Rat Hepatoma Cell Line H4IIE Research Articles

Effect of Acrylamide Treatment on Cyp2e1 Expression and Redox Status in Rat Hepatocytes.

Acrylamide (AA) toxicity is associated with oxidative stress. During detoxification, AA is either coupled to gluthatione or biotransformed to glycidamide by the enzyme cytochrome P450 2E1 (CYP2E1). The aim of our study was to examine the hepatotoxicity of AA in vivo and in vitro. Thirty male Wistar rats were treated with 25 or 50 mg/kg b.w. of AA for 3 weeks. Qualitative and quantitative immunohistochemical evaluation of inducible nitric oxide synthase (iNOS), CYP2E1, catalase (CAT), superoxide dismutase 1 (SOD1), and SOD2 expression in liver was carried out. Bearing in mind that the liver is consisted mainly of hepatocytes, in a parallel study, we used the rat hepatoma cell line H4IIE to investigate the effects of AA at IC20 and IC50 concentrations on the redox status and the activity of CAT, SOD, and glutathione-S-transferase (GST), their gene expression, and CYP2E1 and iNOS expression. Immunohistochemically stained liver sections showed that treatment with AA25mg induced a significant decrease of CYP2E1 protein expression (p < 0.05), while treatment with AA50mg led to a significant increase of iNOS protein expression (p < 0.05). AA treatment dose-dependently elevated SOD2 protein expression (p < 0.05), while SOD1 protein expression was significantly increased only at AA50mg (p < 0.05). CAT protein expression was not significantly affected by AA treatments (p > 0.05). In AA-treated H4IIE cells, a concentration-dependent significant increase in lipid peroxidation and nitrite levels was observed (p < 0.05), while GSH content and SOD activity significantly decreased in a concentration-dependent manner (p < 0.05). AA IC50 significantly enhanced GST activity (p < 0.05). The level of mRNA significantly increased in a concentration-dependent manner for iNOS, SOD2, and CAT in AA-treated H4IIE cells (p < 0.05). AA IC50 significantly increased the transcription of SOD1, GSTA2, and GSTP1 genes (p < 0.05), while AA IC20 significantly decreased mRNA for CYP2E1 in H4IIE cells (p < 0.05). Obtained results indicate that AA treatments, both in vivo and in vitro, change hepatocytes; drug-metabolizing potential and disturb its redox status.

Two new octahydronaphthalene derivatives, trichodermic acids C and D produced by Trichoderma sp. HN-1.1

Two new octahydronaphthalene derivatives, trichodermic acid C (1) and trichodermic acid D (2), along with known analogs, trichodermic acid (3), trichodermic acid A (4) and trichodermic acid B (5), were isolated from an ethyl acetate extract of endophytic strain Trichoderma sp. HN-1.1. The structures of compounds 1 and 2 were elucidated using spectroscopic methods including UV, IR, HRESITOFMS, ECD, 1 D and 2 D NMR. The cytotoxic activity of the isolated compounds was evaluated on the rat hepatoma cell line H4IIE, using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. According to the results, only compound 3 showed a significant reduction of H4IIE cells from 75 to 21% (p < 0.01) with an IC50 value of 50% cell survival estimated as 143.1 µM, which indicated weak cytotoxic activity.

Polythosides A and B, two new triterpenoid saponins from the roots of Acacia polyacantha Willd. (Mimosaceae)

• Two new triterpenoid saponins were isolated from Acacia polyacantha . • Their structures were elucidated by analysis of 1D and 2D-NMR studies. • They were not significantly cytotoxic to H4IIE cells even at 100 μM. Two new oleanane-type triterpene saponins, named polythosides A and B ( 1 and 2 ), together with a known compound, silphioside E ( 3 ), were isolated from the roots of Acacia polyacantha . Their structures were elucidated by analysis of 1D and 2D-NMR experiments, and mass spectrometry (HR-ESITOF-MS) as oleanolic acid 3- O - β - d -galactopyranosyl-(1→2)- β - d -xylopyranosyl-(1→6)-[ β - d -xylopyranosyl-(1→4)]-[ β - d -xylopyranosyl-(1→2)]- β - d -galactopyranoside ( 1 ), 3- O -[ β - d -galactopyranosyl-(1→2)- β - d -xylopyranosyl-(1→6)-[ β - d -xylopyranosyl-(1→4)]-[ β - d -xylopyranosyl-(1→2)]- β - d -galactopyranosyl] oleanolic acid-28- O - β - d -glucopyranosyl ester ( 2 ) and 3- O - β - d -glucopyranosyl-(1→2)- β - d -glucopyranosyl] oleanolic acid 28- O - β - d -glucopyranosyl ester ( 3 ). The cytotoxic effect of isolated saponins was evaluated on the H4IIE rat hepatoma cell line. The results show that none of the compounds (assayed at 100 μM) showed cytotoxicity against H4IIE cells.

Triterpene saponins from the roots of Acacia senegal (L.) Willd.

Six new triterpenoid saponins, named senegalosides A–F (1–6) were isolated from the seedpods and roots of Acacia senegal (Mimosaceae). Their structures were elucidated using 1D and 2D-NMR spectroscopic analysis and mass spectrometry. Compound 1 possesses an unusual sapogenin, 3β-hydroxy-21-oxo-olean-12-en-28-oic acid (machaeric acid), and was reported here in its natural form for the first time within the genus Acacia. Senegaloside A is the first example of a machaeric-type triterpenoid glycoside in the plant kingdom. The cytotoxic effect of isolated saponins was evaluated on the H4IIE rat hepatoma cell line. As a result, compounds 1, 3–6 were not significantly cytotoxic to H4IIE cells even at 200 μM. Compound 2 was suppressed cell viability at 50–200 μM.

Mechanisms of NADPH - cytochrome P450 oxidoreductase induction by dexamethasone in the H4IIE rat hepatoma cell line.

Expression of NADPH - cytochrome P450 oxidoreductase (POR), electron donor for microsomal P450s, is induced in rat liver by dexamethasone (DEX), an activator of the glucocorticoid receptor (GR) and the pregnane X receptor (PXR). DEX induction of POR in rat liver is primarily PXR-mediated, although GR may contribute to mRNA effects. We examined the role of GR and PXR in the DEX induction of POR mRNA and protein in the H4IIE rat hepatoma cell line. The DEX EC50 for a PXR target, CYP3A23, exceeded that for the GR targets tyrosine aminotransferase and PXR as well as POR itself. POR protein levels were induced 3- and 4-fold, respectively, by DEX concentrations activating GR selectively (100 nM) or both GR and PXR (10 μM). POR was induced by triamcinolone acetonide, a selective GR agonist, but not pregnenolone-16α-carbonitrile, a selective PXR agonist. POR induction was blocked by the GR antagonist RU486 but minimally influenced by the PXR antagonist FLB-12. The half-life for POR mRNA was prolonged by DEX at both 100 nM and 10 μM. GR is more important in DEX-induced POR expression in H4IIE cells compared to rat liver in vivo, calling into question the suitability of this cell model for mechanistic studies.

Determination of the CYP1A-inducing potential of single substances, mixtures and extracts of samples in the micro-EROD assay with H4IIE cells

This protocol describes a quantitative and robust 96-well-plate-reader-based assay for the measurement of ethoxyresorufin-O-deethylase (EROD) activity using the rat hepatoma cell line H4IIE. The assay can be used to determine the cytochrome P450 subfamily 1A (CYP1A)-inducing potential of single substances, as well as of mixtures and extracts of samples. It is based on the aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 enzymes (subfamily 1A) in cells after exposure to dioxins and dioxin-like compounds. One enzymatic reaction catalyzed by CYP1A is the deethylation of the exogenous substrate 7-ethoxyresorufin to the fluorescent product resorufin, which is measured as EROD activity in the assay. The CYP1A-inducing potential of a sample can be reliably quantified by comparing the EROD activity with the concentration-response curve of the standard substance 2,3,7,8-tetrachlorodibenzo-p-dioxin, which can be detected at concentrations down to the picogram per liter range. A researcher familiar with the procedure can process up to 160 samples with four wells each within 3 d. The series described uses four plates with three concentrations per sample, which can be easily scaled to accommodate different sample sizes.

Mechanistic role of PDE4/cAMP in the development of HIV‐PI induced FasL mediated hepatotoxicity

HIV protease inhibitors (HIV‐PIs) have been successfully used in the anti‐retroviral therapy in the treatment of HIV‐1 infection in the past two decades. Hence it is highly relevant to study the underlying mechanisms in HIV‐PIs induced hepatotoxicity that can negatively impact treatment outcomes. Hence, activation of hepatotoxic Fas/FasL signaling in HIV‐PI induced hepatotoxicity was investigated.Clinically relevant combination of HIV‐PIs [Ritanovir (Rit)+Lopinavir (Lop)] significantly up‐regulated FasL and Fas expression in H4IIE rat hepatoma cell line as well as primary rat hepatocytes. Our recent work showed that PDE4 expression/cAMP metabolism plays a significant role in hepatic inflammation and injury. Hence, we examined the potential role of PDE4 expression in the HIV‐PIs mediated activation of Fas and FasL expression. Commensurate with the increase in FasL and Fas, PDE4 expression was also increased with Rit+Lop treatment. To address the causal role of PDE4 expression, cells were treated with a highly specific PDE4 inhibitor rolipram which markedly inhibited Fas and FasL mRNA and protein expression induced by HIV PIs.Increased PDE4 decreases cellular cAMP levels and downstream cAMP effector molecules (cAMP‐PKA and ‐EPAC). Hence, effect of H‐89 (PKA inhibitor) and ESI‐09 (EPAC inhibitor) was examined in hepatocytes treated with HIV‐PI and rolipram. Both inhibitors significantly reversed the rolipram protective effect leading to re‐expression of Fas and FasL. Overall, this data strongly support and identify a mechanistic role for PDE4 regulated cAMP‐EPAC/PKA in the development of HIV‐PI induced FasL mediated hepatotoxicity.

P32—Effects of PFCs on gene expression of the H4IIE rat hepatoma cell line

P32—Effects of PFCs on gene expression of the H4IIE rat hepatoma cell line

Effects of cerium oxide nanoparticles to fish and mammalian cell lines: An assessment of cytotoxicity and methodology

Two cerium oxide nanoparticles (CeO2 NPs) and one micro-sized CeO2 particle were thoroughly characterized in their pristine form, in water and in cell culture medium. The particles were tested for cytotoxicity to the H4IIE rat hepatoma cell line or the RTG-2 rainbow trout gonadal cell line by means of four standard cytotoxicity assays. Nominal concentrations were verified by inductively coupled plasma mass spectrometry (ICP-MS) and methods were assessed for their suitability to detect reliably adverse effects due to particle exposure. All three particles showed aggregation in water and media. In the H4IIE cell line, the MTT cytotoxicity test revealed that negative effects could be observed for the CeO2 NPs after 24h and for all particles after 72h of exposure, making the effects size, concentration and time dependent. No negative effect for the concentrations tested was detected for the remaining three assays and the RTG-2 cell line, making the MTT assay and the H4IIE cell line an appropriate system to assess adverse effects of CeO2 NPs. A verification of the nominal concentration through ICP-MS revealed that there was a discrepancy between nominal and measured concentration depending on concentration and particle tested. Interferences of particles with assays were found to be present and need to be taken into consideration.

Abstract LB-25: Activation of AMPK by ATM in reponse to AICAR and metformin

Abstract AMPK is an AMP-activated protein kinase that plays a key role in regulating cellular energy homeostasis. Metabolic stress, such as heat shock and glucose starvation, causes energy deficiency in the cell and leads to elevated levels of intracellular AMP. This results in the phosphorylation and activation of AMPK. LKB1, a tumor suppressor, has been known as an upstream kinase of AMPK. We found that in response to treatment with 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside (AICAR), a LKB1-null cancer cell line, HeLa, still exhibited AMPK phosphorylation. This indicates the existence of an LKB1-independent AMPK protein kinase. ATM is a protein kinase that is deficient in the disease ataxia telangiectasia (A-T). We measured the activation of AMPK by AICAR in the normal mouse embryo fibroblast (MEF) cell line, A29, and the MEF cell line lacking the ATM protein, A38. In A38 cells, the level of AICAR-induced AMPK phosphorylation was dramatically lower than that found in A29 cells. Furthermore, phosphorylation of AMPK in HeLa and A29 cells was inhibited by an ATM specific inhibitor, KU-55933. Our results demonstrate that AICAR treatment could lead to phosphorylation of AMPK in an ATM-dependent and LKB1-independent manner. Similar to the results of AICAR treatment, our work also revealed that the metformin-induced phosphorylation of AMPK in A38 cells is significantly lower than that in A29 cells. We also examined the effect of ATM on AMPK in rat hepatoma cell line H4IIE. Inhibition of ATM by KU-55933 dramatically reduced the phosphorylation of AMPK in response to metformin in this cell line. Thus, ATM may act as a potential AMPK kinase in response to both AICAR and metformin treatment. Metformin is the most commonly prescribed pharmaceutical drug for type 2 diabetes (T2D). In addition, patients with T2D or obesity/insulin resistance who take metformin have reduced rates of cancer occurrence, including breast cancer. However, the mechanism underlying metformin's effect on cancer prevention and/or treatment is unclear (Nature, New &amp; views, Feb. 2011; Science, News Focus, Jan. 2012). While ATM acts as a sensor of DNA damage by controlling cell cycle progression, it also has glucose regulatory function. Our results put ATM as a novel player in the AMPK signaling pathway in response to metformin. These findings provide new insights into the mechanisms by which metformin functions as a preventive and/or therapeutic agent for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-25. doi:1538-7445.AM2012-LB-25

De-Risking of Stilbazulenyl Nitrone (STAZN), a Lipophilic Nitrone to Treat Stroke Using a Unique Panel of In Vitro Assays

In the present study, we used a comprehensive panel of in vitro assays to evaluate the efficacy and safety of stilbazulenyl nitrone (STAZN) as a lead compound to treat acute ischemic stroke. First, we measured neuroprotection in vitro using two different HT22 hippocampal nerve cell assays. Secondly, to de-risk drug development, we used CeeTox analysis with the H4IIE rat hepatoma cell line to determine the acute toxicity profile of STAZN. Third, STAZN was tested in microsomes from four species for measures of metabolic stability. Last, we determined the Ames test genotoxicity profile of STAZN using Salmonella typhimurium TA989 and TA100. In vitro, STAZN was neuroprotective against toxicity induced by iodoacetic acid, and oxytosis-induced glutathione depletion was initiated by glutamate, with an EC(50) value of 1-5 μM. Secondly, using CeeTox analysis, the estimated C(Tox) value (i.e., sustained concentration expected to produce toxicity in a rat 14-day repeat dose study) for STAZN was calculated to be 260 μM. Third, the half-life of STAZN in humans, dogs, and rats was 60-78 min. Last, the genotoxicity profile showed that STAZN did not induce bacterial colony growth under any conditions tested, indicating the lack of mutagenicity with this compound. STAZN appears to be a multi-target neuroprotective compound that has an excellent safety profile in both the CeeTox and Ames mutagenicity assays. STAZN may have significant potential as a novel neuroprotective agent to treat stroke and should be pursued in clinically relevant embolic stroke models.

The suitability of rat hepatoma cell line H4IIE for evaluating the potentials of compounds to induce CYP3A23 expression

To investigate the suitability of H4IIE cells for detecting cytochrome P450 (CYP) induction in vitro, we compared CYP induction by typical CYP inducers in H4IIE cells and rat primary hepatocytes by examining gene expression and enzyme activity, and by immunocytochemistry. The cells were preincubated with 0.1 μM of dexamethasone (DEX) for 24 h, followed by 48 h of exposure to 10 μM of beta-naphthoflavone (bNF), 100 μM of phenobarbital (PB) and 10 μM of DEX. Cyp1a1, Cyp2b1/2 and Cyp3a23/3a1 (Cyp3a23) expressions in H4IIE cells were up-regulated 280-, 1.5- and 65-fold relative to those in vehicle-treated cells, respectively. The fold inductions of those expressions in rat primary hepatocytes were 80-, 33- and 152-fold, respectively. Comprehensive gene expression analysis using DNA microarrays showed that Cyp3a23, Gsta2, Ugt2b12, Udpgt and Sult2a1 expressions were up-regulated in H4IIE cells exposed to 10 μM of DEX. CYP3A activity was not increased, but some H4IIE cells exposed to DEX were stained strongly with anti-CYP3A antibody. We cloned these cells and obtained cloned H4IIE (cH4IIE) cells with expression level of Cyp3a23 higher than those of vehicle-treated cells. It was confirmed that preincubation with 0.1 μM of DEX increased pregnane X receptor (Pxr) expression level and enhanced the Cyp3a23 induction effects of test compounds significantly. Retrospective examination of in vitro CYP induction assay using cH4IIE cells resulted in 80% correlation with the data from in vivo rat toxicity studies. These results suggested that cH4IIE cells are suitable for evaluating the potentials of a compound to induce CYP3A23 expression.

Generation of a Monoclonal Antibody Specifically Reacting with Undifferentiated Liver Progenitor Cells

An adult rat liver progenitor cell line Lig-8 was established. In the induction by sodium butyrate, these cells were shown to be able to differentiate into both hepatocytes and bile duct cells expressing albumin and cytokeratin-19, the markers of respective cell types. In order to generate Lig-8 specific antibody for further studies, we produced a monoclonal antibody using the whole Lig-8 cells as immunogen. The yielded monoclonal antibody, named Ligab, belongs to IgG subclass G1 and kappa light chain. It specifically stained on Lig-8 cells in the cytoplasm but not on a rat hepatoma cell line H4IIE. Its immunoreaction against Lig-8 cell lysate on dot blots diminished as the concentration of sodium dodecyl sulfate (SDS) in the lysate increased to 2%, a level in the sample buffer of standard SDS-polyacrylamide gel electrophoresis (PAGE). Not surprisingly, Ligab failed to detect its reacting antigen in Lig-8 cell lysate by standard SDS-PAGE-based immunoblotting. It could detect this antigen only by native PAGE-based immunoblotting. These characteristics suggested that the antigenic epitope for Ligab is likely a molecular structure instead of a peptide sequence. More interestingly, expression of Ligab-reacting antigen in Lig-8 cells declined as the cells were induced to differentiate by sodium butyrate. This antigen is very likely a differentiation-related marker for these cells, and this monoclonal antibody may help study the molecular mechanisms of liver progenitor cell differentiation.

Effect-directed analysis of contaminated sediment from the wastewater canal in Pancevo industrial area, Serbia

Wastewater canal (WWC) in Pancevo industrial area in Serbia, whose main environmental receptor is the River Danube, is a well known hot-spot of contamination. WWC sediments have been assessed by UNEP based on chemical target analysis. However, integrative biological data on exposure to hazardous compounds are only provided by the present study which aims at evaluating whether the monitored compounds sufficiently reflect potential hazards and to suggest additional compounds to include in monitoring and hazard assessment by applying effect-directed analysis (EDA) based on arylhydrocarbon receptor-mediated activity and cytotoxicity. Multistep NP-HPLC fractionation provided 18 fractions co-eluting with polychlorinated biphenyls (PCBs), polychlorinated dibenzo- p-dioxins (PCDDs), polycyclic aromatic hydrocarbons (PAHs) and more polar compounds. PAHs fractions exhibited great potencies to induce ethoxyresorufin- o-deethylase (EROD) in H4IIE rat hepatoma cell line expressed as 2,3,7,8-tetrachlorodibenzo- p-dioxin equivalents (TCDD-EQ) (0.1–34.6 × 10 3 pg g −1 dry weight). Chemical analysis of the most active fractions revealed great concentrations of PAHs (up to 292 × 10 2 ng g −1 sediment equivalents (SEQ)), methylated PAHs (up to 900 × 10 2 ng g −1 SEQ), and other alkyl-substituted PAHs. Only minor portions of biologically derived TCDD-EQs could be attributed to monitored PAHs with known relative potencies (REPs). We hypothesize that a major part of the activity is due to non-monitored alkylated and heterocyclic PAHs. Results of the cell cytotoxicity/proliferation assay on H4IIE cell line suggest the presence of sediment pollutants with pronounced potency to disturb cell growth.

Induction of DNA Double-Strand Breaks in the H4IIE Cell Line Exposed to Environmentally Relevant Concentrations of Copper, Cadmium, and Zinc, Singly and in Combinations

Xenobiotics, including heavy metals, exist in nature as complex mixtures of compounds with possible interactions. Induction of DNA damage such as DNA strand breaks may exert detrimental consequences to both individuals and populations. In this study, the induction of DNA double-strand breaks was assessed using the H4IIE rat hepatoma cell line following exposure to high and environmentally relevant concentrations of chloride salts of the metals cadmium (Cd), copper (Cu), and zinc (Zn), both singly and in combination. DNA strand break analysis was performed using agarose gel electrophoresis. Median molecular lengths were calculated from fragment size distributions acquired from gel image data and were used as a quantitative measure of DNA double-strand break induction. Exposure to high concentrations of Cu and Cd in combination produced a significant increase in the occurrence of DNA strand break. However, exposing cells to high concentrations of Cu, Cd, and Zn in combination resulted in significantly lower DNA double-strand break compared to control cells. Addition of low Zn to the Cd/Cu mixture restored DNA damage level back to that of the control. Environmentally relevant concentrations of Cd, Cu, and Zn did not appear to induce DNA strand breaks in the H4IIE cell line.

In vitro antidiabetic activities of five medicinal herbs used in Chinese medicinal formulae

Fructus Corni, Fructus Schisandrae Chinensis, Poria, Rhizoma Alismatis and Rhizoma Dioscoreae are commonly used in traditional Chinese medicine for diabetes treatment. They are also the component herbs of an antidiabetic foot ulcer formula with demonstrated clinical efficacy. Although some of these herbal extracts were previously shown to possess in vivo antidiabetic effects (i.e. lowering blood glucose levels), the underlying mechanisms remain elusive. The objective of this study is to investigate the possible antidiabetic mechanisms of these individual herbs, using a systematic study platform which includes four in vitro tissue models: glucose absorption into intestinal brush border membrane vesicles (BBMV), gluconeogenesis by rat hepatoma cell line H4IIE, glucose uptake by human skin fibroblasts cell line Hs68 and mouse adipocytes 3T3-L1. All tested herbs showed significant in vitro antidiabetic effects in at least two models. Fructus Schisandrae Chinensis, Poria, Rhizoma Alismatis and Rhizoma Dioscoreae showed significant inhibitory effects in the BBMV glucose uptake assay. All tested herbs showed significant stimulatory effects to the glucose uptake of Hs68 and 3T3-L1 cells, except Poria and Rhizoma Dioscoreae which were not effective to Hs68 and 3T3-L1 respectively. However, none of the tested herbs inhibited hepatic gluconeogenesis. In conclusion, the five herbs exhibited distinct antidiabetic mechanisms in vitro and hence our investigations provided scientific evidence to support the traditional usage of these herbs for diabetic treatment in medicinal formulae.

Resistin induces insulin resistance, but does not affect glucose output in rat-derived hepatocytes

The aim of the present study was to observe the effects of resistin on insulin sensitivity and glucose output in rat-derived hepatocytes. The rat hepatoma cell line H4IIE was cultured and stimulated with resistin; supernant glucose and glycogen content were detected. The insulin receptor substrate (IRS)-1 and IRS-2, protein kinase B/Akt, glycogen synthase kinase-3beta(GSK-3 beta), the suppressor of cytokine signaling 3 (SOCS-3) protein content, as well as the phosphorylation status were assessed by Western blotting. Specific antisense oligodeoxynucleotides directed against SOCS-3 were used to knockdown SOCS-3. Resistin induced insulin resistance, but did not affect glucose output in rat hepatoma cell line H4IIE. Resistin attenuated multiple effects of insulin, including insulin-stimulated glycogen synthesis and phosphorylation of IRS, protein kinase B/Akt, as well as GSK-3beta. Resistin treatment markedly induced the gene and protein expression of SOCS-3, a known inhibitor of insulin signaling. Furthermore, a specific antisense oligodeoxynucleotide directed against SOCS-3 treatment prevented resistin from antagonizing insulin action. The major function of resistin on liver is to induce insulin resistance. SOCS-3 induction may contribute to the resistin-mediated inhibition of insulin signaling in H4IIE hepatocytes.

APPLICATION OF STATISTICAL EXPERIMENTAL DESIGN AND MULTIVARIATE DATA ANALYSIS FOR EVALUATION OF MIXTURES USING CYTOCHROME P4501A INDUCTION

Cytochrome P4501A (CYP1A) induction was evaluated in the rat hepatoma cell line H4IIE as a biomarker of exposure to organic compounds. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay was performed to assess the viability of cells exposed to environmentally relevant concentrations of cadmium. Cadmium concentrations greater than approximately 0.7 microM were found to affect cell viability. Cells were exposed to environmentally relevant concentrations of 3,3',4,4'-tetrachlorobiphenyl (PCB 77) or benzo[a]pyrene (BaP) and to combinations of PCB 77, BaP, and cadmium based on a statistical experimental design. Quantification of CYP1A proteins using Western blot analysis showed that both BaP and PCB 77 induced CYP1A in a concentration-dependent manner up to 5 microM. Response surface modeling for evaluation of the combined effect of compounds was conducted using the multivariate regression model projection to latent structures (PLS). Analysis of response surface models for the ternary mixtures indicated antagonistic interactions between BaP and PCB 77 and a possible inhibitory effect of cadmium on PCB 77- induced CYP1A. Use of CYP1A induction in the H4IIE cell line with immunodetection of CYP1A proteins, combined with the application of response surface design and PLS, is shown to be a suitable strategy for evaluating combined effects in pollutant mixtures.

A paradox: Insulin inhibits expression and secretion of resistin which induces insulin resistance

To confirm whether insulin regulates resistin expression and secretion during differentiation of 3T3-L1 preadipocytes and the relationship of resistin with insulin resistance both in vivo and in vitro. Supernatant resistin was measured during differentiation of 3T3-L1 preadipocytes. L6 rat myoblasts and hepatoma cell line H4IIE were used to confirm the cellular function of resistin. Diet-induced obese rats were used as an insulin resistance model to study the relationship of resistin with insulin resistance. Resistin expression and secretion were enhanced during differentiation 3T3-L1 preadipocytes. This cellular differentiation stimulated resistin expression and secretion, but was suppressed by insulin. Resistin also induced insulin resistance in H4IIE hepatocytes and L6 myoblasts. In diet-induced obese rats, serum resistin levels were negatively correlated with insulin sensitivity, but not with serum insulin. Insulin can inhibit resistin expression and secretion in vitro, but insulin is not a major regulator of resistin in vivo. Fat tissue mass affects insulin sensitivity by altering the expression and secretion of resistin.

Identification of Ah Receptor Agonists in Soil of E-waste Recycling Sites from Taizhou Area in China

In recent years, increasing concern has surrounded the consequences of improper electric and electronic waste (e-waste) disposal. In order to mitigate or remediate the potentially severe toxic effects of e-waste recycling on the environment, organisms, and humans, many contaminated sites must first be well-characterized. In this study, soil samples were taken from Taizhou city, one of the largest e-waste disposal centers in China, which was involved in recycling for nearly 30 years. The extracts of the samples were assayed for aryl hydrocarbon receptor (AhR)-mediated ethoxyresorufin-O-deethylase (EROD) induction in the rat hepatoma cell line H4IIE. Some of the target AhR agonists, including polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs), were instrumentally analyzed as well. The cause-effect relationship and dose-response relationship between the chemical concentrations of AhR agonists and observed EROD activity were examined. The results showed that soil extracts could induce AhR activity significantly, and the chemically derived 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TEQcal) were perfectly correlated to bioassay-derived TCDD equivalents (TEQbio; R = 0.96, P < 0.001), which indicated that the known AhR agonists could account for the observed responses. Among different contributors, PCBs accounted for 87.2-98.2% and PCDD/Fs contributed 1.7-11.6% of TEQcal, while the contribution of PAHs could almost be neglected. Under these conditions, a quantitative dose-effect relationship between TEQ(PCB) and EROD activity could be evaluated, suggesting that the observed AhR effect was mainly caused by PCBs. Further source identification by congener profiles analysis showed that the crude dismantling of electric power devices and open burning of electric wires and printed circuit boards may be the main sources of these dioxin-like compounds. This study suggests that the combination of in vitro bioassay and chemical analysis is useful to screen, identify, and prioritize AhR agonists in soil from e-waste recycling areas.