- Email: [email protected]
20 Integrating genomics, proteomics and metabonomics
s8
Symposium S2. Developmental immunotoxicology
discussed together with the application of NMR-based metabonomic approaches to investigate toxicological and functional genomic problems and the integration of transcriptomic and metabonomic datasets. A new probabilistic modelling theory for integrative metabolism is also proopised that could help in the understanding of idiosyncratic toxicity. 20
INTEGRATING GENOMICS, PROTEOMICS AND METABONOMICS
T.G. Klenø 1 , D. Baunsgaard 1 , L.L. Rønnedal 1 , S.M. Laursen 2 , T.S. Sørensen 1 . 1 Applied Trinomics, Novo Nordisk A/S, Måløv, Denmark; 2 Toxicology, Novo Nordisk A/S, Måløv, Denmark Genomics, proteomics and metabonomics are rapidly developing technologies which have given researchers new ways of gaining information of toxicity, pharmacological effects and mechanisms of drug treatments. Gene expression micro arrays allow us to study regulation of mRNAs encoded by the genome in response to a drug perturbation. Likewise, proteomics can be used to reveal changes in the expression pattern of hundreds of proteins simultaneously, including posttranslational modifications important for cell function. Information of how regulation is reflected in biochemical pathways can further be elucidated by metabonomics measuring endogenous metabolites. The three technologies are thus complementary and can be interpreted separately. When combining the data valuable information can be obtained. However, tools to handle, reduce and integrate the large amount of information obtained by the three omics techniques are required to identify correlated events caused by drug treatment. Examples are presented where the three “omics” technologies (gene expression micro arrays, proteomics and metabonomics) have been applied to studies of toxicity of hydrazine. Hydrazine is a commonly used model hepatotoxin in animal studies. A major effect of hydrazine is accumulation of fat in the liver leading to liver steatosis. Hydrazine is affecting numerous cellular processes; however, the biochemical mechanism of toxicity of hydrazine is not yet fully understood.
PL Gerhard Zbinden Memorial Lecture 21
adducts from exogenous compounds, although the overall relative biological significance of each type of damage is unknown. The use of DNA and protein adduct measurements in molecular epidemiology will be illustrated. The examples will include the detection of DNA and protein damage from occupational carcinogens, environmental pollution, foodstuffs, and cigarette smoking, and the modification of DNA bases by endogenous hydroxyl radicals and methylating species.
DNA AND PROTEIN ADDUCTS AS MARKERS OF GENOTOXICITY
P.B. Farmer. Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester, University Road, Leicester, LE1 7RH, UK Determination of the interaction products (adducts) of a carcinogen with DNA or protein indicates the amount of genotoxic material that has reached the tissue under study and provides invaluable information for molecular epidemiological studies. Protein adducts are not repaired and are considered simply as exposure monitors, but DNA adducts may also give further information with regard to the mutagenic significance of the exposure. The sensitivity and applicability of the analytical methods for the detection and quantification of carcinogen adducts has improved dramatically in recent years, and DNA damage levels as low as one adduct per 108 nucleotides can now routinely be measured. Although 32 Ppostlabelling has previously been the most sensitive technique for detection of non-radioactive carcinogen adducts with DNA, mass spectrometric approaches using LC-MS-MS are now reaching similar sensitivities. Under normal living conditions, it has been shown that human DNA and protein are exposed to a wide variety of chemicals which have the potential to modify their structures. One of the significant findings resulting from this work has been the discovery of ‘background’ modifications of DNA and protein in supposedly unexposed individuals. These background modifications arise from endogenous processes, often associated with oxidative damage. In human DNA, the extent of oxidative DNA damage is generally greater than the extent of formation of covalently-linked DNA
S2 Developmental immunotoxicology 22
IMMUNOTOXICOLOGICAL CONSEQUENCES OF PERINATAL CHEMICAL EXPOSURES
Henk van Loveren, Aldert Piersma. Laboratory for Toxicology, Pathology and Genetics, National Institute for Public Health and the Environment RIVM, P.O.Box 1, 3720 BA Bilthoven, The Netherlands T lymphocytes play a crucial role in immunocompetence. Maturation of T lymphocytes takes place in the thymus. During the differentiation of progenitor T cells into mature T lymphocytes the repertoire of antigen specificities is generated, and desired specificities are positively selected, while undesired specificities are deleted. During the maturation also differentiation into different subpopulations with their respective regulatory or effector functions take place. Building the repertoire of B cells does not take place in the thymus, but more systemically, including in the bone marrow. Even if such processes probably take place during the entire life, most of these processes are completed at an early stage in life. Immunocompetence starts to develop in utero, and is largely completed early in life. This is illustrated by the involution of the thymus, that progresses with progressing age. It is therefore likely that effects of exposure to immunotoxic chemicals may have important consequences especially during developmental stages, i.e. starting in utero. The immune system of the fetus and neonates is characterized by Th2 type of interleukins. It is suggested that the reason for this is to minimize the development of reactions of the fetal immune system to maternal tissue antigens in the placenta, that would be mediated by Th1 type immune responses. In the early postnatal period the immune system matures to provide a balanced Th1/Th2 state, facilitating resistance to infections, at a time when adverse reactions to maternal components is no longer an issue. It has been shown that infections and vaccinations, that may influence the Th1/Th2 balance, have an impact on the maturation of the immune system. In addition to in utero developmental stages of the immune system, the post-natal period is therefore likely to be another vulnerable period during which immunotoxic chemicals may have relatively pronounced consequences. A number of chemicals that have immunotoxic consequences if exposure takes place during developmental stages of the immune system have been identified. These include TCDD, PCB’s, HCH, heavy metals, steroid hormones and DES, and cytostatic agents. These findings have prompted further activities towards inclusion of developmental toxicity testing in guideline-based protocols. Current predictive immunotoxicity testing is mainly done in the context of general toxicity, according to OECD guideline 407. There is a number of test systems that address effects of exposure during development. This pertains to OECD 414, 415, and 416 respectively, that have all their specific design. Some of these may be adequate for addressing potential effects of immunotoxicants on the developing immune system. In OECD 414 developmental toxicity protocol, given the time of necropsy at one day before birth, inclusion of immunotoxicological parameters is not readily feasible. Although the OECD 415 and 416 generation studies allow for inclusion of immunotoxicological parameters, both these tests are already laborious. A less expensive test that does include exposure during all developmental stages of the immune system, i.e. from day 6 of gestation until weaning of the offspring is described in the draft guideline OECD 426. This test is aimed at detecting developmental neurotoxicity and includes behavioral tests at young adult age. Littermates could be used for immunotoxicity testing.
Symposium S2. Developmental immunotoxicology
discussed together with the application of NMR-based metabonomic approaches to investigate toxicological and functional genomic problems and the integration of transcriptomic and metabonomic datasets. A new probabilistic modelling theory for integrative metabolism is also proopised that could help in the understanding of idiosyncratic toxicity. 20
INTEGRATING GENOMICS, PROTEOMICS AND METABONOMICS
T.G. Klenø 1 , D. Baunsgaard 1 , L.L. Rønnedal 1 , S.M. Laursen 2 , T.S. Sørensen 1 . 1 Applied Trinomics, Novo Nordisk A/S, Måløv, Denmark; 2 Toxicology, Novo Nordisk A/S, Måløv, Denmark Genomics, proteomics and metabonomics are rapidly developing technologies which have given researchers new ways of gaining information of toxicity, pharmacological effects and mechanisms of drug treatments. Gene expression micro arrays allow us to study regulation of mRNAs encoded by the genome in response to a drug perturbation. Likewise, proteomics can be used to reveal changes in the expression pattern of hundreds of proteins simultaneously, including posttranslational modifications important for cell function. Information of how regulation is reflected in biochemical pathways can further be elucidated by metabonomics measuring endogenous metabolites. The three technologies are thus complementary and can be interpreted separately. When combining the data valuable information can be obtained. However, tools to handle, reduce and integrate the large amount of information obtained by the three omics techniques are required to identify correlated events caused by drug treatment. Examples are presented where the three “omics” technologies (gene expression micro arrays, proteomics and metabonomics) have been applied to studies of toxicity of hydrazine. Hydrazine is a commonly used model hepatotoxin in animal studies. A major effect of hydrazine is accumulation of fat in the liver leading to liver steatosis. Hydrazine is affecting numerous cellular processes; however, the biochemical mechanism of toxicity of hydrazine is not yet fully understood.
PL Gerhard Zbinden Memorial Lecture 21
adducts from exogenous compounds, although the overall relative biological significance of each type of damage is unknown. The use of DNA and protein adduct measurements in molecular epidemiology will be illustrated. The examples will include the detection of DNA and protein damage from occupational carcinogens, environmental pollution, foodstuffs, and cigarette smoking, and the modification of DNA bases by endogenous hydroxyl radicals and methylating species.
DNA AND PROTEIN ADDUCTS AS MARKERS OF GENOTOXICITY
P.B. Farmer. Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester, University Road, Leicester, LE1 7RH, UK Determination of the interaction products (adducts) of a carcinogen with DNA or protein indicates the amount of genotoxic material that has reached the tissue under study and provides invaluable information for molecular epidemiological studies. Protein adducts are not repaired and are considered simply as exposure monitors, but DNA adducts may also give further information with regard to the mutagenic significance of the exposure. The sensitivity and applicability of the analytical methods for the detection and quantification of carcinogen adducts has improved dramatically in recent years, and DNA damage levels as low as one adduct per 108 nucleotides can now routinely be measured. Although 32 Ppostlabelling has previously been the most sensitive technique for detection of non-radioactive carcinogen adducts with DNA, mass spectrometric approaches using LC-MS-MS are now reaching similar sensitivities. Under normal living conditions, it has been shown that human DNA and protein are exposed to a wide variety of chemicals which have the potential to modify their structures. One of the significant findings resulting from this work has been the discovery of ‘background’ modifications of DNA and protein in supposedly unexposed individuals. These background modifications arise from endogenous processes, often associated with oxidative damage. In human DNA, the extent of oxidative DNA damage is generally greater than the extent of formation of covalently-linked DNA
S2 Developmental immunotoxicology 22
IMMUNOTOXICOLOGICAL CONSEQUENCES OF PERINATAL CHEMICAL EXPOSURES
Henk van Loveren, Aldert Piersma. Laboratory for Toxicology, Pathology and Genetics, National Institute for Public Health and the Environment RIVM, P.O.Box 1, 3720 BA Bilthoven, The Netherlands T lymphocytes play a crucial role in immunocompetence. Maturation of T lymphocytes takes place in the thymus. During the differentiation of progenitor T cells into mature T lymphocytes the repertoire of antigen specificities is generated, and desired specificities are positively selected, while undesired specificities are deleted. During the maturation also differentiation into different subpopulations with their respective regulatory or effector functions take place. Building the repertoire of B cells does not take place in the thymus, but more systemically, including in the bone marrow. Even if such processes probably take place during the entire life, most of these processes are completed at an early stage in life. Immunocompetence starts to develop in utero, and is largely completed early in life. This is illustrated by the involution of the thymus, that progresses with progressing age. It is therefore likely that effects of exposure to immunotoxic chemicals may have important consequences especially during developmental stages, i.e. starting in utero. The immune system of the fetus and neonates is characterized by Th2 type of interleukins. It is suggested that the reason for this is to minimize the development of reactions of the fetal immune system to maternal tissue antigens in the placenta, that would be mediated by Th1 type immune responses. In the early postnatal period the immune system matures to provide a balanced Th1/Th2 state, facilitating resistance to infections, at a time when adverse reactions to maternal components is no longer an issue. It has been shown that infections and vaccinations, that may influence the Th1/Th2 balance, have an impact on the maturation of the immune system. In addition to in utero developmental stages of the immune system, the post-natal period is therefore likely to be another vulnerable period during which immunotoxic chemicals may have relatively pronounced consequences. A number of chemicals that have immunotoxic consequences if exposure takes place during developmental stages of the immune system have been identified. These include TCDD, PCB’s, HCH, heavy metals, steroid hormones and DES, and cytostatic agents. These findings have prompted further activities towards inclusion of developmental toxicity testing in guideline-based protocols. Current predictive immunotoxicity testing is mainly done in the context of general toxicity, according to OECD guideline 407. There is a number of test systems that address effects of exposure during development. This pertains to OECD 414, 415, and 416 respectively, that have all their specific design. Some of these may be adequate for addressing potential effects of immunotoxicants on the developing immune system. In OECD 414 developmental toxicity protocol, given the time of necropsy at one day before birth, inclusion of immunotoxicological parameters is not readily feasible. Although the OECD 415 and 416 generation studies allow for inclusion of immunotoxicological parameters, both these tests are already laborious. A less expensive test that does include exposure during all developmental stages of the immune system, i.e. from day 6 of gestation until weaning of the offspring is described in the draft guideline OECD 426. This test is aimed at detecting developmental neurotoxicity and includes behavioral tests at young adult age. Littermates could be used for immunotoxicity testing.