Tools and Technologies
In Chapter 2, the committee provided an overview of its vision for toxicity testing, and Chapter 3 described the main components of the vision. Here, tools and technologies that might be used to apply the committee’s vision are briefly discussed. The tools and technologies will evolve and mature over time, but many are already available. The committee emphasizes that technologies are evolving rapidly, and new molecular technologies will surely be available in the near future for mapping toxicity pathways, assessing their functions, and measuring dose-response relationships.
TOOLS AND TECHNOLOGIES FOR CHEMICAL CHARACTERIZATION
A variety of computational methods are available for chemical characterization. The discussion here focuses on structure-activity relationship (SAR) analyses, which use physical and chemical properties to predict the biologic activity, potential toxic-
ity, and metabolism of an agent of concern. All are conceptually based on the similar-property principle, that is, that chemicals with similar structure are likely to exhibit similar activity (Tong et al. 2003). Accordingly, biologic properties of new chemicals are often inferred from properties of similar existing chemicals whose hazards are already known. Specifically, SAR analysis involves building mathematical models and databases that use physical properties (such as solubility, molecular weight, dissociation constant, ionization potential energies, and melting point) and chemical properties (such as steric properties, presence or absence of chemical moieties or functional groups, and electrophilicity) to predict biologic or toxicologic activity of chemicals. SAR analyses can be qualitative (for example, recognition of structural alerts, that is chemical functional groups and substructures) or quantitative (for example, use of mathematical modeling to link physical, chemical, and structural properties with biologic or toxic end points) (Benigni 2004). Key factors in the successful application of SAR methods include proper representation and selection of structural, physical, and chemical molecular features; appropriate selection of the initial set of compounds (that is, the “training set”) and methods of analysis; the quality of the biologic data; and knowledge of the mode or mechanism of toxic action (McKinney et al. 2000).
Current applications of SAR analyses include soft drug design, which involves improving the therapeutic index of a drug by manipulating its steric and structural properties (Bodor 1999); design and testing of chemotherapeutic agents (van den Broek et al. 1989); nonviral gene and targeted-gene delivery (Congiu et al. 2004); creating predictive models of carcinogenicity to replace animal models (Benigni 2004); predicting the toxicity of chemicals, particularly pesticides and metals (Walker et al. 2003a); and predicting the environmental fate and ecologic effects of industrial chemicals (Walker et al. 2003b). Among the available predictive-toxicity systems, the most widely used are statistically based cor-
relative programs (such as CASE/MultiCASE and TOPKAT) and rule-based expert systems (such as DEREK and ONCOLOGIC) (McKinney et al. 2000).
There are many examples of successful applications of SAR and quantitative SAR (QSAR) analysis. One successful application of SAR analysis in risk assessment is the modeling of Ah-receptor-binding affinities of dioxin-like compounds, including the structurally related polychlorinated dioxins, dibenzofurans, and biphenyls. Specifically, SAR methods were used to establish a common mechanism of action for toxic effects and in the further development of toxic equivalency factors in risk assessments involving exposure to complex mixtures of those compounds (van der Berg et al. 1998). Other successful applications have examined how structural alterations influence toxicity. For example, toxic effects of nonpolar anesthetics are mediated by a nonspecific action on cell membranes and have been shown to be directly correlated to their log octanol-water partition coefficient (log Kow). However, the polar anesthetics—which include such chemicals as phenols, anilines, pyridines, nitrobenzenes, and aliphatic amines—generally show an anesthetic potency 5-10 times higher than expected on the basis of their log Kow alone (Soffers et al. 2001).
Much effort has been directed toward the modeling and prediction of specific toxicities, particularly mutagenicity and carcinogenicity because of the importance of these end points, the cost and length of full rodent assays for carcinogenesis, and the availability of high-quality data for modeling purposes. Experimental observation has led to the identification of several structural alerts that can cause both mutation and cancer, including carbonium ions (alkyl-, aryl-, and benzylic-), nitrenium ions, epoxides and oxonium ions, aldehydes, polarized double bonds (alpha and beta unsaturated carbonyls or carboxylates), peroxides, free radicals, and acylating intermediates (Benigni and Bossa 2006). The structural alerts for mutagenicity and carcinogenicity have
been incorporated into expert systems for predicting toxic effects of chemicals (Simon-Hettich et al. 2006).
A number of structural alerts also have been associated with developmental toxicity. They were identified on the basis of known developmental responses to environmental agents, such as valproic acids, hydrazides, and carbamates (Schultz and Seward 2000; Cronin 2002; Walker et al. 2004). Studies have demonstrated that the presence of a hydroxyl group is required for estrogenic activity of biphenyls; symmetric derivatives are 10 times more active than nonsymmetric ones (Schultz et al. 1998). The relationship between the size and shape of the nonphenolic moiety and estrogenic potency among para-substituted phenols demonstrated the trend of increasing estrogenicity with increased molecular size (Schultz and Seward 2000). Thus, although predictive models for some toxic end points, such as mutagenicity, already exist, more mechanistically complex end points—such as acute, chronic, or organ toxicity—are more difficult to predict (Schultz and Seward 2000; Simon-Hettich et al. 2006).
One final application of SAR analysis is in predicting absorption, distribution, metabolism, and excretion. Qualitative SARs, QSARs, and the related quantitative structure-property relationships have been successfully used to estimate such key properties as permeability, solubility, biodegradability, and cytochrome P-450 metabolism (Feher et al. 2000; Bugrim et al. 2004); to predict drug half-life values (Anderson 2002); and to describe penetration of the blood-brain barrier (Bugrim et al. 2004).
As indicated above, the predictive ability of different models depends on selecting the correct molecular descriptors for the particular toxic end points, the appropriate mathematical approach and analysis, and a sufficiently rich set of experimental data. The ability to adapt existing models continuously by building on larger and higher-quality datasets is crucial for the improvement and ultimate success of these approaches.
MAPPING TOXICITY PATHWAYS
As discussed in Chapters 2 and 3, the key component of the committee’s vision is the evaluation of perturbations in toxicity pathways. Many tools and technologies are available that can aid in the identification of biologic signaling pathways and the development of assays to evaluate their function. Recent advances in cellular and molecular biology, -omics technologies, and computational analysis have contributed considerably to the understanding of biologic signaling processes (Daston 1997; Ekins et al. 2005). Within the last 15 years, multiple cellular response pathways have been evaluated in increasing depth as is evidenced by the progress in the basic knowledge of cellular and molecular biology (Fernandis and Wenk 2007; Lewin et al. 2007). Moreover, systems biology constitutes a powerful approach to describing and understanding the fundamental mechanisms by which biologic systems operate. Specifically, systems biology focuses on the elucidation of biologic components and how they work together to give rise to biologic function. A systems approach can be used to describe the fundamental biologic events involved in toxicity pathways and to provide evolving biologic modeling tools that describe cellular circuits and their perturbations by environmental agents (Andersen et al. 2005a). A longer-term goal of systems biology is to create mathematical models of biologic circuits that predict the behavior of cells in response to environmental agents qualitatively and quantitatively (Lander and Weinberg 2000). Progress in that regard is being made in developmental biology (Cummings and Kavlock 2005; Slikker et al. 2005). The sections that follow outline tools and technologies that will most likely be used to elucidate the critical toxicity pathways and to develop assays to evaluate them.
In Vitro Tests
The committee foresees that in vitro assays will make up the bulk of the toxicity tests in its vision. In vitro tests are currently used in traditional toxicity testing and indicate the success of developing and using in vitro assays (Goldberg and Hartung 2006). In vitro tests include the 3T3 neural red uptake phototoxicity assay (Spielman and Liebsch 2001), cytotoxicity assays (O’Brien and Haskins 2007), skin-corrosivity tests, and assays measuring vascular injury using human endothelial cells (Schleger et al. 2004). Many tests have been validated by the European Centre for the Validation of Alternative Methods. The committee notes that the current in vitro tests originated as alternatives to or replacements of other toxicity tests. In the committee’s vision, in vitro assays will evaluate biologically significant perturbations in toxicity pathways and thus are not intended to serve as direct replacements of existing toxicity tests.
The committee envisions the use of human cell lines for the in vitro assays. Cell lines have been used for a long time in experimental toxicology and pharmacology. Human cell lines are readily available from tissue-culture banks and laboratories and are particularly attractive because they offer the possibility of working with a system that maintains several phenotypic and genotypic characteristics of the human cells in vivo (Suemori 2006). Differentiated functions, functional markers, and metabolic capacities may be altered or preserved in cell lines, depending on culture conditions, thereby allowing testing of a wide array of agents in different experimental settings. Other possibilities include using animal cells that are transfected to express human genes and proteins. For example, various cell lines—such as V79, CHO, COS-7, NIH3T3, and HEPG2—have been transfected with complementary DNA (cDNA, DNA synthesized from mature mRNA) coding for human enzymes and used in mutagenesis and
drug-metabolism studies (Potier et al. 1995). Individual enzymes have also been stably expressed to identify the major human isoenzymes, such as cytochromes P-450 and UDP-glucuronosyltransferases, responsible for the metabolism of potential therapeutic and environmental agents. The metabolic in vitro screens with human enzymes are usually conducted as a prelude to clinical studies.
A major limitation of using human cell lines is the difficulty of extrapolating data from the simple biologic system of single cells to the complex interactions in whole animals. Questions have also been raised concerning the stability of cell lines over time, the reproducibility of responses over time, and the ability of cell lines to account for genetic diversity of the human population. Nonetheless, cell lines have been used as key tools in the initial screening and evaluation of toxic agents and the characterization of properties of cancer cells (Suzuki et al. 2005) and in gene profiling with microarrays (Wang et al. 2006). The high-throughput methods now becoming more common will allow the expansion of the methods to larger numbers of end points, wider dose ranges, and mixtures of agents (Inglese 2002; Inglese et al. 2006).
A critical feature of the committee’s vision is the use of high-throughput methods that will allow economical screening of large numbers of chemicals in a short period. The pharmaceutical industry provides an example of the successful use of high-throughput methods. Optimizing drug-candidate screening is essential for timely and cost-effective development of new pharmaceuticals. Without effective screening methods, poor drug candidates might not be identified until the preclinical or clinical phase of the drug-development process, and this could lead to high costs and low productivity for the pharmaceutical industry
(Lee and Dordick 2006). Pharmaceutical companies have turned to high-throughput screening, which allows automated simultaneous testing of thousands of chemical compounds under conditions that model key biologic mechanisms (Fischer 2005). Such technologies as hybridization, microarrays, real-time polymerase chain reaction, and large-scale sequencing are some of the high-throughput methods that have been developed (Waring and Ulrich 2000). High-throughput assays are useful for predicting several important characteristics related to the absorption, distribution, metabolism, excretion, and toxicity of a compound (Gombar et al. 2003). They can predict the interaction of a compound with enzymes, the metabolic degradation of the compound, the enzymes involved in its biotransformation, and the metabolites formed (Masimirembwa et al. 2001). That information is integral for selecting compounds to advance to the next phase of drug development, especially when many compounds may have comparable pharmacologic properties but differing toxicity profiles (Pallardy et al. 1998). High-throughput assays are also useful for rapid and accurate detection of genetic polymorphisms that could dramatically influence individual differences in drug response (Shi et al. 1999).
Microarray technologies have allowed the development of the field of toxicogenomics, which evaluates changes in genetic response to environmental agents or toxicants. These technologies permit genomewide assessments of changes in gene expression associated with exposure to environmental agents. The identification of responding genes can provide valuable information on cellular response and some information on toxicity pathways that might be affected by environmental agents. Some of the tools and technologies are described below.
Microarrays are high-throughput analytic devices that provide comprehensive genome-scale expression analysis by simultaneously monitoring quantitative transcription of thousands of genes in parallel (Hoheisel 2006). The Affymetrix GeneChip Human Genome U133 Plus 2.0 Array provides comprehensive analysis of genomewide expression of the entire transcribed human genome on a single microarray (Affymetrix Corporation 2007). Whole-genome arrays are also available for the rat and mouse. The use of the rat arrays will probably increase as the relationships between specific genes and markers on the arrays become better understood.
Protein microarrays potentially offer the ability to evaluate all expressed proteins in cells or tissues. Protein-expression profiling would allow some understanding of the relationship between transcription (the suite of mRNAs in the cell) and the translational readout of the transcripts (the proteins). Protein microarrays have diverse applications in biomedical research, including profiling of disease markers and understanding of molecular pathways, protein modifications, and protein activities (Zangar et al. 2005). However, whole-cell or tissue profiling of expressed proteins is still in the developmental stage. These techniques remain expensive, and the technology is in flux.
Differential gene-expression experiments use comparative microarray analysis to identify genes that are upregulated or downregulated in response to experimental conditions. The large-scale investigation of differential gene expression attaches functional activity to structural genomics. Whole-genome-expression experiments involve hundreds of experimental conditions in which patterns of global gene expression are used to classify disease specimens and discover gene functions and toxicogenomic targets (Peeters and Van der Spek 2005). Gene-expression profiling will have a role in identifying toxicity pathways in whole-animal studies but is not expected to be the staple technology for identifying and mapping the pathways.
High-Throughput Functional Genomics1
Large-scale evaluations of the status of gene expression and protein concentrations in cells allow understanding of the integrated biologic activities in tissues and can be used to catalog changes after in vivo or in vitro treatment with environmental agents. However, evaluation of the organization and interactions among genes in toxicity pathways requires approaches referred to as functional genomics, which encompass a different suite of molecular tools (Brent 2000). The tools are designed to catalog the full suite of genes that are required for optimal activity of a toxicity pathway. The evaluation of the readout of those functional screens with bioinformatic analysis provides key data about the organization of toxicity pathways and guides computational methods that model the consequences of perturbation of the pathways by environmental agents.
Functional analysis requires a cell-based assay that provides a convenient, automated cell-based measure of functioning of a toxicity pathway (Akutsu et al. 1998; Michiels et al. 2002; Chanda et al. 2003; Lum et al. 2003; Berns et al. 2004; Huang et al. 2004) and requires the ability to automate treatment of the cells with individual cDNAs or small interfering RNAs (siRNAs), which are relatively short RNA oligomers that appear to play important roles in inhibiting gene expression (Hannon 2002; Meister and Tuschl 2004; Mello and Conte 2004; Hammond 2005). Treatment of the cells with a particular cDNA causes overexpression of the gene (and presumably the protein) that is coded by it. In contrast, treatment with gene-specific siRNA causes knockdown of specific proteins by enhancing degradation of the mRNA from the gene.
High-throughput methods permit automation of such cell-based assays by the use of robots and libraries of cDNAs and siRNAs. The screens show which genes increase and which decrease activity of the toxicity pathway.
Computational biology uses computer techniques and mathematical modeling to understand biologic processes. It is a powerful tool to cope with the ever-increasing quantity and quality of biologic information on genomics, proteomics, gene expression, gene varieties, genotyping techniques, and protein and cell arrays (Kriete and Eils 2006). Computational tools are used in data analysis, data mining, data integration, network analysis, and multiscale modeling (Kitano 2005). Computational biology is particularly useful for systems biology in understanding structural, regulatory, and kinetic models (Barabasi and Oltvai 2004); in modeling signal transduction (Eungdamrong and Iyengar 2004); and in analyzing genome information and its structural and functional properties (Snitkin et al. 2006). Furthermore, computational biology is used to predict toxic effects of chemical substances (Simon-Hettich et al. 2006), to understand the toxicokinetics and toxicodynamics of xenobiotics (Ekins 2006), to determine gene-expression profiling of cancer cells (Katoh and Katoh 2006), to help in the development of genomic biomarkers (Ginsburg and Haga 2006), and to design virtual experiments to replace or reduce animal testing (Vedani 1999). In drug design and discovery, novel computational technologies help to create chemical libraries of structural motifs relevant to target proteins and their small molecular ligands (Balakin et al. 2006; O’Donoghue et al. 2006).
Cellular signaling circuits handle an enormous variety of functions. Apart from replication and other functions of individual cells, signaling circuits must implement the complex logic of
development and function of multicellular organisms. Computer models are helpful in understanding that complexity (Bhalla et al. 2002). Recent studies have extended such models to include electrical, mechanical, and spatial details of signaling (Bhalla 2004a,b). The mitogen-activated protein kinase (MAPK) pathway is one of the most important and extensively studied signaling pathways; it governs growth, proliferation, differentiation, and survival of cells. A wide variety of mathematical models of the MAPK pathway have led to novel insights and predictions as to how it functions (Orton et al. 2005; Santos et al. 2007).
Predictive computational models derived from experimental studies have been developed to describe receptor-mediated cell communication and intracellular signal transduction (Sachs et al. 2005). Physicochemical models attempt to describe biomolecular transformations, such as covalent modification and intermolecular association, with physicochemical equations. The models make specific predictions and work mostly with pathways that are better understood. They can be viewed as translations of familiar pathway maps into mathematical forms (Aldridge et al. 2006). Integrated mechanistic and data-driven modeling for multivariate analysis of signaling pathways is a novel approach to understanding multivariate dependence among molecules in complex networks and potentially can be used to identify combinatorial targets for therapeutic interventions and toxicity-pathway targets that lead to adverse responses (Hua et al. 2006).
In Vivo Tests
As discussed in Chapters 2 and 3, in vivo tests will most likely be used in the foreseeable future to evaluate the formation of metabolites and some mechanistic aspects of target-organ responses to environmental agents, including genomewide
evaluation of gene expression. Chapter 3 noted that careful design of those studies could substantially increase the value of information obtained. For example, evaluation of cellular transcriptomic patterns from tissues of animals receiving short-term exposures may provide clues to cellular targets of environmental agents and assist in target-tissue identification. (See Chapter 3 for further discussion of protocol changes that could increase the value of toxicity tests.) Moreover, technologic advances in detection and imaging have the potential for improving in vivo testing. For example, positron-emission tomography (PET) is an imaging tool that can determine biochemical and physiologic processes in vivo by monitoring the activity of radiolabeled compounds (Paans and Vaalburg 2000). Because PET can detect the activity of an administered compound at the cellular level, its use in animal models can result in the incorporation of mechanistic processes and an understanding of the pathologic effects of a candidate compound (Rehmann and Jayson 2005).
TOOLS AND TECHNOLOGIES FOR DOSE-RESPONSE AND EXTRAPOLATION MODELING
As discussed in Chapters 2 and 3, two types of modeling will be critical for implementing the committee’s vision: physiologically based pharmacokinetic (PBPK) models and dose-response models of perturbations of toxicity pathways. PBPK models will allow dose extrapolation from in vitro conditions used for assessing toxicity-pathway perturbations to projected human exposures in vivo. Mechanistic models of perturbations of toxicity pathways should aid in developing low-dose extrapolation models that consider the biologic structure of the cellular circuitry controlling pathway activation.
Physiologically Based Pharmacokinetic Models
Assessing the risk associated with human chemical exposure has traditionally relied on the extrapolation of data from animal models to humans, from one route of exposure to another, and from high doses to low doses. Such extrapolation attempts to relate the extent of external exposure to a toxicant to the internal dose in the target tissue of interest. However, differences in biotransformation and other pharmacokinetic processes can introduce error and uncertainty into the extrapolation of toxicity from animals to humans (Kedderis and Lipscomb 2001).
PBPK models provide a physiologic basis for extrapolating between species and routes of exposure and thus allow estimation of the active form of a toxicant that reaches the target tissue after absorption, distribution, and biotransformation (Watanabe et al. 1988). However, PBPK results can differ significantly in the hands of different modelers (Hattis et al. 1990), and improved modeling approaches for parameter selection and uncertainty analysis are under discussion. PBPK models might also be useful for estimating the effect of exposure at different life stages, such as pregnancy, critical periods of development, and childhood growth (Barton 2005). Interindividual differences can be incorporated into PBPK models by integrating quantitative information from in vitro biotransformation studies (Bois et al. 1995; Kedderis and Lipscomb 2001).
The more pervasive use of PBPK approaches in the new strategy for toxicity testing will be in basing dosimetry extrapolations on estimates of partitioning, metabolism, and interactions among chemicals derived from in vitro measurements or perhaps even from SAR or QSAR techniques. Those extrapolations will require some level of validation that might require data from kinetic studies in volunteers or from biomonitoring studies in human populations. In the committee’s vision for toxicity testing, the development of PBPK models from SAR predictions of partition-
ing and metabolism would decrease animal use, and continued improvements in in vitro to in vivo extrapolations of kinetics will support the translation from test-tube studies of perturbations to predictions.
Dose-Response Models of Toxicity Pathways
Dose-response modeling of toxicity pathways involves the integration of mechanistic and dosimetric information about the toxicity of a chemical into descriptive mathematical terms to provide a quantitative model that allows dose and interspecies extrapolation (Conolly 2002). New techniques in molecular biology, such as functional genomics, will play a key role in the development of such models because they provide more detailed information about the organization of toxicity pathways and the dose-response relationships of perturbations of toxicity pathways by environmental agents. Dose-response models have been developed for cell-signaling pathways and used in risk assessment (Andersen et al. 2002). They have found important applications in studying chemical carcinogenesis (Park and Stayner 2006). In particular, models of cancer formation have been developed to describe the induction of squamous-cell carcinomas of the nasal passage in rats exposed to formaldehyde by inhalation, taking into account both tissue dosimetry and the nonlinear effects of cellular proliferation and formation of DNA-protein cross-links (Slikker et al. 2004a, 2004b; Conolly et al. 2004). However, alternative implementations of the formaldehyde model gave substantially different results (Subramaniam et al. 2006). Emerging developments in systems biology allow modeling of cellular and molecular signaling networks affected by chemical exposures and thereby produce an integrated modeling approach capable of predicting dose-response relationships of pathway perturbations by developmental and reproductive toxicants (Andersen et al. 2005b).
In the next decades, the dose-response modeling tools for perturbations should progress relatively rapidly to guide low-dose extrapolations of initial interactions of toxic compounds with biologic systems. The quantitative lineage of early perturbations with apical responses is likely to develop more slowly. For the foreseeable future, the continued refinement of biologic models of signaling circuitry should guide the extrapolation approaches necessary for conducting risk assessment with the toxicity-pathway tests as the cornerstone of toxicity-testing methods.
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