Different alterations of cytochrome P450 3A4 isoform and its gene expression in livers of patients with chronic liver diseases

AIM: To determine whether parenchymal cells or hepaticcytochrome P450 protein was changed in chronic liverdiseases, and to compare the difference of CYP3A4 enzymeand its gene expression between patients with hepaticcirrhosis and obstructive jaundice, and to investigate thepharmacologic significance behind this difference.METHODS: Liver samples were obtained from patientsundergoing hepatic surgery with hepatic cirrhosis (n=6) andobstructive jaundice (n=6) and hepatic angeioma (controls,n=6). CYP3A4 activity and protein were determined by Nashand western bloting using specific polychonal antibody,respectively. Total hepatic RNA was extracted andCYP3A4cDNA probe was prepared according the methodof random primer marking, and difference of cyp3a4expression was compared among those patients byNorthern blotting.RESULTS: Compared to control group, the CYP3A4 activityand protein in liver tissue among patients with cirrhosis wereevidently reduced. (P＜0.01) Northern blot showed the samechange in its mRNA levels. In contrast, the isoenzyme andits gene expression were not changed among patients withobstructive jaundice.CONCLUSION: Hepatic levels of P450s and its CYP3A4isoform activity were selectively changed in different chronicliver diseases. CYP3A4 isoenzyme and its activity declinedamong patients with hepatic cirrhosis as expression of cyp3a4gene was significantly reduced. Liver's ability to eliminatemany clinical therateutic drug substrates would declineconsequently, These findings may have practical implicationsfor the use of drugs in patients with cirrhosis and emphasizethe need to understand the metabolic fate of therapeuticcompounds. Elucidation of the reasons for these differentchanges in hepatic CYP3A4 may provide insight into morefundamental aspects and mechanisms of imparied liverfunction.

Expression of cytochrome P4502E1 gene in hepatocellular carcinoma

AIM: To investigate cytpchrome P4502E1 (CYP2E1) gene expression in occurrence and progression of hepatocellular carcinoma (HCC). METHODS: The human liver arrayed library was spotted onto the nylon membranes to make cDNA array. Hybridization of cDNA array was performed with labeled probes synthesized from RNA isolated from HCC and adjacent liver tissues. Sprague-Dawley rats were administrated diethyInitrosamine (DENA) to induce HCC. CYP2E1 expression was detected by the method of RT-PCR and Northern blot analysis. RESULTS: CYP2E1 was found by cDNA array hybridization to express differently between HCC and liver tissues. CYP2E1 only expressed in liver, but did not express in HCC tissues and expressed lowly in cirrhotic tissues. In the progression of cirrhosis and HCC, the expression level of CYP2E1 was gradually decreased and hardly detected until the late stage of HCC. CONCLUSION: Using arrayed library to make cDNA arrays is an effective method to find differential expression genes. CYP2E1 is a unique gene expressing in liver but did not express in HCC. CYP2E1 expression descended along with the initiation and progression of HCC, which is noteworthy further investigations in its significance in the development of HCC.

Potential inhibition of cytochrome P450 3A4 by propofol in human primary hepatocytes

AIM: Hepatic cytochrome P450 isoenzymes constitute a superfamily of hemoproteins that play a major role in the metabolism of endogenous compounds and in the detoxification of xenobiotic molecules. P450 3A4 is one of the most important forms in human being, and mediates the metabolism of around 70% of therapeutic drugs and endogenous compounds. Propofol, a widely used intravenous anesthetic drug, is known to inhibit cytochrome P450activities in isolated rat hepatocytes. The goal of this study was to evaluate the potential efficacy of propofol on P4503A4 in a dose-dependent manner to understand its drugdrug interaction.METHODS: Hepatocytes were isolated from liver specimens from hepatic angioma patients undergone hepatic surgery.Primary incubated hepatocytes were treated with 0, 0.01,0.05, 0.1, 0.5, and 1.0 mM propofol for 24 hours. P450 3A4activity was measured with Nash′s colorimetry. The protein expression was assessed by Western blot analysis.RESULTS: A dose-dependent inhibitory effect of propofol was observed in cytochrome P450 3A4 activity. A minimal dosage of propofol (0.01 mM) induced a significant inhibition of P450 3A4 activity, although its regular dosages (0.01-0.1mM) showed no inhibitory effect on the cellular protein expression of P450 3A4.CONCLUSION: Propofol may be a potential CYP3A4 inhibitor as this anesthetic can inhibit isoenzyme activity significantly and reduce the metabolic rate of CYP3A4 substrates. This inhibition occurs at post-expression level, and concentration of propofol used clinically does not affect CYP3A4 protein expression. propofol may thus induce drug interaction of cytochrome P450 3A4 activity at the dosage used clinically.

Isoflurane preserves energy balance in isolated hepatocytes during in vitro anoxia/reoxygenation

AIM: To investigate the protective effect of isoflurane on energy balance in isolated hepatocytes during in vitro anoxia/reoxygenation, and to compare isoflurane with halothane.METHODS: Hepatocytes freshly isolated from fed rats were suspended in Krebs-Henseleit buffer, and incubated in sealed flasks under O2/CO2 or N2/CO2 (95%/5%, V/V)for 30 or 60 min, followed by 5 or 10 min of reoxygenation,with an added volatile anesthetic or not. ATP, ADP, and adenosine monophosphate in hepatocytes were determined by high performance liquid chromatography, and energy charge was calculated.RESULTS: During 30 min of anoxia, the energy charge and total adenine nucleotide steadily increased with the isoflurane dose from 0 to 2 minimum alveolar anesthetic concentration (MAC), then decreased from 2 to 3 MAC.In short incubations (30-35 min) at 1 MAC isoflurane, energy charge modestly decreased during anoxia, which was partially prevented by isoflurane and completely reversed by reoxygenation, and total adenine nucleotide did not decrease. In long incubations (60-70 min), both energy charge and total adenine nucleotide greatly decreased during anoxia, with partial and no reversal by reoxygenation,respectively. Isoflurane partly prevented decreases in both energy charge and total adenine nucleotide during anoxia and reoxygenation. In addition, 1 MAC isoflurane obviously increased ATP/ADP, which could not be changed by 1MAC halothane.CONCLUSION: Isoflurane partially protects isolated hepatocytes against decreases in both energy charge and total adenine nucleotide during short (reversible) or long (irreversible) anoxia.

Effect of insulin on hyperkalemia during anhepatic stage of liver transplantation

AIM: To investigate the effectiveness of insulin on decreasing serum potassium concentration during anhepatic stage of orthotopic liver transplantation.METHODS: Sixteen patients with serum potassium concentrations greater than 4.0 mmol/L at the onset of anhepatic stage were randomized into two groups. The patients in control group (n = 8) received no treatment,while those in treatment group (n = 8) received an intravenous bolus injection of regular insulin (20 U) 10 min into the anhepatic stage, followed by a glucose infusion(500 mL 50 g/L dextrose) over 15 min.RESULTS: In control group, potassium concentration underwent no changes whereas in treatment group, it decreased from 4.8±0.48 mmol/L to 4.19±0.55 mmol/L(mean±SD) within 15 min and to 3.62±0.45 mmol/L 60 min after the therapy. The potassium concentration was lower in treatment group than in control group within 30 min of treatment (3.94+0.57 vs 4.47±0.42 mmol/L,respectively; P<0.05), and increased similarly 30 s after graft reperfusion in both groups of patients, but remained lower in treatment group (5.81±1.78 vs7.44±1.75 mmol/L,respectively; P<0.05). The potassium concentration returned to pre-reperfusion levels within 5 min after graft reperfusion.CONCLUSION: In patients undergoing orthotopic liver transplantation, the administration of insulin rapidly decreases serum potassium concentration even in the absence of the liver, suggesting an important contribution by extrahepatic tissues in insulin-stimulated uptake of potassium.