Download Application of Complementary DNA Microarray

[CANCER RESEARCH 59, 4759 – 4760, October 1, 1999]
Perspectives in Cancer Research
Application of Complementary DNA Microarray Technology to Carcinogen
Identification, Toxicology, and Drug Safety Evaluation
Cynthia A. Afshari, Emile F. Nuwaysir, and J. Carl Barrett1
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Abstract
One major challenge facing today’s cancer researchers and toxicologists is the development of new approaches for the identification of
carcinogens and other environmental hazards. Here, we describe the
potential impact of emerging technologies for measuring gene expression
profiles on carcinogen identification and on the general field of toxicology.
An example of one of these technologies is the use of cDNA microarray
chips. We provide an overview to the key questions that are confronting
investigators charged with determining the relative safety of natural or
synthetic chemicals to which humans are exposed, followed by a discussion
of how cDNA microarray technology may be applied to these questions.
Gene chip technology is still a relatively new technology, and only a
handful of studies have demonstrated its utility. However, as the technical
hurdles to development are passed, the use of this methodology in addressing the questions raised here will be critical to increase the sensitivity
of detection of the potential toxic effects of environmental chemicals and
to understand their risks to humans.
Introduction
Identification of the causes of cancer and other diseases is the first
step in disease prevention. Many diseases are influenced by environmental factors, which include tens of thousands of synthetic and
natural chemicals, radiation, viruses, diet, and poorly defined conditions such as socioeconomic status. Although most of these chemicals
are harmless, it is a tremendous challenge to determine which chemicals contribute to influence disease susceptibility or occurrence in
humans.
Traditionally, toxicologists have used rodent bioassays to identify
potentially hazardous substances, including carcinogens, reproductive
toxins, immunotoxins, and neurotoxins. These assays require high
doses, often take years to complete, and are expensive. It was originally intended that such assays would be the first step in carcinogen
hazard identification and that further studies on mechanisms of action,
species extrapolation, and effects at low doses would be subsequently
performed to determine the risk of chemicals to humans. Unfortunately, because the task of performing all of these subsequent studies
is large, in most cases, the information gained from rodent bioassays
is used to regulate chemicals to which humans are exposed. This
approach is appropriate from a public health perspective but may lead
to incorrect assumptions of hazards to humans. Thus, the application
of testing of chemicals in rodents to humans has recently been
challenged (1).
To further complicate this approach, the confirmation of results
from rodent assays in humans using epidemiological studies is difficult because of the retrospective nature and limited sensitivity of such
Received 2/18/99; accepted 8/6/99.
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1
To whom requests for reprints should be addressed, at National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709. Phone:
(919) 541-3205; Fax: (919) 541-7784; E-mail: [email protected].
studies. Nonetheless, adverse effects in humans could be prevented if
rodent tests were more reliable. Thus, alternative approaches to identify toxic chemicals and carcinogens in humans are needed. It is
possible that new advances in molecular medicine can lead to disease
prevention through the identification of environmental causes as well
as to new approaches for disease treatment.
cDNA microarray technology, which can be used to analyze
changes in genome-wide patterns of gene expression (2, 3), is one new
methodological advance that may revolutionize the way some toxicological problems are investigated (Table 1). The application of a
large number of genes or expressed sequence tags in a condensed
array on glass slides or nylon filters comprises a cDNA microarray (2,
3). Alternatively, specific oligonucleotides that are complementary to
known genes or expressed sequence tags are deposited on a miniature
matrix by a photolithographic process to create an oligonucleotidebased microarray (4). Either cDNA microarrays or oligonucleotidebased chips may be used for gene expression analysis. Oligonucleotide-based DNA chips are also used for analyzing sequence variations
in genomic DNA for screening individuals for DNA mutations
and polymorphism variations. This approach has been recently reviewed (5).
Changes in gene expression in a tissue may result from differences
in physiology, developmental stage, pathology, or environmental exposure. These changes can now be measured using cDNA or oligonucleotide-based microarrays, which are used to compare directly the
gene expression profiles of two RNA samples that are simultaneously
hybridized to the chip (6, 7). The potential analysis of the expression
of thousands of genes in one experiment now allows investigators to
consider addressing some important biological questions that have not
been easily addressed with traditional expression-based technologies,
such as Northern blots, in situ hybridization, or RNase protection
assays, which examine gene expression changes of only a few genes
at a time. The ability to examine thousands of genes (potentially all of
the genes in a given cell type) provides new insights into the effects
of chemical or drugs on biological systems. Microarray technology
will be useful to identify toxic substances individually or in mixtures,
to determine whether toxic effects occur at low doses, and to extrapolate effects from one species to another. Potential applications of this
technology to toxicology problems are listed in Table 2. Assuming
that exposures to different classes of toxicants result in distinct patterns of altered gene expression, in addition to common changes
associated with the subsequent toxic response, microarray technology
can be used to categorize and classify these effects through the direct
comparison of gene expression signatures in exposed and control
samples.
One example of the use of cDNA microarrays is in the process of
drug development (8). Given that advances in genomics and combinatorial chemistry are leading to the discovery of many new potential
drugs, surrogate markers of efficacy and safety are needed to expedite
clinical trials. Gene expression profiles can be used as a proof of
principle assay to show an effect of a candidate drug in vivo. Furthermore, cDNA microarrays can be used to detect toxic responses in
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cDNA MICROARRAY TECHNOLOGY
target and nontarget tissues in rodents and humans. The dose of a drug
that maximizes the therapeutic index can potentially be determined
from such measurements, which will improve optimization of lead
compound development. By the use of cDNA microarrays, toxic or
unanticipated responses in humans may be determined early in a
clinical trial prior to overt tissue toxicity, providing a rapid, sensitive
surrogate of safety, which is essential for improved clinical trials.
Also, microarrays may help identify susceptible individuals who
respond to a treatment or who exhibit adverse effects to drugs.
In the area of environmental health sciences, cDNA microarray
technology can be used in the identification of potential hazards. It
should be relatively easy to establish model systems, both in vitro and
in vivo, to examine gene expression changes as indications of chemical effect. In these defined model systems, treatment with known
agents, such as polycyclic aromatic hydrocarbons, peroxisome proliferators, oxidant stress, or estrogenic chemicals, agents that lead to
activation of signaling pathways will provide a gene expression “signature” on a cDNA microarray, which represents the cellular or tissue
response to these agents. It is likely that the molecular response to
different agents will induce changes in expression of many genes that
are indicative of a general toxic response, but a subset of genes
expressed is predicted to be unique for a particular class of compounds, especially at low doses. Once the subsets of prototypic
response genes are defined for known agents in established models,
treatment of these same systems with unknown, suspect agents may be
used to determine whether one or more of these standard signatures is
elicited. This approach may flag certain compounds as potential
carcinogens/toxicants and will help elucidate the agent’s mechanism
of action by identification of the activated signal transduction pathways (9). Indeed, this approach has already been demonstrated in a
recent study investigating the signature response for drug exposure in
wild-type yeast compared with yeast that harbor a mutation in genes
that are potential targets for compound action (10). Another important
application for cDNA microarrays is in the determination of cross-talk
between combinations or mixtures of agents.
Specific cDNA microarray chips may be designed for the purpose
of studying toxicant action (9) in humans and in a variety of model
organisms, including mouse, rat, and yeast. These cDNA chips will
allow the simultaneous monitoring of gene expression changes for
receptor-mediated responses, xenobiotic metabolizing enzymes, cell
cycle components, oncogenes, tumor suppressor genes, DNA repair
genes, estrogen-responsive genes, oxidative stress genes, and genes
known to be involved in apoptotic cell death. The advantage of this
technology is that expression changes may be easily assessed over a
range of doses as well as times of exposure. However, the bioinformatic analysis of these gene expression changes over time and dose is
complex and needs to be further developed.
It is possible to use cDNA microarrays to measure biomarkers of
exposure or effect in humans. However, these applications will require extensive investigation before they become feasible. Traditional
assays measure metabolites of the toxicant, putative tissue damage
induced by the toxicant, or DNA adducts present in peripheral blood.
One major hurdle in using a gene expression approach for these assays
is to obtain tissue samples at a time when it would be most informative as a biomarker. It may be difficult to obtain tissues that exhibit
Table 1 Key questions in carcinogen identification and toxicology
●
●
●
●
●
How to determine potential toxicity of tens of thousands of chemicals?
How to determine the effects of chemicals at low doses?
How to extrapolate effects of toxins from one species to another?
How to study interactions of mixtures of chemicals?
How to determine the relative contribution of environmental and natural causes of
diseases?
● How to predict the toxic side effects of drugs?
Table 2 Potential uses of microarrays in toxicology
● Define surrogates of safety for use in drug clinical trials
● Identify toxicants on the basis of tissue specific patterns of gene expression by
establishing molecular signatures for chemical exposures
● Elucidate mechanisms of action of environmental agents through the identification
of gene expression networks
● Use toxicant-induced gene expression as a biomarker to assess human exposure
● Extrapolate effects of toxicants from one species to another
● Study the interactions of mixtures of chemicals
● Examine the effects of low-dose exposures versus high-dose exposures
gene expression changes at the mRNA level to assess exposure for the
purpose of determining that an exposure occurred prior to the onset of
pathological symptoms. This, however, is when exposure should
ideally be determined to allow intervention and prevention of disease.
The combined use of chips for measuring DNA sequence and
polymorphisms and cDNA based microarrays might also be used to
identify susceptible individuals. Currently, polymorphism studies are
used to assess individuals that have “susceptible” alleles for gene
implicated in disease. Whereas it is not known initially what effect
these polymorphisms have on gene function, microarrays might be
useful to examine the link between disease susceptibility and individual variability in gene expression (11). However, large studies on
control populations are first needed to understand the intrinsic variability in normal gene expression. Events such as prior exposures,
health, and diet of the individual might influence these levels and will
need to be taken into account.
In summary, the application of cDNA microarray analysis to the field
of toxicology, carcinogen identification, and drug safety provides an
opportunity to change and improve the way environmental factors and
therapeutics are currently investigated. cDNA microarrays may be used
to identify new environmental carcinogens and toxic effects of drugs, to
improve the current testing models, and to also understand the mechanism of action of these agents. Defining the mechanisms of action of
toxic agents can greatly assist in species extrapolation and risk assessment. This should also lead to the identification of new genes/targets
involved in environmentally caused diseases, including cancer and diseases of the immune, nervous, and pulmonary/respiratory systems.
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer
Research.
Application of Complementary DNA Microarray Technology to
Carcinogen Identification, Toxicology, and Drug Safety
Evaluation
Cynthia A. Afshari, Emile F. Nuwaysir and J. Carl Barrett
Cancer Res 1999;59:4759-4760.
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