NCT Intramural Research

Current NCT research aims to formally discriminate between 'chemical signatures' reflecting early adaptive or pharmacological responses with no ensuing pathology and 'effects signatures' that entail altered tissue steady state, toxicity, histopathology, or disease (Bartosiewicz et al. 2001). We are therefore in the process of developing learning sets of genomic profiling data for various classes of agents, with doses ranging from those that are pharmacologic to those that are toxic. We also intend to perform comparative studies that address cross-species differences in toxicological responses as well as susceptibility differences in human subgroups.

The combined and integrated data on gene/protein/metabolite changes collected in the context of dose, time, target tissue, and phenotypic severity across species will provide the interpretive information needed to define the molecular basis for chemical toxicity and to model the resulting toxicological and pathological outcomes (Boor-man et al. 2002). It should then be feasible to search for evidence of exposure or injury prior to any clinical or pathological manifestation, facilitating identification of early biomarkers of exposure, toxic injury, or susceptibility. It is anticipated that toxi-cogenomics research will lead to the identification, measurement, and evaluation of biomarkers that are more accurate, quantitative, and specific. These biomarkers will be recognized as important factors in a sequence of key events that will help to define the way in which specific chemicals or environmental exposures cause disease. In other words, toxicogenomics should help to delineate the mode of action of various classes of agents and the unique attributes of certain species and population subgroups that make them susceptible to toxicants, as an important step in comparatively assessing potential human health risk (Farland 1992).

NCT intramural scientists are now performing additional proof-of-concept experiments that are designed to establish how 'effects signatures' can be defined and to link the patterns of altered gene expression to specific parameters of well defined conventional indices of toxicity. For example, experiments can be designed to correlate gene expression patterns with liver pathologies such as hepatomegaly, hepatocel-lular necrosis, or inflammation. It is also possible to look for correlative patterns, for example, in enzyme levels in liver and other tissues or cells such as blood. Changes in serum enzymes provide diagnostic markers of organ function that are commonly used in medicine and toxicology. This 'phenotypic anchoring' of gene expression data to conventional indices removes some of the subjectivity of conventional molecular expression analyses and helps to distinguish the toxicological signal from other gene expression changes that may be unrelated to toxicity, such as the varied pharmacological or therapeutic effects of a compound (Tennant 2002).

Future NCT studies will define molecular perturbations caused by environmental chemicals in terms of phenotypic severity, dose, and time (Hamadeh 2002 c). We will explore quantitative or absolute gene expression profiling (Dudley et al. 2002) and consider combining such an approach with physiologically based pharmacokinetic (PB/PK) and pharmacodynamic modelling. PB/PK modelling can be used to derive a quantitative estimate of target tissue dose at any time after treatment, thus creating the possibility to anchor molecular expression profiles in internal dose, as well as in time and phenotypic severity. Relationships among gene, protein, and metabolite expression may then be described as a function of the applied dose of an agent and the ensuing kinetic and dynamic dose-response behaviour in various tissue compartments. In addition, the species under study and interspecies interindividual differences must be taken into account. With the aid of the knowledge systematically generated and assembled (Zweiger 1999) through literature mining, comparative analysis, and iterative biological modelling of molecular expression datasets over time, the adaptive responses of biological systems will be differentiated from those changes that are associated with or precede clinical or visible adverse effects. We anticipate that our understanding of mechanisms of toxicity and disease will improve as these new methods are used more extensively and toxicogenomics databases are developed more fully. The expected result will be the emergence of toxicology as an information science that will enable thorough analysis, iterative modelling, and discovery across biological species and chemical classes. CEBS will be designed to meet the information and modelling requirements of an integrated systems toxicology, as illustrated conceptually in Figure 10.2.

Key priorities for NCT intramural toxicogenomics studies are the profiling of specific compounds and disease processes that lead to target organ toxicities (e.g., he-pato- and nephrotoxicity). These studies will entertain the following considerations, and emphasis will be placed on the early steps in the disease processes. Multiple compounds that elicit a particular hepato- or nephrotoxicity will be studied at mul-

Fig. 10.2 Interpretation of molecular expression profiles with literature mining, phenotypic anchoring, and iterative biological modelling for systems toxicology. ADME refers to absorption, distribution, metabolism and excretion.

tiple sampling times following exposure. Subtoxic as well as toxic doses will be used, and nontoxic isomers and related compounds will be included to assess the specificity of effects observed. Drugs and chemicals will be selected for study based on criteria such as human exposure and recent toxicology studies demonstrating consistent cross-species effects. Ideally, a drug will show a therapeutic effect and chemicals will display mechanism(s) of toxicity that are prototypical for other agents, including those in our proof-of-concept studies. For example, acetaminophen or paracetamol is the first agent to be studied comprehensively by the NCT. Its selection was based on an extensive literature (Bessems and Vermeulen 2001) showing that its liver toxicity is a common response in rodents and in humans, its metabolism is similar in rodents and in humans, it displays both therapeutic and toxic effects, and there are opportunities for clinical investigation. Furthermore, it has been studied using toxicogenomic methods by several laboratories (Cunningham et al. 2000; Reilly et al. 2001 a,b; Ruepp et al. 2002; Yamazaki et al. 2002), offering the possibility of comparative assessment of observed molecular expression, toxicology, and pathology.

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