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Biomedical subjects

G C Farrell

Publications and source records attributed to G C Farrell.

At least 37 records · Page 2Linked to original sources
PubMed1996

Effects of metyrapone on expression of CYPs 2C11, 3A2, and other 3A genes in rat hepatocytes cultured on matrigel.

Hepatocytes cultured on matrigel express many liver-specific functions, but the levels and activities of the predominant male-specific rat hepatic CYPs, 3A2 and 2C11, decline rapidly in culture. Metyrapone maintains the level of total cytochrome P450 of rat hepatocytes in primary culture, but the mechanism underlying this effect has not been completely elucidated. The present study sought to determine whether metyrapone acts solely to stabilise CYP proteins in rat hepatocytes cultured on matrigel, or whether it also influences mRNA levels of the encoding genes. Metyrapone maintained the level of total cytochrome P450 in cultured hepatocytes so that values were > 200% of those found in untreated control cells 24 hr after isolation. At this time, CYP3A2-mediated testosterone 6 beta-hydroxylation was approximately 7-fold higher in hepatocytes cultured in the presence of metyrapone than in control cells, and CYP2C11-dependent testosterone 2 alpha- and 16 alpha-hydroxylation activities were between 2 and 3-fold greater. The results inferred from catalytic activities were supported by immunoquantitation of CYP3A and 2C11 proteins. The trend of increased CYP protein levels in metyrapone-treated cells continued throughout the 48-hr culture period. In control cells, CYP3A2 and 2C11 mRNA levels fell abruptly in culture to reach values at 24 hr that were < 30% of those in freshly isolated cells; addition of metyrapone failed to arrest this fall. However, treatment of cells with metyrapone considerably elevated levels of one or more CYP3A subfamily mRNA species, as detected by a riboprobe based on the cDNA for CYP3A1 ("CYP3A1-like mRNA') that were demonstrated, by another riboprobe, not to be CYP3A2 or RNCYP3AM. RT-PCR of mRNA prepared from cultured hepatocytes, followed by restriction mapping of the cloned cDNAs was used to characterise the CYP3A induced by metyrapone. This revealed that elevated levels of the CYP3A1-like mRNA were attributable to induction of RL33/cDEX mRNA; there were no CYP3A1 cDNAs isolated from these cells. These data are interpreted as indicating that metyrapone stabilises the expression of cytochrome P450 in culture by both pre- and posttranslational mechanisms. The particular mechanism employed is gene-specific, whereby even the highly homologous genes CYP3A2, RL33/cDEX and, possibly, RNCYP3AM are subject to different types of regulation in the presence of metyrapone.

Animals↗
PubMed1996

Impaired bile flow and disordered hepatic calcium homeostasis are early features of halothane-induced liver injury in guinea pigs.

To characterize the early events in liver injury produced by halothane, experiments were performed in genetically susceptible guinea pigs 19 hours after halothane exposure. Serum bile acid concentrations were fourfold increased in halothane-exposed animals compared with controls. In isolated perfused liver experiments, livers from halothane-exposed animals did not differ in hepatic oxygen uptake or in perfusion pressure at the end of experiments, but bile flow and biliary bile salt concentrations were reduced. Hepatic calcium content was increased in halothane-exposed guinea pigs compared with controls, and further experiments were performed to explore the reason for this. As determined by infusion of 45Ca to steady-state perfusate concentrations, hepatic calcium clearance was increased in halothane-exposed guinea pigs compared with controls (0.37 +/- 0.06 vs. 0.28 +/- 0.02 mL/min, P < .01). Decreased biliary excretion of calcium was also noted and was entirely attributable to reduced bile flow. However, although decreased excretion contributed to hepatic accumulation of calcium, it was quantitatively less important than enhanced hepatic uptake. As indicated by passage of a bolus of horseradish peroxidase from perfusate into bile, hepatic tight junction permeability was increased five-fold after halothane exposure. It is concluded that cholestasis, as exemplified by reduced bile flow, is an early feature of the liver injury produced by halothane in guinea pigs and is associated with increased tight junction permeability. Although the decrease in bile flow contributes to an early increase in hepatic calcium content, entry of calcium from the perfusion compartment is quantitatively more important.

Animals↗
PubMed1996

Interferon gamma down-regulates cytochrome P450 3A genes in primary cultures of well-differentiated rat hepatocytes.

Administration of interferons of both the gamma and alfa/beta classes down-regulates hepatic cytochrome P450 (CYP) genes when administered to humans or rats. In male rats, interferons decrease expression of CYP3A2 at a pretranslational level, but because interferons also release other cytokines in vivo, it is unclear whether this is a direct effect on hepatocytes. We therefore examined the effects of rat recombinant interferon gamma (IFN-gamma) on CYP3A2, other 3A genes, and 2C11 in stable primary cultures of male rat hepatocytes. Hepatocytes were cultured on matrigel in Williams' E, and messenger RNAs (mRNAs) for 3A2, 3A1-like CYPs, and 2C11 mRNA were determined by RNase protection assays. CYP3A and 2C11 proteins were immunoquantified, and their catalytic activities were estimated by testosterone hydroxylation pathways. In control cells, 3A2 mRNA decreased initially but then recovered, and stable levels (15% of freshly isolated cells) were attained between days 3 and 7. Phenobarbital increased 3A2 mRNA to 60-120% values of freshly isolated cells, and mRNA for 3A1-like CYPs were increased 20-fold. In both control and phenobarbital-treated hepatocytes, rat recombinant IFN-gamma (33 U/mL) reduced mRNA for 3A2 and 3A1-like CYPs, as well as 3A protein and testosterone 6 beta-hydroxylase activity. Interferon had no effect on CYP2C11 at mRNA or protein levels in untreated cells, although a reduction in 2C11 protein was evident in phenobarbital-treated cultures. It is concluded that interferon directly alters expression of constitutive and inducible CYP3A genes in well-differentiated male rat hepatocytes in culture, but has no effect on constitutive expression of CYP2C11.

Animals↗
PubMed1996

The central role of sinusoidal endothelial cells in hepatic hypoxia-reoxygenation injury in the rat.

The role of individual cell types in hepatic hypoxia-reoxygenation (reperfusion) injury has not been completely defined. We therefore examined the effects of hypoxia and hypoxia-reoxygenation on the viability of rat hepatocytes, Kupffer cells, and sinusoidal endothelial cells (SECs) in primary culture and whether direct exposure to hypoxia followed by reoxygenation activated Kupffer cells. Cultures of hepatocytes (purity > 99%), Kupffer cells (97%), and endothelial cells (> 93%) were established as single-cell types and as cocultures. Hypoxia was achieved by culturing cells under 95% N2/5% CO2, and cell viability was estimated by lactate dehydrogenase (LDH) leakage and Trypan blue exclusion. Kupffer cells and endothelial cells were more resistant to hypoxia than hepatocytes. Following 4-8 hours of hypoxia, reoxygenation accentuated cell death in endothelial cells. In contrast, reoxygenation did not accentuate cell death in hepatocytes or in resting Kupffer cells. The activation of Kupffer cells by the addition of lipopolysaccharide failed to alter their response to hypoxia-reoxygenation. The addition of phorbol myristate acetate to Kupffer cells stimulated the production of superoxide as expected, and the medium from these activated cells augmented the cellular injury of hypoxic hepatocytes. In contrast, hypoxia-reoxygenation did not stimulate Kupffer cells to produce superoxide or other hepatotoxic products. Moreover, Kupffer cells in coculture with hepatocytes did not augment hepatocyte injury after hypoxia-reoxygenation. Likewise, in cocultures of Kupffer cells and SECs, the presence of the Kupffer cell failed to enhance endothelial injury following hypoxia-reoxygenation, and these cocultures did not produce superoxide after reoxygenation. Thus, despite other evidence that Kupffer cells are activated in the intact liver during reperfusion injury, when present in isolation, only endothelial cells possess the innate capacity to undergo hypoxia-reoxygenation injury. Furthermore, changes in oxygen tension alone are not sufficient to activate Kupffer cells to secrete superoxides or other cell products that are toxic to hepatocytes or endothelial cells. It is concluded that SECs play a central role in hypoxia-reoxygenation injury, and the factors that activate Kupffer cells in vivo require further study.

Animals↗
PubMed1996

Two years versus six months of interferon therapy for chronic hepatitis C.

Short-term (end-of-treatment) responses (ETR) to interferon (IFN) therapy for chronic hepatitis C are encouraging; however, the relapse rate is high, and long-term response is obtained in only 12-25% of patients. The Australian Hepatitis C Study Group conducted a trial of 230 patients that compared the standard 3 MU three times a week six-month course of IFN-alpha2b with 5 MU three times a week for six months (5 MU group) or 3 MU three times a week for two years (two-year group). ETR (normalization of serum aminotransferase level until the end of treatment) rates based on an intent-to-treat analysis were 64% for the 5 MU group and 58% for the combined 3 MU groups. After six months of treatment, the overall relapse rate was 71%, and the long-term response (LTR; continued normal aminotransferase until six month follow up) rate did not differ significantly between the 3 MU (17% of all treated, 27% ETR) and 5 MU groups (20% of all treated, 31% ETR). In contrast, among the 46 patients who exhibited an ETR in the two-year group, 27 (59%) had a LTR to IFN, resulting in an overall LTR rate of 33% for all patients treated for up to two years (P < 0.001 compared with 3 MU group). Among these 46 subjects, 11 did not complete the full two-year course, including eight who withdrew due to adverse effects. Nine of these 11 patients had received at least 12 months of therapy. All 18 LTR subjects tested (irrespective of treatment group) were serum HCV-RNA negative at the 12-month follow-up evaluation. Improvement in hepatic inflammation was significantly greater among those treated for two years compared with six months, but there was no reduction in fibrosis score in any group. Among the entire study group, treatment duration, liver histology, and liver function (assessed by antipyrine clearance test) were the only independent predictors of ETR, although HCV genotype was closely related to histological severity (eg, cirrhosis was present in 60% of type 1 and 18% of type 3). Viral load and duration of infection were additional predictors of LTR; however, there were insufficient data to determine whether prolonging treatment beyond six months overcomes the negative impact of these predictors. Continuing IFN therapy for at least 12 months decreases the relapse rate by 50% and thereby improves the LTR rate compared with a six-month treatment course. However, our experience of 24 months of treatment indicates that initial IFN treatment courses of this duration are not well tolerated by approximately 20% (8/46) of patients and are unlikely to improve the results obtained with 12-18 months of treatment.

Chronic Disease↗
PubMed1996

Increased hepatocyte CYP2E1 expression in a rat nutritional model of hepatic steatosis with inflammation.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis is morphologically identical to alcoholic hepatitis and has multiple etiologic associations and an unknown pathogenesis. The present study used a rat nutritional model of hepatic steatosis with inflammation to test the hypothesis that induction of the alcohol-inducible hepatic cytochrome P450 (CYP) 2E1 is associated with production of steatohepatitis. METHODS: Rats received a diet devoid of methionine-choline. CYP2E1 protein was detected in liver sections by immunohistochemistry and in hepatic microsomal fractions by immunoblotting; CYP2E1 activity was detected by N-demethylation of N,N-dimethylnltrosamine (NDMA). CYP2E1 messenger RNA was analyzed by Northern blotting and slot blot hybridization. RESULTS: After 4 weeks of methionine-choline devoid diet, macrovesicular steatosis and an inflammatory infiltrate were prominent in hepatic acinar zone 3. CYP2E1 immunostaining was increased and had a more extensive acinar distribution corresponding to that of the steatosis. Microsomal CYP2E1 protein, NDMA activity, and hepatic CYP2E1 messenger RNA levels were all correspondingly increased. CONCLUSIONS: CYP2E1 is induced, partly at a pretranslational level, in this experimental form of steatohepatitis. The finding of biochemical and histological similarities between this nutritional model of hepatic steatosis with inflammation and alcoholic hepatitis indicates possible clues to common pathogenetic mechanisms. The relevance of this finding to human nonalcoholic steatohepatitis remains uncertain and requires further investigation of human liver specimens.

Animals↗
PubMed1996

Virus and host factors are both important determinants of response to interferon treatment among patients with chronic hepatitis C.

Virus and host factors have both been linked to the response to interferon treatment among patients with chronic hepatitis C but their relative importance and potential interactions are unclear. Hepatitis C virus genotype and level of viraemia were determined in pretreatment sera from 65 Australian patients treated with interferon-alpha 2b (IFN-alpha 2b), 3 MU tiw for 6 months. Hepatitis C viraemia was quantitated by a competitive reverse transcription-polymerase chain reaction (RT-PCR) method and genotype was determined by a line probe assay. By univariate analysis, there were positive associations between initial (short-term) responses to IFN treatment and younger age (P = 0.004), absence of cirrhosis (P = 0.01), and injecting drug use as risk factor for infection (P = 0.05) but not gender, duration of infection, or level of viraemia. Genotype appeared to be important (P = 0.06) but failed to reach statistical significance. By multivariate analysis, absence of cirrhosis was the only significant independent predictor of treatment response (P = 0.01). Among initial responders, the factors associated with long-term response were the pretreatment HCV RNA titre and the duration of infection. There was a close association between viral genotype, but not viral load, and the severity of liver disease. An interplay of factors determines the outcome of a 6-month course of interferon treatment for hepatitis C. Severity of liver disease, but not the viral load, is the most crucial determinant of initial response to interferon, and histological severity appeared to be influenced by the viral genotype. The level of hepatitis C virus (HCV) viraemia and the duration of infection are independent determinants of long-term response by affecting the relapse rate after interferon treatment.

Chronic Disease↗
PubMed1996

Influence of clinicopathological variables on CYP protein expression in human liver.

Drug metabolism is usually impaired in malnourished patients with decompensated cirrhosis, but the separate influence of clinicopathological variables, including nutritional status, on the expression of hepatic cytochrome P450 proteins has not been well characterized. We determined the hepatic content of CYP1A2, CYP2C8/10, CYP2E1 and CYP3A proteins in 71 subjects, 21 with histologically normal livers and 50 with chronic liver disease, and then tested for potential relationships between patient variables and individual CYP proteins by multivariate linear regression analysis. Variables analysed included nutritional status (determined by experienced clinicians), serum albumin and bilirubin concentrations, prothrombin time, the grade of ascites and hepatic encephalopathy, and the Child-Pugh score. Impaired nutrition and cachexia were associated with reductions of CYP2C8/10 levels of approximately 19 and 39%, respectively, relative to cases in which nutrition was replete. Similarly, CYP2E1 protein was reduced by approximately 13 and 26%, according to the apparent severity of nutritional impairment. In contrast, nutritional status did not contribute to variability in expression of CYP1A2 or CYP3A proteins. Of the clinicopathological variables analysed, only serum bilirubin was shown to have an independent influence on CYP protein content. Thus, elevated serum bilirubin concentrations were associated with significant declines in the contents of CYP1A2 and CYP2C8/10 but not CYP3A or CYP2E1. The mechanisms for the effects of nutritional status and serum bilirubin concentration on the levels of CYP proteins are unclear, but could be mediated by factors such as cytokines, dietary composition and alterations in the level of serum bile acids. Knowledge of the influence of clinicopathological factors and nutritional status on CYP expression should lead to more rational drug prescribing in patients with hepatic disease.

Bilirubin↗
PubMed1996

Sequential changes in serum levels of individual bile acids in patients with chronic cholestatic liver disease.

In order to determine the value of serum bile acids in predicting the course of chronic cholestatic liver diseases, we measured individual serum bile acids serially, using high-performance liquid chromatography, over a 4 year observation period in 12 patients with primary biliary cirrhosis and six patients with primary sclerosing cholangitis. The changes in individual serum bile acids and the ratios thereof, conventional liver tests and Child-Turcotte and Mayo scores were compared between survivors (n = 10) and patients who underwent liver transplantation for (n = 3) or died of the liver disease (n = 5). Patients with a serum total chenodeoxycholic acid concentration at study entry that exceded 15 mumol/L were 10 times more likely to die or need a liver transplant in the following 4 years than those with chenodeoxycholic acid levels < 15 mumol/L (P < 0.05). None of the other biochemical parameters or clinicopathological scores could similarly discriminate between the two groups at entry. Time-dependent analyses for the cholic acid/chenodeoxycholic acid ratio, serum total bilirubin and albumin concentrations and Child-Turcotte and Mayo scores were able to differentiate between primary sclerosing cholangitis patients who died or were transplanted and those who were not, whereas age of the patients and other parameters did not. The taurocholic acid/taurochenodeoxycholic acid ratio fell during progression of primary biliary cirrhosis but rose in temporal relationship with primary sclerosing cholangitis. This differential pattern of change was unique compared with other clinical and laboratory indices. In conclusion, serum chenodeoxycholic acid levels and the cholic acid/chenodeoxycholic acid ratio in both diseases were independent indices that allowed for the prediction of survival or the need for liver transplantation. These indices are worthy of further examination in a larger group of patients as prognostic criteria for chronic cholestatic liver disease and in the assessment of the efficacy of therapeutic interventions, including liver transplantations.

Adult↗
PubMed1996

Bile acids produce a generalized reduction of the catalytic activity of cytochromes P450 and other hepatic microsomal enzymes in vitro: relevance to drug metabolism in experimental cholestasis.

In bile duct-ligated male rats, there is a reduction of total hepatic microsomal cytochrome P450 (P450) levels and of NADPH-cytochrome P450 reductase (P450-reductase) activity, but the changes in activity of individual microsomal enzymes are nonuniform. We have proposed that the initial effect of cholestasis on microsomal proteins is a non-specific reduction caused by bile acid-mediated destruction, whereas the disproportionate lowering of male-specific P450 enzymes results from secondary down-regulation of some cytochrome P450 (CYP) genes. We report herein the results of experiments to test the first part of this hypothesis, at least as indicated by enzyme inhibition. Hepatic microsomal fractions from normal male rats were incubated at 37 degrees C with increasing concentrations of a range of bile acids selected for their varying physicochemical properties. The endpoints were catalytic activity of three individual CYP proteins, CYP 2A1 (measured as testosterone 7 alpha-hydroxylase activity), 2C11 (testosterone 2 alpha-hydroxylase and 16 alpha-hydroxylase) and 3A2 (testosterone 6 beta-hydroxylase), and the non-CYP enzymes, steroid 17 beta-dehydrogenase and P450-reductase. With 0.25 mmol/L cholic acid, a concentration exceeded in serum following bile duct ligation, there was a significant reduction in the activity of all enzymes at 4 h. Cholic acid-mediated inhibition was dose-dependent and there was no difference in inhibitory activity towards the male sex-dependent CYP 2C11 and 3A2 and the non-sex-dependent CYP 2A1 and other microsomal enzymes. Taurocholic acid was twice as potent an inhibitor as unconjugated cholic acid, the respective apparent I50 values being approximately 0.6 mmol/L compared with approximately 1.2 mmol/L. The dihydroxy bile acids, chenodeoxycholic acid and deoxycholic acid, were also more potent inhibitors than cholic acid, exhibiting I50 values in the range of 0.3-0.5 mmol/L, but the monohydroxy bile acid, lithocholic acid, was the most potent inhibitor (I50 approximately 0.2 mmol/L). Thus, the inhibitory potential of bile acids towards microsomal enzymes was inversely related to their extent of hydroxylation, while taurine conjugation enhanced the inhibitory potential of cholic acid. These data confirm the potential of bile acids to inhibit the activity of microsomal enzymes in livers of bile ductligated rats and indicate that such changes can occur with concentrations of bile acids that are physiologically relevant. Further, the results are consistent with the proposal that the disproportionately greater reduction of the male sex-dependent CYP, 2C11 and 3A2, is not explained by a destructive mechanism.

Animals↗
PubMed1996

Career paths for clinical scientists.

In the 16 years since James Wyngaarden proclaimed the clinical investigator to be an endangered species, attempts to revive this fragile beast have met with limited success in North America, the UK and Australia. The situation may be more healthy in some western European countries and Japan, but in many parts of Asia clinical investigators have vanished without trace. An analysis of the Australian context during the past 16 years suggests a gradual decline in absolute numbers of clinical scientists reaching maturation, coupled with an extraordinary diminution of their research fertility relative to that of basic scientists. In the present review, it is argued that clinical scientists have a vital role to play in medical research and, particularly, in clinical research. The reason why fewer medical graduates are entering and even fewer are being retained in medical research careers cannot be attributed to restrictions at entry, according to the availability of and competition for training scholarships. Other possible explanations include the late age of entry and the negative influence of role modelling. The latter operates directly by the attitudes and pathways of peers and indirectly through the biases of peer review. There is also a perception, possibly a realistic one, that insuperable barriers exist to obtaining a stable career position at the end of training. Finally, there is real concern about whether clinical investigators will be able to compete successfully with basic researchers for research grants. If this summations is correct, the solutions include active recruitment for research training at an earlier age, simultaneous research and medical training and truncation of postgraduate clinical training in the medical specialties with earlier introduction of medical research. In addition to these strategies, the proper training of clinical scientists must afford them generic research skills and social adaptation to a team approach with basic scientists.

Career Choice↗
PubMed1996

Halothane-induced liver injury in guinea-pigs: importance of cytochrome P450 enzyme activity and hepatic blood flow.

The basis for susceptibility to halothane-induced liver necrosis in guinea-pigs was examined. In hepatic microsomes, the following were similar in susceptible and resistant animals: total cytochrome (CYP) P450 (P450), phenobarbital-inducible pathways of mixed function oxidation (androstenedione 6 beta- and 16 beta-hydroxylase activities) and the CyP2E1-catalysed pathway of N-nitrosodimethylamine N-demethylase activity. Similarly, immunohistochemical staining of CYP2E1 protein was equivalent in livers from susceptible and resistant guinea-pigs. Prior treatment with the P450-inhibitors, metyrapone and SKF-525A ameliorated halothane-induced liver damage in susceptible animals. Conversely, in resistant guinea-pigs, stimulation of hepatic CYP2E1 activity by treatment with 4-methylpyrazole produced severe hepatotoxicity after re-exposure to halothane. These results confirm the conclusions of others, that P450-mediated metabolism produces halothane-induced liver necrosis in the guinea-pig model but, as in other work, the data fail to explain why no difference in activity of these enzymes could be found between susceptible and resistant guinea-pigs. To establish whether a differential effect on hepatic blood flow between susceptible and resistant guinea-pigs could explain this paradox, studies were performed using a radiolabelled microsphere technique. The effect of halothane on lowering cardiac output was identical in both groups of animals and halothane significantly reduced hepatic arterial but not portal blood flow. The effect on arterial blood flow was more profound in susceptible guinea-pigs (0.67 +/- 0.17% of injected microspheres) than in resistant animals (0.99 +/- 0.13%; P < 0.005). It is concluded that P450-catalysed metabolism and reduced hepatic blood flow are both necessary to produce halothane-induced liver injury in susceptible guinea-pigs, but it is the effect of halothane on hepatic arterial blood flow that differs between susceptible and resistant animals.

Animals↗
PubMed1995

Age but not gender selectively affects expression of individual cytochrome P450 proteins in human liver.

Multivariate linear regression analysis was used to examine the influence of age, gender and environmental variables on the hepatic content of cytochromes P450 (CYP, P450) 1A2, 2C, 2E1 and 3A in 71 subjects; 21 with histologically normal livers and 50 with chronic liver disease. There was a clear negative association between age and total P450 content, NADPH-cytochrome c reductase activity and levels of 2E1 and 3A proteins. 1A2 and 2C proteins were unaltered with advancing age. Gender did not influence the expression of any of the CYP proteins. Cigarette smoking was associated with enhanced levels of 1A2, but effects of drug ingestion and alcohol consumption were not apparent in this study, probably because of case selection. It is concluded that age but not gender is a constitutional factor that influences the hepatic content of cytochrome P450 and selected CYP proteins.

Aging↗
PubMed1995

Pre-translational regulation of cytochrome P450 genes is responsible for disease-specific changes of individual P450 enzymes among patients with cirrhosis.

We have recently reported that disease-specific differential alterations in the hepatic expression of xenobiotic-metabolizing cytochrome P450 (CYP P450) enzymes occur in patients with advanced liver disease. In order to determine whether the observed changes in CYP proteins are modulated at pre- or post-translational levels, we have now examined the hepatic levels of mRNA for CYPs 1A2, 2C9, 2E1 and 3A4 by solution hybridization in the same livers of 20 controls (surgical waste from histologically normal livers), 32 cases of hepatocellular and 18 of cholestatic severe chronic liver disease. CYP1A2 mRNA and CYP1A immunoreactive protein were both reduced in livers with hepatocellular and cholestatic types of cirrhosis. In contrast, CYP3A4 mRNA and protein were reduced only in livers from patients with hepatocellular diseases. For 1A2 and 3A4 there were significant correlations between mRNA species and the respective protein contents (rS1A2 = 0.74, rS3A4 = 0.64, P < 0.0001). CYP2C9 mRNA was reduced in patients with both cholestatic and hepatocellular types of liver disease, but 2C protein was reduced only in patients with cholestatic dysfunction. The correlation between CYP2C9 mRNA and protein, was also significant (rs = 0.36, P < 0.005) but mRNA levels accounted for only 13% of the variability in protein rankings. This is probably a consequence of other CYP2C proteins apart from 2C9 being detected by the anti-2C antibody. CYP2E1 mRNA and protein were reduced in patients with cholestatic liver disease, but in hepatocellular disease the expression of only CYP2E1 mRNA was decreased. CYP2E1 mRNA was significantly correlated with CYP2E1 protein but accounted for only 18% of the variability in protein rankings (rs = 0.43, P < 0.0005). Taken collectively these data indicate that the disease-specific alterations of xenobiotic-metabolizing CYP enzymes among patients with cirrhosis is due, at least in part, to pre-translational mechanisms. The lack of a strong correlation between CYP2E1 mRNA and protein suggests that this gene, like its rat orthologue, may be subject to pre-translational as well as translational and/or post-translational regulation.

Cytochrome P-450 Enzyme System↗
PubMed1995

Interferon alfa for chronic active hepatitis B. Long term follow-up of 62 patients: outcomes and predictors of response.

OBJECTIVE: To evaluate the response to treatment with interferon alfa and the long term outcome of patients with chronic active hepatitis B. METHODS: Sixty-two patients with chronic active hepatitis B (43 males, 19 females; age range, 10-67 years) who were treated with interferon alfa at Westmead Hospital between 1984 and 1992 were followed up (mean period of follow-up, 44 months). Thirty-nine patients were treated with interferon alfa-2a and 23 with interferon alfa-2b for a mean of 22.5 weeks. Interferon was given three times a week with a dose range of 3-21 million U. We evaluated pretreatment predictors of response (patient's age, sex, ethnic origin, presence of cirrhosis, serum levels of alanine aminotransferase [ALT] and hepatitis B virus DNA [HBV-DNA]) and the effect of dose and type of interferon. RESULTS: Nine patients had a complete response to treatment with interferon alfa (loss of hepatitis B surface antigen), 26 had a partial response (permanently HBV-DNA negative, hepatitis B e antigen to anti-hepatitis Be seroconversion), eight had a transient response and 19 had no response. All patients with a complete response had normal ALT levels at last follow-up. Histological evidence of hepatic inflammation was significantly reduced in responders. A high pretreatment ALT level and a low HBV-DNA titre were both positive predictors of a favourable response. We found no significant difference in the response to different types of interferon or to high or low dose regimens, or in the responses of patients with cirrhosis. CONCLUSION: Treatment with interferon alfa was associated with prolonged suppression of HBV replication in over half these patients and 14% appear to have been cured of the infection. Suppression of HBV replication is associated with sustained abatement of liver disease.

Adult↗
PubMed1995

Effects of extracellular Ca2+ and HCO3- on epidermal growth factor-induced DNA synthesis in cultured rat hepatocytes.

BACKGROUND/AIMS: The elevation of cytosolic free calcium concentration ([Ca2+]i) and intracellular pH mediate the growth factor-initiated proliferation of many cells, but it is not known if they trigger mitosis in resting hepatocytes. The maintenance of [Ca2+]i and intracellular pH depends partly on extracellular calcium concentration ([Ca2+]e) and extracellular bicarbonate concentration ([HCO3-]e). Therefore, the effects of [Ca2+]e and [HCO3-]e on hepatocyte proliferation were examined. METHODS: Epidermal growth factor induced proliferation in primary cultures of rat hepatocytes. [3H]thymidine incorporation into DNA and nuclear labeling indices were measured. RESULTS: Between 0.2 and 0.9 mmol/L of [Ca2+]e, the proliferative response to epidermal growth factor increased, and total hepatocellular Ca2+ content was increased. Increasing [HCO3-]e also stimulated DNA synthesis in a concentration-dependent manner, maximal at 35 mmol/L. Using optimal [Ca2+]e (0.9 mmol/L) and [HCO3-]e (35 mmol/L), a synergistic stimulation of hepatocellular DNA synthesis was shown. Voltage-dependent Ca2+ channel blockers failed to inhibit hepatocyte proliferation when administered in concentrations that inhibit proliferation in other cell types. CONCLUSIONS: [Ca2+]e and [HCO3-]e are both essential for hepatocyte proliferation, and their effects are synergistic. The entry of extracellular Ca2+ is critical for epidermal growth factor-induced DNA synthesis in hepatocytes, but this is not mediated by voltage-dependent Ca2+ channels.

Animals↗
PubMed1995

Pretranslational down-regulation of cytochromes P450 2C11 and 3A2 in male rat liver by tumor necrosis factor alpha.

BACKGROUND & AIMS: The cytokine tumor necrosis factor alpha (TNF-alpha) is a primary inflammatory mediator after liver injury. Several cytokines impair the regulation of cytochrome P450 (CYP) genes in liver, but the specificity of these effects remains unclear. This study investigated the effects of recombinant murine TNF-alpha on the expression of specific constitutive CYPs in male rat liver. METHODS: Microsomal steroid hydroxylation was used to indicate the activities of specific CYPs after TNF-alpha treatment and immunoblotting to correlate CYP activities with protein contents. CYP messenger RNA levels were measured by solution hybridization. RESULTS: Testosterone 2 alpha/16 alpha- and 6 beta-hydroxylations, mediated respectively by CYPs 2C11 and 3A2, were decreased after TNF-alpha treatment, whereas 7 alpha-hydroxylation (CYP 2A1) was unchanged. Similarly, progesterone 2 alpha/16 alpha- (CYP 2C11) and 6 beta-hydroxylations (CYP 3A2), but not 21-hydroxylation (CYP 2C6), were decreased after TNF-alpha treatment. 2C11 and 3A2 apoproteins and messenger RNAs, but not 2A1 apoprotein, were decreased after TNF-alpha treatment; changes in messenger RNAs were evident 4 hours after treatment. CONCLUSIONS: TNF-alpha down-regulates CYPs 2C11 and 3A2 in male rat liver at a pretranslational level, whereas two other constitutive CYPs, 2A1 and 2C6, seem refractory to TNF-alpha. Thus, impaired CYP regulation by TNF-alpha resembles the combined effects of autologous interferons (on 3A2) and interleukins (on 2C11).

Analysis of Variance↗