The Future of Regulatory Toxicology: Impact of the Biotechnology Revolution
James T. MacGregor 1
United States Food and Drug Administration, National Center for Toxicological Research, Rockville, Maryland 20857
The molecular biology revolution and the advent of genomic and proteomic technologies are facilitating rapid advances in our understanding of the molecular details of cell and tissue function. These advances have the potential to transform toxicological and clinical practice, and are likely to lead to the supplementation or replacement of traditional biomarkers of cellular integrity, cell and tissue homeostasis, and morphological alterations that result from cell damage or death. New technologies that permit simultaneous monitoring of many hundreds, or thousands, of macro- and small molecules ("-omics" technologies) promise to allow functional monitoring of multiple (or perhaps all) key cellular pathways simultaneously. Elucidation of cellular responses to molecular damage, including evolutionarily conserved inducible molecular defense systems, suggests the possibility of new biomarkers based on molecular responses to functional perturbations and cellular damage. Our improved understanding of the molecular basis of various pathologies suggests that monitoring specific molecular responses may provide improved prediction of human outcomes. Responses that can be monitored directly in the human should provide "bridging biomarkers" that may eliminate much of the current uncertainty in extrapolating from laboratory models to human outcome. Another aspect of genomics is our enhanced ability to associate DNA sequence variations with biological outcomes and individual sensitivity. The human genome sequence has revealed that sequence variations are very common, and may be an important determinant of variation in biological outcomes. The impending availability of a complete human haplotype map linked to standard genetic markers greatly facilitates identification of genetic variations that convey sensitivity or resistance to chemical exposures. Genetic approaches have already linked a large number of genetic variants (polymorphisms) with human diseases and adverse reactions from exposure to drugs or toxicants, suggesting an important role in sensitivity to drugs and environmental agents, disease susceptibilities, and therapeutic responses. As these opportunities are transformed into reality, regulatory toxicological practice is likely to be shaped in the future by the combination of conventional pathology, toxicology, molecular genetics, biochemistry, cell biology, and computational bio-informatics—resulting in the broad application of molecular approaches to monitoring functional disturbances.
Key Words: toxicity; biomarkers; microarray; proteomics; genomics; metabonomics; polymorphism; haplotype; validation.
Toxicological Sciences 75, 236-248 (2003).
Many of us currently engaged in the practice of toxicology have had the privilege of witnessing the impact of one of the greatest scientific advances of all time—the discovery of the mechanism of genetic inheritance and subsequent elucidation of the mode of genetic control over cellular functions. In the span of less than 60 years since the historic demonstration by Avery and colleagues (1944) that DNA was the genetic material, a comprehensive understanding of the structure of DNA, the mechanism of DNA replication, the genetic code for synthesis of the proteins and enzymes that control cell structure and function—and even the ability to manipulate genetic information and move it among organisms—has been achieved (see MacGregor, 1994). These advances were based on the rapid development of ever-improving technologies for identifying, manipulating, and monitoring DNA sequences and gene products. These advances have resulted in a wealth of knowledge about cell and gene function, and the availability of technologies to characterize gene sequences and gene products simultaneously in many hundreds or thousands of genes in assays that require only µg or pg quantities of analate. These advances are currently driving a marked transformation of the field of regulatory toxicology, which is evolving from a descriptive science toward a discipline based on molecular genetic and biochemical mechanistic understanding.
All evidence suggests that the pace of these advances will continue to accelerate (Cantor, 2000). The purpose of this article is to discuss the potential impact of this knowledge, and these technologies, on the practice of regulatory toxicology.
In view of the pace of advances in the biological sciences, it may come as a surprise to those new in the field—and especially new graduates with an orientation toward molecular genetics—that the principals of safety evaluation delineated by Lehman et al.(1949) more than 50 years ago still describe relatively accurately current regulatory toxicological practice as it applies to the evaluation of general organ and tissue damage. This historical approach, expanded upon in the classical review of Barnes and Denz (1954; see also Paget, 1970), consists of assessment of the effect of a substance on growth and tissue mass (body and organ weights), measurements of serum biomarkers (aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), etc.), hematological evaluation, observation of behavior and appearance, and histopathology assessment. This is essentially the approach practiced today by major product industries, including the pharmaceutical and food industries (Table 1).Although toxicological practice has been relatively stable for the past few decades, with the exception of the introduction of specialized tests for genetic and reproductive damage and some other special functional assessments (e.g., immunological, neurological, electrophysiological), all indications are that the field is now poised for major change. A glance at the program of recent annual meetings of the Society of Toxicology, or at the ever-increasing schedule of toxico- or pharmacogenomics meetings, leaves little doubt that toxicologists in industry, academia, and government are intensively evaluating modern molecular technologies.
As we anticipate how these technologies may impact current practice, the basic elements of the current strategy should first be considered. Essentially, in vivo toxicological assessment of organ and tissue damage involves the assessment of three basic types of "biomarkers" that indicate adverse biological effects on the organism—markers of (1) function and homeostasis, (2) cell and tissue integrity, and (3) cell and tissue damage or damage-response. The key elements of this strategy are sound, but the biotechnology revolution has presented major opportunities to improve the biomarkers that comprise these three classes. The focus below is on the potential for improved biomarkers within these classes, both the molecular markers themselves and also the methodologies for monitoring those markers.
Table 2 summarizes some of the opportunities for improved toxicological assessment created by the advances in molecular technologies and our enhanced knowledge of the molecular basis of tissue damage and response. These include opportunities for improved biomarkers, better technologies for monitoring biomarkers, and new laboratory models that incorporate human biochemical characteristics. Examples of implementation of each of these opportunities already exist. The discussion below focuses on the impact of the recent revolution in genetics and biotechnology on strategies for improved biomarker development and application, and on assessment of the role of genetic variation in determining or modifying toxicological outcomes.
New technologies of molecular biology are being applied in several ways to assess the function and structure of the major organ and tissue systems. Much attention is currently focused on the potential of DNA microarrays to identify either inducible damage responses or shifts in genetic expression patterns that are characteristic of specific molecular insults to the cell. This focus is driven by the convergence of two factors: (1) the availability of technology to monitor the expression of many genes simultaneously using very small samples of DNA or RNA, and (2) the recently developed knowledge that molecular evolution has resulted in specific inducible defense systems and regulatory control pathways for key cell functions. Additional opportunities include the potential (1) to develop comprehensive panels of biomarkers of cell and tissue integrity through proteomic technologies, (2) for monitoring functional pathways using metabonomic technologies, (3) for development of mechanism-based models of human disease (including short-term models of carcinogenesis), (4) to identify genetic alterations that lead to human disease, and (5) for application of imaging technologies to noninvasive monitoring. Each of these opportunities merits discussion.
One of the mainstays of toxicological practice is the set of biomarkers that indicate a loss of cellular integrity. Typically, these are cellular constituents, such as cytoplasmic enzymes, that leak from damaged or dying cells and can be monitored in blood (e.g., AST, ALT, ALP, creatine kinase [CK], etc.). Differences in tissue content and differences among isoforms in different tissues allow monitoring of general tissue damage as well as acquisition of information about relative damage in different tissues. These biomarkers have been chosen well and have served the field well for decades. Nonetheless, the modern technologies discussed above provide a major opportunity to systematically identify additional biomarkers that can provide greater sensitivity and tissue specificity.
Some specific examples of relatively new biomarkers, developed through conventional biochemical techniques, may illustrate the potential of a systematic approach to developing a complete new set of such biomarkers. One example is the use of glutathione S-transferase isoforms (GSTs) to identify and monitor hepatic and renal damage (Kilty et al., 1998). These biomarkers have recently come into more widespread use as cell-specific markers of damage in these organs because they are contained at higher levels than AST and ALT, have lower molecular weights and shorter half-lives in plasma, and exist as specific isoforms that are localized to particular cell populations in liver and kidney: the a form in hepatocytes and proximal tubular cells and the p form in bile ducts and distal tubular cells. This makes GSTs more useful for monitoring short-term pathological change, such as liver damage during periods of immunological rejection and response to immunosuppressive therapies, with more sensitivity and specificity than the longer-lived AST and ALT. Thus, GSTs have become important biomarkers for clinical monitoring of transplant rejection and clinical response to immunosuppressive therapy (Backman et al., 1988; Polak et al., 1999; Trull et al., 1994). This does not suggest that either GSTs or AST/ALT should be considered as a generally preferred biomarker, but rather that each has advantages for specific applications. For example, the shorter half-life of a-GSTs is an asset for monitoring response to immunosuppressive therapy and development of rejection over short periods of intensive monitoring following liver transplantation, whereas the longer-lived AST and ALT would be advantageous in a sporadic longer-term sampling strategy for liver toxicity. The use of GSTs is now being extended to use a- and p-GSTs in plasma, urine, and bile to differentially identify and monitor hepatocellular, biliary, proximal tubular renal, and distal tubule renal damage (Kilty et al., 1998).
Another example is the use of cardiac troponins as markers of cardiac damage (Herman et al., 1999). These biomarkers offer more specificity and sensitivity than earlier markers of cardiac and muscle damage. The cardiac troponins T (cTnT) and I (cTnI) are now in clinical use for monitoring damage due to myocardial infarction and for monitoring pediatric chemotherapy with anthracycline compounds in order to minimize cardiac damage (Lipshultz et al., 1997). Indeed, the high specificity and sensitivity of the blood level of cardiac troponins as an indicator of myocardial damage has recently resulted in a redefinition of myocardial infarction that places a heavy reliance on this biomarker for diagnosis of myocardial infarction (Apple and Wu, 2001).
Modern high-throughput technologies for proteins or small molecular weight products offer a major opportunity to systematically identify sensitive and specific plasma or urine biomarkers that could serve as an index of damage specific to each of the important internal organs and tissues. Although not a trivial exercise, it is conceptually straightforward to systematically induce tissue-specific damage and to identify tissue-specific markers through the use of these technologies. In principle, a moderate-sized battery of such markers, perhaps 150 or so, should allow monitoring of all major classes of tissue or organ damage, and permit identification of specific tissues in which damage is occurring.
Inducible Defense Systems
As molecular genetics has revealed the structure and function of key functional systems within the cell, it has become evident that defense mechanisms to protect these key functions have coevolved with essentially all the major functional systems. Just as key structural elements that confer function to protein molecules and gene products have been conserved during evolution, the defenses that protect or repair these systems have also been highly conserved. This fact—that they are highly conserved—attests to their importance in limiting pathological damage to these systems. Thus, understanding these key protective and defense response systems provides the opportunity to introduce measures of these responses as biomarkers of potentially pathological damage.
This type of biomarker represents a new class of molecular markers not currently used in toxicological practice. Among those endpoints used routinely in toxicological practice, only cellular host-defense responses (e.g., lymphocyte or macrophage infiltration, increase in leukocyte count, etc.) fall within the category of damage-induced biomarker. Thus, the opportunity exists to introduce a new class of biomarker—one with the marked advantage that many of these markers are based on molecular responses that occur prior to extensive cell and tissue damage. This provides the potential that "pre-pathological" events can be monitored—a major need for human studies. Examples of some of the defense systems with the potential to be used in this manner are given in Table 3. The examples included are from organisms ranging from procaryotes to mammals, including humans, illustrating the conservation of damage-response systems during evolution.A prime example of the coevolution of functional and defensive systems is the system for replicating DNA. The basic replicative DNA polymerase has coevolved a proofreading function that corrects mispairing that occurs during DNA replication (Watson et al., 1987). Additionally, specific damage-recognition and repair molecules have evolved to protect the fidelity of replication, and maintenance of the integrity of DNA after replication (Lindahl and Wood, 1999). These include enzymes such as those involved in excision repair of bulky adducts, as well as systems that recognize damage and control overall processes of cell replication and cell death pathways to prevent highly damaged cells from replicating under conditions that would induce extreme damage, such as p53 and associated pathways. More than 125 specific human DNA repair genes are now known (Ronen and Glickman, 2001).
Similar examples of the coevolution of protective functions along with functional activity can be found for essentially all of the major functional systems of cells and tissues. Thus, the functional molecules that control protein integrity have coevolved to respond to protein damage by using these same molecular systems (chaperones and proteasomes) to destroy or re-fold structurally damaged proteins (Wickner et al., 1999). These same molecules control protein folding for normal function and export, protein destruction during the cell cycle control and tissue remodeling, antigen processing, and other functions, and also play a major role in the control and repair of protein damage. In the case of cellular energetics, defense systems have evolved to scavenge potentially toxic oxidative by-products and to respond to perturbations that increase oxidative species within cells (e.g., Pinkus et al., 1996). Coupled with our new knowledge of damage-class-specific molecular responses, the recent availability of the powerful tools of molecular biology that allow high-throughput measurement of gene expression, cellular protein products, and metabolites presents a unique opportunity to introduce new and efficient biomarkers in highly efficient technical formats.
These damage and defense responses, monitored in "global" formats such as DNA or protein arrays, offer the potential to identify and monitor specific types of cellular damage very efficiently. Figure 1 illustrates how a DNA array might be used to provide information about tissue-specific mechanisms of cellular damage. The example given imagines probes for damage-inducible gene transcripts in horizontal rows and tissue samples in vertical columns, allowing multiple samples to be processed on individual chips such that tissue specificities and dose-response relationships could be determined on fewer chips than required by the common practice of using an entire chip per sample (Fig. 1).
Perturbation of Critical Metabolic and Control Pathways: Genomic, Proteomic, and "Metabonomic" Technologies Can Provide a Global View
The availability of the new "global" technologies of genomics (Aardema and MacGregor, 2002), expression profiling (Hamadeh et al., 2002), proteomics (Anderson et al., 2000; Bichsel et al., 2001; Hermann et al., 2001; Huang et al., 2001; Steiner and Anderson, 2000; Wolters et al., 2001; Yates, 1998, 2001), and metabonomics (Nicholson et al., 1999, 2002) promises to make routine the monitoring of many, or in some cases all, of the components of key control and metabolic pathways. These powerful technologies, termed "-omic" technologies because of their potential to monitor complete classes of structural or functional molecules within tissues or organisms (Lederberg and McCray, 2001), provide the potential to assess the functional activity of biochemical pathways through a single simultaneous analysis of the many cellular components controlled by a particular pathway. The potential of these "-omic" technologies to revolutionize the current approach to toxicological assessment has recently been addressed (Aardema and MacGregor, 2002). Among the classes of molecules that are currently thought to be addressable through "-omics" technologies are mRNAs, proteins and peptides, and small molecular weight intermediary metabolites.
Each of the technologies available currently has particular advantages and disadvantages for specific applications. DNA arrays are powerful tools for direct monitoring of increases or decreases in gene transcripts from large numbers of genes in comparative samples. Thus, this technology will play an important role in identifying those genes induced in response to specific types of damage or to identify global shifts in gene expression that result from pathological alterations within cells and tissues. However, it is likely that this technology will play mainly a "discovery" role with respect to biomarkers for in vivo monitoring, because invasive procedures are required to obtain sufficient nucleic acid samples from internal tissues and organs. Thus, it is likely that proteins or peptides, or small molecules controlled by gene expression, will emerge as those biomarkers of functional status or damage response used in routine toxicological practice. When key gene products are identified using nucleic acid array technologies, methods are now available to construct protein-based assays for monitoring those products. These methods include phage-antibody libraries coupled with high-throughput selection of antigen-antibody interactions that can identify high-affinity binding molecules to almost any protein (Holt et al., 2000). Once suitable antibodies or other binding substrates are identified, protein-binding arrays can be constructed for efficient analysis of the protein products (Huang, 2001).
Proteomic and metabonomic approaches are suitable for identification of gene products and cellular constituents in accessible body fluids and tissue compartments, and will likely lead to new biomarkers for in vivo monitoring. Among the advances in the technologies of identifying proteins and peptides are improvements in classical 2-dimensional (2D) gel electrophoresis coupled with sophisticated mass spectroscopic identification of protein sequences (Anderson et al., 2000; Steiner and Anderson, 2000), matrix-assisted (MALDI) or surface-enhanced laser desorption ionization (SELDI) techniques that allow rapid characterization of proteins, protein fragments, or polypeptides (e.g., Bichsel et al., 2001; Hermann et al., 2001), and multidimensional chromatographic/mass spectroscopic methodologies (Wolters et al., 2001; Yates, 1998, 2001). These technologies have the potential to identify accessible markers in body fluids as well as measures of functional and structural proteins and peptides within tissues and cells. Protein and antibody arrays (Cahill, 2001; Huang et al., 2001) and bead-capture methodologies (Nolan and Mandy, 2001) also offer advantages for certain applications.
Metabonomics employs NMR technology to identify intermediary metabolites that provide an index of metabolic state (Nicholson et al., 1999). This technology has proven effective at characterizing metabolic shifts associated with a variety of pathologies and functional alterations (Nicholson et al., 2002), including renal and hepatic toxicity (Robertson et al., 2000). This technology has the major advantage that it is based on non- or minimally-invasive measurements in urine or plasma, making it directly applicable to studies in humans or animals in vivo.
Various strategies to identify inducible (and suppressible) biomarkers of pathology are possible. Whatever the strategy employed, effects of specific well-characterized pathologies on genes, proteins, and small molecules within the cell will need to be characterized to determine the relationship between these potential markers and specific types of damage. Once key elements of damage response and/or pathological perturbation are characterized, appropriate low-cost technologies that allow these identified changes to be monitored inexpensively on a routine basis will then need to be validated for regulatory purposes.
"Molecular Fingerprinting": Characterization of Biological Effects by Patterns of Perturbations in Cellular Levels of Macro and Small Molecules
An important aspect of the ability to monitor patterns of perturbations in key pathways through global analysis of cellular levels of molecular components is the ability to develop "fingerprints" of cellular responses to classes of chemicals with known common biological effects (e.g., Hamadeh et al., 2002; Robertson et al., 2000; Murata et al., 1999). Such fingerprints have the potential: (1) to allow classification of chemicals based on the biological responses they elicit, (2) to provide mechanistic information about the cellular perturbations and responses elicited by specific exposures (through comparison with responses associated with previously characterized mechanisms), and (3) to identify biomarkers specific to particular classes of molecular damage.
It should become possible to develop a compendium of chemical class-specific cellular perturbations, and to introduce a new system of biological classification of chemicals based on similarities in their mechanisms of interaction with key cell receptors and response elements (Hughes et al., 2000). Such a classification would have several practical applications. In product development, for example, the biological fingerprints of new candidates for development could be compared with previously characterized agents with known beneficial or detrimental properties. Complex mixtures, such as environmental samples or product impurities, could be analyzed to determine if they produce patterns that indicate adverse biological effects—and the patterns observed would provide valuable mechanistic information about the nature of the expected effects as well as the chemical class likely to be associated with a particular pattern. Such fingerprints would also predict particular mechanisms of action, guiding subsequent studies designed to provide confirmatory evidence. This ability to predict mechanism based on "fingerprints" of biomarker and pathway responses, and to categorize chemicals based on patterns of effect produced by well-characterized agents, should facilitate selection of agents for product development and greatly increase the efficiency of toxicology testing strategies (e.g., Ulrich and Friend, 2002).
A comprehensive histopathological evaluation of the major organs and tissues is generally considered to be the most reliable metric by which adverse toxicological effects are determined. As knowledge of the molecular alterations that underlie the morphological and histochemical changes scored by the anatomic pathologist has increased, molecular techniques have become increasingly used in conjunction with traditional histological evaluation. For example, as the molecular basis of apoptosis has become elucidated, molecular assays that visualize the externalization of membrane proteins (van Engeland et al., 1998) or DNA strand breakage (Thiry, 1992; Wijsman et al., 1993) characteristic of the apoptotic process have been found to be more sensitive and more specific endpoints than traditional evaluation of nuclear and cellular morphological characteristics. Recently, adaptations of the annexin assay have been used to image apoptotic cell populations noninvasively in the human in vivo (Blankenberg et al., 1999; Reutelingsperger et al., 2002).
Application of histochemical and immunohistochemical techniques to visualize (e.g., Bullock and Petrusz, 1986) or quantitatively analyze (Wilson et al., 1990), specific small molecules or proteins in cells and tissues has, of course, long been used to identify specific cellular and extra-cellular constituents associated with normal functions and pathological conditions. In general, these molecular techniques have been used as an adjunct to traditional morphological evaluation using light microscopy. Although it may still be premature to consider a comprehensive reassessment of "standard" regulatory histopathological practice, it is not difficult to envision the development of methodologies suitable for quantitative monitoring of endpoints that are currently evaluated in a qualitative or semi-quantitative manner by visual observation.
Host-defense cell infiltration of damaged tissue is one example of a process that has been characterized to an extent that suggests improved strategies for evaluation. Currently, the degree of infiltration by host-defense cells such as lymphocytes or macrophages is evaluated via a qualitative judgement by the pathologist during visual screening of tissue sections. Objective quantitative approaches that use labeling of well-characterized surface markers of these leukocytic cell populations (Zola, 1992) are easily envisioned, but apparently have not yet been developed or proposed for regulatory testing applications. Likewise, the chemokine and cytokine signals generated in response to tissue damage might serve as biomarkers of cellular responses to tissue damage. Much is now known about the chemokine and cytokine signals that activate and recruit host-defense cells, and immunoassays for leukocyte subclasses are employed routinely in research and clinical testing (Borish and Steinke, 2003; Olson and Ley, 2002).
Figure 2 illustrates the cellular signals and resulting cellular recruitment that are characteristic of tissue damage, and suggests quantitative biomarkers that could be used to characterize the associated pathology. Objective quantitative assays of damage-related host-defense cell signaling and host-defense cell accumulation would be expected to be more sensitive and more objective than the visual screening currently employed (Fig. 2). Additionally, probes for specific cell populations that could be visualized by noninvasive imaging techniques could allow noninvasive monitoring of these responses, as discussed in the section below.
Now that the sequence of the human genome has been determined, we know that polymorphic sequence variations occur in every individual at a frequency of approximately one in 1000 base pairs (Venter et al., 2001). Because mammalian genes generally contain thousands of base pairs, it is to be expected that genetic variability among individuals will occur at most, if not all, genes—and therefore in most, or all, molecular targets for toxicants. This suggests that genetic variation may to be found to be a major cause, or perhaps the major cause, for variation in susceptibility to toxicant exposure. This possibility is supported by the exponentially growing list of spontaneous pathologies (diseases) associated with genetic variants (Ashton et al., 2002; Balmain et al., 2003; Botstein and Risch, 2003) as such variants would be expected to be involved with both spontaneous and chemically induced pathologies.
Of course, the knowledge that genetic variation can influence sensitivity to toxicants is not new. Classic examples are the sensitivity to fava bean toxicity among Mediterranean populations with glucose-6-phosphate dehydrogenase deficiency (G6PD) and the sensitivity to isoniazide among subpopulations with N-acetylase variants (Kalow, 1965; Weber, 1999). Historically, the term "pharmacogenetics," and by extrapolation "toxicogenetics," was applied to the study of the influence of genetic variation on pharmacological or toxicological response (Kalow, 1968). However, advances in the technologies of sequencing and identification of sequence variants have resulted in a set of linkage markers that now make it possible to efficiently identify highly penetrant polymorphisms that modify biological outcomes (Roses, 2002). This was made possible because of the realization that sequence variations in the human are linked in chromosomal blocks that have not been fully randomized during the course of evolutionary crossing-over, a phenomenon known as linkage disequilibrium (Dawson et al., 2002; Goodman, 2002; Stumpf, 2002). Thus, a set of linkage markers covering the entire genome is now available (Roses, 2002). Because of this linkage, it is possible to first determine whether a biological outcome is associated with one of these blocks and then, knowing that the outcome has a genetic basis, to identify the specific sequence variations responsible for the observed effect (Fig. 3). This strategy, coupled with ever-improving technologies for efficient haplotype screening (see, e.g., Buetow et al., 2001; De La Vega et al., 2002), will greatly diminish the time and effort required to identify associations between specific genetic variations and biological outcomes. Thus, the traditional field of pharmacogenetics is now being transformed into pharmacogenomics—the study of the effects of genetic variants across the entire genome rather than one gene at a time (Cantor, 1999).
Examples are now known in which genetic polymorphisms, in addition to the classical metabolic polymorphisms, render individuals sensitive to specific forms of toxicity. For example, polymorphisms in the structural proteins of the cardiac potassium channels are known to render individuals sensitive to drugs that induce prolongation of the cardiac Q-T interval, with an attendant risk of fatal cardiac arrhythmia (Larsen et al., 2001; Weber, 2001). The ability to identify individuals with such polymorphisms opens the door to identifying the genetic factors that place individuals into high-risk categories. This has strong implications for designing drugs that minimize effects in sensitive individuals, individualizing treatment therapies, and designing initial clinical trials to include subjects with common (and less than common) polymorphisms that may influence the action of particular classes of drugs.
As genomic technologies become more available, it is not unrealistic to expect that an individual’s genotype for key genes associated with disease susceptibility, metabolic capacity, and drug sensitivity might become a routine part of one’s medical record and be used in diagnosis, selection of appropriate drugs, and adjustment of drug dosages on an individual basis. In recent editorials, Roses (2001) and Cantor (1999) speculate about the potential impact of such approaches on medical diagnosis and therapy, suggesting that genetically based medicine and pharmaceutical development may soon be commonplace.
In addition to providing a better understanding of the role of genetic variation in interindividual responses among humans, these same technologies will allow genetic characterization of laboratory animal model systems and their comparison with human systems. This should significantly improve our ability to extrapolate quantitatively across species. Further, genetic technologies have provided the capability of "humanizing" laboratory animal and cellular models. Thus, as important human targets for drug and toxicant interactions are identified and characterized, analogous laboratory models that allow these interactions to be studied in laboratory species can be constructed. Such models have already been created, demonstrating the feasibility of this approach. Examples include animal models of sickle-cell disease (Fabry, 1993), cell lines engineered to express the human cytochrome p450 drug-metabolizing enzymes (Crespi and Penman, 1997; Crespi et al., 1993), and animal and laboratory models containing human receptors or enzymes, and/or knockouts of specific genes of interest (Gonzalez, 2002; Nakazawa and Ohno, 1999).
The birth of the field of genetic toxicology and modern molecular genetics were essentially contemporaneous, with the demonstration by Avery et al.(1944) that DNA was the genetic material and Auerbach’s demonstration (1946) that chemicals can exert powerful mutagenic effects. It was recognized immediately that modification by chemical exposure of the genetic code that controlled all life’s functions would have adverse consequences, and that product safety assurance should include studies to define the potential for such genetic effects. The need for in vivo methods that allow quantitative risk assessment was recognized at the time regulatory genetic toxicology testing requirements were introduced (Department of Health Education and Welfare, 1977), but until recently such methods have been too laborious and costly to be used as part of the routine evaluations used for product development. Hence, current regulatory guidelines for product development still rely primarily on in vitro cellular methods for detecting mutagenesis (MacGregor et al., 2000). Advances in technology may soon make in vivo measurements more practical. The development of transgenic animal models containing neutral reporter genes that are easily recovered and screened in vitro for mutations following exposure in vivo (Heddle et al., 2000; Mirsalis et al., 1995), new models for measurement of mutation at endogenous gene loci (Dobrovolsky et al., 1999; Stambrook et al., 1996; Wijhoven et al., 1998), and new techniques that may allow direct measurement of DNA sequence changes in tissues in vivo at the sensitivity required to observe small changes in spontaneous rates of mutation (Parsons and Heflich, 1998) are now either available or in advanced stages of development. These new techniques may make possible the integration of mutation measurements into conventional toxicity evaluations, including regulatory toxicity assays and clinical trials in humans. Also, the development of practical biomarkers of genetic damage, such as induction of DNA repair, gene responses to DNA damage, measures of adduct formation and DNA strand breaks, etc., provide other means for monitoring genetic damage in practical assays suitable for incorporation into animal and human studies (Hoffmann, 1996).
As the molecular basis of carcinogenesis becomes understood, monitoring key genetic alterations associated with carcinogenesis will play an ever-increasing role in toxicological evaluation. Also, molecular biological techniques can be used to construct animal models that include key molecular features of the human carcinogenesis process. Such approaches to carcinogenesis evaluation have already begun to be used in regulatory practice, and they can be expected to play a more prominent role in the future. For example, the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use recently adopted testing guidelines that allow mechanistic short-term models of carcinogenesis to be incorporated into regulatory submission for approval of new pharmaceuticals (DeGeorge, 1998). EPA draft carcinogenicity evaluation guidelines that encourage incorporation of mechanistic data (i.e., related to mode of action) into cancer risk assessments are currently pending adoption (Federal Register, 2003). Mechanistically based animal carcinogenicity models currently under evaluation currently include the rasH2 mouse model that incorporates the normal human ras protooncogene into a mouse model, the p53 mouse model, based on the recognition of the importance in the human of changes in p53 function for expression of carcinogenesis, and several other mechanistically based models including some with inactivated defense or repair genes (e.g., Robinson and MacDonald, 2001). Thus, the tools of genetics are already being employed to construct appropriate "humanized" genetic models for carcinogenicity testing and also to monitor genetic changes associated with human and animal carcinogenesis as a part of product development. As the specific processes involved in human carcinogenesis, including the role of defense and repair systems, become better understood, it can be expected that animal models will be selected or constructed that allow more accurate and earlier prediction of induction of events likely to result in human cancers. Differences in these factors between humans and rodents are already becoming understood for a limited number of well-studied cases such as UV-induced mutation and cancer (Tang et al., 2000). This is likely to make feasible the monitoring in animal models those specific genetic events associated with human cancer, and also may permit monitoring of those same genetic events directly in the human.
Perhaps the greatest single limitation of modern toxicological practice has been the uncertainty of quantitative extrapolation from laboratory models to the human. Although the similarities in biochemistry and molecular biology among living species has permitted a wide variety of useful laboratory models for the study of toxicological effects, there is generally much uncertainty about quantitative exposure-response relationships in the human compared with laboratory model systems. Quantitative differences almost always exist in dose-response relationships between humans and model species, and in extreme cases biological responses to a given exposure may differ qualitatively. Thus, one of the great needs in the field is to have biomarkers of damage that can be used to compare toxic responses among species. In addition, those to be used in the human should indicate that a given pathological condition is being approached—before it actually becomes manifest. The inducible defense and damage-response molecules discussed above, and new tissue-specific serum and urine biomarkers that reflect damage to specific cell populations, have the potential to serve as such "bridging" biomarkers. The combination of new biological knowledge about defense and damage response systems, coupled with the revolution in molecular biological technology that allows inexpensive multiple-endpoint assays in microscale formats, provides an unprecedented opportunity to develop a comprehensive new set of bridging biomarkers. This may make it possible, for the first time, to routinely "cross-calibrate" among model systems—telling the toxicologist whether similar mechanistic patterns of damage and response are occurring in the human and in laboratory models, and defining the levels of exposure at which they occur in each case.
Various technologies will be needed to achieve these goals. It is likely that nucleic acid array technology will be a major tool for elucidating key inducible defense and damage-response molecules that are characteristic of specific pathological mechanisms and chemical classes. Proteomic and "metabonomic" technologies will be valuable tools for identification of accessible biomarkers (proteins and small molecules from accessible fluids and compartments) that reflect cellular status and function (Anderson et al., 2000; Jones et al., 2002; Nicholson et al., 2002). Ultimately, sets of biomarkers that can be sampled in accessible compartments such as blood, urine, and other body fluids will need to be made available in low-cost formats for routine application in biomonitoring studies.
The opportunities for improved regulatory practice discussed above are exciting, and surely the future will bring other unforeseen opportunities. Translation of these opportunities into practical methods and approaches suitable for routine application in product development and regulation is, however, not a trivial exercise. Key elements of the necessary evaluation and validation process include:
Satisfactory demonstration of these elements is difficult and time-consuming even for a single-endpoint assay. Defining these elements for highly multiplexed assays capable of monitoring many hundreds or thousands of endpoints simultaneously presents a significant challenge. "Biological validation" will need to include studies in important model species, and must include demonstration of an understanding of the relationship of biomarkers employed to cell and tissue injury. For example, it is important to distinguish whether a biomarker is a measure of a rate-limiting defense process that will prevent pathology until a defined threshold is passed, whether it is a marker that indicates that a specific type of damage has already occurred, etc. It is also important to understand the differences and commonalties in such responses among well-established laboratory animal and cellular models, and the human. The principles of assay and biomarker validation have been delineated (ICCVAM, 1997, 1999), and will need to be applied to each of the new biomarkers discussed above.
Some in the field have stated that it will take decades to achieve appropriate validation, but regulatory implementation will likely be much more rapid. Though the pace of scientific change often seems slow to those engaged in its practice, reflection on the rapidity of the adoption into practice of the major advances in science and technology during the past century reveals the opposite. For example, the periods from the discovery that DNA was the genetic material to the construction of transgenic organisms with modified genetic information, from first heavier than air flight to well-established commercial aviation, from invention of the transistor to the current prevalence of microelectronic integrated circuits in our society, and from the first descriptions of intracellular enzymes to the use of these enzymes as biomarkers of cellular toxicity were all achieved in a few decades or less. Why then has the approach to toxicological assessment been so stable over a comparable period of time? This likely stems from two key factors: the excellence of the strategy devised by early toxicologists and the need for conservative change associated with the dependence of the economic viability of new product development on well-established and predictable regulatory rules and practices. However, the current intense focus on the areas discussed above suggests that the field is entering a major transition that will employ the impressive technologies of the biological revolution to improve our approaches to product development and regulation.
Among these improvements, we may look forward to reconstitution of the fundamental set of biomarkers used to identify and monitor pathological and toxicological effects, and introduction of a more sensitive and specific set of markers that allows characterization of tissue sites of damage as well as mechanisms of cellular perturbations. Indeed, there is the potential to develop a new quantitative molecular pathology approach to supplement, or in some cases replace, the present semiquantitative histopathological evaluation that is the principal endpoint upon which many safety decisions are currently based. Molecular techniques may prove to be more objective, more quantitative, and more sensitive than the current approach, which relies on human judgements about changes in morphological structures and cell population alterations. The ability to monitor these biomarkers in vivo should allow increased reliance on direct human studies, as biomarker measurements in the human become more possible. This, coupled with bridging biomarkers that allow comparison of responses in the human with those in laboratory animal models, promises to greatly reduce the present uncertainty in quantitative extrapolation of results from laboratory models to human outcomes. Together, these approaches should dramatically improve selection of lead compounds in discovery, evaluation of toxicity in animal models, linkage between animal models and humans, and human monitoring. To reach these goals, applied research will be required to establish the necessary linkage between each new biomarker and the pathologies of interest, as well as to establish the statistical performance characteristics of the system of measurement (reproducibility, robustness, etc.). This will require commitment and collaboration among all sectors involved in product development, regulation, and utilization—the public, industry, and government.
I thank Drs. Roger Ulrich, Bernard Schwetz, Judith MacGregor, and Daniel Casciano for their review of this manuscript, and for their helpful suggestions.
1 E-mail: [email protected] . Fax: 301-827-9104.
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FIG. 1. Determination of mechanism and extent of damage via RNA expression profiling. The example illustrates probes for damage-specific responses in horizontal rows and samples printed in vertical columns. Agent A induces genes in liver associated with DNA and protein damage (and to a lesser extent, DNA damage in lung and kidney). The occurrence of protein damage in conjunction with extensive nucleic acid damage is expected (Dukan et al., 2000; Jelinsky et al., 2000; Taddei et al., 1997). Agent B induces genes in lung, liver, and kidney associated with protein damage and downregulates genes associated with oxidative damage, with little damage to DNA.
FIG. 2. Host-defense cell responses as biomarkers of tissue damage. Knowledge of the mechanisms of host-defense responses to tissue damage, including chemo- and cytokine signals, host-defense cell accumulation, and host-defense cell activation, allows selection of potential biomarkers of tissue injury based on measurement of key chemical signals and cellular responses.
FIG. 3. Identification of genetic variations responsible for adverse outcomes (e.g., drug toxicity). Association of specific haplotype markers with biological outcomes can be used to determine if outcomes are genetically determined prior to searching for the specific polymorphism responsible. The haplotype marker also identifies the chromosomal region that contains the genetic variant of interest. This approach greatly simplifies identification of specific DNA sequence variants that modify biological outcome.
Source: Toxicological Sciences 75, 236-248 (2003).