Primary Cultures Of Rat Hepatocytes Research Articles

Activation of transcription factor HIF inhibits IL-1β-induced NO production in primary cultured rat hepatocytes

Roxadustat and other hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs) have recently been approved for the treatment of chronic renal anemia. In macrophages and monocytes, the activation of HIF-1 by pro-inflammatory cytokines induces iNOS expression and activity through the NF-κB pathway to produce nitric oxide (NO), which causes liver injury when excessively produced. Few studies have reported a relationship between HIF activity and iNOS induction in hepatocytes. We investigated the effect of drug- and hypoxia-induced HIF activations on NO production in primary cultured rat hepatocytes. Roxadustat treatment and hypoxic conditions activated HIF. Contrary to expectations, HIF–PHI treatment and hypoxia inhibited IL-1β-induced NO production. RNA-Seq analysis of mRNA expression in rat hepatocytes showed that roxadustat treatment decreased the expression of genes related to inflammation, and genes in the NF-κB signaling pathway were induced by IL-1β. Moreover, roxadustat suppressed IL-1β-activated signaling pathways in an HIF-dependent manner. GalN/LPS-treated rats were used as in vivo models of hepatic injury, and roxadustat treatment showed a tendency to suppress the death of rats. Therefore, exogenous HIF-1 activation, including HIF–PHI and hypoxia exposures, suppressed IL-1β-induced iNOS mRNA expression and subsequent NO production in hepatocytes, by suppressing the NF-κB signaling pathway. Roxadustat treatment suppresses the expression of pro-inflammatory genes by activating HIF, and thus may exhibit hepatoprotective effects.

Metabolic Activation and Cytotoxicity of Fungicide Carbendazim Mediated by CYP1A2.

Carbendazim (CBZ) is a broad-spectrum fungicide widely used in many nations for foliar spray as well as seed and soil treatment. The resulting contamination and environmental pollution have been drawing public attention. In particular, CBZ was reported to cause liver damage in rats and zebrafish, and the mechanisms of its toxicity have not been clarified. The purposes of this study were to investigate the metabolic activation of CBZ and to determine a possible role of the reactive metabolites in CBZ-induced liver injury reported. One oxidative metabolite (M1), one glutathione conjugate (M2), and one N-acetyl cysteine conjugate (M3) were detected in human and rat liver microsomal incubations fortified with glutathione or N-acetyl cysteine after exposure to CBZ. CYP1A2 was the major enzyme responsible for the metabolic activation of CBZ. Biliary M2 and urinary M3 were detected in rats treated with CBZ. CBZ-derived protein adduction was found in cultured rat primary hepatocytes treated with CBZ. The increase of administration concentration intensified not only the cytotoxicity but also protein adduction induced by CBZ, suggesting a correlation of the cytotoxicity with the observed protein modification. The findings facilitate the understanding of the mechanisms of toxic action of CBZ.

Detection of Decarboxylated Amino Acids after in Vitro Protease Digestion of the Hydrophilic Fraction of Crude Drug Extracts.

Many constituents of crude drugs in Japanese Kampo formulas are thought to function as pro-drugs, whose pharmacological activity is manifested after oral administration. Proteins and peptides in crude drugs may be digested and metabolized in the digestive tract and liver. However, few studies have reported the pharmacological activity of peptides in crude drugs. Here, we applied an analysis using LC-tandem mass spectrometry (LC-MS/MS) to identify the compounds derived from six crude drugs that are assumed to have anti-inflammatory effects. To simulate in vivo protease digestion, each water-soluble fraction of the crude drug extracts was treated with proteases, including endoproteinases and exopeptidases. Amines in the resultant digests were modified by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and analyzed using LC-MS/MS, which demonstrated the presence of four decarboxylated amino acids (primary amines). In the digest of the hydrophilic fraction of the fruit of Ziziphus jujuba Miller var. inermis Rehder (Taiso), isobutylamine, isoamylamine, and 2-methylbutylamine were identified, which may be derived from valinyl, leucinyl, and isoleucinyl residues, respectively. Additionally, tyramine possibly derived from tyrosyl residues was identified in the digests of all the crude drugs. In primary cultured rat hepatocytes treated with interleukin-1β, all these decarboxylated amino acids suppressed the production of nitric oxide, a proinflammatory mediator. Our approach, i.e., in vitro protease digestion and LC-MS/MS analysis, suggests that decarboxylated amino acids may be formed in vivo from peptides and may be responsible for the anti-inflammatory effect of crude drugs included in Kampo medicine.

Signal transduction mechanism for autocrine secretion of insulin-growth factor type 1 by <i>S</i>-allyl-L-cysteine in primary cultures of adult rat hepatocytes.

Signal transduction mechanism for autocrine secretion of insulin-growth factor type 1 by <i>S</i>-allyl-L-cysteine in primary cultures of adult rat hepatocytes.

Effects of <i>S</i>-allyl-L-cysteine on phosphorylation of insulin-like growth factor type-I receptor tyrosine kinase in primary cultures of adult rat hepatocytes.

Effects of <i>S</i>-allyl-L-cysteine on phosphorylation of insulin-like growth factor type-I receptor tyrosine kinase in primary cultures of adult rat hepatocytes.

Omeprazole Increases Survival Through the Inhibition of Inflammatory Mediaters in Two Rat Sepsis Models.

ABSTRACTBackground:Omeprazole (OMZ) is a proton pump inhibitor that is used to reduce gastric acid secretion, but little is known about its possible liver protective effects. This study investigated whether OMZ has beneficial effects in rat septic models of LPS-induced liver injury after D-galactosamine (GalN) treatment and 70% hepatectomy (PH), and to determine the mechanisms of OMZ in an in vitro model of liver injury.Methods:In the in vivo models, the effects of OMZ were examined 1 h before treatments in both models on survival, nuclear factor (NF)-κB activation, histopathological analysis, and proinflammatory mediator expression in the liver and serum. In the in vitro model, primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of OMZ. The influence of OMZ on nitric oxide (NO) product and inducible NO synthase (iNOS) induction and on the associated signaling pathway was analyzed.Results:OMZ increased survival and decreased tumor necrosis factor-alpha, iNOS, cytokine-induced neutrophil chemoattractant 1, IL-6, and IL-1β mRNA expression, and increased IL-10 mRNA expression in the livers of both GaIN/LPS- and PH/LPS-treated rats. Necrosis and apoptosis were inhibited by OMZ in GaIN/LPS rats, but OMZ had no effects on necrosis in PH/LPS rats. OMZ inhibited iNOS induction partially through suppression of NF-κB signaling in hepatocytes.Conclusions:OMZ inhibited the induction of several inflammatory mediators, resulting in the prevention of LPS-induced liver injury after GalN liver failure and PH, although OMZ showed different doses and mechanisms in the two models.

Comparison of the anti-inflammatory activities of furanocoumarins from the roots of Angelica dahurica

Background: The roots of Angelica dahurica Bentham et Hooker filius ex Franchet et Savatier (Apiaceae) have traditionally been used for inflammatory skin diseases. A. dahurica roots (Byakushi) contain furanocoumarins, such as imperatorin and byakangelicin. To elucidate which constituents are responsible for the anti-inflammatory effects, we evaluated the potency of crude A. dahurica root extract fractions by monitoring the production of the inflammatory mediator nitric oxide (NO) in hepatocytes.Methods: The dried roots of A. dahurica were collected in South Korea and extracted with methanol. The resulting extract was fractionated into ethyl acetate (EtOAc)-soluble, n-butanol-soluble, and water-soluble fractions. Primary cultured rat hepatocytes were treated with interleukin (IL)-1β and each fraction for 8 h, and then the NO production and lactate dehydrogenase activity in the medium were measured. The expression of inducible nitric oxide synthase (iNOS) was detected by Western blotting, and its mRNA expression level was measured by quantitative reverse transcription-polymerase chain reaction.Results: Among the three fractions, the EtOAc-soluble fraction markedly suppressed NO production without showing cytotoxicity and decreased iNOS expression in hepatocytes. From this hydrophobic fraction, we isolated five furanocoumarins: isoimperatorin, imperatorin, phellopterin, oxypeucedanin, and oxypeucedanin methanolate. Phellopterin and oxypeucedanin methanolate significantly suppressed NO production and reduced the mRNA expression of iNOS and tumor necrosis factor α. In contrast, the other three constituents did not affect NO production. Comparison of their chemical structures suggests that a methoxy group at carbon 5 and a side chain at carbon 8 in the furanocoumarin skeleton may be essential for NO production suppression.Conclusion: These data imply that phellopterin and oxypeucedanin methanolate, which are hydrophobic furanocoumarins, may contribute to the anti-inflammatory effects of A. dahurica roots by suppressing iNOS gene expression.Keywords: Inflammation, nitric oxide, hepatocyte, coumarin, Kampo medicine

In- vitro Hepatoprotective Action of the Crude Extracts of Luffa amara (Whole Fruit) and Rheum emodi (Rhizomes) in CCl4 Intoxicated Rat Hepatocytes

Aim: The objective of this in-vitro study involves evaluating the protective action of the extracts of L. amara (LA) (whole fruits including seeds) and R. emodi (RE) (rhizomes) at various concentrations on isolated primary rat hepatocytes.
 Methods: The pulverised dried whole fruits of L. amara (LA) and rhizomes of R.emodi (RE) were extracted successively with petroleum ether (PE), ethanol (EE) and distilled water (AE) and vacuum dried. These extracts of LA petroleum ether (PE), ethanolic (EE) and aqueous (AE) extracts and RE obtained were subjected to in vitro studies at doses of 25, 75, 100, and 150 µg/ml and silymarin (250 µg/ml) in CCl4 (1%) intoxicated primary hepatocytes monolayer cultures the hepatoprotective action of all the extracts of both plants at different doses was carried out using isolated rat hepatocytes which were subjected to CCl4 intoxication followed by estimating/ measuring the changes in serum biochemical markers – SGPT, SGOT, ALP, Total proteins (TP), total bilirubin (TB), albumin (ALB) and triglycerides (TGL).
 Results: Hepatoprotective activity against CCl4 was demonstrated in the rat primary monolayer hepatocyte culture using MMT assay with the ethanolic extracts of both plants showing more hepatocyte protective action compared to the aqueous and petroleum ether extracts by reducing the elevated serum marker levels. Alcoholic and aqueous extracts were found to express more protective action towards CCl4 intoxicated isolated primary rat hepatocytes in a dose dependant manner.
 Conclusion: Based on the result, it is suggested that the extract with the most hepatocyte protective action at a dose of 150µg is LA ethanolic extract (viability=88.24%), followed by LA aqueous extract (viability=84.31%), RE ethanolic extract (viability=88.24%) and RE aqueous extract (viability=88.24%) - which are comparable to the reference silymarin with viability at 92.15%. the petroleum ether extracts of both plants showed least hepatic cell viability with LA pet ether extract at 49.02% and RE pet ether extract at 47.85%

A Benchmark analysis of acrylamide-derived DNA adducts in rat hepatocytes in culture measured by a new, highly sensitive method

Acrylamide (AA) is a carcinogen formed during thermal food processing and can cause tumors in rodents while its carcinogenic potency in humans is unclear. Metabolism of AA, preferentially in the liver, leads to glycidamide (GA) forming N7-GA-guanine (N7-GA-Gua) as the major AA-derived DNA adduct in rodents. Here, a novel method allowing high sensitivity by avoidance of major matrix effects was applied to analyze N7-GA-Gua levels in nuclear DNA from rat hepatocytes in primary culture. We could thus for the first time detect a background level of 5–10 adducts/108 nucleosides in untreated hepatocytes. Incubation with AA did not result in a statistically significant increase in adduct levels over background up to a substrate concentration of 500 μM although a trend to slightly higher adduct levels was observed at and above 200 μM AA. At concentrations > 500 μM significant increases in N7-GA-Gua levels were found. When Benchmark concentration (BMC) modeling was applied to the data, non-linear concentration-response curves were obtained suggesting that AA started to cause measurable increases over background of N7-GA-Gua levels above certain concentrations only. Calculation of the composite BMCL10 (Lower Bound of a 95 % confidence interval) of a BMC leading to a 10 % increase of N7-GA-Gua levels over background resulted in a value of 6.35 μM AA after 24 h. A concentration below this value cannot be expected to lead to an increase in N7-GA-Gua of more than 10 % over the background seen in untreated hepatocytes.

Long-term culture of rat hepatocytes using human amniotic membrane as a culture substrate.

Amniotic membrane is attracting attention as a new material for regenerative medicine. We herein report that the culture of primary rat hepatocytes on human amniotic membrane maintained their morphology and their production of albumin for at least two months. Human amniotic membrane was collected during planned cesarean section and kept frozen until usage. Primary rat hepatocytes were plated on human amniotic membrane. Hepatocytes accumulated as colonies on amniotic membrane, and their rat albumin level was maintained for two months. Their three-dimensional structure on extracellular matrix, which is abundant in amniotic membranes might influence the maintenance of the hepatocyte-specific function.

Enhanced ammonia detoxification to urea in hepatocytes transduced with human aquaporin-8 gene.

Hepatic ammonia detoxification to urea is critical for the prevention of hyperammonemia and neurological damage. Hepatocyte mitochondrial aquaporin-8 (AQP8) channels have been involved in ammonia-derived ureagenesis. Herein, we studied whether the adenoviral gene transfer of human AQP8 (hAQP8) to hepatocyte mitochondria enhances ammonia conversion to urea. Using primary cultured rat hepatocytes, we first confirmed the mitochondrial expression of hAQP8 and then, using unlabeled or 15 N-labeled ammonia, we demonstrated that the urea synthesis was significantly enhanced in hAQP8-transduced hepatocytes. Studies using isolated hAQP8-expressing mitochondria also showed an increased ammonia metabolism. hAQP8 transduction was able to recover the impaired ammonia-derived ureagenesis in hepatotoxin-treated hepatocytes. Our data suggest that mitochondrially-expressed hAQP8 enhances and improves hepatocyte ammonia conversion to urea, a finding with potential therapeutic implications for liver disease with impaired ammonia detoxification.

Negative Regulation of Human Hepatic Constitutive Androstane Receptor by Cholesterol Synthesis Inhibition: Role of Sterol Regulatory Element Binding Proteins.

The squalene synthase inhibitor squalestatin 1 (Squal1) is a potent and efficacious inducer of CYP2B expression in primary cultured rat hepatocytes and rat liver. To determine whether Squal1 is also an inducer of human CYP2B, the effects of Squal1 treatment were evaluated in primary cultured human hepatocytes, differentiated HepaRG cells, and humanized mouse livers. Squal1 treatment did not increase CYP2B6 mRNA levels in human hepatocytes or HepaRG cells and only slightly and inconsistently increased CYP2B6 mRNA content in humanized mouse liver. However, treatment with farnesol, which mediates Squal1's effect on rat CYP2B expression, increased CYP2B6 mRNA levels in HepaRG cells expressing the constitutive androstane receptor (CAR), but not in cells with knocked-down CAR. To determine the impact of cholesterol biosynthesis inhibition on CAR activation, the effects of pravastatin (Prava) were determined on CITCO-mediated gene expression in primary cultured human hepatocytes. Prava treatment abolished CITCO-inducible CYP2B6 expression, but had less effect on rifampicin-mediated CYP3A4 induction, and CITCO treatment did not affect Prava-inducible HMG-CoA reductase (HMGCR) expression. Treatment with inhibitors of different steps of cholesterol biosynthesis attenuated CITCO-mediated CYP2B6 induction in HepaRG cells, and Prava treatment increased HMGCR expression and inhibited CYP2B6 induction with comparable potency. Transfection of HepG2 cells with transcriptionally active sterol regulatory element binding proteins (SREBPs) reduced CAR-mediated transactivation, and inducible expression of transcriptionally active SREBP2 attenuated CITCO-inducible CYP2B6 expression in HepaRG cells. These findings suggest that Squal1 does not induce CYP2B6 in human hepatocytes because Squal1's inhibitory effect on cholesterol biosynthesis interferes with CAR activation. SIGNIFICANCE STATEMENT: The cholesterol biosynthesis inhibitor squalestatin 1 induces rat hepatic CYP2B expression indirectly by causing accumulation of an endogenous isoprenoid that activates the constitutive androstane receptor (CAR). This study demonstrates that squalestatin 1 does not similarly induce CYP2B6 expression in human hepatocytes. Rather, inhibition of cholesterol biosynthesis interferes with CAR activity, likely by activating sterol regulatory element binding proteins. These findings increase our understanding of the endogenous processes that modulate human drug-metabolizing gene expression.

LXR and INSIG Act as Differentiators in the Regulation of the Gene Expression of G6PDH and FAS Under Insulin Resistant Conditions

Diabetes, a chronic metabolic disease that affects nearly 10% of the world's population can lead to very serious complications such as renal failure, liver cirrhosis, and heart attacks. The most common type is Type 2 diabetes and is diagnosed when a person has elevated amounts of blood glucose due to insulin resistance. This resistance to insulin leads to problems with glucose transport into tissues for subsequent metabolism. Over the years it has been shown that insulin regulates the expression of several key enzymes in both carbohydrate and fatty acid metabolic pathways via the phosphatidyl inositol 3-kinase (PI3K) pathway. Previously, using glucosamine, a precursor of the hexosamine biosynthetic pathway, we had established a model of insulin resistance in primary rat hepatocytes in culture. Using this primary cell culture model, we showed that under insulin resistant conditions, the expression of glucose 6 phosphate dehydrogenase (G6PDH), a key enzyme in carbohydrate metabolism and fatty acid synthase (FAS), a key enzyme in fat metabolism were differently regulated but the mechanism of this differentiation was unclear. Under this model of insulin resistance, we now show that this differential regulation is due to the liver X receptor (LXR) and insulin induced gene (INSIG).

Metabolic Pattern of Hepatotoxic Pyrrolizidine Alkaloids in Liver Cells

Contamination with 1,2-unsaturated pyrrolizidine alkaloids (PAs) is a serious problem for certain phytomedicines, foods, and animal feeds. Several of these PAs are genotoxic and carcinogenic, primarily in the liver, upon cytochrome P450 (CYP)-catalyzed activation into reactive (pyrrolic and pyrrole-like) metabolites. Here we investigated the metabolism of selected PAs (echimidine, europine, lasiocarpine, lycopsamine, retrorsine, and senecionine) in rat hepatocytes in primary culture and in human CYP3A4-transfected HepG2 cells. The open-chained diesters echimidine and lasiocarpine and the cyclic diester senecionine were extensively metabolized in rat hepatocytes into a broad spectrum of products released into the medium. A large portion of unidentified, possibly irreversibly bound, products remained in the cells while detectable amounts of reactive and other metabolites were found in the incubation media. In HepG2-CYP3A4 cells, lasiocarpine was more extensively metabolized than echimidine and senecionine which also gave rise to the release of pyrrolic metabolites. In human cells, no pyrrolic metabolites were detected in retrorsine or lycopsamine incubations, while no such metabolites were detected from europine in both cell types. Other types of metabolic changes comprised modifications such as side chain demethylation or oxygenation reactions like the formation of N-oxides. The latter, considered as a detoxification step, was a major pathway with cyclic diesters, was less distinctive for echimidine and lycopsamine and almost negligible for lasiocarpine and europine. Our data are in agreement with previously published cyto- and genotoxicity findings and suggests that the metabolic pattern may contribute substantially to the specific toxic potency of a certain congener. In addition, marked differences were found for certain congeners between rat hepatocytes and transfected human HepG2 cells, whereby a high level of bioactivation was found for lasiocarpine, whereas a very low level of bioactivation was observed for monoesters, in particular in human cells.

Growth Hormone Signaling Pathway Leading to the Induction of DNA Synthesis and Proliferation in Primary Cultured Hepatocytes of Adult Rats.

We investigated the signal transduction pathway associated with growth hormone (GH)-stimulated DNA synthesis and proliferation in primary cultured hepatocytes. Adult rat hepatocytes were isolated from normal livers by two-step in situ collagenase perfusion to facilitate disaggregation of the adult rat liver. Then hepatocytes were cultured in serum-free Williams' medium E supplemented with GH (1-100 ng/ml) in the presence or absence of test reagents. GH-induced hepatocyte DNA synthesis and proliferation were determined, and the phosphorylation activities of Janus kinase (JAK) 2 (JAK2) (p125 kDa), p95-kDa RTK, and ERK1/2 were measured by western blotting. Hepatocytes grown in serum-free defined medium proliferated within 5 h of culture in the presence of GH (100 ng/ml) in a concentration- and time-dependent manner (EC50 75 ng/ml). These proliferative effects of GH were almost completely blocked by an anti-GH receptor monoclonal antibody (85 ng/ml) and an anti-insulin-like growth factor (IGF)-I receptor monoclonal antibody. In addition, the proliferative effects of GH were significantly blocked by a JAK2 inhibitor (TG101209, 10-6 M), as well as specific signal-transducing inhibitors of phospholipase C (PLC; U-73122, 10-6 M), RTK (AG538, 10-6 M), phosphoinositide 3-kinase (PI3K; LY294002, 10-6 M), mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK; PD98059, 10-6 M), and mammalian target of rapamycin (mTOR; rapamycin, 10 ng/ml). GH significantly induced the phosphorylations of JAK2 (p125 kDa), p95-kDa IGF-I receptor tyrosine kinase (RTK), and ERK2 in this order according to western blotting analysis. The proliferative action of GH is mediated by two main signaling pathways. One includes activation of the GH receptor/JAK2/PLC/Ca2+ pathway, and the other involves activation of the p95-kDa IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.

Signal transduction mechanism for S-allyl-L-cysteine-induced cell proliferation in primary cultures of adult rat hepatocytes

Signal transduction mechanism for S-allyl-L-cysteine-induced cell proliferation in primary cultures of adult rat hepatocytes

Reproducibility and robustness of high-throughput S1500+ transcriptomics on primary rat hepatocytes for chemical-induced hepatotoxicity assessment

Cell-based in vitro models coupled with high-throughput transcriptomics (HTTr) are increasingly utilized as alternative methods to animal-based toxicity testing. Here, using a panel of 14 chemicals with different risks of human drug-induced liver injury (DILI) and two dosing concentrations, we evaluated an HTTr platform comprised of collagen sandwich primary rat hepatocyte culture and the TempO-Seq surrogate S1500+ (ST) assay. First, the HTTr platform was found to exhibit high reproducibility between technical and biological replicates (r greater than 0.85). Connectivity mapping analysis further demonstrated a high level of inter-platform reproducibility between TempO-Seq data and Affymetrix GeneChip data from the Open TG-GATES project. Second, the TempO-Seq ST assay was shown to be a robust surrogate to the whole transcriptome (WT) assay in capturing chemical-induced changes in gene expression, as evident from correlation analysis, PCA and unsupervised hierarchical clustering. Gene set enrichment analysis (GSEA) using the Hallmark gene set collection also demonstrated consistency in enrichment scores between ST and WT assays. Lastly, unsupervised hierarchical clustering of hallmark enrichment scores broadly divided the samples into hepatotoxic, intermediate, and non-hepatotoxic groups. Xenobiotic metabolism, bile acid metabolism, apoptosis, p53 pathway, and coagulation were found to be the key hallmarks driving the clustering. Taken together, our results established the reproducibility and performance of collagen sandwich culture in combination with TempO-Seq S1500+ assay, and demonstrated the utility of GSEA using the hallmark gene set collection to identify potential hepatotoxicants for further validation.

Fermented Wheat Germ Extract as a Redox Modulator: Alleviating Endotoxin-Triggered Oxidative Stress in Primary Cultured Rat Hepatocytes.

Bioactive compounds such as benzoquinone derivates presented in fermented wheat germ extract (FWGE) have several positive effects on overall health status of humans and animals alike. Since available data regarding the antioxidant activity of FWGE are limited, the aim of our study was to investigate its effects on the cellular redox homeostasis applying primary hepatocyte cell cultures of rat origin. Cultures were challenged to lipopolysaccharide (LPS) treatment for 2 or 8 hours to trigger inflammatory response. Further, culture media were concomitantly supplemented with or without FWGE (Immunovet®, 0.1% and 1%). In order to monitor the metabolic activity of the cell cultures, CCK-8 test was applied, while reactive oxygen species (ROS) production was measured using Amplex Red method. Malondialdehyde concentration of culture media as a specific marker of lipid peroxidation and the activity of glutathione peroxidase in cell lysates were also determined to monitor the redox status of the cultures. Based on our findings, it can be concluded that FWGE did not show cytotoxic effects in any applied concentration in cell cultures. Furthermore, FWGE efficiently decreased cellular ROS production and lipid peroxidation rate in case of LPS-induced inflammatory response. However, without LPS treatment, higher concentration of FWGE increased the rate of both ROS and malondialdehyde synthesis. This observation may refer to the prooxidant activity of high dose FWGE, which is an important beneficial effect regarding tumor cells. However, in case of noninflamed hepatocytes, considering the results of glutathione peroxidase activity, the application of the product did not result in severe oxidative distress. In accordance with the abovementioned findings, FWGE as a redox modulator, applied in the appropriate concentration, can serve as a promising candidate in the supplementary therapy of patients suffering from various inflammatory diseases, decreasing the free radical generation, thus avoiding the occurrence of cytotoxic effects.

Autocrine secretion of insulin-like growth factor-I mediates growth hormone-stimulated DNA synthesis and proliferation in primary cultures of adult rat hepatocytes

The intracellular signaling pathway of growth hormone (GH)-stimulated DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes. DNA synthesis and cell proliferation were detected in hepatocyte parenchymal cells grown in serum-free, defined medium containing GH (100 ng/ml). GH-stimulated hepatocyte DNA synthesis and proliferation were almost completely blocked by TG101209 (10−6 M), a selective Janus kinase (JAK)2 inhibitor, U-73122 (10−6 M), a selective phospholipase C (PLC) inhibitor, and a monoclonal antibody to insulin-like growth factor-I (IGF-I) receptor (100 ng/ml) or anti-secretion agents such as somatostatin (10−6 M) and BAPTA/AM (10−7 M). In addition, blocking monoclonal antibodies to IGF-I, but not transforming growth factor-α, completely inhibited GH-induced hepatocyte DNA synthesis and proliferation. IGF-I levels in the culture medium increased rapidly versus baseline levels within 5 min in response to GH (100 ng/ml), and the maximum IGF-I level (100 pg/ml) was reached 20 min after GH stimulation. Autocrine secretion of IGF-I into the culture medium was inhibited by a growth-inhibitory dose of TG101209, U-73122, somatostatin, or BAPTA/AM. These data indicate that the proliferative mechanism of action of GH is mediated mainly through a GH receptor/JAK2/PLC-stimulated increase in the autocrine secretion of IGF-I by primary cultured hepatocytes, followed by stimulation of the 95 kDa IGF-I receptor tyrosine kinase signaling pathway.

Estragole: DNA adduct formation in primary rat hepatocytes and genotoxic potential in HepG2-CYP1A2 cells

Estragole is a natural constituent in herbs and spices and in products thereof such as essential oils or herbal teas. After cytochrome P450-catalyzed hydroxylation and subsequent sulfation, estragole acts as a genotoxic hepatocarcinogen forming DNA adducts in rodent liver. Because of the genotoxic mode of action and the widespread occurrence in food and phytomedicines a refined risk assessment for estragole is needed. We analyzed the time- and concentration-dependent levels of the DNA adducts N2-(isoestragole-3‘-yl)-2‘-desoxyguanosine (E3′N2dG) and N6-(isoestragole-3‘-yl)-desoxyadenosine (E3′N6dA), reported to be the major adducts formed in rat liver, in rat hepatocytes (pRH) in primary culture after incubation with estragole. DNA adduct levels were measured via UHPLC-ESI-MS/MS using stable isotope dilution analysis. Both adducts were formed in pRH and could already be quantified after an incubation time of 1 h (E3′N6dA at 10 μM, E3′N2dG at 1μM estragole). E3′N2dG, the main adduct at all incubation times and concentrations, could be detected at estragole concentrations < 0.1 μM after 24 h and < 0.5 μM after 48 h. Adduct levels were highest after 6 h and showed a downward trend at later time-points, possibly due to DNA repair and/or apoptosis. While the concentration-response characteristics of adduct formation were apparently linear over the whole concentration range, strong indication for marked hypo-linearity was obtained when the modeling was based on concentrations < 1 μM only. In the micronucleus assay no mutagenic potential of estragole was found in HepG2 cells whereas in HepG2-CYP1A2 cells 1 μM estragole led to a 3.2 fold and 300 μM to a 7.1 fold increase in micronuclei counts. Our findings suggest the existence of a ‘practical threshold’ dose for DNA adduct formation as an initiating key event of the carcinogenicity of estragole indicating that the default assumption of concentration-response-linearity is questionable, at least for the two major adducts studied here.