TOXICOLOGICAL SCIENCES 75, 236 –248 (2003)
DOI: 10.1093/toxsci/kfg197
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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
Received May 21, 2003; accepted July 9, 2003
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 toxico1
logical practice is likely to be shaped in the future by the
combination of conventional pathology, toxicology, molecular
genetics, biochemistry, cell biology, and computational bioinformatics—resulting in the broad application of molecular
approaches to monitoring functional disturbances.
Key Words: toxicity; biomarkers; microarray; proteomics;
genomics; metabonomics; polymorphism; haplotype; validation.
E-mail: jmacgregor@nctr.fda.gov. Fax: 301-827-9104.
Toxicological Sciences 75(2), © Society of Toxicology 2003; all rights reserved.
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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 ␮g 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.
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Current Toxicological Practice
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
TABLE 1
Current Toxicological Practice
Parameters and biomarkers evaluated
Organ and tissue damage
Markers of function or homeostasis
BUN, electrolytes, cell type, ECG, BSP, etc.
Evaluation of organ and tissue growth (organ and body weights)
Markers of cell and tissue integrity
AST, ALT, ALP, CK, troponin, etc.
Markers of damage or damage response
Visible morphologic evidence of damage (gross- and histopathological
observation)
Host defense responses (host-defense cell infiltration, immune cell
response)
Other effects
Reproductive effects
Mutagenesis
Carcinogenesis
Special functional evaluations
Safety pharmacology, EKG, CV
Neurological and behavioral
Immunotoxicology
Pulmonary
Dermal
Ocular
Note. From Lehman et al. (1949); Barnes and Denz (1954); Paget (1970);
D’Arcy et al. (1998; ICH Nonclinical Safety Studies Guidance for Pharmaceuticals); US Food and Drug Administration (2001). AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; CK,
creatine kinase.
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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.
Opportunities for Improved Approaches to Toxicological
Assessment
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
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TABLE 2
Opportunities for Improved Toxicological Assessment
Opportunities
Improved biomarkers of toxicant-induced damage
Damage-inducible markers and defense responses
Specific markers of cell and tissue integrity and homeostasis
“-omic” technologies for global monitoring of multiple pathways
Molecular markers of pathological processes (e.g., cell death, hostdefense cell signaling and infiltration, etc.)
“Fingerprinting” of molecular response and pathway perturbations
“Bridging” biomarkers that link laboratory studies to human outcomes
Influence of genetic variation on toxic response
Metabolic polymorphisms
Receptor and response polymorphisms
Individual vs. population responses
Predictions of interactions and/or susceptibility
“Humanized” laboratory models
Human metabolic characteristics
Human receptors and molecular targets
Human disease models
Short-term carcinogenesis models based on human characteristics
(including in vivo genetic markers, oncogene and suppressor
inactivation models)
Noninvasive pathology and functional monitoring
Bioinformatics and artificial intelligence approaches
alterations that lead to human disease, and (5) for application
of imaging technologies to noninvasive monitoring. Each of
these opportunities merits discussion.
Biomarkers of Cellular Integrity
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 ␣ form in hepatocytes and
proximal tubular cells and the ␲ 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 ␣-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 ␣- and ␲-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 tissuespecific 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
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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 damagerecognition 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
TABLE 3
Some Classes of Damage- or Agent-Inducible Genes
Cellular characteristic
Damage type or inducer class
Examples
References
Protein structure
DNA integrity
Oxidative protectants
Metal inducible
Xenobiotic metabolism
Protein denaturation
DNA damage
Redox balance
Toxic metals
Xenobiotics
HSP70, clpB
p53, GADD153, recA
NF-kB, GST
Metallothionein
CYP1A1, CYP2E1
Wickner et al., 1999
Lindahl and Wood, 1999; Offer et al., 2002
Pinkus et al., 1996
Murata et al., 1999
Parkinson, 1996
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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.
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 ar-
rays 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
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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).
Toward Molecular Pathology: Improving the “Gold
Standard”
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
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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 hostdefense 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 dam-
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.
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age-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.
Noninvasive Imaging
Because biochemical processes and responses in specific
tissues may not always be reflected by biomarkers measurable
in other compartments, a key need in the field of toxicology is
for noninvasive methods of monitoring responses and events
within specific tissues. Advances in imaging technology suggest the potential to develop probes for cellular macromolecules that would permit these technologies to be used to monitor cellular markers noninvasively. Imaging modalities with
potential application for monitoring molecular biomarkers include PET, MRI, fMRI, optical imaging, and x-ray computed
tomography (Frank and Hargreaves, in press; Rudin and
Weissleder, 2003). While each technology may have application for specific purposes, the measurement of radio-labeled
probes using PET is probably the most conceptually straightforward method to adapt in a general way to monitoring
macromolecules in accessible tissues. In principle, this technique could be used to monitor any macromolecule for which
a nontoxic small molecular weight radio-labeled probe could
be devised.
Though not yet in routine use, feasibility studies to demonstrate the applicability of PET imaging to monitoring fundamental cellular responses and their response to toxic insult—
cell death, cell proliferation, gene expression, and protein-protein
interactions—are underway or completed (Blankenberg et al.,
1999; Brown et al., 2002; Herschman et al., 2000; Massoud
and Gambhir, 2003; Paulmurugan et al., 2002; Ray et al.,
2003). The ability of PET to monitor essentially any molecule
for which a suitable radioactive probe can be incorporated is
well-established, and so the use of this technology for monitoring biomarkers in vivo will depend on the ingenuity of
investigators in developing labeled probes to track molecules
within tissues. One obvious approach is the use of labeled
antibodies or other binding proteins that can interact with cell
surface markers, as was done in the Blankenberg et al. (1999)
studies of cell apoptosis.
Significant barriers still exist with regard to the potential to
apply such technologies routinely. Among these barriers are
cost, availability of suitable labeled probes, lack of general
availability of instrumentation, and the time necessary to acquire and process images. Thus, introduction of these approaches can be expected to be significantly slower than the
molecular approaches discussed above. Nevertheless PET,
MRI, and gamma-scintography are already very widely used in
medical applications, and further technical improvements are
likely to increase the availability and capability of such instrumentation.
Individual Genetic Variation and Toxicity
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).
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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).
Mutagenesis
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.
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
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).
Carcinogenesis
As the molecular basis of carcinogenesis becomes understood, monitoring key genetic alterations associated with car-
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cinogenesis 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.
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 “crosscalibrate” 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.
Regulatory Acceptance and Validation
“Bridging” Biomarkers
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:
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 doseresponse relationships between humans and model species, and
in extreme cases biological responses to a given exposure may
● Demonstration of a clear understanding of the relationship
between the endpoint(s) measured and the biological outcome
of interest (biological validation, often referred to as “evaluation”).
● Determination of the performance characteristics of the
assays employed (analytical validation), including sensitivity,
accuracy, and reproducibility within and among laboratories.
● Identification of interfering factors that may modify assay
outcome, yielding “false” or misleading results that may underor over-estimate the biological event of interest.
● Development of consensus among the scientific commu-
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nity and responsible regulatory bodies on appropriate application of methods and approaches.
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.
ACKNOWLEDGMENTS
I thank Drs. Roger Ulrich, Bernard Schwetz, Judith MacGregor, and Daniel
Casciano for their review of this manuscript, and for their helpful suggestions.
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