Effect of the histone deacetylase inhibitor trichostatin A on spontaneous apoptosis in various types of adult rat hepatocyte cultures

Acetylation and deacetylation of histones, catalysed by histone acetyl transferases and histone deacetylases (HDAC), respectively, are known to be involved in gene expression regulation. Here, the effect on the activity and expression of several apoptosis-related proteins of trichostatin A (TSA), a well-known HDAC inhibitor, were studied in short-term (conventional monolayer) and long-term cultured (collagen I gel sandwich cultures and co-cultures) adult rat hepatocytes. No significant effects of TSA on the caspase-3-like activity were seen in rat hepatocytes cultured in a sandwich configuration or in a co-culture with rat liver epithelial cells of primitive biliary origin. In both culture models, the basal level of apoptosis was found to be much lower than in control monolayer cultures. In the latter system, it was found that, after 4 days of culture, TSA decreased the levels of caspase-3 (both proform and p17 fragment) and of the pro-apoptotic protein Bid. No effect of TSA was found on the expression of Bax. As expected, a TSA-mediated increase of acetylated histones H3 and H4 was observed in all culture systems examined. In addition, in the presence of TSA, increased albumin secretion and cytochrome P450 1A1/2 and 2B1-dependent enzyme activities were found in conventional cultures after 7 days. In conclusion, TSA delayed the occurrence of apoptosis and loss of liver specific functions in conventional hepatocyte monolayers. In contrast, in hepatocyte culture models in which spontaneous apoptosis is already minimised through the addition of either extracellular matrix components (sandwich cultures) or non-parenchymal liver cells (co-cultures), TSA did not have any additional anti-apoptotic effect.     

Ginkgolide A contributes to the potentiation of acetaminophen toxicity by Ginkgo biloba extract in primary cultures of rat hepatocytes

The present cell culture study investigated the effect of Ginkgo biloba extract pretreatment on acetaminophen toxicity and assessed the role of ginkgolide A and cytochrome P450 3A (CYP3A) in hepatocytes isolated from adult male Long-Evans rats provided ad libitum with a standard diet. Acetaminophen (7.5-25 mM for 24 h) conferred hepatocyte toxicity, as determined by the lactate dehydrogenase (LDH) assay. G. biloba extract alone increased LDH leakage in hepatocytes at concentrations > or =75 mug/ml and > or =750 mug/ml after a 72 h and 24 h treatment period, respectively. G. biloba extract (25 or 50 mug/ml once every 24 h for 72 h) potentiated LDH leakage by acetaminophen (10 mM for 24 h; added at 48 h after initiation of extract pretreatment). The effect was confirmed by a decrease in [(14)C]-leucine incorporation. At the level present in a modulating concentration (50 mug/ml) of the extract, ginkgolide A (0.55 mug/ml), which increased CYP3A23 mRNA levels and CYP3A-mediated enzyme activity, accounted for part but not all of the potentiating effect of the extract on acetaminophen toxicity. This occurred as a result of CYP3A induction by ginkgolide A because triacetyloleandomycin (TAO), a specific inhibitor of CYP3A catalytic activity, completely blocked the effect of ginkgolide A. Ginkgolide B, ginkgolide C, ginkgolide J, quercetin, kaempferol, isorhamnetin, and isorhamnetin-3-O-rutinoside did not alter the extent of LDH leakage by acetaminophen. In summary, G. biloba pretreatment potentiated acetaminophen toxicity in cultured rat hepatocytes and ginkgolide A contributed to this novel effect of the extract by inducing CYP3A.     

Hepatoprotective and antioxidative effects of total phenolics from Laggera pterodonta on chemical-induced injury in primary cultured neonatal rat hepatocytes

Although Laggera pterodonta as a folk medicine has been widely used for several centuries to ameliorate some inflammatory ailments as hepatitis in China, there have been no studies of the hepatoprotective and antioxidative effects of this plant. In this paper, the hepatoprotective effect of total phenolics from L. pterodonta (TPLP) against CCI₄-, d-GalN-, TAA-, and t-BHP-induced injury was examined in primary cultured neonatal rat hepatocytes. TPLP inhibited the cellular leakage of two enzymes, hepatocyte ASAT and ALAT, caused by these chemicals and improved cell viability. Moreover, TPLP afforded much stronger protection than the reference drug silibinin. Meanwhile, DPPH and superoxide radicals scavenging activities of TPLP were also determined. The present investigation is the first to report chemical-induced injury model in primary cultured neonatal rat hepatocytes and provide evidence for the hepatoprotective and antioxidative effects of L. pterodonta. Neutralizing reactive oxygen species by nonenzymatic mechanisms may be one of main mechanisms of TPLP against chemical-induced hepatocyte injury. Furthermore, The total phenolic content of L. pterodonta and its main component type were quantified, and its principle components isochlorogenic acids were isolated and authenticated. These data support the folkloric uses of L. pterodonta in the treatment of hepatitis.     

Coordinate regulation of UDP-glucuronosyltransferase UGT1A6 induction by 3-methylcholanthrene and multidrug resistance protein MRP2 expression by dexamethasone in primary rat hepatocytes

Concentration-dependent regulation of 3-methylcholanthrene (MC) inducibility of UDP-glucuronosyltransferase UGT1A6 by the synthetic glucocorticoid, dexamethasone (DEX) was studied. Treatment of cultured rat hepatocytes with MC, 0.1, 1, and 10 microM DEX, and MC combined with DEX, resulted in different induction patterns measured in the intact cells compared to that observed in the microsomes prepared from the same cells. DEX treatment in various concentrations caused a concentration-dependent increase in p-nitrophenol (p-NP) conjugation in intact cells (3-, 4-, and 5-fold over control, respectively), and it positively regulated MC induction (4-, 5-, and 6-fold over control, respectively). In contrast, DEX had smaller effect on microsomal p-NP conjugation (115, 200, 220% of control, respectively) and although MC induction was increased significantly by 0.1 microM DEX (520% of control), but higher concentrations of DEX (10 microM) decreased the degree of induction to 410%. Similar results obtained from in vivo experiments showed that at high DEX concentration (100mg/kg), the rate of MC induction (540%) decreased (420%). Permeabilization of the plasma membrane resulted in a 15-fold increase of p-NP conjugation indicating the importance of transport in the rate of overall p-NP elimination, and the induction pattern was similar to that observed in microsomes isolated from cells. Hyper-osmolarity (405 mOsmol/L) led to a 3-fold decrease of p-NP conjugation, the loss of DEX inducibility and reduction of the MRP2 protein level. Our results suggest coordinated regulation of UGT1A6 inducibility and substrate or product transport by DEX.     

A physiologically based biodynamic (PBBD) model for estragole DNA binding in rat liver based on in vitro kinetic data and estragole DNA adduct formation in primary hepatocytes

Estragole has been shown to be hepatocarcinogenic in rodent species at high-dose levels. Translation of these results into the likelihood of formation of DNA adducts, mutation, and ultimately cancer upon more realistic low-dose exposures remains a challenge. Recently we have developed physiologically based biokinetic (PBBK) models for rat and human predicting bioactivation of estragole. These PBBK models, however, predict only kinetic characteristics. The present study describes the extension of the PBBK model to a so-called physiologically based biodynamic (PBBD) model predicting in vivo DNA adduct formation of estragole in rat liver. This PBBD model was developed using in vitro data on DNA adduct formation in rat primary hepatocytes exposed to 1′-hydroxyestragole. The model was extended by linking the area under the curve for 1′-hydroxyestragole formation predicted by the PBBK model to the area under the curve for 1′-hydroxyestragole in the in vitro experiments. The outcome of the PBBD model revealed a linear increase in DNA adduct formation with increasing estragole doses up to 100 mg/kg bw. Although DNA adduct formation of genotoxic carcinogens is generally seen as a biomarker of exposure rather than a biomarker of response, the PBBD model now developed is one step closer to the ultimate toxic effect of estragole than the PBBK model described previously. Comparison of the PBBD model outcome to available data showed that the model adequately predicts the dose-dependent level of DNA adduct formation. The PBBD model predicts DNA adduct formation at low levels of exposure up to a dose level showing to cause cancer in rodent bioassays, providing a proof of principle for modeling a toxicodynamic in vivo endpoint on the basis of solely in vitro experimental data.     

Andrographolide-induced pi class of glutathione S-transferase gene expression via PI3K/Akt pathway in rat primary hepatocytes

Andrographis paniculata is an herb widely used in China, Korea, and India for its anti-hepatotoxic, anti-viral, and anti-inflammatory effects. Andrographolide is the major bioactive diterpene lactone in A. paniculata. The pi class of glutathione S-transferase (GSTP) is one of the phase II biotransformation enzymes. Our previous study indicated that andrographolide upregulates the expression of GSTP. The aim of this study was to investigate the mechanism by which andrographolide induces GSTP gene expression in rat primary hepatocytes. In hepatocytes treated with 40 μM andrographolide, immunoblots showed maximal Akt phosphorylation at 0.5 h and maximal c-jun phosphorylation at 3 h. However, pretreatment with PI3K inhibitors, wortmannin and LY294002, or siPI3K inhibited the andrographolide-induced phosphorylation of c-jun and GSTP protein expression. EMSA showed that pretreatment with wortmannin, LY294002, or siPI3K attenuated the AP-1-DNA-binding activity caused by andrographolide. Results of immunoprecipitation indicated that nuclear c-fos/c-jun heterodimer increases with andrographolide treatment. Addition of antibodies against c-jun and c-fos decreased nuclear protein bound to the AP-1 consensus DNA sequence. In summary, andrographolide induces GSTP gene expression in rat primary hepatocytes through activation of the PI3K/Akt, phosphorylation of c-jun, nuclear accumulation of AP-1, and subsequent binding to the response element in the gene promoter region.     

Effect of naturally occurring phenolic acids on the expression of glutathione S-transferase isozymes in the rat.

Naturally occurring plant phenols, protocatechuic and tannic acids, have been reported to be inhibitors of chemical mutagenesis and carcinogenesis in experimental models. Our previous studies, have shown that these compounds modulate the activity of phases 1 and 2 enzymes in rodents. The aim of the present study was to investigate whether these compounds affect protein levels of rat hepatic and renal glutathione S-transferase (GST) isozymes. Male Wistar rats were treated intraperitoneally with protocatechuic or tannic acid at 50 mg/kg body weight five times during 14 days. 3-Methylcholanthrene (MC) was administered at 20 mg/kg body weight on day 13 (the last treatment with phenolic compounds) and on day 14. Tissues were obtained from rats terminated 24 h after the last treatment. Western blot analysis with specific antibodies showed significant differences in the effect of the phenolic compounds in the liver and kidney. In the liver, protocatechuic acid significantly increased the constitutive GSTmicro, while tannic acid reduced the GSTalpha protein level by 60%. Both plant phenols decreased all classes of constitutive GST isozymes in the kidney including GSTpi, and also the MC-induced GSTalpha and/or pi protein levels. These results, as well as our previous reports, suggest that protocatechuic and tannic acids interfere with the pathways related to xenobiotic toxicities and carcinogenesis. This effect may be important for chemoprotective activity of these plant phenols.     

Influence of isolation procedure,extracellular matrix and dexamethasone on the regulation of membrane transporters gene expression in rat hepatocytes

The influence of the isolation procedure of hepatocytes, extracellular matrix (ECM) configuration and incubation medium supplementation by dexamethasone (DEX) on the cell morphology and on the gene expression of membrane transporters was examined in rat hepatocytes. The mRNA levels were determined using oligonucleotide microarrays, in liver, in suspension and in primary culture in monolayer (CPC), and in collagen gels sandwich (SPC) in absence and presence of DEX (100 and 1000 nM). The results indicated pronounced morphological differences between CPC and SPC in response to DEX demonstrating that the hepatocytes re-formed, as in vivo, multicellular arrays with extensive bile canalicular network only in SPC in presence of DEX. The mRNA levels of membrane transporters were not affected significantly during isolation procedure. However, plating hepatocytes in CPC resulted in a decrease of major basolateral transporters mRNA level whereas mRNA levels of mdr1b and mrp3 were increased (>100-fold). Similar observations were made in SPC in the absence of DEX demonstrating that the ECM configuration alone did not play a critical role in the regulation of membrane transporters. However, adding DEX to the incubation medium in SPC resulted in an up-regulation of mdr2, oatp2 and mrp2 in a concentration-dependent way for the two latter genes, whereas mdr1b and mrp3 expression were maintained to their baseline liver levels. These data suggested therefore that the combination of ECM and DEX supplementation is essential for the formation of the bile canalicular network and is a determinant factor in the regulation of membrane transporters in cultured rat hepatocytes.