Aidi injection altered the activity of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 in normal and diethylnitrosamine-induced hepatocellular carcinoma in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Aidi injection (ADI), a traditional chinese medicine preparation, is widely used in combination with chemotherapy for the treatment of various malignant tumors, such as hepatocellular carcinoma (HCC). Studies have shown that changes in cytochrome P450 (CYP450) activity in disease states would affect the metabolism of drugs in vivo, especially liver diseases. However, the changes of Aidi injection on the activities of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 in normal and HCC states are still unknown. AIM OF THE STUDY: The cocktail probe drugs method was used to investigate the effects of ADI on the activity of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 in normal and HCC rats. MATERIALS AND METHODS: The HCC rats was induced by diethylnitrosamine (DEN). Then, both normal and HCC rats were randomly divided into 2 groups (n = 6). They were given saline or ADI (10 mL/kg/d, i.p) for 2 weeks, respectively. On the fifteenth day, cocktail probe mixing solution, including metoprolol (10 mg/kg), caffeine (1.0 mg/kg), omeprazole (2.0 mg/kg), midazolam (2.0 mg/kg), chlorzoxazone (4.0 mg/kg) and tolbutamide (0.5 mg/kg), was injected into tail vein of all rats in each group. The blood sample was obtained at specified time. After the protein is precipitated, six probe drugs are analyzed by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). RESULTS: Compared with control group, the activity of CYP3A2 and CYP2E1 was significantly lower in the ADI group. Compared with the model group, the activities of CYP1A2, CYP3A2, CYP2E1, and CYP2C11 enzymes in the ADI model group were significantly reduced. Additionally, the activity of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 enzymes in model group was significantly lower than control group. CONCLUSIONS: ADI can inhibit a lot of CYP450 enzyme, so it may reduce the dosage of chemotherapeutic drugs to reach the required plasma concentration of chemotherapeutic drugs, which is of great significance for the combination of anti-tumor chemotherapeutic drugs and is worthy of further in-depth study and clinical attention.

Prevention of rat liver fibrosis and carcinogenesis by coffee and caffeine.

Coffee has been inversely related to the incidence of human liver disease; however, whether caffeine is the component responsible for the beneficial effects of coffee remains controversial. This study evaluated the beneficial effects of coffee or caffeine in a medium-term bioassay for rat liver fibrosis/carcinogenesis induced by diethylnitrosamine (DEN) and carbon tetrachloride (CCl4). One week after the DEN injection, the groups started to receive conventional coffee, instant coffee or 0.1% caffeine ad libitum for 24 weeks. The groups receiving conventional coffee or caffeine presented a significant reduction in collagen content and mRNA expression of collagen I. The groups receiving instant coffee or caffeine had a significant reduction in the size and area of pre-neoplastic lesions and in the mean number of neoplastic lesions. A significant increase in liver bax protein levels was observed in the groups receiving instant coffee or caffeine as compared to the control group. These data indicate that the most pronounced hepatoprotective effect against fibrosis was observed in the groups receiving conventional coffee and 0.1% caffeine, and the greatest effects against liver carcinogenesis were detected in the groups receiving instant coffee and 0.1% caffeine.

In vivo modulation of iNOS pathway in hepatocellular carcinoma by Nigella sativa.

OBJECTIVE: Nitric oxide (NO) and inducible nitric oxide synthase enzyme (iNOS) have been implicated in various tumors. Hepatocellular carcinoma is a highly aggressive form of solid tumor. The lack of effective therapy necessitates the introduction of novel therapeutic strategies to counter this disease. Nigella sativa (NS) has been shown to have specific health benefits. The aim of this study was to investigate the in vivo modulation of the iNOS pathway by NS ethanolic extract (NSEE) and the implications of this effect as an antitumor therapeutic approach against diethylnitrosamine (DENA)-induced hepatocarcinogenesis. METHODS: Rats were divided into four groups, normal control, NSEE control, cancer control, and NSEE-DENA groups. The diagnosis of cancer was based on alpha-fetoprotein (AFP) levels and histological variations. Serum NO, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels and serum iNOS activity were measured. Liver iNOS expression was investigated by reverse transcriptase (RT)-PCR and western blot assays. RESULTS: Serum AFP, NO, TNF-α, and IL-6 levels and iNOS enzyme activity were significantly increased in rats treated with DENA. Significant up-regulation of liver iNOS mRNA and protein expression was also observed. Subsequent treatment with NSEE significantly reversed these effects and improved the histopathological changes in malignant liver tissue which appeared after treatment with DENA, without any toxic effect when given alone. CONCLUSION: These results provide evidence that attenuation of the iNOS pathway and suppression of the inflammatory response mediated by TNF-α, and IL-6 could be implicated in the antitumor effect of NSEE. As such, our findings hold great promise for the utilization of NS as an effective natural therapeutic agent in the treatment of hepatocarcinogenesis.

Review: Exploring anticarcinogenic agents in a rat hepatocarcinogenesis model--focus on selenium and statins.

In this review, we describe a rat model for chemically induced hepatocarcinogenesis that can be used for studying the anticarcinogenic effects of different agents. In this model the process of carcinogenesis can be followed through the different stages of initiation, promotion and progression. Mechanistic studies of anticarcinogenic agents can be carried out and two examples are given by studies on selenium and statins as anticarcinogenic agents. These compounds suppress cancer via different mechanisms. In the case of selenium the induction of glutathione peroxidase 4 and inhibition of lipid peroxidation might be a part of the anticarcinogenic effect. In the case of statins, the inhibition of ubiquinone synthesis, as well as of the selenium-containing enzyme thioredoxin reductase 1 (TrxR1) might explain their anticarcinogenic properties. Interestingly, also in the case of selenium the inhibited carcinogenesis was associated with reduced TrxR activity, indicating an important role for this enzyme in carcinogenesis.

Effects of catechin-rich green tea on gene expression of gluconeogenic enzymes in rat hepatoma H4IIE cells.

Rat hepatoma H4IIE cells were stimulated with dexamethasone and dibutyryl cAMP to increase gene expressions of gluconeogenic enzymes, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Inclusion of catechin-rich green tea beverage (GTB) in the culture medium reduced the up-regulation of these genes as well as that of hepatocyte nuclear factor 4 alpha (HNF4alpha) gene. GTB was fractionated into chloroform-soluble (Fraction I), ethyl acetatesoluble (Fraction II), methanol-soluble (Fraction III) and residual (Fraction IV) fractions. Fractions II and III containing catechins caused an attenuation of the up-regulated expression of these genes as well as the down-regulation of HNF4alpha gene expression. Fraction IV had a synergistic effect on the up-regulation by dexamethasone/dibutyryl cAMP of the PEPCK gene expression and upregulated HNF4alpha gene expression. These results suggest that GTB down-regulated the expression of the HNF4alpha gene to cause the down-regulated gene expression of gluconeogenic enzymes. One reason why GTB did not down-regulate hepatic PEPCK gene expression in previous animal experiments may be that the component(s) acting to up-regulate PEPCK gene expression was more effective in vivo than in cultured cells.

Water extracts of cabbage and kale inhibit ex vivo H(2)O(2)-induced DNA damage but not rat hepatocarcinogenesis.

The chemopreventive potential of water extracts of the Brassica vegetables cabbage and kale was evaluated by administering their aqueous extracts in drinking water ad libitum to Wistar rats submitted to Ito's hepatocarcinogenesis model (CB group and K group, respectively - 14 rats per group). Animals submitted to this same model and treated with water were used as controls (W group - 15 rats). Treatment with the vegetable extracts did not inhibit (P > 0.05) placental glutathione S-transferase-positive preneoplastic lesions (PNL). The number of apoptotic bodies did not differ (P > 0.05) among the experimental groups. Ex vivo hydrogen peroxide treatment of rat livers resulted in lower (P < 0.05) DNA strand breakage in cabbage- (107.6 +/- 7.8 microm) and kale- (110.8 +/- 10.0 microm) treated animals compared with control (120.9 +/- 12.7 microm), as evaluated by the single cell gel (comet) assay. Treatment with cabbage (2 +/- 0.3 microg/g) or kale (4 +/- 0.2 microg/g) resulted in increased (P < 0.05) hepatic lutein concentration compared with control (0.5 +/- 0.07 microg/g). Despite the absence of inhibitory effects of cabbage and kale aqueous extracts on PNL, these Brassica vegetables presented protection against DNA damage, an effect possibly related to increased hepatic lutein concentrations. However, it must be pointed out that the cause-effect relationship between lutein levels and protection is hypothetical and remains to be demonstrated.

Water extracts of cabbage and kale inhibit ex vivo H2O2-induced DNA damage but not rat hepatocarcinogenesis

The chemopreventive potential of water extracts of the Brassica vegetables cabbage and kale was evaluated by administering their aqueous extracts in drinking water ad libitum to Wistar rats submitted to Ito’s hepatocarcinogenesis model (CB group and K group, respectively - 14 rats per group). Animals submitted to this same model and treated with water were used as controls (W group - 15 rats). Treatment with the vegetable extracts did not inhibit (P > 0.05) placental glutathione S-transferase-positive preneoplastic lesions (PNL). The number of apoptotic bodies did not differ (P > 0.05) among the experimental groups. Ex vivo hydrogen peroxide treatment of rat livers resulted in lower (P < 0.05) DNA strand breakage in cabbage- (107.6 ± 7.8 µm) and kale- (110.8 ± 10.0 µm) treated animals compared with control (120.9 ± 12.7 µm), as evaluated by the single cell gel (comet) assay. Treatment with cabbage (2 ± 0.3 µg/g) or kale (4 ± 0.2 µg/g) resulted in increased (P < 0.05) hepatic lutein concentration compared with control (0.5 ± 0.07 µg/g). Despite the absence of inhibitory effects of cabbage and kale aqueous extracts on PNL, these Brassica vegetables presented protection against DNA damage, an effect possibly related to increased hepatic lutein concentrations. However, it must be pointed out that the cause-effect relationship between lutein levels and protection is hypothetical and remains to be demonstrated.

Biological response of hepatomas to an extract of Fagopyrum esculentum M. (buckwheat) is not mediated by inositols or rutin.

Buckwheat contains d-chiro-inositol (D-CI) and myo-inositol (MI), possible insulin-mimetic compounds; thus, this study investigated the insulin-mimetic activities of a buckwheat concentrate (BWC), D-CI, and MI on insulin signal transduction pathways and glucose uptake with H4IIE rat hepatoma cells. BWC stimulated phosphorylation of p42/44 extracellular-related kinase (p42/44 ERK) and its downstream target, p70(S6K), on Thr(421). In contrast, D-CI, MI, rutin, or its agylcone form, quercetin, did not activate these signal transduction proteins. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), another target of insulin, was also up-regulated upon BWC treatment. The effects of BWC on glucose uptake were subsequently investigated using H4IIE cells. Insulin and D-CI stimulated glucose uptake, whereas BWC inhibited basal and insulin-stimulated glucose uptake. Although results from this work suggest that BWC has insulin-mimetic effects on select protein phosphorylation events in H4IIE cells, D-CI and MI were not the active components responsible for the observed effects. The inhibition of glucose uptake by BWC suggests that buckwheat may affect hepatic glucose metabolism, possibly by inhibiting glucose flux. Furthermore, the fact that D-CI and MI stimulated glucose uptake in H4IIE cells suggests that other compounds are responsible for inhibition of glucose uptake by BWC.

Solanum nigrum L. extract inhibits 2-acetylaminofluorene-induced hepatocarcinogenesis through overexpression of glutathione S-transferase and antioxidant enzymes.

Solanum nigrum L. (SN) is a widespread plant and is regarded as a common relish in the east and the south of Taiwan. Our previous study has found that SN water extract (SNWE) alleviated carbon tetrachloride-induced liver damage in rats. However, the effects of SNWE on chemical-induced hepatic injury and hepatocarcinogenesis remain unclear. Therefore, this study aims to investigate the effects of SNWE on hepatic injury and hepatocarcinogenesis by using 2-acetylaminofluorene (AAF) and AAF/NaNO(2) treatment. The serum biomarkers for hepatic injury, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and gamma-glutamyl transferase, and for hepatocarcinogenesis, alpha-fetoprotein, were determined. Our results showed that AAF treatment led to a significant decrease of body weight and an increase of liver/body weight and serum biomarkers for hepatic injury and hepatocarcinogenesis. Interestingly, the SNWE supplement significantly lowered the liver/body weight and the biomarkers but did not affect the body weight. Further investigation revealed that a SNWE supplement increased the expression of glutathione S-transferase-alpha and -mu, the level of transcription factor for protection from oxidative stress, Nrf2, and the level of downstream targets regulated by Nrf2, including glutathione peroxidase, superoxide dismutase-1, and catalase. Moreover, the effects of SNWE on AAF/NaNO(2)-induced hepatoma were also investigated, and the findings revealed that SNWE suppressed the progression of the hepatoma and resulted in a great increase of the survival rate. Our findings indicate that the SNWE supplement significantly alleviated the AAF-induced hepatic injury and early hepatocarcinogenesis as well as the AAF/NaNO(2)-induced lethal hepatoma, which may result from the overexpression of glutathione S-transferases, Nrf2, and antioxidant enzymes.

Green and black tea extracts inhibit HMG-CoA reductase and activate AMP kinase to decrease cholesterol synthesis in hepatoma cells.

Recent studies have demonstrated that green and black tea consumption can lower serum cholesterol in animals and in man, and suppression of hepatic cholesterol synthesis is suggested to contribute to this effect. To evaluate this hypothesis, we measured cholesterol synthesis in cultured rat hepatoma cells in the presence of green and black tea extracts and selected components. Green and black tea decreased cholesterol synthesis by up to 55% and 78%, respectively, as measured by a 3-h incorporation of radiolabeled acetate. Inhibition was much less evident when radiolabeled mevalonate was used, suggesting that the inhibition was mediated largely at or above the level of HMG-CoA reductase. Both extracts directly inhibited HMG-CoA reductase when added to microsomal preparations, although the extent of inhibition was considerably less than the decrease in cholesterol synthesis observed in whole cells. As HMG-CoA reductase activity also can be decreased by enzyme phosphorylation by AMP kinase, the phosphorylation state of HMG-CoA reductase and AMP kinase, which is activated by phosphorylation, was determined in lysates from cells treated with tea extracts. Both extracts increased AMP-kinase phosphorylation and HMG-CoA reductase phosphorylation by 2.5- to 4-fold, but with different time courses: maximal phosphorylation with green tea was evident within 30 min of treatment, whereas with black tea phosphorylation was slower to develop, with maximal phosphorylation occurring > or =3 hours after treatment. These results suggest that both green and black tea decrease cholesterol synthesis in whole cells by directly inhibiting HMG-CoA reductase and by promoting its inactivation by AMP kinase.

Black tea polyphenols modulate xenobiotic-metabolizing enzymes, oxidative stress and adduct formation in a rat hepatocarcinogenesis model.

The present study was designed to investigate the modulatory effects of black tea polyphenols (Polyphenon-B) on phase I and phase II xenobiotic-metabolizing enzymes and oxidative stress in a rat model of hepatocellular carcinoma (HCC). Liver tumours induced in male Sprague-Dawley rats by dietary administration of rho-dimethylaminoazobenzene (DAB) increased cytochrome P450 (total and CYP1A1, 1A2 and 2B isoforms), cytochrome b(5), cytochrome b(5) reductase, glutathione S-transferase (GST total and GST-P isoform) and gamma-glutamyltranspeptidase (GGT) with decrease in quinone reductase (QR). This was accompanied by enhanced lipid and protein oxidation and compromised antioxidant defences associated with increased expression of the oxidative stress markers 4-hydroxynonenal (4-HNE), anti-hexanoyl lysine (HEL), dibromotyrosine (DiBrY) and 8-hydroxy 2-deoxyguanosine (8-OHdG). Dietary administration of Polyphenon-B effectively suppressed DAB-induced hepatocarcinogenesis, as evidenced by reduced preneoplastic and neoplastic lesions, modulation of xenobiotic-metabolizing enzymes and amelioration of oxidative stress. Thus, it can be concluded that Polyphenon-B acts as an effective chemopreventive agent by modulating xenobiotic-metabolizing enzymes and mitigating oxidative stress in an in vivo model of hepatocarcinogenesis.

[Expression of MT1-MMP and its significance in rabbit VX2 tumor tissues after transarterial embolization with hydroxyapatite nanoparticles].

OBJECTIVE: To study location of MT1-MMP and effect of its change in expression on rabbit VX2 tumor tissues after transarterial embolization with hydroxyapatite nanoparticles loaded with lipiodol. METHODS: Sixty rabbits implanted with tumor tissue of cell line VX2 were divided into three groups (control group, lipiodol group, hydroxyapatite nanoparticles loaded with lipiodol group). The transarterial embolization was performed super-selectively via gastro- duodenal artery of rabbits, each rabbit in control group was inserted with 1 ml normal saline,that in lipiodol group was inserted with 0.3 lipiodol ml/kg, also 0.3 ml hydroxyapatite nanoparticles loaded with lipiodol per kg for that in the last group. Results of embolization were detected by using CT scanning 3 days after operation. After two weeks, all tumors were took out as specimens to investigate location of MT1-MMP in VX2 tumor tissues,and also to determine the change of its expression in tumor tissues after embolization with different medicines, with three-step immunohistochemical technique (S-P). MT1-MMP mRNA was measured by RT-PCR to determine whether there were differences in three groups. Western blot technique was performed to determine difference of MT1-MMP protein expression of in three groups. RESULTS: Immunohistochemical results exposed that MT1-MMP was expressed on membrane of tumor cells and in extracellular matrix of tumor cells. Comparison of MT1-MMP expression in control group with that in other two groups, showed a significant lower level in control group (P < 0.05). There was no difference in MT1-MMP expression between lipiodol group, hydroxyapatite nanoparticles loaded with lipiodol group (P > 0.05). Western blot supported this conclusion. RT-PCR detecting MT1-MMP mRNA was found no differences among three groups (P > 0.05). CONCLUSIONS: MT1-MMP was mainly expressed on membrane of tumor cells and in extracellular matrix of tumor cells. There was an increasing tendency on expression of MT1-MMP in tumor tissues and extracellular matrix after transarterial embolization with hydroxyapatite nanoparticles loaded with lipiodol,it might be one of important mechanisms provoking high recurrence rate for hepatocellular carcinoma after treatment embolization.

Novel tumor-ablation device for liver tumors utilizing heat energy generated under an alternating magnetic field.

BACKGROUND AND AIMS: We have developed a novel tumor-ablation device for liver tumors utilizing heat energy induced by magnesium ferrite (MgFe(2)O(4)) particles under an alternating magnetic field (AMF) produced by electric currents. This novel device can repeatedly heat liver tumors at lower temperature than usual heating devices, such as radiofrequency ablation therapy, with slight infliction of pain. This study assesses its heating effect on rat liver tumors as local therapy. METHOD: The small needle was manufactured from MgFe(2)O(4) particles by sintering at 1100 degrees C. After a MgFe(2)O(4) needle was inserted into liver tumors comprising of dRLh-84 cells, the tumors were heated for 30 min under an AMF. We examined cellular activity by using nicotinamide adenine dinucleotide (NADH) diaphorase staining and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) staining, and evaluated the effect of suppressing tumor growth by sequentially comparing the tumor diameter with that of the control group. RESULTS: The mean temperature of the heated tumors was 60.2 +/- 1.8 degrees C. The tumor cells were constricted, and chromatin of nuclei had shrunk immediately after heating. The heat-injury area that contained the tumors was negative for NADH diaphorase activity. After 3 days, the tumor cells in the heat-injury area became positive for TUNEL staining, which detects cell death. At 7 days, the mean tumor diameters were significantly smaller in the heating group than in the control group (6.15 +/- 0.47 mm vs 16.89 +/- 2.69 mm; P < 0.05). CONCLUSION: This device, utilizing heat energy induced by ferromagnetic metal under an AMF, appears useful as local thermotherapy for human liver cancer.

Adenovirus vector-mediated gene therapy using iodized oil esters for hepatocellular carcinoma in rats.

BACKGROUND: When gene therapy is performed for malignant tumors, gene transfer efficiency and selectivity are extremely important. The usefulness of gene therapy by intraarterial injection of an adenovirus vector with iodized oil esters (IOEs) for hepatocellular carcinoma (HCC) was studied. MATERIALS AND METHODS: HCC was induced in rats with diethyl nitrosamine and phenobarbital, after which either adenovirus vector expressing the herpes simplex virus thymidine kinase (AxCAHSVtk) and IOEs or AxCAHSVtk alone was injected through the hepatic artery. On postoperative days 2, 4 and 6, gancyclovir was injected into the peritoneum; blood sampling was performed on day 7. RESULTS: Aspartate aminotransferase and alanine aminotransferase levels in the AxCAHSVtk with IOEs group were lower than in the AxCAHSVtk alone group (p = 0.0274, p = 0.0323). However, the survival rate was not significantly different between groups (p = 0.7122). CONCLUSION: Intra-arterial injection of an adenovirus vector with IOEs can result in cancer-selective but not effective gene therapy for HCC.

[Influence of neoadjuvant chemotherapy on liver integrity and ischemic tolerance]. / Der Einfluss neoadjuvanter Chemotherapie auf Leberintegrität und Ischämietoleranz.

The increasing interest in neoadjuvant chemotherapy of liver metastasis after colorectal carcinoma prior to resection has focussed surgical concerns to the influence of oncological chemotherapy on hepatic tolerance to intraoperative ischaemia. The present study was thus undertaken in order to produce first experimental data on liver function and morphology after neoadjuvant chemotherapy and subsequent ischaemic challenge in a rat model. Male Wistar rats were randomised to receive an intraperitoneal chemotherapy (CH) or placebo (PL) according to the same protocol. Afterwards the animals were subjected to 30 min of total hepatic ischaemia induced by Pringle's manoeuvre and subsequent reperfusion for 1 h or 24 h. Serum activities of hepatic enzymes showed no differences between CH and PL at any time. Bile flow, however, was found to be significantly reduced in CH. In contrast, post-ischaemic up-regulation of PUMA and cleavage of caspase3 was found to be more prominent in PL than in CH, while the antiapoptotic chaperone GRP78 revealed a higher expression in the latter. It is concluded that chemotherapy did not affect ischaemic tolerance of the liver in our model, but promoted a kind of preconditioning, that is likely to counteract cellular induction of apoptosis upon ischaemic challenge.

Modulation of energy metabolic enzyme expression in N-nitrosodiethylamine-mediated hepatocarcinogenesis by Chinese herbs, Huqi San.

AIM: To elucidate the anticancer mechanism of Huqi San by assessing the expression of G-6-Pase, SDH, ATPase and AFP in N-nitrosodiethylamine-mediated hepatocarcinogenesis in rats. METHODS: A Solt-Farber two-step test model of hepatocarcino genesis was established by diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) in rats to investigate the modifying effects of expression of 6-glucosephosphatase (G-6-Pase), succinodehydrogenase (SDH), adenosine triphosphatase (ATPase) in N-nitrosodiethylamine-mediated hepatocarcinogenesis. Hu Qisan compounded by eight medicinal herbs was prepared in glycoprival granules with wich 0.38 g crude herbs/mL solution was prepared for administration. gamma-Glutamy-transpeptidase (gamma-GT), G-6-Pase, SDH and ATPase were immunohistochemically determined. The activity of alpha-fetoglobulin (AFP) in the livers was measured with Immunofluorescence. RESULTS: Huqi San treated rats showed significant decrease in areas of gamma-GT positive foci (P< 0.001). On the other hand, the expression of G-6-Pase, SDH and ATPase has obviously altered in Huqi San treated group. The activity of AFP also significantly decreased after the treatment with Huqi San (8 g/kg body weight or 4 g/kg body weight) or total alkali of mistletoe (0.12 g/kg body weight). CONCLUSIONS: Huqi San can obviously increase these activities of G-6-Pase, SDH and ATPase, and at the same time significantly decrease the expression of gamma-GT and AFP. Therefore, it can obstruct or inhibit the rat's liver preneolastic lesion induced by DEN.

Induction of Nrf2-mediated cellular defenses and alteration of phase I activities as mechanisms of chemoprotective effects of coffee in the liver.

Coffee consumption has been associated with a significant decrease in the risk of developing chronic diseases such as Parkinson disease, diabetes type-2 and several types of cancers (e.g. colon, liver). In the present study, a coffee-dependent induction of enzymes involved in xenobiotic detoxification processes was observed in rat liver and primary hepatocytes. In addition, coffee was found to induce the mRNA and protein expression of enzymes involved in cellular antioxidant defenses. These inductions were correlated with the activation of the Nrf2 transcription factor as shown using an ARE-reporter luciferase assay. The induction of detoxifying enzymes GSTs and AKR is compatible with a protection against both genotoxicity and cytotoxicity of aflatoxin B1 (AFB1). This hypothesis was confirmed in in vitro and ex vivo test systems, where coffee reduced both AFB1-DNA and protein adducts. Interestingly, coffee was also found to inhibit cytochrome CYP1A1/2, indicating that other mechanisms different from a stimulation of detoxification may also play a significant role in the chemoprotective effects of coffee. Further investigations in either human liver cell line and primary hepatocytes indicated that the chemoprotective effects of coffee against AFB1 genotoxicity are likely to be of relevance for humans. These data strongly suggest that coffee may protect against the adverse effects of AFB1. In addition, the coffee-mediated stimulation of the Nrf2-ARE pathway resulting in increased endogenous defense mechanisms against electrophilic but also oxidative insults further support that coffee may be associated with a protection against various types of chemical stresses.

Overexpression of regucalcin suppresses gene expression of insulin signaling-related proteins in cloned rat hepatoma H4-II-E cells: involvement of insulin resistance.

Overexpression of regucalcin has been shown to enhance glucose utilization and lipid production in the cloned rat hepatoma H4-II-E cells in vitro, and it induces insulin resistance. The effect of regucalcin on the gene expression of insulin signaling-related proteins was investigated in the cloned rat hepatoma H4-II-E cells overexpressing regucalcin in vitro. The hepatoma cells (wild-type) and stable regucalcin/ pCXN2-transfected cells (transfectants) were cultured for 72 h in a medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. Cells with subconfluency were cultured for 24, 48, or 72 h in a medium containing either vehicle or insulin (10(-9)-10(-7) M) with or without supplementation of glucose (10, 25, or 50 mg/ml of medium). The expression of rat insulin receptor (Insr), phosphatidylinositol 3-kinase (PI3K), glucose transporter 2 (GLUT 2), or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNAs was examined using reverse transcription-polymerase chain reaction analysis with specific primers. GLUT 2 mRNA expression was significantly increased in the transfectants, while Insr, PI3K, and G3PDH mRNA levels were not significantly changed in the transfectants. Culture with insulin (10(-8) or 10(-7) M) caused a significant increase in PI3K mRNA levels in wild-type cells cultured for 24 or 48 h, while it had no effect on Insr mRNA levels. The supplementation of glucose (10, 25, or 50 mg/ml) caused a significant increase in Insr and PI3K mRNA levels in wild-type cells. The effect of insulin or glucose supplementation on these gene expression levels was not seen in the transfectants. The combination of insulin (10(-7) M) and glucose (50 mg/ml) caused a significant increase in Ins and PI3K mRNA levels in wild-type cells. Such an effect was not seen in the transfectants. Culture with insulin or glucose supplementation failed to have a significant effect on GLUT2 and G3PDH mRNA levels in the wild-type cells or transfectants. This study demonstrates that overexpression of regucalcin suppresses the enhancing effect of insulin or glucose on the gene expression of insulin signaling-related proteins in the cloned rat hepatoma H4-II-E cells.

Hepatoprotective effect of andrographolide against hexachlorocyclohexane-induced oxidative injury.

Many plant products are known to exert antioxidative effects by quenching various free radicals and singlet molecular oxygen. Andrographis paniculata (Kalmegh) is used extensively in the Indian traditional system of medicine as a hepatoprotective and hepatostimulative agent and has been reported to have antioxidant effects against different hepatotoxins. The present study aims to analyze antioxidant properties of an active component, andrographolide (ANDLE), extracted from A paniculata. This study investigates the effect of andrographolide on the hepatocellular antioxidant defense system and lipid peroxidation of control mice, mice treated with hexachlorocyclohexane (BHC) only, and andrographolide + BHC. Glutathione (GSH), glutathione-s-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GSH-Px), gamma-glutamyl transpeptidase (gamma-GTP), superoxide dismutase (SOD), catalase (CAT), and lipid peroxidation (LPO) are studied by spectrophotometric methods. The BHC experimental model forms an irreversible liver tumor in male mice. The activities of GSH, GR, GSH-Px, SOD, and CAT show significant (P