Focus
D N A MICROARRAYS IN TOXICOLOGY
T
oxicology usually brings to mind feeding chemicals to mice or rats and seeing what happens to the tissues or cells. However, thefieldof toxicology is currently undergoing a molecular revolution. The toxicology community is increasingly recognizing that integrating molecular techniques into traditional toxicity tests by analyzing gene expression will not only tell investigators what happened
after a chemical exposure but also how it happened. This revolution has been fueled by advances in genomics (the comprehensive analysis of all genes in an organism) and advances in the associated technology (DNA microarrays), which allows scientists to view gene expression at a genomewide level. DNA microarrays are made by placing single-stranded pieces of DNA—either
Gene expression analysis promises to change toxicology.
short oligonucleotides (oligos) or longer strands of complementary DNA (cDNA)—in a grid pattern on a solid substrate. This substrate is most often glass or silicon but also can be a nylon membrane. The DNA either can be transferred to the surface ("spotted") as intact pieces or synthesized one base at time directly on the substrate using photolithographic techniques inspired by the computer chip industry. Depending on the organism and the size of its genome, the arrays can consist of anywhere from a particular subset of genes up to an entire set of genes from a genome To assess gene expression using the arrays, messenger RNA (mRNA) is extracted from the tissue of interest and converted into DNA by the enzyme reverse transcriptase, which incorporates fluorescent or radioactive nucleotides into the DNA The labeled DNA is then incubated with the array and allowed to hybridize with its immobilized complements. The labels allow the hybridized sequences to be detected by either a fluorescence phosphorimager. The data are then stored as digital images. If it is known which the immobilized represent then the hybridization pattern identifies the genes that are expressed differently in the tissue after chemical Alan J. Robinson of the European Bioinformatics Institute in Hinxton, U.K. (an outstation of the European Molecular Biology Laboratory), says that the ability to measure the expression level of thousands of genes simultaneously has immense implications. "Before the advent of microarray
Celia M. Henry 462 A
Analytical Chemistry News & Features, July 1, 1999
technology, the collection of either qualitative or quantitative data on the expression of genes was an intensive and laborious task. With large-scale gene expression detection technologies, scientists may quickly examine the activity of potentially all the genes in an organism under differing conditions to identify those that are of most interest and merit further study." DNA microarrays have the potential to be used in a variety of ways in toxicology, including identifying biomarkers of exposure and determining how a particular chemical causes toxicity. For such experiments, an animal is dosed with a chemical of interest, the tissue is isolated, and the gene expression is analyzed using the microarrays. "[The experiments] have to be done in such a way that you can correlate the gene expression changes with the toxic response," says Chris Corton of the Chemical Industry Institute of Toxicology (Research Triangle Park NC) All substances have a threshold at which they are toxic. The dose determines the toxicity. "If you go high enough for long enough, you're going to reach some point when you overload the system and induce toxicity," says Corton. "There are a lot of changes that could be going on at lower doses or earlier times that have nothing to do with toxicity. When you look at one dose or one time, you wouldn't see all those changes." The experiments must be carefully designed and include multiple doses or time points to avoid being "fooled", says Corton. Looking at a single time or single dose doesn't reveal the genes that are linked to the toxicity. "There may be genes that are turned on at low doses and early times,
which have nothing to do with toxicity," says Corton. Corton believes that before DNA microarrays will be maximally useful for characterizing the toxic effects of unknown chemicals, a database cataloging the toxic effects of model compounds must be constructed. He says that after a sufficient number of chemicals in a particular class have been analyzed, patterns of genes that are turned on or off for a particular exposure will be revealed. "Each chemical is going to have afingerprint,"says Corton. "If you look at all the genestiiosefingerprintshave in common, you're going to come up wiih a profile or signature pattern. It allows you to say [that] if you see this pattern of gene expres-
sion changes, you're looking at a chemical that falls into this class." Once a gene expression database is established, unknown chemicals will be identified more easily as producing a particular toxic response. "When a new chemical comes along, you put it through the assay and come up with a gene expression profile that will allow toxicologists to determine if the chemical is a peroxisome proliferator, an estrogen receptor agonist, cytotoxic, or falls into some other class. Then you can begin to assess risk on [the basis of] this large body of data, which shows mat chemicals of that particular class behave with a well-defined mode of action " says Corton A database of this sort could be com-
Schematic representation of gene expression analysis using Affymetrix GeneChip expression arrays. (Image courtesy of Affymetrix.) Analytical Chemistry News & Features, July 1, 1999 463 A
Focus bined with microarray analysis to determine potential side effects of drugs. The gene expression profiles of tissues are measured before and after drug treatment. "Although the drug may have the desired effect on some parts of the metabolism, it may also interfere with other parts, which could lead to side effects," says Robinson. "Thus, drugs with possible side effects can potentially be determined before they have moved into animal or human trials."
A project spearheaded by the National Institute of Environmental Health Sciences is working on establishing such a database. The Environmental Genome Project (EGP) is a multidisciplinary, collaborative effort that involves other institutes at NIH and other federal agencies, including the Department of Energy. The stated aim of this project is to "understand the impact and interaction of environmental exposures on human disease".
M a k i n g DNA a r r a y s
There are two general approaches to making DNA arrays for gene expression. In the photolithographic method performed at Affymetrix (Santa Clara, CA), synthetic linkers modified with photochemically removable protecting groups are attached to a silicon surface. Light is directed through a photolithographic mask, removing the protecting groups from specific parts of the surface. The surface is then incubated with one of the four hydroxyl-protected deoxynucleotides, which couple with the DNA strands that have been deprotected. A new mask is then used, so that different parts of the surface are illuminated. The cycle is repeated until the strands are the desired length and sequence. The complete set of oligos of length (TV) (4N different strands) requires 4 x N cycles. For example, the complete set of 5-base oligos consists of 1024 strands and requires 20 cycles. The chemistry generally limits the length of the probes to
