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Finest ambient particles induce a pro-inflammatory response and mucus overexpression in airway epithelial cells
S152
Abstracts / Toxicology Letters 205S (2011) S60–S179
In vitro toxicology P1301 Physiologically based kinetic modeling of bioactivation and detoxification of the alkenylbenzene methyleugenol in human as compared with rat A.A. Al-Subeihi 1,∗ , A. Spenkelink 1 , A. Punt 1 , M. Boersma 1 , P. van Bladeren 2 , I.M.C.M. Rietjens 1 1 2
Toxicology, Wageningen University, Wageningen, The Netherlands, Nestle Research Center, Lausanne, Switzerland
A physiologically based kinetic (PBK) model for the alkenylbenzene methyleugenol in human was defined. With this PBK model the relative extent of bioactivation and detoxification of methyleugenol at dose levels relevant for dietary human intake could be investigated. The outcomes were compared with those of a previously developed PBK model for methyleugenol in male rat (1) to allow evaluation of species differences. The results reveal that formation of 1 -hydroxymethyleugenol glucuronide, which represents the major metabolic pathway in male rat liver, appears to represent a minor metabolic pathway in human liver whereas in human liver a significantly higher formation of 1 -oxomethyleugenol compared with male rat liver is observed. In spite of a significant difference in especially the metabolic pathways of the proximate carcinogenic metabolite 1 -hydroxymethyleugenol between human and male rat, the influence of species differences on the ultimate overall bioactivation of methyleugenol to 1 -sulfooxymethyleugenol, the ultimate carcinogenic metabolite, appears to be limited. Moreover, the PBK model predicted the dose-dependent formation of 1 -sulfooxymethyleugenol in the liver of human and rat to be linear from doses as high as the benchmark dose (BMD10 ) for tumor formation in animal models down to as low as the virtual safe dose (VSD) representing a dose that would give a one in a million cancer risk upon life time exposure. It is concluded that kinetic data do not provide a reason to argue against a linear extrapolation from the rat tumor data to the human situation. (1) Al-Subeihi et al., 2011. Toxicol. In Vitro 25, 267–285. doi:10.1016/j.toxlet.2011.05.535
P1302 Decrease of BSEP expression is associated with oxidative stress in chlorpromazine-induced cholestasis in HepaRG cells S. Anthérieu ∗ , A. Guillouzo Faculté De Pharmacie, INSERM U991 - Université de Rennes 1, Rennes, France Purpose: Cholestasis is a clinical syndrome induced by various factors, including a number of drugs such as chlorpromazine (CPZ). Unfortunately, the mechanisms of drug-induced cholestasis are varied and poorly understood, and current models used for safety assessment in drug development do not accurately predict cholestasis in humans. We have investigated hepatic effects of CPZ using the well differentiated human hepatoma HepaRG cell line. Methods: Bile acid transport was analysed using [H3 ]-taurocholic acid, mRNA quantification by RT-qPCR analysis, CYP3A4 activity by testosterone 6-hydroxylation measurement and oxidative stress by the glutathione assay. All analyses were carried out after CPZ treatment of differentiated HepaRG cells. Results: CPZ induced an accumulation of bile acids, the hallmark of cholestasis, in HepaRG
cells, that was associated with a decreased expression of canalicular efflux transporters BSEP and MDR3. Moreover, increased basolateral transport (MRP4 overexpression), decreased bile acid synthesis (CYP8B1 inhibition) and enchanced bile acid detoxication (CYP3A4 up-regulation) could represent hepatoprotective mechanisms aimed at reducing intrahepatic accumulation of toxic bile acids. Furthermore CPZ was shown to generate reactive oxygen species and blocking this oxidative stress by N-acetyl cysteine strongly prevented bile acid accumulation and BSEP expression decrease but did not affect MDR3 expression. The present study provides the first in vitro analysis of mechanisms involved in CPZinduced cholestasis in a human model and support the view that the HepaRG cell line is an appropriate cell model for a better understanding of drug-induced liver lesions in human. doi:10.1016/j.toxlet.2011.05.536
P1303 Finest ambient particles induce a pro-inflammatory response and mucus overexpression in airway epithelial cells A. Baeza-Squiban 1,∗ , S. Val 1 , I. George 1 , H. Cachier 2 , L. Martinon 3 1
Bfa Laboratory Rmcx, University Paris Diderot-Paris 7, Paris, France, Cea-cnrs, Centre De Saclay, Laboratory of Climate and Environment Sciences, Gif sur Yvette, France, 3 Laboratoire des particules inhalées (LEPI), Paris, France 2
Purpose: This study aims at evaluating the pro-inflammatory potential and mucus expression after different size PM exposure. Method: Coarse, fine and ultrafine PM sampled with Dekati impactors were tested on human bronchial epithelial cells (16HBE, NCI-H292 cell lines and primary cultures NHBE) at non cytotoxic concentrations (max 10 g/cm2 ) for 24 h and different biomarkers were evaluated at the level of mRNA and protein releases. Results of the study: Finest PM (fine and ultrafine) are able to dosedependently induce amphiregulin (AR, an EGFR ligand) expression and release after 24 h exposure in 16HBE or NHBE cells. It is associated to the overexpression of heme oxygenase-1 (HO-1), an antioxidant enzyme, revealing an unbalance in cell redox homeostasis and the increased expression and release of two cytokines, GM-CSF and IL-6, showing the induction of a proinflammatory response. Such responses are size-dependent as the finest particles are always more reactive than coarse ones. Finest particles also exhibit a specific chemical composition characterized by a high amount of carbonaceous compounds. Among them are polyaromatic hydrocarbon (PAH) that become bioavailable as we observed an increased expression of cytochrome P450 1A1 (CYP1A1), known to be specifically induced by PAH as well as of NADPH Quinone-Oxydoreductase (NQO-1). In addition particles modify mucin expression by analysing MUC5AC expression in NCIH292 cells. Finest PM are the most efficient to induce MUC5AC expression and it is dependent on EGFR pathway as it decreased in presence of an inhibitor of EGFR activation (AG1478) and in presence of a neutralizing antibody of EGFR. doi:10.1016/j.toxlet.2011.05.537
Abstracts / Toxicology Letters 205S (2011) S60–S179
In vitro toxicology P1301 Physiologically based kinetic modeling of bioactivation and detoxification of the alkenylbenzene methyleugenol in human as compared with rat A.A. Al-Subeihi 1,∗ , A. Spenkelink 1 , A. Punt 1 , M. Boersma 1 , P. van Bladeren 2 , I.M.C.M. Rietjens 1 1 2
Toxicology, Wageningen University, Wageningen, The Netherlands, Nestle Research Center, Lausanne, Switzerland
A physiologically based kinetic (PBK) model for the alkenylbenzene methyleugenol in human was defined. With this PBK model the relative extent of bioactivation and detoxification of methyleugenol at dose levels relevant for dietary human intake could be investigated. The outcomes were compared with those of a previously developed PBK model for methyleugenol in male rat (1) to allow evaluation of species differences. The results reveal that formation of 1 -hydroxymethyleugenol glucuronide, which represents the major metabolic pathway in male rat liver, appears to represent a minor metabolic pathway in human liver whereas in human liver a significantly higher formation of 1 -oxomethyleugenol compared with male rat liver is observed. In spite of a significant difference in especially the metabolic pathways of the proximate carcinogenic metabolite 1 -hydroxymethyleugenol between human and male rat, the influence of species differences on the ultimate overall bioactivation of methyleugenol to 1 -sulfooxymethyleugenol, the ultimate carcinogenic metabolite, appears to be limited. Moreover, the PBK model predicted the dose-dependent formation of 1 -sulfooxymethyleugenol in the liver of human and rat to be linear from doses as high as the benchmark dose (BMD10 ) for tumor formation in animal models down to as low as the virtual safe dose (VSD) representing a dose that would give a one in a million cancer risk upon life time exposure. It is concluded that kinetic data do not provide a reason to argue against a linear extrapolation from the rat tumor data to the human situation. (1) Al-Subeihi et al., 2011. Toxicol. In Vitro 25, 267–285. doi:10.1016/j.toxlet.2011.05.535
P1302 Decrease of BSEP expression is associated with oxidative stress in chlorpromazine-induced cholestasis in HepaRG cells S. Anthérieu ∗ , A. Guillouzo Faculté De Pharmacie, INSERM U991 - Université de Rennes 1, Rennes, France Purpose: Cholestasis is a clinical syndrome induced by various factors, including a number of drugs such as chlorpromazine (CPZ). Unfortunately, the mechanisms of drug-induced cholestasis are varied and poorly understood, and current models used for safety assessment in drug development do not accurately predict cholestasis in humans. We have investigated hepatic effects of CPZ using the well differentiated human hepatoma HepaRG cell line. Methods: Bile acid transport was analysed using [H3 ]-taurocholic acid, mRNA quantification by RT-qPCR analysis, CYP3A4 activity by testosterone 6-hydroxylation measurement and oxidative stress by the glutathione assay. All analyses were carried out after CPZ treatment of differentiated HepaRG cells. Results: CPZ induced an accumulation of bile acids, the hallmark of cholestasis, in HepaRG
cells, that was associated with a decreased expression of canalicular efflux transporters BSEP and MDR3. Moreover, increased basolateral transport (MRP4 overexpression), decreased bile acid synthesis (CYP8B1 inhibition) and enchanced bile acid detoxication (CYP3A4 up-regulation) could represent hepatoprotective mechanisms aimed at reducing intrahepatic accumulation of toxic bile acids. Furthermore CPZ was shown to generate reactive oxygen species and blocking this oxidative stress by N-acetyl cysteine strongly prevented bile acid accumulation and BSEP expression decrease but did not affect MDR3 expression. The present study provides the first in vitro analysis of mechanisms involved in CPZinduced cholestasis in a human model and support the view that the HepaRG cell line is an appropriate cell model for a better understanding of drug-induced liver lesions in human. doi:10.1016/j.toxlet.2011.05.536
P1303 Finest ambient particles induce a pro-inflammatory response and mucus overexpression in airway epithelial cells A. Baeza-Squiban 1,∗ , S. Val 1 , I. George 1 , H. Cachier 2 , L. Martinon 3 1
Bfa Laboratory Rmcx, University Paris Diderot-Paris 7, Paris, France, Cea-cnrs, Centre De Saclay, Laboratory of Climate and Environment Sciences, Gif sur Yvette, France, 3 Laboratoire des particules inhalées (LEPI), Paris, France 2
Purpose: This study aims at evaluating the pro-inflammatory potential and mucus expression after different size PM exposure. Method: Coarse, fine and ultrafine PM sampled with Dekati impactors were tested on human bronchial epithelial cells (16HBE, NCI-H292 cell lines and primary cultures NHBE) at non cytotoxic concentrations (max 10 g/cm2 ) for 24 h and different biomarkers were evaluated at the level of mRNA and protein releases. Results of the study: Finest PM (fine and ultrafine) are able to dosedependently induce amphiregulin (AR, an EGFR ligand) expression and release after 24 h exposure in 16HBE or NHBE cells. It is associated to the overexpression of heme oxygenase-1 (HO-1), an antioxidant enzyme, revealing an unbalance in cell redox homeostasis and the increased expression and release of two cytokines, GM-CSF and IL-6, showing the induction of a proinflammatory response. Such responses are size-dependent as the finest particles are always more reactive than coarse ones. Finest particles also exhibit a specific chemical composition characterized by a high amount of carbonaceous compounds. Among them are polyaromatic hydrocarbon (PAH) that become bioavailable as we observed an increased expression of cytochrome P450 1A1 (CYP1A1), known to be specifically induced by PAH as well as of NADPH Quinone-Oxydoreductase (NQO-1). In addition particles modify mucin expression by analysing MUC5AC expression in NCIH292 cells. Finest PM are the most efficient to induce MUC5AC expression and it is dependent on EGFR pathway as it decreased in presence of an inhibitor of EGFR activation (AG1478) and in presence of a neutralizing antibody of EGFR. doi:10.1016/j.toxlet.2011.05.537