H4IIE Cell Research Articles

Removal efficiencies and risk assessment of endocrine-disrupting chemicals at two wastewater treatment plants in South China

Endocrine-disrupting chemicals (EDCs) in the effluent from wastewater treatment plants (WWTPs) are an important pollutant sources of the aquatic system. In this study, the removal efficiencies of eight typical EDCs at two domestic WWTPs in Dongguan City, China, are reported based on instrumental analysis and bioassay results. Bioactivities, including steroidogenesis-disrupting effects, estrogen receptor (ER)-binding activity, and aryl hydrocarbon receptor (AhR)-binding activity were evaluated using the H295R, MVLN, and H4IIE cell bioassays, respectively. The potential environmental risks of these residual EDCs were also evaluated. The results of instrumental analysis showed that nonylphenol was the major chemical type present among the eight tested EDCs. Meanwhile, concentrations of estrogen compounds including estrone, 17β-estradiol (E2), estriol, 17α-ethinyl estradiol, and diethylstilbestrol were relatively low. The removal rates of all eight EDCs were relatively high. Although the chemical analysis indicated high removal efficiency, the bioassay results showed that steroidogenesis-disrupting effects as well as ER-binding and AhR-binding activities remained, with E2-equivalent values of effluent samples ranging from 0.16 to 0.9 ng·L−1, and 2,3,7,8-tetrachlorodibenzo-p-dioxin-equivalent values ranging from 0.61 to 4.09 ng L−1. Principal component analysis combined with regression analysis suggests that the chemicals analyzed in this study were partly responsible for these ER and AhR activities. Ecological risk assessment of the residual EDCs showed that estrone was the most hazardous chemical among the eight EDCs tested, with a risk quotient of 1.44–5.50. Overall, this study suggests that, despite high apparent removal efficiencies of typical EDCs, their bioactivities and potential ecological risks cannot be ignored.

Glucagon Activates mTOR1/2 Signaling via an EPAC/Rap1 Signaling Axis in Hepatocyte Cultures

Activation of the protein kinase mTOR (mechanistic target of rapamycin) in complex 1/2 (mTORC1/2) in the liver is repressed during fasting and rapidly stimulated in response to feeding. The effect of a meal on hepatic mTORC1/2 is largely mediated by an increase in plasma levels of nutrients (i.e. amino acids) and growth factors (i.e. insulin). By contrast, fasting is associated with elevated plasma levels of glucagon, which is conventionally viewed as playing a counter-regulatory role to insulin. However, glucagon and insulin secretion are both rapidly increased by consumption of a protein meal. In fact, amino acid-induced glucagon secretion is many times greater than that seen with fasting. Herein we investigated the impact of insulin and glucagon on mTORC1/2 activation in rat H4IIE and human HepG2 cell cultures. As expected, insulin enhanced phosphorylation of the mTORC1 substrates p70S6K1 and 4E-BP1. Surprisingly, the effect of glucagon on mTORC1 signaling was biphasic, wherein there was an acute increase in phosphorylation of p70S6K1 and 4E-BP1 over the first hour of exposure, followed by latent suppression. Notably, the stimulatory effect of glucagon on mTORC1 was not additive with insulin, suggesting a convergent signaling axis. Glucagon-induced mTORC1 activation was associated with enhanced cAMP levels and required activation of the glucagon receptor, PI3K/Akt, and exchange protein activated by cAMP (EPAC). An EPAC-specific cAMP analog as well as PKA inhibition enhanced activation of mTORC1/2. EPAC acts as the guanine nucleotide exchange factor for the small GTPase Rap1, and in H4IIE cultures, transient expression of Rap1 was sufficient to stimulate activation of mTORC1/2. Finally, we found that glucagon addition to culture medium was sufficient to promote Rap1-GTP loading in H4IIE cells. Overall, the findings support that glucagon elicits biphasic regulation of hepatic mTORC1/2 by an EPAC-dependent increase in Rap1-GTP which is followed by a repression of mTORC1.

Ameliorative potential of Lavandula stoechas in metabolic syndrome via multitarget interactions

Ethnopharmacological importanceDecoction and infusion prepared from aerial parts of Lavandula stoechas L. (L. stoechas) have been traditionally used as remedy against several components of metabolic syndrome (MetS) and associated disorders including type II diabetes and cardiovascular diseases by Anatolian people. Aim of the studyThe aim is to elucidate the potential ameliorative effects of L. stoechas aqueous extracts on insulin resistance and inflammation models through multitarget in vitro approaches and also to elucidate mechanism of action by analyzing transcriptional and metabolic responses. Materials and methodsAn aqueous extract was prepared and fractionated to give rise to ethyl acetate (EE) and butanol (BE) extracts. The anti-insulin resistance effects of BE and EE were evaluated on palmitate induced insulin resistance model of H4IIE, C2C12 and 3T3L1 cells by using several metabolic parameters. Specifically, whole genome transcriptome analysis was performed by using microarray over 55.000 genes in control, insulin resistant and EE (25 µg/mL) treated insulin resistant H4IIE cells. Anti-inflammatory effects of both extracts were analyzed in LPS-stimulated RAW264.7 macrophages. ResultsBoth EE and BE at low doses (25–50 µg/mL) significantly decreased hepatic gluconeogenesis in H4IIE cell line by suppressing the expression of PEPCK and G6Pase. In C2C12 myotubes, both extracts increased the insulin stimulated glucose uptake more effectively than metformin. Both extracts decreased the isoproterenol induced lipolysis in 3T3L1 cell line. Moreover, they also effectively increased the expression of lipoprotein lipase protein level in insulin resistant myotubes at low doses. EE increased the protein level of PPARγ and stimulated the activation AKT in insulin resistant H4IIE and C2C12 cell lines. The results obtained from biochemical assays, mRNA/protein studies and whole genome transcriptome analyses were found to be complementary and provided support for the hypothesis that EE might be biologically active against insulin resistance and act through the inhibition of liver gluconeogenesis and AKT activation. Besides, LPS induced inflammation in RAW264.7 macrophages was mainly inhibited by EE through suppression of iNOS/NO signaling, IL1β and COX-2 genes.HPLC-TOF/MS analysis of EE of L. stoechas mainly resulted in caffeic acid, apigenin, luteolin, rosmarinic acid and its methyl ester, 4-hydroxybenzoic acid, vanillic acid, ferrulic acid and salicylic acid. ConclusionData suggest that EE of L. stoechas contains phytochemicals that can be effective in the treatment/prevention of insulin resistance and inflammation. These results validate the traditional use of L. stoechas in Anatolia against several metabolic disorders including metabolic syndrome.

Dioxin-like activity of brominated dioxins as individual compounds or mixtures in in vitro reporter gene assays with rat and mouse hepatoma cell lines

In vitro reporter gene assays detecting dioxin-like compounds have been developed and validated since the middle 1990's, and applied to the determination of dioxin-like activities in various samples for their risk management. Data on characterizing the potency of individual brominated dioxins and their activity in mixture with chlorinated dioxins are still limited on the cell-based assay. This study characterized the dioxin-like activities of the 32 brominated dioxins, such as polybrominated dibenzo-p-dioxins, polybrominated dibenzofurans (PBDFs), coplanar polybrominated biphenyls, mixed halogenated dibenzo-p-dioxins and dibenzofurans (PXDFs), as a sole component or in a mixture by DR-CALUX (dioxin-responsive chemically activated luciferase expression) using the rat hepatoma H4IIE cell line and XDS-CALUX (xenobiotic detection systems–chemically activated luciferase expression) assays using the mouse hepatoma H1L6.1 cell line. The 2,3,7,8-TCDD-relative potencies (REPs) of most of the brominated dioxins were within a factor of 10 of the WHO toxicity equivalency factor (WHO-TEF) for the chlorinated analogues. The REPs of a few PXDFs were an order of magnitude higher than the corresponding WHO-TEFs, indicating their toxicological importance. Results with reconstituted mixtures suggest that the activity of brominated and chlorinated dioxins in both CALUX assays was dose-additive. Thus, obtained results indicated the applicability of the CALUX assays as screening tools of brominated dioxins together with their chlorinated analogues.

Evaluation of AhR-agonists and AhR-agonist activity in sediments of Liaohe River protected areas, China

A total of 9 sediment samples of Liaohe River protected areas were collected to evaluate aryl hydrocarbon receptor agonists (AhR-agonists) and AhR-agonist activity via chemical analysis and in vitro H4IIE cell bioassay. Results indicated that bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (Bio-TEQs) ranged from 89.1 to 251.1pg/g dry weight. Concentrations of 16 EPA polycyclic aromatic hydrocarbons (PAHs), 12 dioxin-like polychlorinated biphenyls (PCBs), and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) ranged from 256.8 to 560.1ng/g, 79.2 to 416.2pg/g, and 199.6 to 538.4pg/g, respectively. According to potency balance analysis, TEQchems based on PAHs, PCBs, and PCDD/Fs could contribute 16.56% to 26.11% of Bio-TEQs. This could be explained by the potential existence of unidentified AhR-agonists and the potential non-additive interactions among AhR-agonists in sediment extracts. Through the different contributions to Bio-TEQs, this study confirms that PCDD/Fs were the main pollutants that induced significantly AhR-agonist activity in sediments of Liaohe River protected areas.

Effect of genistein on lipid accumulation in H4II E cell induced by oleic acid

Objective To observe the effects of genistein(GEN)on oleic acid(OA)induced lipid accumulationin in H4 ⅡE cells and to discuss the possible mechanism of GEN in the pointof AMPK.Methods H4ⅡE cells were cultured in vitro.The control group(NOR),OA treatment group(MOD group)and GEN treatment group were established according to the experimental requirements.The effects of GEN on the proliferation of H4 Ⅱ E cells were measured with MTT assay.The intraeellular TG mass was quantified spectrophotometrically using TG test kit.Cell protein was determined by DCTM Protein Assay kit.The intracellular TG concentration which was used to evaluate lipid accumulation was corrected using protein content as an internal standard.Western blotting was applied to determine the expression of AMPK and P-AMPK (Thr172).Accordingly the phosphorylation levels of AMPK was by means of P-AMPK(Thr172)/AMPK.Results OA treatment can induce lipid accumulation in H4 Ⅱ E cells while GEN treatment can decrease the intracellular TG concentration through up-regulate the phosphorylation levels of AMPK,though the effect was blocked by Compound C that is the inhibitor of AMPK.Conclusion GEN has anti-accumulation of lipid effect in H4ⅡE cell.The mechanism of GEN protective effect is partially due to AMPK. Key words: Genistin ; Oleic acid ; H4ⅡE cell ; Lipid accumulation ; AMPK ;

In vitro dose–response effects of poly(amidoamine) dendrimers [amino-terminated and surface-modified with N-(2-hydroxydodecyl) groups] and quantitative determination by a liquid chromatography–hybrid quadrupole/time-of-flight mass spectrometry based method

This article presents a dose-response study of the effects of two types of third-generation (G3) and fourth-generation poly(amidoamine) (PAMAM) dendrimers on two cell lines (RTG-2 and H4IIE) by in vitro cytotoxicity assays with 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red uptake (NRU), and lactate dehydrogenase (LDH) assays. We particularly investigated the potential cytotoxic effect of positive surface charge, which a cationic amino-terminated PAMAM dendrimer can display, on the marked ability of PAMAM dendrimers to cross the cell membrane compared with PAMAM dendrimers functionalized with chains of N-(2-hydroxydodecyl). Quantification of dose-response effects was performed by use of mass spectrometry analysis. The analytical method using liquid chromatography-hybrid quadrupole/time-of-flight mass spectrometry that we developed allowed characterization of defective dendrimers instead of "ideal structures." Identification was based on accurate mass measurement, assignment of elemental composition, and the fully resolved (13)C/(12)C isotopic clusters of the multiply charged ions of PAMAM dendrimers. Validation of the liquid chromatography-mass spectrometry method made possible reliable and reproducible quantification of the extracellular and intracellular concentration of dendrimers at a micromolar level (limits of detection from 0.14 to 1.34 μM and from 0.43 to 1.82 μM in standard and culture medium, respectively). A higher cytotoxicity was found with the H4IIE cell line for surface-modified PAMAM dendrimers. The LDH assay was significantly more sensitive than the MTT and NRU assays, with half-maximal inhibitory concentrations (IC(50)) of 12.96 and 38.31 μg mL(-1) for surface-modified G3 and G4 dendrimers, respectively. No cytotoxic effects, in terms of IC(50), of amino-terminated PAMAM dendrimers were observed on both H4IIE and RTG-2 cells when the concentration was below 500 μg mL(-1) for G3 and G4 dendrimers.

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 1982: Turmeric oil inhibits hepatocellular carcinoma through abolishing EpCAM expression

Abstract Background: In previous study, we found that turmeric oil, a volatile oil from turmeric, can prevent hepatocellular carcinoma (HCC) in an orthotopic HCC mouse model. Epithelial cell adhesion molecule (EpCAM), a marker of cancer stem cells (CSC), is correlated with HCC initiation, progression, and prognosis. Our resent data showed that turmeric oil can decrease EpCAM expression in esophageal cancer and HCC cell lines. Since the role of EpCAM in HCC carcinogenesis is becoming clarified, it is likely that turmeric oil prevents HCC through EpCAM signaling. Aim: To investigate the mechanism of turmeric oil for HCC prevention. Method: EpCAM expression in the specimens from HCC patients (n=25) was evaluated by immunohistochemical staining. Red fluorescent protein (RFP)-labeled H4IIE cells were inoculated into liver lobe of ACI rats. The rat HCC tumor tissues from RFP-H4IIE cell inoculation were evaluated for the subpopulation cells, which co-expressed RFP and EpCAM. The HCC rats by H4IIE cell inoculation were treated with turmeric oil (100mg/kg, i.p, every 2 days) for 2 weeks. The H4IIE cells treated with turmeric oil as well as with EpCAM siRNA, and then inoculated at 106 into the right flank of nude BALB/c mice for 1 week. Three HCC cell lines from human (HepG-2), mouse (hepal-6), and rat (H4IIE) cells, were treated with turmeric oil to evaluate the cell viability EpCAM expression, and β-catenin in vitro. Results: In human samples, a strong EpCAM expression was found HCC tissues, while EpCAM was barely detected in the healthy controls. In HCC animal models, RFP+ EpCAM+ cells were detected in the formed tumor tissue and in circulation. The incidence of HCC by H4IIE cells inoculation was significantly decreased in rats treated with turmeric oil. Abolishing of EpCAM by turmeric oil caused the H4IIE cells unable to form tumor in nude mice. In cell culture study, turmeric oil significantly decreased EpCAM expression in all three HCC cell lines. Turmeric oil caused relocalization of β-catenin, a component in Wnt/β-catenin pathway, in nuclear and cytosol (Figure 1). Conclusion: 1) EpCAM-positive HCC cells contribute to HCC initiation in animal models; 2) The inhibition of HCC by turmeric oil is through decreasing EpCAM expression; 3). The regulation of EpCAM expression by turmeric oil is associated with Wnt/β-catenin pathway. 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 1982. doi:1538-7445.AM2012-1982

Palmitic and linoleic acids induce ER stress and apoptosis in hepatoma cells

ObjectivesHepatic inflammation and degeneration induced by lipid depositions may be the major cause of nonalcoholic fatty liver disease. In this study, we tried to investigate the effects of saturated and unsaturated fatty acids on hepatoma cell apoptosis.MethodsH4IIE liver cells were treated with palmitic acid, linoleic acid, or both with or without the calcium-specific chelator BAPTA-AM after which the expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis, caspase-3 levels, and calcium flux were measured.ResultsPalmitic or linoleic acid (250 μM) induced H4IIE cell apoptosis, which required calcium flux but not caspase-3. Apoptosis was not observed when cells were co-treated with linoleic acid (125 μM) and palmitic acid (250 μM). Importantly, the release of cytochrome C from mitochondria into cytoplasm during cell apoptosis was specifically detected only when linoleic acid (125 μM), but not palmitic acid (250 μM), was added to the cells. Depletion of intracellular calcium flux by the calcium-specific chelator, BAPTA-AM, abolished linoleic acid-induced apoptosis. Moreover, in the presence of BAPTA-AM, expression of the unfolded protein response (UPR)-associated genes, CHOP, GRP78, and GRP94, was induced by linoleic acid, but not palmitic acid.ConclusionsThe results suggest that linoleic acid promotes cell apoptosis through the release of cytochrome C, only if the intracellular calcium flux is unperturbed and intact. These results confirm that ER stress contributes to fatty acid-induced liver cell apoptosis.

Leptin inhibits hepatocellular carcinoma proliferation via p38-MAPK-dependent signalling

ObjectivesObesity is a significant risk factor for many liver diseases, including hepatocellular carcinoma (HCC). Leptin has been identified as a central mediator of factors that regulate energy intake and expenditure, including appetite, metabolism and fat storage. The role of leptin in the initiation, development and progression of HCC remains poorly understood. The aims of this study were to determine the effect(s) of leptin on HCC cell proliferation and to identify potential signalling mechanism(s) by which leptin exerts these effects. MethodsRat H4IIE HCC cells and H4IIE-derived HCC tumours were analysed for leptin receptor (LR) expression. H4IIE cells were treated with leptin (0–100ng/ml) in the absence or presence of pharmacological inhibitors of p42/p44 mitogen-activated protein kinase (MAPK) (PD98059), p38-MAPK (SB202190) or Janus kinase-signal transducers and activators of transcription (JAK-STAT) (AG490; 10µM) signalling. Cell proliferation was determined and signal pathway activity analysed. ResultsImmunohistochemistry identified increased LR expression in HCC in human tissue. Leptin did not significantly affect H4IIE cell numbers in serum-depleted (0.1% [v/v] foetal bovine serum [FBS]) medium. However, leptin significantly inhibited serum-stimulated (1.0% [v/v] FBS) H4IIE proliferation. Immunoblot analysis demonstrated that leptin significantly activated p42/p44-MAPK, p38-MAPK and STAT3 signalling in a time-dependent manner. Pretreatment of H4IIE cells with SB202190 abrogated leptin-dependent inhibition of H4IIE proliferation, an effect not observed in cells pretreated with PD98059 or AG490. ConclusionsLeptin inhibits HCC cell growth in vitro via a p38-MAPK-dependent signalling pathway. Identifying similar effects on tumour growth in vivo may provide an attractive therapeutic target for slowing HCC progression.

Aplicación del bioensayo EROD-H4IIE para la determinación de dioxinas en carnes de pollos broiler: un estudio de equivalencia con la cromatografía de gases de alta resolución acoplada a espectrometría de masas de alta resolución

The EROD bioassay with H4IIE cell line was applied in this study to determine the equivalence of the results for chicken meat between the ERODH4IIE Bioassay ...

Enhanced siRNA delivery using cationic liposomes with new polyarginine-conjugated PEG-lipid

Gene therapy based on small interfering RNA (siRNA) has emerged as an exciting new therapeutic approach. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of cationic liposomes that contain a new PEG-lipid. The new lipid, poly- l-arginine-conjugated polyethylene glycol (PLR–PEG), was synthesized. To confirm the synthesis of the amino acid-conjugated PEG-lipid, 1H NMR and gel permeation chromatography (GPC) were performed. Cationic liposomes as non-viral vectors were formulated using the cationic lipids 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl- sn-glycero-3-phosphoethanolaminepropane (DOPE), cholesterol (Chol) and PLR–PEG. Physicochemical properties of cationic liposomes were investigated. A GFP siRNA was used as a model siRNA to test the efficiency of cationic liposome-mediated siRNA delivery. The liposomes could enhance delivery efficiency and decrease cytotoxicity at an optimized lipid composition. The new cationic liposome formulation using a new PEG-lipid (PLR–PEG) showed not only enhanced intracellular delivery of siRNA but also decreased cytotoxicity in H4II-E and HepG2 cell lines. The GFP siRNA delivered by new cationic liposomes using PLR–PEG was effective in reducing the GFP protein expression levels of the gene. These results suggest that the new cationic liposomes could be used for efficient delivery of siRNA therapeutics.

Species-specific activation of nuclear receptors correlates with the response of liver drug metabolizing enzymes to EMD 392949 in vitro

We previously reported on the species-specific effects on drug metabolizing enzymes (DME), in particular cytochrome P450-dependent monooxygenases (P450s), by the drug development candidate EMD 392949 (EMD) in vitro and in vivo. Induction of P450s occurs via activation of specific transcription factors such as the arylhydrocarbon receptor (AhR) and the nuclear xenobiotic receptors (NXRs). We analyzed whether the reported species-specific P450 induction by EMD could be related to a specific activation of the CYP1A regulator AhR and the CYP3A regulator pregnane X receptor (PXR) in human and rat cell lines. The human HepG2 and rat H4IIE cell lines exhibited inducibility of CYP1A and 3A and expressed functional AhR as well as PXR. CYP3A was induced by EMD in human HepG2 cells exceeding the level induced by rifampicin, but was not induced in rat H4IIE cells. Regulation of P450s was not related to expression levels of their respective transcription factor, but EMD treatment resulted in a significant reporter gene activation in xenobiotic response enhancer module (XREM)-transfected HepG2 but not H4IIE cells indicating activation of human but not rat PXR. In summary, we showed that the P450 inducing properties of EMD were perfectly reflected by its ability to activate AhR or PXR in a species-specific manner. These findings support the tight correlation of species-specific nuclear receptor activation with P450 induction and foster the use of nuclear receptor activation as a complementary screen to identify cytochrome P450 inducers.

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.

Effects of DTPA on zinc flux in leukemia cell lines

Previous studies from our laboratory with Zn‐65 have shown that H4IIE (rat hepatoma) and GH3 (rat pituitary tumor) cell lines reduce zinc efflux in the presence of the extracellular metal chelator DTPA (diethylenetriaminepentaacetic acid). Primary rat hepatocytes and anterior pituitary cells give out more zinc under the same conditions. This may reflect the greater zinc requirements of the rapidly dividing transformed cells. Here, we examined the effects of DTPA on radioactive Zn‐65 flux in C58(NT) (rat thymic lymphoma) and HL‐60 (human promyelocytic leukemia) cell lines. When these cells were co‐incubated with DTPA (50 µM) and Zn‐65, uptake of label was reduced. But when cells were pre‐labeled with Zn‐65 and then incubated with DTPA, the chelator increased efflux of Zn‐65. Despite being transformed, these cell lines behaved similarly to primary cells. Since the leukemia cells grow in suspension unlike the monolayer cell lines previously examined, we determined whether non‐adherence contributed to the difference in response to zinc deprivation. Experiments were repeated with H4IIE cells kept in suspension by growth in roller bottles. However, even under these conditions, DTPA enhanced retention of cellular zinc in the hepatoma cells. Thus, leukemia cells differ from other transformed cell lines in their homeostatic response to zinc deprivation and this is not due to their inability to form a monolayer.

Regulation of Cpt1b Gene Transcription in liver and muscle cell lines

Regulation of mitochondrial fatty acid oxidation resides at the outer membrane enzyme carnitine palmitoyltransferase‐1 (Cpt1). The Cpt1b isoform is expressed in skeletal muscle cells while liver cells express Cpt1a. Fatty acids stimulate expression of Cpt1 genes through PPARa response elements (PPRE). We found a similar expression spectrum of Cpt1b‐luciferase activities in C2C12, L6 and H4IIE cell lines. WY‐14,643 (WY) stimulated expression in H4IIE cells, but not in C2C12 or L6 cells. Measurement of PPARa mRNA by real‐time PCR indicated the muscle cell lines had low levels of PPARa as well as PGC1a. After cotransfection of L6 cells with PPARa and PGC1a expression vectors, luciferase activity was increased by PPARa and additionally enhanced by PGC1a. WY further stimulated expression in cells with PPARa overexpression. Mutation of the PPRE in the ‐391/+80 Cpt1b construct eliminated WY stimulation of transcription, but co‐transfection with the PGC1a still increased luciferase activity in the mutant construct. Results show that activation through PPRE is highly dependent on PPARa binding, but PGC1a can stimulate through PPARa and independently of PPARa.

Bioactivity of POPs and their effects in mosquitofish in Sydney Olympic Park, Australia

The site of the 2000 Olympic Games (Sydney Olympic Park (SOP), Sydney, Australia) was contaminated by persistent organic pollutants (POPs) prior to remediation in the 1990s. This study investigates the bioactivity of POPs in the sediment and water of wetlands across SOP by in vitro 2,3,7,8 -TCDD equivalence (TCDDeq) measurement (H4IIE cell line bioassay). Further, it examines whether disturbance of these sediments is likely to mobilise ligands for this receptor into the water column. Exposure to aryl hydrocarbon receptor (AhR) ligands was measured in vivo using hepatic cytochrome P4501A (CYP1A) induction (EROD) in the mosquitofish ( Gambusia holbrooki). Aqueous TCDDeq ranged from 0.013 to 0.057 pM in SOP wetlands which was significantly ( p < 0.05) less that in urban reference sites. These concentrations were not correlated to physical or chemical characteristics of the wetlands. In the sediments, TCDDeq ranged from 0.0016 to 7.06 µg/kg and these were not significantly ( p ≥ 0.05) different to that measured in urban reference sites. Simulated disturbance of small quantities of sediment in water samples significantly ( p < 0.05) increased the levels of TCDDeq measured in the water. Sediment TCDDeq was correlated to sediment ΣPAH concentration in 2006 and sediment ΣPCB, ΣDDT concentrations and fine sediment grain size in 2005. While fish at one SOP wetland had hepatic EROD activity elevated above the estimated basal level for this species, these were at the lower end of the range measured in urban impacted, non-remediated wetlands. EROD activity was positively correlated with both the sediment ΣPCB load and aqueous TCDDeq. Increased catchment size was correlated with increased EROD activity suggesting an even spread of POPs throughout the residential areas of the Sydney metropolitan area. The concentration of bioactive POPs in the wetlands of SOP is therefore low relative to urban reference sites demonstrating the ongoing success of the remediation program.

Characterization of aryl hydrocarbon receptor agonists in sediments of Wenyu River, Beijing, China

Aryl hydrocarbon receptor agonistic (Ah-agonistic) effects of 23 sediments from Wenyu River in Beijing, China were evaluated using the H4IIE cell bioassay. Five samples were selected for chemical analysis of most concerned Ah-agonists, i.e. polychlorinated dibenzo- p-dioxins (PCDDs), dibenzofurans (PCDFs), biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs). All raw sediment extracts induced significant Ah-agonistic effects, and the bioassay-derived 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) equivalents of raw extracts (TEQ raws) ranged from 8.5 to 336.0 pg/g dry weight (dw). Chemical analysis-derived TEQs (TEQ chems) ranged from 20.5 to 64.8 pg/g dw. When raw extracts were purified by sulphuric acid silica gel column to derive acid stable fraction, the TCDD equivalents in this fraction (TEQ stables) ranged from 2.7 to 63.8 pg/g dw. PCBs, PCDDs and PCDFs contributed about 51.4–72.1%, 1.2–7.3%, and 16.4–34.8% of TEQ stables, respectively, and the sum was 69.1–108.6%. Therefore, about 65.0% of TEQ raws could be attributed to acid labile fraction and only 3.3–9.6% to PAHs. These observations suggested that acid stable fraction and labile fraction contributed together to total Ah-agonistic effects in the sediments, and PCBs and PCDFs might be the two main components in acid stable Ah-agonists. The proposed approach using both H4IIE cell bioassay and chemical analysis could be used for characterization and prioritization of Ah-agonists in river sediments and helpful to the following progression of ecological remediation.

Zinc retention differs between primary and transformed cells in response to zinc deprivation

Previous studies in our laboratory have demonstrated that reducing the availability of zinc with the extracellular metal chelator DTPA (diethylenetriaminepentaacetate) enhances, rather than inhibits, the thyroid hormone induction of growth hormone mRNA in GH3 rat anterior pituitary tumor cells. To understand the actions of the chelator on cellular zinc status, we observed the effects of DTPA on 65Zn uptake and retention. DTPA reduced the uptake of 65Zn by GH3 cells from the medium, but when GH3 cells were prelabeled with 65Zn, it resulted in greater retention of the isotope. In primary hepatocytes, DTPA both reduced the uptake of 65Zn from the medium and increased efflux from prelabeled cells. To investigate this difference, we studied the effects of DTPA on radioactive zinc flux in the H4IIE (rat hepatoma), MCF-7 (human breast cancer) and Hs578Bst (nontransformed human mammary) cell lines and in rat primary anterior pituitary cells. DTPA reduced the uptake of 65Zn in all cell lines examined. DTPA increased the retention of 65Zn in prelabeled H4IIE, MCF-7 and Hs578Bst cells but reduced it in primary pituitary cells. Time course experiments showed that 65Zn efflux is shut down rapidly by DTPA in transformed cells, whereas the chelator causes greater efflux from primary hepatocytes over the first 6 h. Experiments with 14C-labeled DTPA confirmed that this chelator does not cross cell membranes, showing that it operates entirely within the medium. Expression of ZnT-1, the efflux transporter, was not affected by DTPA in H4IIE cells. Thus, zinc deprivation enhanced zinc retention in established cell lines but increased efflux from primary cells, perhaps reflecting differing requirements for this mineral.