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Do uncertainty factors take into account toxicokinetic interactions?
S90
Abstracts / Toxicology Letters 180S (2008) S32–S246
these studies to replace the UF for TK variability in humans on a case-by-case basis. As an example, PRUFs for polymorphic CYP2D6 metabolism were above the current TK default factor. Here we present an innovative approach for meta-analysis, based on full Bayesian inference and applied to the polymorphic CYP2D6 pathway. Geometric means and geometric standard deviations for TK parameters were collected for healthy adults. Hierarchical modeling with covariates, combined with compartmental models allowed to account for inter-individual, inter-compound and inter-study variability. In addition, a sensitivity analysis with respect to the model assumptions has been carried out to assess their impact on the analysis of variability and uncertainty. PRUFs for the CYP2D6 pathway were derived for subgroups of the population (healthy adults, extensive and poor metabolisers) and compared to our previous published analysis. The advantages of this method which takes into account population variability and uncertainty are discussed in relation to introducing more transparency and chemical-specific data in human risk assessment. doi:10.1016/j.toxlet.2008.06.477 K03 Do uncertainty factors take into account toxicokinetic interactions? Conclusions and recommendations from the sixth framework project NOMIRACLE Jean Lou C.M. Dorne ∗ , Alex Papadopoulos University of Southampton; Institute of Human Nutrition, Southampton, United Kingdom One of the objectives of the 6th framework EU project NOMIRACLE (http://viso.jrc.it/nomiracle/) is to improve the scientific basis of uncertainty factors (UFs) for chemical mixtures. Current UFs allowing for human variability in toxicokinetics (3.16) and toxicodynamics (3.16) do not take into account potential interaction between mixtures at either or both levels. Recently, toxicokinetic UFs have been shown to underestimate human variability in polymorphic CYP metabolism (CYP2C9, CYP2D6, CYP2C19) for contaminants eliminated via these routes. Literature searches have been performed using on-line databases to identify human studies quantifying interaction between major probe substrates of polymorphic CYP and specific inhibitors or inducers. Overall 200 studies were retrieved and analysed. For each study, changes in internal dose for markers of chronic/acute exposure were quantified and UFs were calculated (95th, 97.5th and 99th percentiles) using a previously published method. Overall, the current UF for toxicokinetic would not cater for such interactions. This human database, although based on therapeutic doses higher than current contaminant levels in food, represents a very useful dataset to quantify human variability for toxicokinetic interactions. Many contaminants are known to be substrates for CYP enzymes as well as inhibitors or inducers at relatively low doses in mammals depending on their potency. Hence, further research is needed to characterise the potential effects of such contaminants on individual CYP at their current level of exposure. Such information can be obtained routinely in the laboratory using recombinant technology and toxicokinetic assays. doi:10.1016/j.toxlet.2008.06.478
K04 Characterisation of oxidative deaminase activity in the reconstructed human epidermis EpiskinTM Joan Eilstein ∗ , Eric Arbey, Florence Canivet, Jean-Roch Meunier, Jacques Leclaire, Daniel Duché L’Oréal Research and Development, Aulnay sous Bois, France The skin is not just a passive physical barrier but is also a biological structure involved in a wide range of metabolic activities. The reconstructed human epidermis EpiskinTM was a tool developed for studying, in vitro, the skin toxicity of chemicals and their metabolism as alternative methods to animal experimentations. This implies that the model is metabolically active. Adenosine deaminase (ADA) and adenylate deaminase (AMPDA) are enzymes involved in the irreversible oxidative deamination of adenosine and adenosine derivatives to inosine and inosine derivatives, respectively. These enzymes, which are present in normal human epidermis, have a critical role in the adenosine homeostasis and potentially in the proliferation and maturation of certain types of mammalian cells. In this work, we reported the characterization of oxidative deaminase activity in EpiskinTM using adenosine derivatives as enzyme substrates (2 ,3 -O-isopropylidene adenosine). Substrate disappearance and the corresponding inosine derivatives apparition were detected by UV-HPLC and demonstrated that ADA and/or AMPDA are present and active in EpiskinTM . doi:10.1016/j.toxlet.2008.06.479 K05 CYP450 characterization of the reconstructed human epidermis EpiskinTM involved in xenobiotic metabolism Joan Eilstein 1,∗ , Pierre-Alain Meunier 1 , Maria Manso 2 , Jean Pachot 2 , Van Luu-The 3 , Fernand Labrie 3 , Jean-Roch Meunier 1 , Jacques Leclaire 1 , Daniel Duché 1 1
L’Oréal Research & Development, Aulnay sous Bois, France, Oroxcell, Romainville, France, 3 Oncology and Molecular Endocrinology Research Center, Laval University Hospital Research Center (CRCHUL) and Laval University, Québec, Canada 2
The main role of human skin is its barrier function to environment and exogenous chemicals. However, it is not merely an inert organ but it performs a wide range of metabolic activities and detoxification reactions. Previous studies have shown that the reconstructed human epidermis EpiskinTM expressed numerous isoforms of phase I metabolizing-enzyme mRNA such as cytochromes P450 (CYP). However, the presence of mRNA material is not the proof that proteins are translated or active. A western blot methodology applied to EpiskinTM microsomes, allowed us to probe CYP 1A1, 1B1, 2C18, 2D6, 2E1, 2J2, 3A4, 3A5 and 3A7, which are the main CYP mRNA involved in the xenobiotic metabolism expressed in skin and the model. In addition, to confirm the epidermal CYP functionality, we measured the catalytic activities of CYP 1A1/1B1, 2B6/2C18/2E1 and 3A isoforms by using fluorogenic substrates. Results showed that CYP1A1, 2D6, 2E1, 3A4, 3A5 and 3A7 proteins were present in EpiskinTM . CYP 1A1/1B1 and CYP3A isoforms were the main activities measured in the epidermis. CYP 2B6/2C18/2E1 activities were also detected but at a too weak level to be quantified. Consequently, no real correlation was observed between the CYP mRNA expression, translation into protein and their activity levels. This demonstrates that catalytic activity studies are necessary steps to prove the real metabolic capabilities of a tissue. In conclusion these findings confirm that EpiskinTM is a metabolically active skin model for studying in vitro xenobiotic phase I metabolism
Abstracts / Toxicology Letters 180S (2008) S32–S246
these studies to replace the UF for TK variability in humans on a case-by-case basis. As an example, PRUFs for polymorphic CYP2D6 metabolism were above the current TK default factor. Here we present an innovative approach for meta-analysis, based on full Bayesian inference and applied to the polymorphic CYP2D6 pathway. Geometric means and geometric standard deviations for TK parameters were collected for healthy adults. Hierarchical modeling with covariates, combined with compartmental models allowed to account for inter-individual, inter-compound and inter-study variability. In addition, a sensitivity analysis with respect to the model assumptions has been carried out to assess their impact on the analysis of variability and uncertainty. PRUFs for the CYP2D6 pathway were derived for subgroups of the population (healthy adults, extensive and poor metabolisers) and compared to our previous published analysis. The advantages of this method which takes into account population variability and uncertainty are discussed in relation to introducing more transparency and chemical-specific data in human risk assessment. doi:10.1016/j.toxlet.2008.06.477 K03 Do uncertainty factors take into account toxicokinetic interactions? Conclusions and recommendations from the sixth framework project NOMIRACLE Jean Lou C.M. Dorne ∗ , Alex Papadopoulos University of Southampton; Institute of Human Nutrition, Southampton, United Kingdom One of the objectives of the 6th framework EU project NOMIRACLE (http://viso.jrc.it/nomiracle/) is to improve the scientific basis of uncertainty factors (UFs) for chemical mixtures. Current UFs allowing for human variability in toxicokinetics (3.16) and toxicodynamics (3.16) do not take into account potential interaction between mixtures at either or both levels. Recently, toxicokinetic UFs have been shown to underestimate human variability in polymorphic CYP metabolism (CYP2C9, CYP2D6, CYP2C19) for contaminants eliminated via these routes. Literature searches have been performed using on-line databases to identify human studies quantifying interaction between major probe substrates of polymorphic CYP and specific inhibitors or inducers. Overall 200 studies were retrieved and analysed. For each study, changes in internal dose for markers of chronic/acute exposure were quantified and UFs were calculated (95th, 97.5th and 99th percentiles) using a previously published method. Overall, the current UF for toxicokinetic would not cater for such interactions. This human database, although based on therapeutic doses higher than current contaminant levels in food, represents a very useful dataset to quantify human variability for toxicokinetic interactions. Many contaminants are known to be substrates for CYP enzymes as well as inhibitors or inducers at relatively low doses in mammals depending on their potency. Hence, further research is needed to characterise the potential effects of such contaminants on individual CYP at their current level of exposure. Such information can be obtained routinely in the laboratory using recombinant technology and toxicokinetic assays. doi:10.1016/j.toxlet.2008.06.478
K04 Characterisation of oxidative deaminase activity in the reconstructed human epidermis EpiskinTM Joan Eilstein ∗ , Eric Arbey, Florence Canivet, Jean-Roch Meunier, Jacques Leclaire, Daniel Duché L’Oréal Research and Development, Aulnay sous Bois, France The skin is not just a passive physical barrier but is also a biological structure involved in a wide range of metabolic activities. The reconstructed human epidermis EpiskinTM was a tool developed for studying, in vitro, the skin toxicity of chemicals and their metabolism as alternative methods to animal experimentations. This implies that the model is metabolically active. Adenosine deaminase (ADA) and adenylate deaminase (AMPDA) are enzymes involved in the irreversible oxidative deamination of adenosine and adenosine derivatives to inosine and inosine derivatives, respectively. These enzymes, which are present in normal human epidermis, have a critical role in the adenosine homeostasis and potentially in the proliferation and maturation of certain types of mammalian cells. In this work, we reported the characterization of oxidative deaminase activity in EpiskinTM using adenosine derivatives as enzyme substrates (2 ,3 -O-isopropylidene adenosine). Substrate disappearance and the corresponding inosine derivatives apparition were detected by UV-HPLC and demonstrated that ADA and/or AMPDA are present and active in EpiskinTM . doi:10.1016/j.toxlet.2008.06.479 K05 CYP450 characterization of the reconstructed human epidermis EpiskinTM involved in xenobiotic metabolism Joan Eilstein 1,∗ , Pierre-Alain Meunier 1 , Maria Manso 2 , Jean Pachot 2 , Van Luu-The 3 , Fernand Labrie 3 , Jean-Roch Meunier 1 , Jacques Leclaire 1 , Daniel Duché 1 1
L’Oréal Research & Development, Aulnay sous Bois, France, Oroxcell, Romainville, France, 3 Oncology and Molecular Endocrinology Research Center, Laval University Hospital Research Center (CRCHUL) and Laval University, Québec, Canada 2
The main role of human skin is its barrier function to environment and exogenous chemicals. However, it is not merely an inert organ but it performs a wide range of metabolic activities and detoxification reactions. Previous studies have shown that the reconstructed human epidermis EpiskinTM expressed numerous isoforms of phase I metabolizing-enzyme mRNA such as cytochromes P450 (CYP). However, the presence of mRNA material is not the proof that proteins are translated or active. A western blot methodology applied to EpiskinTM microsomes, allowed us to probe CYP 1A1, 1B1, 2C18, 2D6, 2E1, 2J2, 3A4, 3A5 and 3A7, which are the main CYP mRNA involved in the xenobiotic metabolism expressed in skin and the model. In addition, to confirm the epidermal CYP functionality, we measured the catalytic activities of CYP 1A1/1B1, 2B6/2C18/2E1 and 3A isoforms by using fluorogenic substrates. Results showed that CYP1A1, 2D6, 2E1, 3A4, 3A5 and 3A7 proteins were present in EpiskinTM . CYP 1A1/1B1 and CYP3A isoforms were the main activities measured in the epidermis. CYP 2B6/2C18/2E1 activities were also detected but at a too weak level to be quantified. Consequently, no real correlation was observed between the CYP mRNA expression, translation into protein and their activity levels. This demonstrates that catalytic activity studies are necessary steps to prove the real metabolic capabilities of a tissue. In conclusion these findings confirm that EpiskinTM is a metabolically active skin model for studying in vitro xenobiotic phase I metabolism