophosphoglyceroyl-ATP (OPG-ATP). Purification followed by assay with a new specific 3' phosphodiesterase confirmed this. The quantities present were 5-10-fold those found previously and comparable to total soluble nucleotides. OPG-ATP was also purified from the mitochondrial fraction, shown to co-distribute with succinate dehydrogenase and can be co-purified with an enzyme confined to intermembrane space.

Delayed calcium channel blockade with diltiazem reduces paracetamol hepatotoxicity in mice.

1. Diltiazem (30 mg kg-1 body weight, intraperitoneally) given to mice 9 h after paracetamol (450 mg kg-1, orally) reduced liver damage, as judged by plasma aspartate aminotransferase activity (median 186, range 6-602 IU 1(-1), n = 18 vs 466, range 23-3872 IU 1(-1) in 18 saline-treated controls; P less than 0.05) with comparable reductions in mortality (14% vs 33%, respectively; NS). 2. Regenerative activity, as judged by mitotic figures in tissue removed at 30 h after paracetamol, was significantly higher in mice treated at 9 h with diltiazem (median 0.83 per high power field vs 0.1 in saline-treated controls; P less than 0.05). 3. Diltiazem administered earlier or later than 9 h showed reduced efficacy and in some cases potentiated toxicity, as did nifedipine (40 mg kg-1 in divided doses up to 9 h).