Better Living Through Toxicogenomics?

Toxicologists traditionally use animals to test the toxicity of chemicals and other substances. But the brand new field of toxicogenomics, which applies a whole-genome approach to toxicology questions, is changing all that. Still in its infancy, this field is destined to change the way toxicologists think and act, and could even help optimize the drug-development process. "It holds great promise for the future," says Jay Goodman, a Michigan State University toxicologist. "Toxicogenomics is a tool that can improve the assessment of potential toxicity." Phil Iannaccone, a researcher at Northwestern University Medical School and invited author of a recent Environmental Health Perspective editorial on the new technology,1 agrees: "The hope is that the observed patterns will be characteristic of a class of toxicants, such as polycyclic aromatic hydrocarbons versus peroxysome proliferators. Eventually one might hope for specificity allowing actual identification of the chemical," he says. "For now it is exciting enough that one might be able to determine if an unknown chemical is likely to behave as a certain class of toxicants or not." The new field is based on the premise that tumors, disease, and other physical responses to toxic chemicals find their origins in gene expression, which depends on the environment—chemical or otherwise. Determining how genes respond to a toxicant, then, could be a direct measure of toxicity. It's faster, cheaper, and more accurate than animal testing, which often takes years to perform; examining gene expression takes only days or months. That's welcome news, as there are over 80,000 substances being used in commerce such as drugs, food additives, cosmetics, and chemicals, but only a fraction has been thoroughly tested for toxicity, says Michael McClure, Chief, Organs and Systems Toxicology Branch, Office of Program Development, National Institute of Environmental Health Sciences (NIEHS). Toxicogenomics could accelerate the speed at which these substances are tested. Microarrays Fit the Bill The key toxicogenomicist's tool is the microarray, which allows scientists to simultaneously assess the expression of thousands of genes and produce a profile, or signature, for each toxin. Like traditional biomarkers, such as blood enzyme levels, microarray profiles can flag substances as hazardous, but this new biomarker provides greater precision in identifying hazards. Each chip's probe set is manufacturer-dependent (see sidebar), but they typically include genes implicated in DNA replication and repair, and apoptosis; transcription factors; signaling molecules; and genes known to respond to various cellular insults. Toxicologists use these microarrays in several ways. Instead of groping in the dark for a few genes that might be involved in the toxicant response, scientists can use toxicogenomics to get a complete picture of all affected genes, and then focus on the promising candidates. This knowledge provides clues as to why animals develop cancer, liver damage, heart problems, or birth defects in response to a toxin. Researchers also can screen an environment for toxins by assessing the gene expression characteristics of organisms living in that environment. And they can measure the gene expression profiles of clinical trial participants to determine a drug's toxicity, its effects on the body, and the effects of different dosages. These applications could change the way pharmaceutical companies operate. Drug discovery methods such as combinatorial chemistry have greatly increased the number of drug leads, but taking one compound through the development process often costs millions of dollars. If, during clinical trials, that compound is found to be toxic, the money would be wasted. Pharmaceutical companies can reduce R&D costs by running the compound through microarray testing early on in the process. A Complement, Not a Replacement The NIEHS established the National Center for Toxicogenomics (NCT) in September 2000. According to the Center's mission statement, its goal is "to use the methodologies and information of genomics science to significantly improve our understanding of basic biological responses to environmental stressors/toxicants." NCT director Raymond Tennant explains that toxicogenomics fundamentally changes the way toxicologists carry out their work: "In football you try to identify the person carrying the ball and tackle him. With global gene expression, you can tackle the entire football team at one time and throw out the players until you find the ball." Of course, it helps to have the entire team on one chip. With all kinds of genes assembled on one array, probes can exclude those genes that don't play a role in the toxic response. But knowing which genes change their expression in the presence of toxicants is of only limited value. Scientists must be able to correlate these changes with something tangible, such as a tumor. Otherwise, the fact that certain genes become up- or down-regulated doesn't mean much. "If you take a pesticide and throw it at a cell culture, you're going to get a response, but you don't know what the significance of the response is," says Peter Spencer, director, Center for Research on Occupational and Environmental Toxicology at Oregon Health Sciences University. Goodman adds, "In order to make progress, this new tool must be linked to basic principles of toxicology—for example, dose- and time-response relationships—and we need to understand that a simple change in gene expression is not necessarily indicative of toxicity." So, researchers will continue to perform animal testing, while toxicologists anchor gene expression profiles to actual phenotypic effects. Starting with chemicals already known to cause cancer, a gene expression profile could be correlated with carcinogenicity. Toxicologists could then compare the signature of an unknown chemical to that of known toxicants. A matching signature then offers clues to that compound's effects. Bioassays that don't use animals can also help interpret gene expression profiles, measure toxicant-induced DNA damage, and can determine neurotoxicity, immunotoxicity, reproductive and developmental toxicology, and genetic toxicology. The unknown chemical's signature can help the researcher determine if the toxicant induces a biological response. Then, the most relevant bioassay can be selected. This makes sense, as some bioassays can be extremely expensive and time consuming, such as the rodent cancer bioassay, which requires four years, 1,200 animals, and millions of dollars to execute and analyze.2 Courtesy of PHASE-1 Molecular Toxicology Inc.An Eye Towards the Future: Gene expression profiling may one day replace traditional bioassays, but not until scientists can correlate the data with a tangible, physical effect. Microarrays offer several advantages, despite their reliance on bioassays for data validation. The approach could enhance a bioassay's sensitivity and interpretability, possibly reducing its cost. In addition, because low doses of chemicals can affect gene expression, bioassays can be performed using less of the potential toxicant. This produces a more realistic picture of how humans might be affected by the chemicals because everyday exposures normally occur in small doses. Microarray testing also allows scientists to observe how humans react to a toxicant rather than having to extrapolate data from rats, mice or other animals. Scientists can also observe how acute exposure to a chemical might differ from chronic exposure. In a recent study, researchers in England exposed human hepatoma cell line HepG2 to two hepatotoxins, carbon tetrachloride and ethanol, and collected gene expression data using microarrays.3 After two hours, the scientists found that gene expression hadn't changed much, but after 24 hours, carbon tetrachloride caused an increase in expression of genes involved in extracellular transport and cell signaling, whereas ethanol exposure caused a decrease in the expression of genes involved in the stress response and metabolism. A Sea of Data Analyzing these results is not easy. Microarrays generate giant lists of numbers representing changes in gene expression. "It's one thing to run an array. That's the easy part," Tennant says. "Coming to understand what those data are telling you about is a substantial effort." That's where bioinformatics comes in, says McClure. Bioinformaticians can help toxicologists make meaningful conclusions out of the deluge of data points. But the trial of wading through the data is nothing compared to the effort it took to study how toxicants affected genes before toxicogenomics came along. At that time, it was, "One gene, one protein at a time," observes Leona Samson, a toxicologist at Massachusetts Institute of Technology. Now, "our eyes are being opened to a multitude of responses that are helpful to the cell for recovery." Using software to archive, compare, and interpret microarray data, researchers can now begin the task of compiling databases against which new compounds can be tested. For example, Santa Fe, NM-based PHASE-1 Molecular Toxicology Inc., is developing a database that will hold the gene expression profiles of a whole spectrum of chemicals. The NCT is also building a database. In going forward with that task, the NCT announced in November the formation of the Toxicogenomics Research Consortium. Organized by an NIEHS team led by McClure and Ben Van Houten, the Consortium is a $37 million effort. To make a useful database, which can serve as a universal reference, the researchers must establish standards, fine tuning environmental factors and experimental conditions. They'll have to consider dosages, and finely control how they culture cells and treat animals—including such details as lighting, nutrition and feeding schedules, and handling effects. This will ensure that any changes in gene expression result from the tested chemical and not from the ambient environment. In the future, toxicogenomics will likely play an important role in personalized medicine, says Erik Jongedijk, marketing and sales director at PHASE-1's Belgium headquarters. Yet despite its promise, very few companies offer arrays specifically for toxicological applications, and fewer still have developed technology that's affordable and accessible to individual academic researchers. Toxicogenomics is big-ticket science; most of the players are pharmaceutical and biotech companies. But, as Iannaccone points out, "if you have access to a biotech center, which most universities have now, then the individual investigator absolutely can do this. Most of the interesting work in the past few years can be traced to a single postdoc in the lab that published the work. Generally this person picked this up as a project without prior experience. They then wind up getting job offers in drug companies and are whisked out of the lab!" Laura Lane (lanelaura@hotmail.com) is a freelance writer in San Francisco. Leslie Pray (lpray@nasw.org) is a freelance writer in Leverett, Mass. References 1. P.M. Iannaccone, "Toxicogenomics: 'The call of the wild chip,'" Environmental Health Perspectives, 109:A8-11, January 2001. 2. E.F. Nuwaysir et al., "Microarrays and toxicology: The advent of toxicogenomics," Molecular Carcinogenesis, 24[3]:153-9, 1999. 3. H.M. Harries et al., "The use of genomics technology to investigate gene expression changes in cultured human liver cells," Toxicology In Vitro, 15[4-5]:399-405, August-October 2001. Chip Makers IN FOCUS | Laura Lane & Leslie Pray ToxChip1 was the prototype toxicology chip, says Cynthia Afshari, one of the chip's developers at the National Institute of Environmental Health Sciences (NIEHS) Microarray Center in Research Triangle Park, NC (www.niehs.nih.gov/nct). In developing the chip, Afshari and colleagues consulted with experts to identify toxicologically relevant genes; the expertise they rallied in developing the chip is one of ToxChip's unique features, says Afshari. Initially comprised of 2,000 genes, the chip now holds 12,000 genes—including 6,000 unknown, which is more than any of the commercially available toxicology-targeted microarrays. For academicians, the more genes examined, the better, says Afshari, because it is impossible to predict all toxicologically relevant genes. Also, not every gene that seems important based on past research will necessarily be informative in an expression-based array. RNA from exposed animals and humans is a very valuable resource, observes Afshari, and researchers want to get as much information from it as they can. The ToxChip isn't for sale, though the gene list is free and accessible on the Internet. Researchers can, however, collaborate with NIEHS after submitting a proposal for in-house review (dir.niehs.nih.gov/microarray/). Currently, NIEHS has over 50 microarray-based collaborations in both basic and toxicology-based studies. The group hopes that by making the gene list public, other researchers will alert them to important genes that should be added to the ToxChip, so that its usefulness will continue to evolve. For commercial toxicogenomics applications, companies are required to obtain a license from Discovery Partners International Inc. (DPII) of San Diego. DPII's subsidiary, Xenometrix, owns and has exclusive rights to US patent 5,811,231, "Methods and Kits for Eukaryotic Gene Profiling," issued in 1998. According to Xenometrix president Pauline Gee, the patent is fundamental to toxicogenomics. It covers "the creation of many multi-gene expression profiles resulting from exposure of eukaryotic cells ... to pharmaceutical compounds and other chemical entities," according to a press release announcing the patent's issuance. Several companies offer "tox chips" commercially. Santa Clara, Calif.-based Affymetrix Inc. (www.affymetrix.com) introduced its Rat Toxicology U34 Array, containing over 850 genes and ESTs, in 1999. Gaithersburg, Md.-based Gene Logic (www.genelogic.com) used this chip, plus the Human Genome U95 array, to develop a reference database, the GeneExpress® ToxExpress™ Module. ToxExpress is a "reference set of sentinel genes and their gene expression patterns that are predictive" of toxicity, says Bob Burrows, Gene Logic's corporate communications director. The data are based on profiling selected pharmaceuticals in rats and human cell lines, at both therapeutic and toxic doses, to examine gene expression changes following exposure to a toxic compound. Companies that already have their own in-house toxicogenomics programs can use ToxExpress as a "predictive backdrop, or library, of classic toxicity patterns," says Burrows. Currently, the company has 10 pharmaceutical and biotech subscribers. Palo Alto, Calif.-based CLONTECH Laboratories (www.clontech.com) offers the Atlas™ Human, Mouse, and Rat Toxicology 1.2 nylon-based arrays. Each contains over 1,000 genes selected by scientists at the Environmental Protection Agency (EPA) for their involvement in cellular responses to toxicant exposure. Will Scovill, Atlas™ product manager, says CLONTECH's products are accessible to individual researchers because they are less expensive than similar products on the market and do not require any specialized techniques or equipment. The only extra hardware that the user needs for a complete Atlas-based toxicogenomic study is a phosphorimager to read the hybridization profile. CLONTECH's arrays contain a relatively small number of genes, but each has established importance in the toxicology field, says Scovill, which allows researchers to focus on the most relevant genes. "It makes the bioinformatics end of toxicogenomics much easier," he adds. Users receive free access to the AtlasInfo™ Bioinformatics Database, which includes information such as alternative names and known functions for each gene, journal citations, and links to related databases. One of Santa Fe, NM-based PHASE-1 Molecular Toxicology's (www.phase1tox.com) primary functions is to provide pre-clinical toxicogenomic services to biotech and pharmaceutical companies, with a flexible, customized approach. Smaller companies often submit their samples and say "we want you to do everything," says Erik Jongedijk, marketing and sales director at PHASE-1's Belgium headquarters. But larger companies sometimes prefer to do their own work, in which case they buy PHASE-1's microarrays and access to its database and software, but perform their own analyses. PHASE-1 produces the Rat 700, Canine 140, and Custom arrays. Like CLONTECH's arrays, these chips contain far fewer probes than the ToxChip, but from a regulatory perspective, it may make more sense to use the smaller chips, says Afshari. The more genes there are, the larger the data set, and the more unexplained data. This can cause trouble with the FDA, because the agency requires that every data point be explained. Most of the genes on PHASE-1's arrays are picked using empirical validation, says Jongedijk, unlike other toxicology arrays on the market, which are knowledge-based. In other words, the gene sets have been identified experimentally by PHASE-1 scientists, and not culled from peer-reviewed literature. Expression profile data are analyzed using PHASE-1 software and compared to gene expression patterns of known compounds in PHASE-1's TOXbank™ database, and to small sets of proprietary organ-specific toxicity biomarkers. TOXbank was developed from thousands of experiments on the effects of over 150 compounds in rats. "All data are generated in-house in a very strict protocol," says Jongedijk. Because they don't use external data, the company's reference database is not confounded by the kind of variation expected in databases made up of multiple sources, he says. PHASE-1 also produces software that can generate, store, and interpret the mounds of data that arise from molecular toxicology experiments. One of the programs, called MATRIXexpress™, enables the manipulation and extraction of relevant data from the company's toxicology profile database, including sorting and analysis, annotation, and clustering analysis to enhance the predictive value of the inherent data. 1. E.F. Nuwaysir, "Microarrays and toxicology: The advent of toxicogenomics," Molecular Carcinogenesis, 24:153-9, 1999.

Better Living Through Toxicogenomics?

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