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Relative bioavailability of coumarin from cinnamon and cinnamon-containing foods compared to isolated coumarin: A crossover study in human volunteers
Abstracts / Toxicology Letters 205S (2011) S36–S59
OS7-5 Investigating danger signals in the KeratinoSens cell line: The potential link between Nrf2-activation and other signaling cascades A. Natsch 1,∗ , G. Ellis 2 , R. Emter 1 1
Bioscience, Givaudan Schweiz AG, Dübendorf, Switzerland, 2 Global Toxicology, Givaudan Schweiz AG, Vernier, Switzerland
Purpose: There is high interest in developing cell-based in vitro assays to predict skin sensitizers. Most assays measure innate responses to sensitizers, which may reflect the response of the cells to the ‘danger signal’, a signal thought to be a necessary second stimulus for the skin sensitization reaction. In the published studies, several key signaling pathways have been identified, which are triggered by skin sensitizers. The most eminent pathways are (i) the Nrf2-pathway, (ii) the MAPK p38 kinase pathway and (iii) oxidative stress pathways. In addition, the activation of the inflammasome/caspase-1 and the formation of heat-shock proteins appear to be important innate reactions, but the links between these pathways are not known. Method: We have developed the KeratinoSens assay based on Nrf2-signaling to screen for skin sensitizers. Using this cell line we have now studied in detailed dose-response analysis the possible links between activation of the Nrf2-pathway and (a) oxidative stress/redox signaling (as H2 O2 formation), (b) glutathione depletion, and (c) p38-activation. Results: This analysis indicates that different pathways become important depending on the dose of the allergens added, with redox signaling and p38 activation affecting the Nrf2-response to sensitizers at high doses close to cytotoxic concentrations only. Based on these studies, potential links between different danger signals will be discussed. doi:10.1016/j.toxlet.2011.05.191
OS7-6 Endotoxin, IgE antibody responses and allergenicity assessment R. Dearman 1,∗ , S. McClain 2 , I. Kimber 1 University of Manchester, Manchester, UK, 2 Syngenta Biotech, Research Triangle Park, USA
1
One approach for assessing the potential allergenicity of novel proteins is the measurement in mice of specific IgE antibody induced by intraperitoneal exposure to protein. This method has shown promise, with IgE responses to various proteins correlating with allergenic potential. Given that endotoxin (lipoplysaccharide; LPS) has been used as an adjuvant for IgE, the endogenous endotoxin content of such proteins may impact on induced IgE responses. We have explored antibody responses to proteins in BALB/c (high IgE responder) and C3HHeJ (endotoxinresistant/mutant TLR4) strain mice. Animals were immunized intraperitoneally with protein and after 14 days serum samples were analyzed for specific IgE, IgG, IgG1 and IgG2a antibody production. Mice were exposed to various doses (0.1–1%) of d-ribulose 1,5-diphosphate carboxylase (RUBISCO) from spinach, a protein that is believed to be non-allergenic and which contains high levels of endogenous endotoxin (3885 EU/mg). In both mouse strains treatment with RUBISCO provoked vigorous IgE, IgG1 and IgG2a antibody responses. In addition, C3HHeJ mice were immunized with the food allergen ovalbumin (OVA) alone and in the presence of equivalent amounts of exogenous endotoxin (20 microg
S49
LPS) to that received in 1% RUBISCO. Lipopolysaccharide had an adjuvant effect on IgG1, IgG2a and IgE antibody responses. These data demonstrate that the potential adjuvant effect of endogenous endotoxin must be taken into consideration when interpreting IgE antibody responses to novel proteins. Furthermore, despite having a defective TLR4, LPS has an adjuvant effect in C3HHeJ mice, therefore, use of this strain will not obviate effects of endogenous endotoxin. doi:10.1016/j.toxlet.2011.05.192
OS8 Food Safety
OS8-1 Relative bioavailability of coumarin from cinnamon and cinnamon-containing foods compared to isolated coumarin: A crossover study in human volunteers K. Abraham ∗ , M. Pfister, F. Woehrlin, A. Lampen Federal Institute for Risk Assessment, Berlin, Germany Purpose: Cassia cinnamon contains high levels (up to 1%) of coumarin. Heavy consumption of this spice may result in a dose exceeding the tolerable daily intake (TDI) which is derived from toxicological data on isolated coumarin (animal experiments and medicinal drug in humans). In this context, the question was raised whether coumarin in the plant matrix of cinnamon has the same bioavailability as isolated coumarin. Methods: A crossover study was performed, in which the same dose of 12 mg coumarin was administered to 24 healthy volunteers. They received four different formulations: isolated coumarin in a capsule (reference), cinnamon in capsules, cinnamon tea prepared from the same cinnamon, and cinnamon in rice pudding as a typical meal. The relative extent of absorption was measured as urinary excretion of the main metabolite 7-hydroxycoumarin (7OHC) within 8 h after application. In order to monitor the velocity of absorption, 7OHC plasma levels were measured for 105 min after administration. Results: Plasma levels of 7OHC revealed a fast absorption of coumarin especially from cinnamon tea leading to the highest peak concentrations. Mean urinary excretion of 7OHC was found to be 62.8% for isolated coumarin in a capsule, 56.0% for cinnamon in capsules, 66.1% for cinnamon tea, and 54.7% for cinnamon in rice pudding. Therefore, the relative extent of absorption of coumarin from powder of cassia cinnamon was only slightly lower than that of isolated coumarin, and the TDI of 0.1 mg/kg bw can be used for risk assessment of coumarin exposure from cinnamon-containing meals. doi:10.1016/j.toxlet.2011.05.194
OS7-5 Investigating danger signals in the KeratinoSens cell line: The potential link between Nrf2-activation and other signaling cascades A. Natsch 1,∗ , G. Ellis 2 , R. Emter 1 1
Bioscience, Givaudan Schweiz AG, Dübendorf, Switzerland, 2 Global Toxicology, Givaudan Schweiz AG, Vernier, Switzerland
Purpose: There is high interest in developing cell-based in vitro assays to predict skin sensitizers. Most assays measure innate responses to sensitizers, which may reflect the response of the cells to the ‘danger signal’, a signal thought to be a necessary second stimulus for the skin sensitization reaction. In the published studies, several key signaling pathways have been identified, which are triggered by skin sensitizers. The most eminent pathways are (i) the Nrf2-pathway, (ii) the MAPK p38 kinase pathway and (iii) oxidative stress pathways. In addition, the activation of the inflammasome/caspase-1 and the formation of heat-shock proteins appear to be important innate reactions, but the links between these pathways are not known. Method: We have developed the KeratinoSens assay based on Nrf2-signaling to screen for skin sensitizers. Using this cell line we have now studied in detailed dose-response analysis the possible links between activation of the Nrf2-pathway and (a) oxidative stress/redox signaling (as H2 O2 formation), (b) glutathione depletion, and (c) p38-activation. Results: This analysis indicates that different pathways become important depending on the dose of the allergens added, with redox signaling and p38 activation affecting the Nrf2-response to sensitizers at high doses close to cytotoxic concentrations only. Based on these studies, potential links between different danger signals will be discussed. doi:10.1016/j.toxlet.2011.05.191
OS7-6 Endotoxin, IgE antibody responses and allergenicity assessment R. Dearman 1,∗ , S. McClain 2 , I. Kimber 1 University of Manchester, Manchester, UK, 2 Syngenta Biotech, Research Triangle Park, USA
1
One approach for assessing the potential allergenicity of novel proteins is the measurement in mice of specific IgE antibody induced by intraperitoneal exposure to protein. This method has shown promise, with IgE responses to various proteins correlating with allergenic potential. Given that endotoxin (lipoplysaccharide; LPS) has been used as an adjuvant for IgE, the endogenous endotoxin content of such proteins may impact on induced IgE responses. We have explored antibody responses to proteins in BALB/c (high IgE responder) and C3HHeJ (endotoxinresistant/mutant TLR4) strain mice. Animals were immunized intraperitoneally with protein and after 14 days serum samples were analyzed for specific IgE, IgG, IgG1 and IgG2a antibody production. Mice were exposed to various doses (0.1–1%) of d-ribulose 1,5-diphosphate carboxylase (RUBISCO) from spinach, a protein that is believed to be non-allergenic and which contains high levels of endogenous endotoxin (3885 EU/mg). In both mouse strains treatment with RUBISCO provoked vigorous IgE, IgG1 and IgG2a antibody responses. In addition, C3HHeJ mice were immunized with the food allergen ovalbumin (OVA) alone and in the presence of equivalent amounts of exogenous endotoxin (20 microg
S49
LPS) to that received in 1% RUBISCO. Lipopolysaccharide had an adjuvant effect on IgG1, IgG2a and IgE antibody responses. These data demonstrate that the potential adjuvant effect of endogenous endotoxin must be taken into consideration when interpreting IgE antibody responses to novel proteins. Furthermore, despite having a defective TLR4, LPS has an adjuvant effect in C3HHeJ mice, therefore, use of this strain will not obviate effects of endogenous endotoxin. doi:10.1016/j.toxlet.2011.05.192
OS8 Food Safety
OS8-1 Relative bioavailability of coumarin from cinnamon and cinnamon-containing foods compared to isolated coumarin: A crossover study in human volunteers K. Abraham ∗ , M. Pfister, F. Woehrlin, A. Lampen Federal Institute for Risk Assessment, Berlin, Germany Purpose: Cassia cinnamon contains high levels (up to 1%) of coumarin. Heavy consumption of this spice may result in a dose exceeding the tolerable daily intake (TDI) which is derived from toxicological data on isolated coumarin (animal experiments and medicinal drug in humans). In this context, the question was raised whether coumarin in the plant matrix of cinnamon has the same bioavailability as isolated coumarin. Methods: A crossover study was performed, in which the same dose of 12 mg coumarin was administered to 24 healthy volunteers. They received four different formulations: isolated coumarin in a capsule (reference), cinnamon in capsules, cinnamon tea prepared from the same cinnamon, and cinnamon in rice pudding as a typical meal. The relative extent of absorption was measured as urinary excretion of the main metabolite 7-hydroxycoumarin (7OHC) within 8 h after application. In order to monitor the velocity of absorption, 7OHC plasma levels were measured for 105 min after administration. Results: Plasma levels of 7OHC revealed a fast absorption of coumarin especially from cinnamon tea leading to the highest peak concentrations. Mean urinary excretion of 7OHC was found to be 62.8% for isolated coumarin in a capsule, 56.0% for cinnamon in capsules, 66.1% for cinnamon tea, and 54.7% for cinnamon in rice pudding. Therefore, the relative extent of absorption of coumarin from powder of cassia cinnamon was only slightly lower than that of isolated coumarin, and the TDI of 0.1 mg/kg bw can be used for risk assessment of coumarin exposure from cinnamon-containing meals. doi:10.1016/j.toxlet.2011.05.194