Hepatic covalent adduct formation with zomepirac in the CD26-deficient mouse

Background and Aims: Zomepirac (ZP), a non-steroidal anti-inflammatory drug (NSAID), has been reported to cause immune-mediated liver injury. In vivo, ZP is metabolized to a chemically reactive acyl glucuronide conjugate (ZAG) which can undergo covalent adduct formation with proteins. Such acyl glucuronide-derived drug-protein adducts may be important in the development of immune and toxic responses caused by NSAID. We have shown using immunoabsorptions that the 110 kDa CD26 (dipeptidyl peptidase IV) is one of the hepatic target proteins for covalent modification by ZAG. In the present study, a CD26-deficient mouse strain was used to examine protein targets for covalent modification by ZP/metabolites in the liver. Methods and Results: The CD26-deficient phenotype was confirmed by immunohistochemistry, flow cytometry analysis, RT-PCR, enzyme assay and immunoblotting. Moreover, by using monoclonal antibody immunoblots, CD26 was not detected in the livers of ZP-treated CD26-deficient mice. Immunoblots using a polyclonal antiserum to ZP on liver from ZP-treated mice showed three major sizes of protein bands, in the 70, 110 and 140 kDa regions. Most, but not all, of the anti-ZP immunoreactivity in the 110 kDa region was absent from ZP-treated CD26-deficient mice. Conclusion: These data definitively showed that CD26 was a component of ZP-modified proteins in vivo. In addition, the data suggested that at least one other protein of approximately 110 kDa was modified by covalent adduct formation with ZAG. (C) 2002 Blackwell Science Asia Pty Ltd.

Hepatic xenobiotic metabolism of cylindrospermopsin in vivo in the mouse

Cylindrospermopsin (CYN) is a hepatotoxin isolated from the blue-green alga Cylindrospermopsis raciborskii. The role of both glutathione (GSH) and the cytochrome P450 enzyme system (P450) in the mechanism of toxicity of CYN has been previously investigated in in vitro systems. We have investigated the role of GSH and P450 in vivo in mice. Mice pre-treated with buthionine sulphoximine and diethyl maleate to deplete hepatic GSH prior to dosing with 0.2 mg/kg CYN showed a seven-day survival rate of 5/13 while the control group rate was 9/14. Dosing mice with 0.2 mg/kg CYN produced a small decrease in hepatic GSH with a characteristic rebound effect at 24 h, The magnitude of this effect is however small and combined with the non-significant difference in survival rates after GSH depletion suggest depletion of GSH by CYN could not be a primary mechanism for CYN toxicity, Conversely, pro-treatment with piperonyl butoxide, a P450 inhibitor, protected mice against CYN toxicity giving a survival rate of 10/10 compared with 4/10 in the control group (p < 0.05 Chi squared) and was protective at doses up to 0.8 mg/kg, suggesting activation of CYN by P450 is of primary importance in the mechanism of action. (C) 2002 Elsevier Science Ltd. All rights reserved.

A model to investigate hepatic extraction of oxygen during anaesthesia in the dog

Measurement of hepatic oxygen extraction was performed on six healthy Greyhound dogs over a two hour period. The Greyhounds were anaesthetised and a right subcostal surgical incision performed. Ultrasonic flow transducers were used to measure flow rate in the hepatic artery and the portal vein. The blood oxygen tensions in arterial blood and in the portal and hepatic veins were also measured. Hepatic oxygen extraction remained stable throughout the study, despite a steady decline in arterial blood pressure. The methodology described in this study provides a direct measure of oxygen uptake by the liver in the dog and could readily be used to investigate hepatic uptake of drugs. (C) 2003 Elsevier Ltd. All rights reserved.

Fatty acid binding protein is a major determinant of hepatic pharmacokinetics of palmitate and its metabolites

Disposition kinetics of [H-3] palmitate and its low-molecular-weight metabolites in perfused rat livers were studied using the multiple-indicator dilution technique, a selective assay for [H-3] palmitate and its low-molecular-weight metabolites, and several physiologically based pharmacokinetic models. The level of liver fatty acid binding protein (L-FABP), other intrahepatic binding proteins (microsomal protein, albumin, and glutathione S-transferase) and the outflow profiles of [H-3] palmitate and metabolites were measured in four experimentalgroups of rats: 1) males; 2) clofibrate-treated males; 3) females; and 4) pregnant females. A slow-diffusion/bound model was found to better describe the hepatic disposition of unchanged [H-3] palmitate than other pharmacokinetic models. The L-FABP levels followed the order: pregnant female > clofibrate-treated male > female > male. Levels of other intrahepatic proteins did not differ significantly. The hepatic extraction ratio and mean transit time for unchanged palmitate, as well as the production of low-molecular-weight metabolites of palmitate and their retention in the liver, increased with increasing L-FABP levels. Palmitate metabolic clearance, permeability-surface area product, retention of palmitate by the liver, and cytoplasmic diffusion constant for unchanged [H-3] palmitate also increased with increasing L-FABP levels. It is concluded that the variability in hepatic pharmacokinetics of unchanged [H-3] palmitate and its low-molecular-weight metabolites in perfused rat livers is related to levels of L-FABP and not those of other intrahepatic proteins.

Identification and characterization of the hepatic stellate cell transferrin receptor

Activated hepatic stellate cells have been implicated in the fibrogenic process associated with iron overload, both in animal models and in human hemochromatosis. Previous studies have evaluated the role of ferritin/ferritin receptor interactions in the activation of stellate cells and subsequent fibrogenesis; however, the role of transferrin in hepatic stellate cell biology is unknown. This study was designed to identify and characterize the stellate cell transferrin receptor and to evaluate the influence of transferrin on stellate cell activation. Identification and characterization of the stellate cell transferrin receptor was determined by competitive displacement assays. The effect of transferrin on stellate cell activation was assessed using western blot analysis for alpha-smooth muscle actin expression, [H-3]Thymidine incorporation, and real-time RT-PCR for procollagen 1(I) mRNA expression. A specific receptor for rat transferrin was observed on activated but not quiescent stellate cells. Transferrin significantly increased the expression of alpha-smooth muscle actin, but caused a decrease in proliferation. Transferrin induced a significant increase in procollagen alpha1(I) mRNA expression. In conclusion, this study has demonstrated for the first time a specific, high affinity receptor for rat transferrin on activated hepatic stellate cells, which via interaction with transferrin regulates stellate cell activation. This suggests that transferrin may be an important factor in the activation of hepatic stellate cells in conditions of iron overload.

In overweight patients with chronic hepatitis C, circulating insulin is associated with hepatic fibrosis: implications for therapy

Background/Aims: Host factors such as increased body mass index (BMI) and genotype-specific viral factors contribute to the development of steatosis in patients with chronic hepatitis C (HCV). We hypothesized that host metabolic factors associated with increased BMI may play a role in disease progression. Methods: Fasting serum was collected from 160 patients with chronic HCV at the time of liver biopsy and 45 age, gender and BMI matched controls, and assessed for levels of insulin, c-peptide and leptin. Results: Patients with viral genotype 3 had more severe steatosis (P = 0.0001) and developed stages 1 and 2 fibrosis at a younger age (P < 0.05) than patients with genotype 1. For both genotypes, overweight patients had significantly more steatosis and increased insulin and leptin levels. In contrast to lean patients, there was a statistically significant increase in circulating insulin levels with increasing fibrosis in overweight patients with chronic HCV (P = 0.03). Following multivariate analysis, insulin was independently associated with fibrosis (P = 0.046) but not inflammation (P = 0.83). There was no association between serum leptin levels and stage of fibrosis. Conclusions: Increasing circulating insulin levels may be a factor responsible for the association between BMI and fibrosis in patients with HCV, irrespective of viral genotype. (C) 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Tumor progression in hepatocellular carcinoma: Relationship with tumor stroma and parenchymal disease

Background: Encapsulation in hepatocellular carcinoma is associated with decreased invasiveness and improved survival in several series. Although active fibrogenesis by myofibroblasts has been demonstrated in the capsule, it is unclear if the capsule results from a general increase in peritumoral fibrosis, or an inherently less invasive tumor phenotype. The relationship between collagen deposition within tumor stroma, presence of cirrhosis and invasiveness also needs clarification. Methods: We performed immunohistochemistry for collagens I, III, IV and VI on sections of encapsulated and non-encapsulated hepatocellular carcinoma, arising in cirrhotic and non-cirrhotic livers. Staining was graded semi-quantitatively in tumor stromal elements and adjacent parenchymal sinusoids. The relationship of this staining with encapsulation, cirrhosis, and vascular invasion was analyzed. Results: Formation of a discrete capsular layer was associated with reduced vascular invasion, but not with a pervasive increase in peritumoral fibrosis. Increased collagen I content of tumor stroma and adjacent parenchymal sinusoids was associated with non-encapsulated tumors and vascular invasion. The presence of cirrhosis had little effect on capsule composition. Conclusions: Encapsulation of hepatocellular carcinoma reflects reduced invasiveness, rather than increased peritumoral collagen synthesis, which may instead enhance invasion. Increased intratumoral collagen I protein is also associated with increased tumor invasiveness. Pre-existing cirrhosis has little effect on tumor progression, possibly because the characteristics of cirrhosis are overwhelmed by tumor-induced changes in the adjacent parenchyma.(C) 2003 Blackwell Publishing Asia Pty Ltd.

Ion-trapping, microsomal binding, and unbound drug distribution in the hepatic retention of basic drugs

This study investigated the relative contribution of ion-trapping, microsomal binding, and distribution of unbound drug as determinants in the hepatic retention of basic drugs in the isolated perfused rat liver. The ionophore monensin was used to abolish the vesicular proton gradient and thus allow an estimation of ion-trapping by acidic hepatic vesicles of cationic drugs. In vitro microsomal studies were used to independently estimate microsomal binding and metabolism. Hepatic vesicular ion-trapping, intrinsic elimination clearance, permeability-surface area product, and intracellular binding were derived using a physiologically based pharmacokinetic model. Modeling showed that the ion-trapping was significantly lower after monensin treatment for atenolol and propranolol, but not for antipyrine. However, no changes induced by monensin treatment were observed in intrinsic clearance, permeability, or binding for the three model drugs. Monensin did not affect binding or metabolic activity in vitro for the drugs. The observed ion-trapping was similar to theoretical values estimated using the pHs and fractional volumes of the acidic vesicles and the pK(a) values of drugs. Lipophilicity and pK(a) determined hepatic drug retention: a drug with low pK(a) and low lipophilicity (e.g., antipyrine) distributes as unbound drug, a drug with high pK(a) and low lipophilicity (e.g., atenolol) by ion-trapping, and a drug with a high pK(a) and high lipophilicity (e.g., propranolol) is retained by ion-trapping and intracellular binding. In conclusion, monensin inhibits the ion-trapping of high pK(a) basic drugs, leading to a reduction in hepatic retention but with no effect on hepatic drug extraction.

Haemodynamic assessment following inferior vena cava resection without replacement

Background: Treatment of bulky retroperitoneal malignancy may require en bloc resection of the infrarenal inferior vena cava. A number of reconstructive options are available to the surgeon but objective haemodynamic assessment of the peripheral venous system following resection without replacement is lacking. The aim of the present paper was thus to determine the symptomatic and haemodynamic effects of not reconstructing the resected infrarenal inferior vena cava. Methods: A retrospective descriptive study was carried out at Princess Alexandra Hospital in Queensland. Five patients underwent resection of the thrombosed infrarenal inferior vena cava as part of retroperitoneal lymph node dissection for testicular cancer (n = 3), radical nephrectomy for renal cell carcinoma (n = 1) and thrombosed inferior vena cava aneurysm (n = 1). Clinical effects were determined via the modified venous clinical severity score and venous disability score. Haemodynamic data were obtained postoperatively using venous duplex ultrasound and air plethysmography. Results: None of the present patients scored >2 (out of 30) on the modified venous clinical severity score or >1 (out of 3) on the venous disability score. Haemodynamic studies showed only minor abnormalities. Conclusions: Not reconstructing the resected thrombosed infrarenal inferior vena cava results in minor signs and symptoms of peripheral venous hypertension and only minor abnormalities on haemodynamic assessment.

Surgical resection of retrobulbar infiltrative lipomas in two dogs

Two dogs were presented for referral with nonpainful exophthalmia due to a retrobulbar mass. Incisional biopsies confirmed a histological diagnosis of infiltrative lipoma in both cases. Infiltrative lipomas are rare tumours of soft tissues and are invasive to adjacent tissue. Wide surgical resection is required to prevent local recurrence of the disease. Computed tomography of the head was performed in both cases to evaluate the extent of the masses and to plan surgical resection. Both dogs underwent a partial orbitectomy and were disease free 24 and 21 months later. The cosmetic appearance was good and both dogs maintained vision in the affected eyes.

Acute cadmium chloride administration induces hepatic and renal CYP2A5 mRNA, protein and activity in the mouse: involvement of transcription factor NRF2

Modulation of the cytochrome P450 (CYP) monooxygenase system by cadmium was investigated in male, adult DBA/2J mice treated with a single dose (16 mumol/kg body weight, i.p.) of cadmium chloride (CdCl2). Total CYP content of liver and kidney microsomes decreased maximally (56% and 85%, respectively) 24 and 18 h, respectively, after CdCl2 treatment. Progressive increases of hepatic coumarin 7-hydroxylase (COH) activity; indicative of CYP2A5 activity, relative to the total CYP content were seen at 8 h (2-fold), 12 h (3-fold), 18 h (12-fold), and 24 h (15-fold). Similar changes were seen in the kidney. Liver and kidney CYP2A5 mRNA levels increased maximally 12 and 4 h after treatment and decreased to almost half 6 h later. In contrast, kidney and liver CYP2A5 protein levels increased maximally at 18 and 24 h. The CYP2A5 mRNA levels in the kidney and liver increased after Cd treatment in Nrf2 +/+ but not in Nrf2 -/- mouse. This study demonstrates that hepatic and kidney CYP2A5 is upregulated by cadmium with a somewhat faster response in the kidney than the liver. The strong upregulation of the CYP2A5 both at mRNA and enzyme activity levels, with a simultaneous decrease in the total CYP concentration suggest an unusual mode of regulation of CYP2A5 in response to cadmium exposure, amongst the CYP enzymes. The observed decrease in the mRNA but not in protein levels after maximal induction may suggest involvement of post-trancriptional mechanisms in the regulation. Upregulation of CYP2A5 by cadmium in the Nrf2 +/+ mice but not in the Nrf2 -/- mice indicates a role for this transcription factor in the regulation. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Effects of cigarette smoking and exposure to cadmium and lead on phenotypic variability of hepatic CYP2A6 and renal function biomarkers in men

Effects of cigarette smoking and exposure to dietary cadmium (Cd) and lead (Pb) on urinary biomarkers of renal function and phenotypic variability of cytochrome P450 2A6 (CYP2A6) were investigated in a group of 96 healthy Thai men with mean age of 36.7 year (19-57 years). In non-smokers, Cd burden increased with age (r = 0.47, P < 0.001). In current smokers, Cd burden increased with both age (r = 0.45, P = 0.01) and number of cigarettes smoked per day (r = 0.32, P = 0.05). Cd-linked renal tubular dysfunction was seen in both smokers and non-smokers, but Pb-linked glomerular dysfunction was seen in smokers only, possibly due to more recent exposure to high levels of Cd and Pb, as reflected by 30-50% higher serum Cd and Pb levels in smokers than non-smokers (P < 0.05). Exposure to dietary Cd and Pb appeared to be associated with mild tubular dysfunction whereas dietary exposure plus cigarette smoking was associated with tubular plus glomerular dysfunction. Hepatic CYP2A6 activity in non-smokers showed a positive association with Cd burden (adjusted P = 0.38, P = 0.006), but it showed an inverse correlation with Pb (adjusted beta = -0.29, P = 0.003), suggesting opposing effects of Cd and Pb on hepatic CYP2A6 phenotype. In contrast, CYP2A6 activity in current smokers did not correlate with Cd or Pb, but it showed a positive correlation with serum ferritin levels (r = 0.45, P = 0.01). These finding suggest that Pb concentrations in the liver probably were too low to inhibit hepatic synthesis of heme and CYP2A6 and that the concurrent induction of hepatic CYP2A6 and ferritin was probably due to cigarette smoke constituents other than the Cd and Pb. (C) 2004 Elsevier Ireland Ltd. All rights reserved.