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Perspectives in Cancer Research |
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 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 oligonucleotide-based microarray (4)
. Either cDNA microarrays or oligonucleotide-based 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)
.
|
. 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.
|
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 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.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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: barrett{at}niehs.nih.gov 
Received 2/18/99. Accepted 8/ 6/99.
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