\-\w j Toxicogenomics Applications of new functional genomics technologies in toxicology Wilbert H.M. Heijne Proefschrift ter verkrijging vand egraa dva n doctor opgeza gva nd e rector magnificus vanWageninge n Universiteit, Prof.dr.ir. L. Speelman, in netopenbaa r te verdedigen op maandag6 decembe r200 4 des namiddagst e half twee ind eAul a - Table of contents Abstract Chapter I. page 1 General introduction [1] Chapter II page 21 Toxicogenomics of bromobenzene hepatotoxicity: a combined transcriptomics and proteomics approach[2] Chapter III page 48 Bromobenzene-induced hepatotoxicity atth etranscriptom e level PI Chapter IV page 67 Profiles of metabolites and gene expression in rats with chemically induced hepatic necrosis[4] Chapter V page 88 Liver gene expression profiles in relation to subacute toxicity in rats exposed to benzene[5] Chapter VI page 115 Toxicogenomics analysis of liver gene expression in relation to subacute toxicity in rats exposed totrichloroethylen e [6] Chapter VII page 135 Toxicogenomics analysis ofjoin t effects of benzene and trichloroethylene mixtures in rats m Chapter VII page 159 Discussion and conclusions References page 171 Appendices page 187 Samenvatting page 199 Dankwoord About the author Glossary Abbreviations List of genes Chapter I General introduction Parts of this introduction were publishedin : Molecular Biology in Medicinal Chemistry, Heijne etal., 2003 m NATO Advanced Research Workshop proceedings, Heijne eral., 2003 81 Chapter I 1. General introduction 1.1 Background /.1.1 Toxicologicalrisk assessment 1.1.2 Currenttoxicological methods for hazard identification 1.1.3 Newfunctional genomics technologies in toxicology 1.2Application s of functional genomics technologies in toxicology 1.2.1 Studyingmechanisms oftoxicity 1.2.2 Identificationof toxicity markers 1.2.3 Interspeciesand intraspecies extrapolation 1.2.4 Exposureto mixtures ofcompounds 1.2.5 Toxicogenomicsin combination within vitrotesting 1.2.6 Reduction,refinement and replacement ofanimals testing 1.3Functiona l genomics technologies 1.3.1 Genomics 1.3.2 Transcriptomics 1.3.3 Proteomics 1.3.4 Metabolomics(metabolite profiling) 1.3.5 Dataprocessing andbioinformatics 1.3.6 Biologicalinterpretation and hypothesis generation 1.4Objective s of this thesis 1.5Outlin e of the thesis General introduction 1.1.Backgroun d 1.1.1 Toxicologicalrisk assessment Toxicology investigates adverse effects on organisms, induced by substances in their environment. To assess whether a substance forms a health risk, the potential hazards of the substance have to be identified. This involves empirical toxicity studies and investigation of the mechanism of toxic action (figure 1.1) Substances in a mixture may cause unexpected effects (interactions). Extrapolations are needed to interpret short-term and high dose studies to real-life long term exposures to low doses, /nfraspecies extrapolation accounts for differences in response between individuals, /nferspecies extrapolation is required to translate the results obtained in animal toxicity studies to the human situation. The factors are taken into account to determine an uncertainty factor, which is applied in combination with exposure levels to assess human health risk. Human health risk assessment Exposure levels t Uncertainty factor Chronic studies Lowdose s /interspecies extrapolation Acute studies Highdose s /ntraspecies extrapolation Mixtures studies interactions Mechanism ofactio n Figure1.1 Schematicoverview ofhuman health risk assessment. The identification of hazard involves empirical testing in acute and chronic toxicity studies, and assessment of (unexpected) effects of mixtures. Mechanism of toxic action of the compound may beinvestigated ,t oenabl eintra- and/nferepecie sextrapolations .Th efactor sar etake n intoaccoun t to determine an uncertainty factor, which is applied in combination with exposure levels toasses s humanhealt hrisk . 1.1.2 Currenttoxicological methods for hazard identification To determine potential hazards of a substance, a set of toxicity studies has to be performed according to international standards (EC, OECD, FDA). In these studies, the acute and chronic toxicity of the compound will be established, based on a number of parameters, such as (histo)pathology, clinical chemistry and hematology. Pathology and histopathology form the basis of routine toxicity studies. Pathological examination may reveal changes in body and organ weights and macroscopic abnormalities. Histopathological techniques enable the microscopic examination of tissues, to detect toxic effects at the cellular level. Clinical chemistry, and hematology are used to determine changes in blood that correlate with 1 Chapter I certain types of toxicity. Most of these parameters were established empirically. They are the result of aberrant cellular processes and late effects of toxicity, rather than components of mechanisms leading to toxicity. For instance, leakage of enzymes through disrupted cell membranes is observed in blood by clinical chemistry, and is clearly a secondary process in a late stage of cell death. Dependent onth e complexity of the study, biochemical analyses of tissue samples and urine analysis may be required to provide complementary information about the compound under study. However, most of the routine toxicity studies determine adverse effects empirically and provide few insights in mechanisms of toxicity. The development of small-scale molecular biological techniques enabled to determine the composition and expression of specific genes and proteins, relevant to mechanisms of toxicity. These methods (eg. RT-PCR, Northern and Western blotting, ELISA, see table 1.1) are intended to study mechanisms of toxicity and interindividual or interspecies differences, at the molecular level. Specific molecular markers can distinguish different forms of toxicity within an organism or even within a tissue. Also, specific molecular endpoints may be detected early and at lower exposure levels compared to histopathology, clinical chemistry or hematology. However, these small-scale methods are applied only if a preconceived notion exists on the possible mode of action of the substance. Moreover, they will only reveal effects on single molecules. 1.1.3 New functionalgenomics technologiesin toxicology New technologies are being developed that could enable cell-wide and detailed analysis of mechanisms of toxicity, without the need of a priori knowledge on the mode of toxic action. As a result of the recent elucidation of complete genome sequences, functional genomics disciplines have emerged. These disciplines study the functions of the (complete) genome in relation to its composition, and measure gene expression (transcriptomics), protein levels (proteomics) or metabolite contents (metabolomics). Toxicogenomics can be defined as the application of the functional genomics technologies in toxicology. Toxicogenomics studies the adverse effects of xenobiotic substances in relation to the structure and activity of the genome [9]. The different disciplines and technologies are described in detail at the end of this chapter. First, applications of the functional genomics technologies in toxicology will be discussed. 1.2 Applications offunctiona l genomics technologies intoxicolog y Toxicology is likely to benefit from the newfunctiona l genomics technologies, as discussed in various publications [10-20].Toxicogenomic s methods may enhance health risk assessment in various ways. Detailed investigation of mechanisms of toxicity may enable to identify critical effects and the sequence of events at the molecular level (section 1.2.1). Adverse effects in (animal) toxicity studies could be detected earlier and more sensitively than with the current methods (section 1.2.2). Toxicogenomics results may enable more accurate extrapolations and the identification or even prediction of interactions upon exposure to mixtures (sections 1.2.3, 1.2.4). Eventually, toxicogenomics could enable to determine or predict the implications of the (individual's) genome composition for the toxic effects caused by substances. In vitro models in combination with toxicogenomics methods might allow to reduce, refine and possibly even replace in vivotestin g (section 1.2.5,1.2.6) . 1.2.1 Studyingmechanisms oftoxic action Changes in protein and gene expression play a role in signal transduction mechanisms, metabolic pathways and protective responses in a cell. In reaction to an insult, the changes at the protein and gene expression level in the cell precede the response at the tissue or even organism level. Thus, mechanisms leading to pathologically observed endpoints may be recognised through observation of changes in expression of genes and proteins. The functional genomics technologies could be useful to elucidate the critical steps and the sequence of events inth e development oftoxicit y after exposure to toxic substances. A toxic compound, by itself, or via reactive metabolites after bioactivation, elicits a cellular response upon interaction with a target molecule. This target may be a receptor molecule that initiates a response that involves many other molecules. The primary target of the toxicant might not always be identified directly through changes in gene or protein expression. However, the downstream cascade of effects may help to find the initial targets atth e origin of the response. As thousands of molecules are investigated simultaneously, the chance of identifying (unexpected) molecules affected by the xenobiotic