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High throughput screening for thyroid hormone receptor mediated effects in a newly developed gh3.tre-luc reporter gene assay
Abstracts / Toxicology Letters 205S (2011) S60–S179
and winter PM10. Duration of cells exposure was 6 or 24 h. IL-1 levels were determined in culture medium by sandwich ELISA. An inhibitor of caspases-1 was used to evaluate its role in the activation of IL-1 release. Quantitative Western blot was performed to determine early endosomes expression antigen 1 (EEA1), involved in the TLR pathway and lysosomal damage. Results: IL-1 release was significantly increased after exposure to summer PM10, while winter PM10 produced no effect. Cells, pre-incubated with Caspasi-1, TLR2 and 4 inhibitors and then exposed to summer PM10, did not show an increase in IL-1 release. Furthermore in exposed macrophages the expression of NF-B and EEA1 increased after 6 h of summer PM10 treatment. This result confirms the ability of PM10 to activate the inflammosome pathway with subsequent release of IL-1. This result explains the increase in inflammatory lung diseases during summer season with high levels of PM10. doi:10.1016/j.toxlet.2011.05.563
P1330 Ready-to-use in vitro blood–brain barrier model D. Hallier-Vanuxeem 1,∗ , L. Dehouck 2 , E. Sevin 2 , Y. Delplace 1 , R. Cecchelli 2 R&D, Cellial Technologies, Lens, France, 2 Lbhe, Université Lille Nord de France, Lens, France
1
Cell models based on neural cell lines have shown promise for assessing neurotoxicity in vitro. However, in order to correlate with in vivo studies, these models must integrate biokinetic parameters that considered the extent and rate of CNS distribution such as Blood–Brain Barrier (BBB) permeability. Located at the level of brain capillaries, the BBB is a dynamic interface which regulates exchanges between blood and brain. In the early 90’s, Cecchelli et al. did pioneering work in establishing a highly predictive BBB model, consisting in the co-culture of bovine brain capillary endothelial cells (BBCEC) and rat glial cells. By modifying this well-validated co-culture model, a BBB system more easy to use, quick and suitable for High-Throughput Screening (HTS) has been developed. Available in frozen ready-to-use format, this model comprises BBCEC frozen onto collagen-coated 24-well cell culture insert plates. Upon thawing, the cells form a confluent endothelial cells monolayer after only 4 days of culture and then show the typical expression and localisation of tight junction proteins. The tightness of endothelial cell junctions was confirmed by a reproducible low permeability to Lucifer Yellow, a non permanent marker. Presence and functionality of the P-glycoprotein efflux transporter have also been demonstrated. To further characterise the model, drug permeability assays were performed using more than 35 CNS marketed drugs. These investigations demonstrated that data produced by the “Ready to use” BBB model were similar to the standard coculture model and can be easily used “in house” for HTS. doi:10.1016/j.toxlet.2011.05.564
S161
P1331 Use of three dimensional perfused liver models to study drug hepatotoxicity T. Hart 1,∗ , J. Yang 2 , E. Sceats 1 , J. Anderton 1 , D. Hughes 1 , Z. Li 1 , Z. Cui 3 , A. Rossi 2 1
Zyoxel Ltd., Oxford, UK, 2 Investigative Toxicology, Pfizer Ltd., Sandwich, UK, 3 Institute of Biomedical Engineering, University of Oxford, Oxford, UK Hepatotoxicity is a leading cause of drugs being removed from the market, which strongly suggests that existing pre-clinical models for hepatotoxic risk assessment must be improved. Conventional hepatocyte cultures suffer from impaired structure, function and genetic characteristics meaning that drug responses may be difficult to rationalise with in vivo observations. A potential solution is three dimensional (3D) culture, which offers a more in vivo-like environment for the growth and maintenance of cells. Here we describe the development and characterisation of several 3D perfused liver cultures and assess their suitability for toxicity testing. Scaffold-free and scaffold-based 3D cultures of HepG2 cells and primary rat hepatocytes were developed in TissueFlex, a perfusion microbioreactor. Liver-specific factors were analyzed by immunohistochemistry staining and imaged by fluorescence microscopy. Lactate dehydrogenase (LDH), urea and albumin secretion were also evaluated. Conventional (2D) hepatocyte cultures were characterized for comparative purposes. Under optimised 3D culture conditions high viability and liver-specific function was routinely achieved for more than 14 days for both cell line and primary cell cultures. Primary hepatocyte cultures were HNF4 positive, which indicates highly preserved cell differentiation. Meanwhile, albumin, cytokeratin-18, ZO-1 and Mrp-2 expression were all confirmed. Our results suggest that 3D perfusion culture may be an improved model for in vitro hepatotoxicity studies, particularly chronic toxicity studies, due to the extended maintenance of morphological and physiological integrity compared to 2D cultures. doi:10.1016/j.toxlet.2011.05.565
P1332 High throughput screening for thyroid hormone receptor mediated effects in a newly developed gh3.tre-luc reporter gene assay D. Hernández-Moreno 1,∗ , J. Freitas 1 , J.H.J. van den Berg 1 , C. Craig-Veit 2 , M.L. Goodson 3 , J.D. Furlow 2 , A.J. Murk 1 1
Toxicology, Wageningen University, Wageningen, The Netherlands, Neurobiology, Physiology and Behavior, University of California-Davis, Davis, USA, 3 Molecular and Cellular Biology, University of California-Davis, Davis, USA 2
The thyroid endocrine axis is of remarkable importance as the biologic effects of the thyroid hormones (TH), which are mainly mediated through the thyroid hormone receptors (TR), are essential for growth, development, cellular differentiation and metabolism. During the last years, a great effort has been put in the design and establishment of integrated and intelligent testing strategies for the evaluation of the potential adverse effects of chemicals on the TH mode of action, particularly in the development of in vitro assays. In this study, a stable reporter gene assay was developed using the THresponsive rat pituitary GH3 cell line, that constitutively expresses both TR isoforms. The functional test system proved highly repro-
S162
Abstracts / Toxicology Letters 205S (2011) S60–S179
ducible with triiodothyronine and thyroxine effect concentrations in the picomolar range that can already be quantified after 24 h of exposure. The GH3.TRE-Luc assay was subsequently applied for screening the US Library of Pharmacologically Active Compounds (LOPAC, 1281 chemicals) and the National Toxicology Program (NTP1408, 1408 chemicals) libraries in an automated 1536-well plate quantitative High Throughput Screening platform. Again the assay proved to be very robust and reproducible, and although the usually most active hydroxylated metabolites of poly halogenated aromatic compounds were not included in these chemicals collections, a number of potential agonists and antagonists were identified. Authors thank The Netherlands Organisation for Health Research and Development (ZonMW)–NOW Grant (11.400.0075) for subsidizing the research and Junta de Extremadura (Spain) and FEDER funds of the European Union for funding attendance of the congress. doi:10.1016/j.toxlet.2011.05.566
P1333 Hepatotoxic pyrrolizidine alkaloids—Bioavailability and cellular effects on human HEPG2 cells S. Hessel 1,∗ , C. Gottschalk 2 , H.S. Klaffke 2 , L. Heinze 1 , A. Preiss-Weigert 2 , M. Lahrssen-Wiederholt 2 , A. Lampen 1 1
Food Safety, Federal Institute for Risk Assessment, Berlin, Germany, Department of Safety IN THE Food Chain, Federal Institute for Risk Assessment, Berlin, Germany 2
Purpose: 1,2-Unsaturated pyrrolizidine alkaloids (PA) belong to the most toxic compounds. These substances are found in several plants such as Asteraceae and Boraginaceae families. Acute PA poisoning via food contamination causes severe damage to the liver; long-term, sub-lethal doses may cause cumulative damage or cancer. Methods: In this study, we analyzed the toxicological effects (cytotoxicity, ATP-content, and oxidative stress) of selected PA (retrorsine, senecionine, and seneciphylline) on the hepatocarcinoma cell line HepG2 by using a bioactivating system (S9-fraction). Furthermore, we characterized the uptake of senecionine and its N-oxidated form across the intestinal barrier by using the wellcharacterized human Caco-2 cell Transwell-model as an in vitro system for the human gut barrier. The PA content was analyzed by LC–MS/MS. Results: The metabolic bioactivation of PA with the S9-fraction of rat liver homogenate resulted in a weak increase of cellular cytotoxicity including a slight reduction of cellular ATPcontent, as well as in a weak increase of the apoptosis rate and of oxidative stress. The Caco-2 cell absorption studies revealed a fast passage of senecionine from the apical into the basolateral compartment suggesting that active transport mechanisms are involved in the uptake and excretion of the substance. In contrast, the oxidized metabolite senecionine-N-oxide did not cross the differentiated Caco-2 monolayer from the apical to the basolateral side. In addition, we observed the conversion of senecionine into the oxidated form, as well as the reduction of senecionine-Noxide. This indicates that Caco-2 cells are able to metabolize this compound. doi:10.1016/j.toxlet.2011.05.567
P1334 Time dependent evaluation of the cytotoxicity and carcinogenicity of bioremediated petroleum hydrocarbon contaminated soil D.H.T. Ho 1,∗ , A.S. Ball 2 , B.J. Sanderson 1 , L. Schmidt 1 1 2
Medical Biotechnology, Flinders University, Bedford Park, Australia, Biological Sciences, Flinders University, Bedford Park, Australia
Bioremediation is a process which aims to reduce the amount of chemical contaminants in an environmental sample through biological activity. One goal of bioremediation is to reduce the hazard posed by the contaminated sample. However, the toxicity or carcinogenicity of the sample may vary during this remediation process due to the breakdown of various contaminants into their metabolites. This study monitored the changes in toxicity and carcinogenicity of petroleum hydrocarbon contaminated soil extracts in HepG2 cells before, during and after bioremediation by a combination of bioaugmentation and biostimulation over a period of 0–12 weeks. Two in vitro cell viability assays, the MTT assay and the Crystal Violet assay were used to evaluate the cytotoxicity of the soil extracts in HepG2 cells. Chemical analysis of the extracts was carried out using GC analysis. To determine potential carcinogenicity, western blotting was employed to analyse changes in the levels of apoptosis and carcinogenesis related proteins p53 or Mdm2 in HepG2 cells in response to exposure to a sublethal dose of soil extract. Results will be presented comparing changes in petroleum hydrocarbons to changes in cell viability, p53 and Mdm2 protein expression. This will show if there is any relationship between toxicity or carcinogenicity and the nature of contamination and/or breakdown products of the contaminating material during the bioremediation process. doi:10.1016/j.toxlet.2011.05.568
P1335 An investigation into the comparative toxicity of glyphosate formulations and household detergents N.J. Hodges ∗ , S. Jondhale School of Biosciences, University of Birmingham, Birmingham, UK Purpose: Studies have reported that glyphosate and glyphosateformulations are toxic when tested in vitro at high concentrations. This investigation compared the toxicity of the glyphosate formulation R400 to a glyphosate free-formulation blank and three household detergents; sodium dodecyl sulphate (SDS), benzalkonium chloride (BAC) and biosoft (BOS). Methods: HEK293 and JEG3 cell lines and primary human vein cord epithelial cells (HUVEC) were cultured and treated with test materials (0–0 062%, v/v) for 24 h. Cell viability (release of adenylate kinase), mitochondrial function (MTT assay) and caspase 3/7 activity in cell lysates were measured. Results: R400 and the formulation blank had similar toxicity profiles in both the MTT and adenylate kinase release assays in all cells, comparable with SDS and BOS. BOC was approximately an order of magnitude more toxic to cells. Only weak activation of caspase 3/7 was observed in HEK293 and JEG3 cells suggesting that necrosis is the primary mode of cell death. In contrast, test materials induced a marked activation of caspase 3/7 in primary HUVEC cells but no statistically significant difference between the response of R400, the formulation blank, SDS and BOS. BAC was an order of magni-
and winter PM10. Duration of cells exposure was 6 or 24 h. IL-1 levels were determined in culture medium by sandwich ELISA. An inhibitor of caspases-1 was used to evaluate its role in the activation of IL-1 release. Quantitative Western blot was performed to determine early endosomes expression antigen 1 (EEA1), involved in the TLR pathway and lysosomal damage. Results: IL-1 release was significantly increased after exposure to summer PM10, while winter PM10 produced no effect. Cells, pre-incubated with Caspasi-1, TLR2 and 4 inhibitors and then exposed to summer PM10, did not show an increase in IL-1 release. Furthermore in exposed macrophages the expression of NF-B and EEA1 increased after 6 h of summer PM10 treatment. This result confirms the ability of PM10 to activate the inflammosome pathway with subsequent release of IL-1. This result explains the increase in inflammatory lung diseases during summer season with high levels of PM10. doi:10.1016/j.toxlet.2011.05.563
P1330 Ready-to-use in vitro blood–brain barrier model D. Hallier-Vanuxeem 1,∗ , L. Dehouck 2 , E. Sevin 2 , Y. Delplace 1 , R. Cecchelli 2 R&D, Cellial Technologies, Lens, France, 2 Lbhe, Université Lille Nord de France, Lens, France
1
Cell models based on neural cell lines have shown promise for assessing neurotoxicity in vitro. However, in order to correlate with in vivo studies, these models must integrate biokinetic parameters that considered the extent and rate of CNS distribution such as Blood–Brain Barrier (BBB) permeability. Located at the level of brain capillaries, the BBB is a dynamic interface which regulates exchanges between blood and brain. In the early 90’s, Cecchelli et al. did pioneering work in establishing a highly predictive BBB model, consisting in the co-culture of bovine brain capillary endothelial cells (BBCEC) and rat glial cells. By modifying this well-validated co-culture model, a BBB system more easy to use, quick and suitable for High-Throughput Screening (HTS) has been developed. Available in frozen ready-to-use format, this model comprises BBCEC frozen onto collagen-coated 24-well cell culture insert plates. Upon thawing, the cells form a confluent endothelial cells monolayer after only 4 days of culture and then show the typical expression and localisation of tight junction proteins. The tightness of endothelial cell junctions was confirmed by a reproducible low permeability to Lucifer Yellow, a non permanent marker. Presence and functionality of the P-glycoprotein efflux transporter have also been demonstrated. To further characterise the model, drug permeability assays were performed using more than 35 CNS marketed drugs. These investigations demonstrated that data produced by the “Ready to use” BBB model were similar to the standard coculture model and can be easily used “in house” for HTS. doi:10.1016/j.toxlet.2011.05.564
S161
P1331 Use of three dimensional perfused liver models to study drug hepatotoxicity T. Hart 1,∗ , J. Yang 2 , E. Sceats 1 , J. Anderton 1 , D. Hughes 1 , Z. Li 1 , Z. Cui 3 , A. Rossi 2 1
Zyoxel Ltd., Oxford, UK, 2 Investigative Toxicology, Pfizer Ltd., Sandwich, UK, 3 Institute of Biomedical Engineering, University of Oxford, Oxford, UK Hepatotoxicity is a leading cause of drugs being removed from the market, which strongly suggests that existing pre-clinical models for hepatotoxic risk assessment must be improved. Conventional hepatocyte cultures suffer from impaired structure, function and genetic characteristics meaning that drug responses may be difficult to rationalise with in vivo observations. A potential solution is three dimensional (3D) culture, which offers a more in vivo-like environment for the growth and maintenance of cells. Here we describe the development and characterisation of several 3D perfused liver cultures and assess their suitability for toxicity testing. Scaffold-free and scaffold-based 3D cultures of HepG2 cells and primary rat hepatocytes were developed in TissueFlex, a perfusion microbioreactor. Liver-specific factors were analyzed by immunohistochemistry staining and imaged by fluorescence microscopy. Lactate dehydrogenase (LDH), urea and albumin secretion were also evaluated. Conventional (2D) hepatocyte cultures were characterized for comparative purposes. Under optimised 3D culture conditions high viability and liver-specific function was routinely achieved for more than 14 days for both cell line and primary cell cultures. Primary hepatocyte cultures were HNF4 positive, which indicates highly preserved cell differentiation. Meanwhile, albumin, cytokeratin-18, ZO-1 and Mrp-2 expression were all confirmed. Our results suggest that 3D perfusion culture may be an improved model for in vitro hepatotoxicity studies, particularly chronic toxicity studies, due to the extended maintenance of morphological and physiological integrity compared to 2D cultures. doi:10.1016/j.toxlet.2011.05.565
P1332 High throughput screening for thyroid hormone receptor mediated effects in a newly developed gh3.tre-luc reporter gene assay D. Hernández-Moreno 1,∗ , J. Freitas 1 , J.H.J. van den Berg 1 , C. Craig-Veit 2 , M.L. Goodson 3 , J.D. Furlow 2 , A.J. Murk 1 1
Toxicology, Wageningen University, Wageningen, The Netherlands, Neurobiology, Physiology and Behavior, University of California-Davis, Davis, USA, 3 Molecular and Cellular Biology, University of California-Davis, Davis, USA 2
The thyroid endocrine axis is of remarkable importance as the biologic effects of the thyroid hormones (TH), which are mainly mediated through the thyroid hormone receptors (TR), are essential for growth, development, cellular differentiation and metabolism. During the last years, a great effort has been put in the design and establishment of integrated and intelligent testing strategies for the evaluation of the potential adverse effects of chemicals on the TH mode of action, particularly in the development of in vitro assays. In this study, a stable reporter gene assay was developed using the THresponsive rat pituitary GH3 cell line, that constitutively expresses both TR isoforms. The functional test system proved highly repro-
S162
Abstracts / Toxicology Letters 205S (2011) S60–S179
ducible with triiodothyronine and thyroxine effect concentrations in the picomolar range that can already be quantified after 24 h of exposure. The GH3.TRE-Luc assay was subsequently applied for screening the US Library of Pharmacologically Active Compounds (LOPAC, 1281 chemicals) and the National Toxicology Program (NTP1408, 1408 chemicals) libraries in an automated 1536-well plate quantitative High Throughput Screening platform. Again the assay proved to be very robust and reproducible, and although the usually most active hydroxylated metabolites of poly halogenated aromatic compounds were not included in these chemicals collections, a number of potential agonists and antagonists were identified. Authors thank The Netherlands Organisation for Health Research and Development (ZonMW)–NOW Grant (11.400.0075) for subsidizing the research and Junta de Extremadura (Spain) and FEDER funds of the European Union for funding attendance of the congress. doi:10.1016/j.toxlet.2011.05.566
P1333 Hepatotoxic pyrrolizidine alkaloids—Bioavailability and cellular effects on human HEPG2 cells S. Hessel 1,∗ , C. Gottschalk 2 , H.S. Klaffke 2 , L. Heinze 1 , A. Preiss-Weigert 2 , M. Lahrssen-Wiederholt 2 , A. Lampen 1 1
Food Safety, Federal Institute for Risk Assessment, Berlin, Germany, Department of Safety IN THE Food Chain, Federal Institute for Risk Assessment, Berlin, Germany 2
Purpose: 1,2-Unsaturated pyrrolizidine alkaloids (PA) belong to the most toxic compounds. These substances are found in several plants such as Asteraceae and Boraginaceae families. Acute PA poisoning via food contamination causes severe damage to the liver; long-term, sub-lethal doses may cause cumulative damage or cancer. Methods: In this study, we analyzed the toxicological effects (cytotoxicity, ATP-content, and oxidative stress) of selected PA (retrorsine, senecionine, and seneciphylline) on the hepatocarcinoma cell line HepG2 by using a bioactivating system (S9-fraction). Furthermore, we characterized the uptake of senecionine and its N-oxidated form across the intestinal barrier by using the wellcharacterized human Caco-2 cell Transwell-model as an in vitro system for the human gut barrier. The PA content was analyzed by LC–MS/MS. Results: The metabolic bioactivation of PA with the S9-fraction of rat liver homogenate resulted in a weak increase of cellular cytotoxicity including a slight reduction of cellular ATPcontent, as well as in a weak increase of the apoptosis rate and of oxidative stress. The Caco-2 cell absorption studies revealed a fast passage of senecionine from the apical into the basolateral compartment suggesting that active transport mechanisms are involved in the uptake and excretion of the substance. In contrast, the oxidized metabolite senecionine-N-oxide did not cross the differentiated Caco-2 monolayer from the apical to the basolateral side. In addition, we observed the conversion of senecionine into the oxidated form, as well as the reduction of senecionine-Noxide. This indicates that Caco-2 cells are able to metabolize this compound. doi:10.1016/j.toxlet.2011.05.567
P1334 Time dependent evaluation of the cytotoxicity and carcinogenicity of bioremediated petroleum hydrocarbon contaminated soil D.H.T. Ho 1,∗ , A.S. Ball 2 , B.J. Sanderson 1 , L. Schmidt 1 1 2
Medical Biotechnology, Flinders University, Bedford Park, Australia, Biological Sciences, Flinders University, Bedford Park, Australia
Bioremediation is a process which aims to reduce the amount of chemical contaminants in an environmental sample through biological activity. One goal of bioremediation is to reduce the hazard posed by the contaminated sample. However, the toxicity or carcinogenicity of the sample may vary during this remediation process due to the breakdown of various contaminants into their metabolites. This study monitored the changes in toxicity and carcinogenicity of petroleum hydrocarbon contaminated soil extracts in HepG2 cells before, during and after bioremediation by a combination of bioaugmentation and biostimulation over a period of 0–12 weeks. Two in vitro cell viability assays, the MTT assay and the Crystal Violet assay were used to evaluate the cytotoxicity of the soil extracts in HepG2 cells. Chemical analysis of the extracts was carried out using GC analysis. To determine potential carcinogenicity, western blotting was employed to analyse changes in the levels of apoptosis and carcinogenesis related proteins p53 or Mdm2 in HepG2 cells in response to exposure to a sublethal dose of soil extract. Results will be presented comparing changes in petroleum hydrocarbons to changes in cell viability, p53 and Mdm2 protein expression. This will show if there is any relationship between toxicity or carcinogenicity and the nature of contamination and/or breakdown products of the contaminating material during the bioremediation process. doi:10.1016/j.toxlet.2011.05.568
P1335 An investigation into the comparative toxicity of glyphosate formulations and household detergents N.J. Hodges ∗ , S. Jondhale School of Biosciences, University of Birmingham, Birmingham, UK Purpose: Studies have reported that glyphosate and glyphosateformulations are toxic when tested in vitro at high concentrations. This investigation compared the toxicity of the glyphosate formulation R400 to a glyphosate free-formulation blank and three household detergents; sodium dodecyl sulphate (SDS), benzalkonium chloride (BAC) and biosoft (BOS). Methods: HEK293 and JEG3 cell lines and primary human vein cord epithelial cells (HUVEC) were cultured and treated with test materials (0–0 062%, v/v) for 24 h. Cell viability (release of adenylate kinase), mitochondrial function (MTT assay) and caspase 3/7 activity in cell lysates were measured. Results: R400 and the formulation blank had similar toxicity profiles in both the MTT and adenylate kinase release assays in all cells, comparable with SDS and BOS. BOC was approximately an order of magnitude more toxic to cells. Only weak activation of caspase 3/7 was observed in HEK293 and JEG3 cells suggesting that necrosis is the primary mode of cell death. In contrast, test materials induced a marked activation of caspase 3/7 in primary HUVEC cells but no statistically significant difference between the response of R400, the formulation blank, SDS and BOS. BAC was an order of magni-