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ABSTRACT: Ras activation is a frequent event in human hepatocarcinoma that may contribute to resistance towards apoptosis. Salirasib is a ras and mTOR inhibitor that induces a pro-apoptotic phenotype in human hepatocarcinoma cell lines. In this work, we evaluate whether salirasib sensitizes those cells to TRAIL-induced apoptosis. Cell viability, cell death and apoptosis were evaluated in vitro in HepG2, Hep3B and Huh7 cells treated with DMSO, salirasib and YM155 (a survivin inhibitor), alone or in combination with recombinant TRAIL. Our results show that pretreatment with salirasib sensitized human hepatocarcinoma cell lines, but not normal human hepatocytes, to TRAIL-induced apoptosis. Indeed, FACS analysis showed that 25 (Huh7) to 50 (HepG2 and Hep3B) percent of the cells treated with both drugs were apoptotic. This occurred through activation of the extrinsic and the intrinsic pathways, as evidenced by a marked increase in caspase 3/7 (five to ninefold), caspase 8 (four to sevenfold) and caspase 9 (eight to 12-fold) activities in cells treated with salirasib and TRAIL compared with control. Survivin inhibition had an important role in this process and was sufficient to sensitize hepatocarcinoma cells to apoptosis. Furthermore, TRAIL-induced apoptosis in HCC cells pretreated with salirasib was dependent on activation of death receptor (DR) 5. In conclusion, salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis by a mechanism involving the DR5 receptor and survivin inhibition. These results in human hepatocarcinoma cell lines and primary hepatocytes provide a rationale for testing the combination of salirasib and TRAIL agonists in human hepatocarcinoma.
Cell Death & Disease 01/2013; 4:e471. · 5.33 Impact Factor
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ABSTRACT: Aberrant activation of oncogenes, such as Ras, likely contributes to the development of hepatocarcinoma (HCC).
We evaluated in vivo the effect of intraperitoneal injections of the Ras inhibitor S-trans, trans-farnesylthiosalicyclic acid (FTS) on Ras activation and the development of preneoplastic liver lesions in rats receiving weekly diethylnitrosamine (DEN) injections for 16weeks. Western blotting, quantitative PCR, immunohistochemistry, Tunel and caspase activity assays were used.
FTS prevents liver nodule formation and reduces foci expressing the tumour marker GSTp. FTS abrogates DEN-induced Ras membrane activity, increases Tunel positive cells in transformed, GSTp-expressing hepatocytes, up-regulates caspase 3 and 8 activity, induces Fas, Fas ligand and JNK phosphorylation that occurs independently of TNFalpha and Trail. Cytochrome C release, Bax, Bcl2, Bcl-xl, Ki67 and nuclear cyclin D expression is not affected by FTS.
FTS inhibits Ras activation and prevents preneoplastic liver nodule development by inducing apoptosis in transformed hepatocytes through activation of the Fas/Fas ligand system. FTS might be new molecule for HCC treatment.
European journal of cancer (Oxford, England: 1990) 06/2009; 45(11):2050-60. · 4.12 Impact Factor
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ABSTRACT: Hepatocellular carcinoma (HCC) is increasing worldwide and is the fifth main cause of cancer-related death. HCC develops on a preneoplastic organ, the cirrhotic liver. Therefore, chemoprevention could play a role in the therapy of HCC. We evaluated the preventive effects of pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, on the induction of early carcinogenic events. We monitored pre-neoplastic foci induced by a two-stage initiation/promotion model of hepatocarcinogenesis in rats, using diethylnitrosamine and acetylaminofluorene. Pioglitazone treatment was initiated the day after the first diethylnitrosamine injection. By quantitative morphometry and Western blot, we showed that pioglitazone significantly decreases the size of pre-neoplastic foci. Analysis of proliferation and apoptosis, assessed by immunohistochemistry, demonstrated decreased proliferation but no effect on cell death in rats treated with pioglitazone. These events were associated with an increased expression of the cyclin-dependent kinase inhibitor p27(kip1), compared to the non treated group. In conclusion, pioglitazone inhibits early carcinogenic transformation in a two-step rat model. As pioglitazone has a low toxicity profile, we believe it would be interesting to evaluate its effect in chemoprevention of HCC in humans in a clinical setting.
European Journal of Cancer 08/2007; 43(11):1755-63. · 5.54 Impact Factor
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ABSTRACT: Hypervitaminosis A-related liver toxicity may be severe and may even lead to cirrhosis. In the normal liver, vitamin A is stored in hepatic stellate cells (HSC), which are prone to becoming activated and acquiring a myofibroblast-like phenotype, producing large amounts of extracellular matrix.
In order to assess the relationship between vitamin A intake, HSC activation and fibrosis, we studied nine liver biopsies from patients belonging to a well-characterized series of 41 patients with vitamin A hepatotoxicity.
Fibrosis was underlined by Sirius-red staining, whereas activated HSC were immunohistochemically identified using an antibody against alpha smooth muscle actin. The volume density (Vv) of sinusoidal and total fibrosis and of sinusoidal and total activated HSC was quantified by the point-counting method.
Morphology ranged from HSC hypertrophy and hyperplasia as the sole features to severe architectural distortion. There was a significant positive correlation between Vv of perisinusoidal fibrosis and the daily consumption of vitamin A (P=0.004).
The close correlation between the severity of perisinusoidal fibrosis and the daily dose of the retinol intake suggests the existence of a dose-effect relationship.
Liver international: official journal of the International Association for the Study of the Liver 04/2006; 26(2):182-6. · 3.82 Impact Factor
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ABSTRACT: Hypervitaminosis A-related liver toxicity may be severe and may even lead to cirrhosis. In the normal liver, vitamin A is stored in hepatic stellate cells (HSC), which are prone to becoming activated and acquiring a myofibroblast-like phenotype, producing large amounts of extracellular matrix.Aims: In order to assess the relationship between vitamin A intake, HSC activation and fibrosis, we studied nine liver biopsies from patients belonging to a well-characterized series of 41 patients with vitamin A hepatotoxicity.Methods: Fibrosis was underlined by Sirius-red staining, whereas activated HSC were immunohistochemically identified using an antibody against smooth muscle actin. The volume density (Vv) of sinusoidal and total fibrosis and of sinusoidal and total activated HSC was quantified by the point-counting method.Results: Morphology ranged from HSC hypertrophy and hyperplasia as the sole features to severe architectural distortion. There was a significant positive correlation between Vv of perisinusoidal fibrosis and the daily consumption of vitamin A (P=0.004).Conclusion: The close correlation between the severity of perisinusoidal fibrosis and the daily dose of the retinol intake suggests the existence of a dose–effect relationship.
Liver international: official journal of the International Association for the Study of the Liver 02/2006; 26(2):182 - 186. · 3.82 Impact Factor
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ABSTRACT: CYP3A is responsible for the metabolism of numerous endogenous and exogenous compounds. Several substrates of CYP3A have been investigated to assess the CYP3A-metabolizing capacity of an individual in an attempt to predict the rate of metabolism of other CYP3A substrates. Two such tests of CYP3A activity are the midazolam plasma clearance after its intravenous administration and the 6beta-OH cortisol urinary ratio. Possible correlations between these 2 tests were investigated before and after treatment with rifampin in a group of healthy volunteers.
Pharmacokinetic parameters of midazolam and 6beta-OH cortisol urinary ratio were evaluated in 8 volunteers before and after 6 days treatment with rifampin, a potent inducer of CYP3A, and after cessation of rifampin treatment.
Midazolam systemic clearance and the 6beta-OH cortisol urinary ratio were significantly higher at Days 7 and 10 than at Day 0. There was a strong positive correlation between these 2 parameters (r = 0.70, p < 0.001). In contrast, no correlation was observed between the ratio of the AUCs of 1'-OH midazolam vs. midazolam (AUC0-1(1'-OH)/AUC0-t(MDZ)) or the ratio of plasma concentration of 1'-OH midazolam vs. midazolam (C30 min(1'-OH)/C30 min(MDZ)) and the 6beta-OH cortisol urinary ratio (r = 0.05, p = 0.82; r = 0.04, p = 0.88, respectively). Considering only data obtained before or after treatment with rifampin, however, no correlation was observed between midazolam systemic clearance and the 6beta-OH cortisol urinary ratio.
These data demonstrate that there is a strong positive correlation between systemic midazolam clearance and 6beta-OH cortisol urinary ratio before and after induction. This suggests that the 6beta-OH cortisol urinary ratio test is a non-invasive alternative to the use of systemic midazolam clearance for monitoring the time-course of CYP3A induction.
International journal of clinical pharmacology and therapeutics 08/2001; 39(7):293-9. · 1.18 Impact Factor
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ABSTRACT: In rats, partial ligation of portal branches produces atrophy of the deprived lobes and hypertrophy of the intact lobes. The hepatocyte proliferation observed in the nondeprived lobes is viewed as a compensatory hyperplasia, implying that the atrophy somewhat precedes the initiation of the proliferative response. As this has not been demonstrated, the time course and magnitude of those two sequences of events were investigated and compared with the well-defined response to a partial hepatectomy.
The portal branch feeding the anterior liver lobes was ligated in male Wistar rats. One-third and two-thirds partial hepatectomies were also performed. Liver weight, the aminopyrine demethylation rate, an index of the liver mass, the DNA content and various indices of cell proliferation were measured.
Resection of the anterior lobes (PH) or ligation of their portal blood supply (PBL) induced a marked DNA synthesis in the posterior lobes (3H-thymidine incorporation) reaching its maximum 24 h after both interventions. This response can even be accelerated by performing a sham operation 6 h before the PBL. The process leading to DNA synthesis thus seems to start as early after PBL as after a PH, although the weight of the liver or the aminopyrine demethylation rate was nearly unchanged 2 h following PBL. The initiation of the proliferative response clearly precedes and is thus independent of the reduction of the liver mass. On the other hand, the progressive reduction of the liver mass seems to determine the magnitude of the proliferative response, which is, for instance, greatly increased following the excision of the deprived lobes, as late as 10 h after ligation of their portal branches. In comparison with the results obtained after a 113 PH, the peak of DNA synthesis at the 24th hour is greater than predicted by the liver weight loss, but this parameter could underestimate the reduction of the functional liver mass.
The proliferative response following a PBL can be divided into an early phase occurring independently of the reduction of the liver mass and a late phase controlled by this reduction. The paradox of the proliferative response which seems to start before the atrophy to be compensated is resolved by this hypothesis.
Journal of Hepatology 07/2000; 32(6):940-5. · 9.26 Impact Factor
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ABSTRACT: Dietary habits are often considered as a pathogenic factor for fatty liver. The impact of dietary intake and steatosis on drug metabolism remains poorly investigated. Our aim was to assess the effect of dietary intake on in vivo cytochrome P450 (CYP) activities in eleven patients with abnormal liver function tests potentially due to fatty liver and associated with a high-sugar diet. Liver function tests, liver volume, aminopyrine breath test (ABT) and chlorzoxazone (CZ) pharmacokinetics (area under the curve, AUC) which are known to reflect CYP2E1 activity were evaluated before and after 2 months restriction of dietary sugar intake. Features at inclusion were an increased BMI (30.3 (SD 3.2) kg/m2), high hepatic volume (1.96 (SD 0.48) litres), hyperechogenic liver parenchyma, elevated liver enzyme activities (alanine aminotransferase (EC 2.6.1.2) 58.6 (SD 17.4) IU/1 with alanine aminotransferase: aspartate aminotransferase (EC 2.6.1.1) ratio > 1), together with a normal ABT value (0.68 (SD 0.21)% specific activity of administered dose of [14C]aminopyrine in breath after 1 h) and a high CYP2E1 activity (CZ AUC 20.3 (SD 7.1) micrograms/ml per h). A dietary sugar restriction was prescribed. On the basis of repeated interviews by the same dietitian, unaware of any clinical and biochemical data, six patients remained complaint to the diet and exhibited reductions in BMI (P < 0.001), serum alanine aminotransferase (P = 0.008), liver volume (P = 0.002) and CYP2E1 activity (P = 0.007), a significant increase in ABT (P < 0.001) together with the disappearance of liver hyperechogenicity at ultrasound. In contrast, the five non-compliant patients did not show any significant change in any of these variables. In conclusion, CYP2E1 activity is induced in patients with perturbations of liver function tests potentially due to fatty liver. In these patients, effective dietary sugar restriction is associated with a reduction in liver volume, a reduction in CYP2E1 activity and an increased aminopyrine metabolism rate.
British Journal Of Nutrition 10/1999; 82(4):257-62. · 3.01 Impact Factor
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ABSTRACT: We have developed a HPLC method which allows the determination of chlorzoxazone and its hydroxy metabolite in rat liver microsomes and in human plasma. We found that dehalogenated chlorzoxazone or 2-benzoxazolinone was a convenient and stable internal standard. Proteins were precipitated with diluted perchloric acid and the supernatant was extracted with ethyl acetate. Complete resolution of the peaks was achieved within 20 min with a Spherisorb ODS-1 column. The inter-day R.S.D.s were 6.5% at 0.5 microgram/ml of hydroxychlorzoxazone and 5.8% at 1 microgram/ml of chlorzoxazone in human plasma. The reproducibility of the method has been demonstrated for a large number of samples over a long period.
Journal of Chromatography 01/1999; 828(1-2):291-6. · 4.53 Impact Factor
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ABSTRACT: To investigate the effect of watercress on the metabolism of chlorzoxazone, an in vivo probe for CYP2E1, the oral pharmacokinetics of chlorzoxazone was studied in 10 healthy volunteers before and after a single ingestion of a watercress homogenate (50 gm). A third chlorzoxazone pharmacokinetic study was performed after a 1-week treatment with isoniazid (300 mg/day), a well-known CYP2E1 inhibitor. Ingestion of watercress or isoniazid did not affect the oral absorption of chlorzoxazone. The area under the chlorzoxazone plasma concentration-time curve was significantly increased by 56% (p < 0.05) after watercress ingestion and by 135% (p < 0.001) with isoniazid treatment. Similarly, chlorzoxazone elimination half-life was prolonged after watercress (53%; p < 0.05) and isoniazid (104%; p < 0.01) administration. These results show that a single ingestion of watercress inhibits the hydroxylation of chlorzoxazone, an in vivo probe for CYP2E1.
Clinical Pharmacology & Therapeutics 08/1998; 64(2):144-9. · 6.04 Impact Factor
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ABSTRACT: Fatty liver has been associated with an increased risk of primary graft non-function and drug toxicity. However, these effects have been observed mainly in fatty liver with inflammation, a situation characterized by an overall reduction in cytochrome P-450 (CYP)-dependent activities as well as a contrasting increase in CYP2E1 activity. Our aim was to examine the impact of liver-fat accumulation on CYP in two animal models of fatty liver without necroinflammation.
Ducks were force-fed with a high-glucidic diet and male Wistar rats, after 48 h fasting, were refed a high-glucidic, fat-free diet for 48 h. Total CYP, aminopyrine- (AND), erythromycin-N-demethylase (END) and chlorzoxazone hydroxylase (CZOHase) activities as well as CYP2E1 and CYP3A proteins were quantified on microsomal proteins.
Livers from force-fed ducks exhibited significant decreases in total CYP, AND, END and CZOHase activities, inversely correlated with fat-liver content. Refeeding male Wistar rats a high-glucidic, fat-free diet after 48 h fasting, resulting in a 235% increased liver fat content, was associated with a decrease in total CYP (55%), AND (78%), END (55%) and CZOHase (62%) activities as well as in CYP3A (70%) and CYP2E1 (80%) protein content. A significant inverse correlation was observed between CYP and total lipid content.
In these models of steatosis induced by nutritional manipulations, fat liver accumulation was associated with a significant decrease in CYP activities and in CYP protein expression. Furthermore, the decreases in both CYP content and related activities were correlated with the degree of liver fat content.
Journal of Hepatology 03/1998; 28(3):410-6. · 9.26 Impact Factor
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ABSTRACT: Although the monoethylglycinexylidide (MEGX) test defined as a single determination of MEGX plasma concentration after lidocaine injection has been proposed as a liver function test, some discrepancies appeared in assessing the quality of liver donor for transplantation as well as the severity of liver disease. The present study used a severe ischemia-reperfusion liver injury (IRI) in rat to evaluate the various factors able to influence the level of MEGX. The metabolism of lidocaine was studied on microsomes isolated from intact rats and from rats submitted to this liver injury. A significant reduction of the various pathways transforming lidocaine but also MEGX was demonstrated. Lidocaine inhibited the MEGX transformation both in intact and injured liver microsomes. In vivo, plasma MEGX concentrations, determined by high-performance liquid chromatography (HPLC), were lower in IRI than in controls up to 80 minutes after lidocaine injection but not later. By contrast, using the usual commercial fluorescence polarization immunoassay (FPIA), MEGX concentrations were paradoxically higher in IRI than in controls. Moreover, MEGX values obtained using FPIA were threefold higher in controls and ninefold higher in IRI than with HPLC. It was shown that these differences were related to the detection by FPIA of free and mainly of conjugated hydroxy-MEGX that accumulated in plasma from rats submitted to an IRI. These data emphasize the complexity of factors influencing the appearance and disappearance of MEGX because of delayed MEGX formation with liver injury but also to inhibition of its further metabolization. The choice of the sampling time for MEGX determination is critical and has to be optimized in every type of liver injury. Moreover, a specific technique, such as HPLC, will avoid cross-reactivity with other metabolites, which may be particularly abundant when the biliary excretion is impaired.
Hepatology 12/1997; 26(5):1182-8. · 11.66 Impact Factor
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ABSTRACT: A method to determine the activity of the cytochrome P-450 1A1 enzyme, by measuring 7-ethoxyresorufin-O-deethylase (EROD) activity using high-performance liquid chromatography (HPLC) with fluorescence or with visible absorbance detection of resorufin, is described. The lowest quantifiable activity (0.2 pmol/mg min) is obtained by incubation of 0.3 mg of human duodenal microsomal proteins using HPLC fluorescence detection. Using HPLC with visible absorbance detection, sensivity was ten times lower. However, the equipment for this last method is available in most laboratories. The use of both HPLC assays allows determination of the low EROD activity level in samples of small size, such as two or three human duodenal biopsies obtained by routine endoscopy. These methods will be a useful tool to study the role of drug intestinal metabolism by cytochrome P-450 1A1.
Journal of chromatography. B, Biomedical applications 07/1996; 681(2):227-32.
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ABSTRACT: Serum alanine aminotransferase (ALT) level is the most common screening test as part of a routine evaluation of liver damage. In order to determine the factors influencing this liver function test in normal subjects, the relationship between ALT level and gender, age and body mass index (BMI) was studied in a large population of healthy blood donors.
This population included 9,420 volunteer blood donors (4,488 men and 4,932 women aged from 18 to 70 years) selected on the basis of negative answers to a detailed medical questionnaire including past medical history, drug and alcohol consumption, on the absence of clinical signs of liver disease, on the negativity of serological testing for hepatitis B and C virus and HIV.
In the overall population, the mean serum ALT value was 21.8 I.U./L and the mean BMI was 24.4 kg/m2. There was a positive significant correlation between serum ALT level and BMI (Pearson r = 0.54; p < 0.001) and between ALT and age (Pearson r = 0.25; p < 0.001). A major sex-difference in ALT value was observed, the mean ALT value being higher in men than in women (26.8 +/- 13.6 vs. 17.2 +/- 8.1 I.U./L, p < 0.0001). In both sexes, ALT level was significantly correlated with BMI (Pearson r = 0.45 in men and r = 0.37 in women; p < 0.001). In women a consistent rise in BMI and ALT value with increasing age was observed whereas in men BMI and ALT level only increased with age up to the fifth decade.
There was a significant positive correlation between ALT and BMI regardless the gender in a population of healthy volunteer blood donors. Moreover, at the same age and the same BMI, ALT was significantly lower in women than in men suggesting that the normal range for ALT value should be adjusted for gender. So gender and BMI have to be considered in the interpretation of ALT values.
Acta gastro-enterologica Belgica 62(1):16-20. · 0.64 Impact Factor
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ABSTRACT: We report the case of a patient who exhibited severe acute hepatitis with symptomatic cholestasis for more than 3 months and bile duct injury following the prescription of atorvastatin. After withdrawal the drug, the patient's wellbeing slowly improves and biological features normalize in 4 months. Therapy aimed at treating severe liver steatosis and hypercholesterolemia. Atorvastatin is a highly effective 3-hydroxy-3 methylglutamyl- coenzyme A reductase (statin) used to lower low-density lipoprotein. Reported frequent adverse events of the medication include nausea, depression, myalgia, abdominal pain and abnormal liver function tests. Although abnormal liver function tests is not an uncommon side effect of the medication, more serious liver injury is rare. In a recent literature review, about ten cases of serious hepatotoxicity have been documented. In the typical presentation, the duration of exposure prior to hepatic toxicity is variable. Liver injury is generally of the mixed type. A prolonged cholestasis for more than 3 months has been seldom reported. Morphological changes includes canalicular cholestasis, feathery degeneration but no cholangiolitis nor cholangitis under the form of cytological and inflammatory changes at the level of interlobular bile ducts. This case report provides further evidence that among statins, atorvastatin may be implicated in drug-induced liver injury and indicates for the first time that such liver injury may be followed by prolonged cholestasis and interlobular bile duct injury. Atorvastatin has thus to be added to the list of medication potentially responsible for bile duct injury.
Acta gastro-enterologica Belgica 71(3):318-20. · 0.64 Impact Factor
