J BIOCHEM MOLECULAR TOXICOLOGY
Volume 15, Number 5, 2001
Discovery in Toxicology: Mediation by Gene Expression
Array Technology
Hisham K. Hamadeh, Pierre Bushel, Richard Paules, and Cynthia A. Afshari
National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA; E-mail: afshari@niehs.nih.gov
Received 24 April 2001; accepted 27 April 2001
ABSTRACT: Toxicogenomics is a term that represents
the merging of toxicology with novel genomics tech-
niques. Data generated in the new-age era of toxico-
logy is relatively complex, requires new bioinformat-
ics tools for adequate interpretation, and allows for the
rapid generation of testable hypotheses. Hazard iden-
tification and risk assessment processes will advance
from the use of genomics techniques, which will lead
to greater understanding of mechanism(s) of action of
toxicants, development of novel biomarkers of expo-
sure and effect, and better identification of sensitive
subpopulations. ° C 2001 John Wiley & Sons, Inc.∗ J
Biochem Mol Toxicol 15:231–242, 2001
KEY WORDS: Toxicogenomics, Genomics, Toxicology,
Microarrays, cDNA, Single Nucleotide Polymor-
phisms, Pattern Recognition, Bioinformatics, Gene
Expression, Biomarkers
INTRODUCTION
The field of toxicology has traditionally focused on
assessing toxicity or investigating the mechanisms of
action of potentially hazardous agents. The evolution of
new, innovative technologies coupled with the advance
of genomic information is revolutionizing the future of
toxicological analyses. This review will describe the ap-
plication of microarray technology to facilitate discov-
ery of mechanistic pathways associated with toxicant
action as well as identification of novel targets for de-
velopment of susceptibility models.
DNA MICROARRAYS
Classically, toxicologists assess potential adverse
health outcomes resulting from chemical exposure by
using gross endpoints such as body and organ weight
changes and histopathological observations. However,
Correspondence to: Cynthia A. Afshari.
c 2001 John Wiley & Sons, Inc. ∗ This article is a US Government work
° generate the sequence order in the synthesis. The re-
and, as such, is in the public domain in the United States of America.
the analysis of histopathological or biochemical mark-
ers often does not provide information about a toxi-
cant’s mechanism of action. In addition, gross patho-
logical changes are insensitive for detecting toxicity at
low doses or early time points leading to incorrect as-
sessment of potential toxicity or safety. In order to better
evaluate the hazard associated with exposure to chem-
icals at varying doses and exposure, one needs to un-
derstand the specific mechanism of action of a toxicant.
Gene expression changes associated with signal
pathway activation can provide chemical-specific in-
formation on the mechanistic and biological effects
of a chemical. A standard method used to study the
changes in gene expression is the Northern blot. This
method, however, is relatively labor intensive and is
only practical for examining expression changes for a
limited number of genes. However, the advancements
in genome sequencing present researchers with the
opportunity to study genome-wide expression alter-
ations. New technologies, including DNA microarrays,
will facilitate studies that glean insight into the cellu-
lar response to chemical exposure. While traditional
assays, like Northern blots, measure the expression of
only one gene at a time, DNA microarrays measure the
expression of tens of thousands of genes in an equiva-
lent amount of time. In addition, array technology al-
lows newly discovered or unidentified genes to be in-
cluded in the analysis, providing a remarkable potential
to accelerate the discovery of toxicant targets.
DNA microarrays [1] provide a revolutionary plat-
form to perform genome-wide gene expression analy-
ses through comparison of virtually any two biological
samples. There are two basic types of microarrays used
in gene expression analyses: oligonucleotide-based ar-
rays and cDNA arrays. Both types yield compara-
ble results, though they differ in their methodology.
Oligonucleotide arrays are made using specific chemi-
cal synthesis steps by means of a series of photolitho-
graphic mask [2], light [3], or other methods [4] to
sult of these processes is the generation of high density
231
232 HAMADEH ET AL.
arrays of short oligonucleotides (∼20 bases) probes that
are synthesized in predefined positions [2]. Oligonu-
cleotide arrays may be used for expression studies as
well as sequence analysis.
cDNA microarrays can be used to study differences
in gene expression in different cells or tissues. Partial
cDNAs (0.5–2 kb in length) that correspond to unique
gene sequences are usually spotted onto the surface of
treated glass slides using high speed robotic printers [1]
that allow the user to configure the placement of cD-
NAs on a glass substrate or chip. Spotted cDNAs can
represent either sequenced genes of known function or
collections of partially sequenced cDNA derived from
expressed sequence tags (ESTs) corresponding to
messenger RNAs of genes of unknown function. It is
possible to print greater than 25,000 spots of cDNAs
on a single chip. To adhere to the surface of a glass
slide, the cDNA can be covalently linked or bound
to a coated surface by electrostatic interaction. After
the DNA is spotted on the chip, it is dried, UV cross-
linked, and blocked. While UV cross-linking attaches
the spotted DNA more firmly to the slide, blocking
prevents fluorescently labeled target cDNA from non-
specifically binding to the slide coating during hy-
bridization. Blocking is important for minimizing the
background around the spotted DNA in order to op-
timize the signal-to-noise ratio. A low noise-to-signal
ratio maximizes the sensitivity of detection. Optimal
microarray measurements can detect changes as small
as 1.2-fold increase or decrease in gene expression.
Any biological sample from which high quality
RNA can be isolated may be used for microarray anal-
ysis. For example, one may compare tissue types such
as normal versus diseased tissue, or untreated versus
treated tissue. Gene expression analysis using cDNA
microarrays is conducted by the simultaneous com-
parison of at least two biological samples. Differential
gene expression measurements are achieved by a com-
petitive, simultaneous hybridization using a two-color
fluorescence labeling approach. Isolated RNA is con-
verted to fluorescently labeled “targets” by a reverse
transcriptase reaction using a modified nucleotide, typ-
ically dUTP or dCTP conjugated with a chromophore.
The two RNAs being compared are labeled with differ-
ent fluorescent tags, traditionally either Cy3 or Cy5, so
that each RNA has a different energy emission wave-
length or color when excited by dual lasers. The fluo-
rescently labeled targets are mixed and hybridized on
a microarray chip. The array is scanned at two wave-
lengths using independent laser excitation of the two
fluors at 632 and 532 nm wavelengths for the red (Cy5)
and green (Cy3) labels, respectively. Thus, one RNA can
be regarded as labeled in “red” and the other in “green.”
The intensity of the signals relative to each other and
the background can be optimized by adjusting the gains
Volume 15, Number 5, 2001
of the photomultiplier tubes associated with each laser.
A simplified overview of the protocol for generating
gene expression data using microarrays is illustrated
in Figure 1.
Data acquired from scanning the microarray chips
are converted to 16-bit TIFF images corresponding to
each of the individual fluors. Specialized image pro-
cessing software overlays the two images and assigns a
color intensity to each spot representing the amount of
fluorescence. The result is a red–green pseudo-color ra-
tio image with colors for each cDNA signal ranging be-
tween green and red. For example, if a gene is expressed
in equal amounts in both the control and treated sam-
ples, the cDNA spot is yellow, indicating an equal ratio
of both fluors hybridizing with that gene. If a gene were
highly expressed in one of the samples, then the spot
would be predominantly red or green (Figure 2).
The sensitivity of microarray data is dynamic and
depends on the confidence level associated with the tar-
get definition and image analysis processes. Statistical
analysis at low confidence levels yields an increased
sensitivity for detecting subtle changes in gene expres-
sion levels but inherently results in the inclusion of false
positives as significantly regulated genes. Conversely,
high confidence levels decrease the sensitivity of detec-
tion and might omit genes with significant expression
changes (false negatives). A successful strategy to de-
termine significant gene expression responses utilizes
lower confidence levels in combination with biological
and hybridization replicates. Replicate data sets are an-
alyzed using a binomial distribution [5]. Table 1 demon-
strates the correlation between confidence levels and
false negative genes.
ANALYSIS TOOLS
FOR DNA MICROARRAYS
The amount of data derived from microarray anal-
ysis is relatively difficult to analyze without the utiliza-
tion of computational tools that can efficiently handle
large volumes of data. Thus the field of bioinformatics
has emerged. Bioinformatics in gene expression analy-
sis deals with tasks including spot location, definition,
and intensity calculations from raw scanned images,
transformation of data sets into more easily quantifi-
able forms, application of analysis tools to extract asso-
ciations between gene expression levels, and extraction
of information from analyses that facilitate the devel-
opment of new testable hypotheses.
Changes in gene expression are quantified from
raw images using image analysis algorithms and data
acquisition procedures to detect target regions, com-
pute feature pixel intensity values, subtract background
intensity, and compare the pixel intensity values of a
Volume 15, Number 5, 2001 GENE ARRAY TECHNOLOGY IN TOXICOLOGY 233

FIGURE 1. High-density gene expression analysis using cDNA microarrays. This illustration shows mRNA samples derived from in vivo
TCDD-treated rats or in vitro TCDD-treated liver cells used to prepare fluorescently labeled cDNA by reverse transcription in the presence of
fluorescently labeled (Cy3 or Cy5) nucleotide precursors. Competitive hybridization of the untreated and treated labeled RNA samples allows
for measuring the relative abundance of the transcripts from each gene as reflected by the ratio of “red” to “green” fluorescence from each spot
on the array. The abundance of mRNA copies corresponding to CYP1A1, 1A2, and 1B1 is greater in the TCDD-treated biological models than in
the unexposed counterparts. This is evidenced by the mostly “red” fluorescence measured at the spots representing those genes on the array.

cDNA between two samples. Normalization is regu-
larly performed during data analysis to adjust and cor-
rect the variability in the intensity values obtained from
the scanned image. This is achieved by using the inten-
sity values for either a standard set of control genes
or for the entire collection of the targets represented
on the DNA chip. A final ratio pixel intensity value is
calculated for each gene target and processed into a
standard 24-bit pseudo-color composite image for vi-
sualization of the gene expression profile.
Pixel intensity values, calculated ratio, and statis-
tics for each gene are stored as records in files for subse-
quent data management and analysis purposes. For in-
stance, the microarray database ArrayDB was designed
234 HAMADEH ET AL. Volume 15, Number 5, 2001

FIGURE 2. Color gradation of microarray spots resulting from competitive hybridization of two fluorescently tagged cDNA populations on a
DNA microarray. This colorimetric representation of the ratio of the “red” signal to the “green” signal indicates that if the transcript copies of a
gene were found exclusively in one of the samples of the comparison pair, the color of the respective spot would be at one end of the spectrum.
A gene whose transcripts are found in approximately equal amounts in both samples would be indicated as yellow.

as a relational database management system to store,
analyze, and associate gene expression data with infor-
mation from remote biological resources [6]. A more
comprehensive database has recently been developed
to correlate gene expression patterns with information
about pharmacological compounds [7].
Inherent with microarray gene expression data is
the nonlinearity nature of the data and the low qual-
ity or unreliability of data points that are generated
from below threshold detection levels, anomalies on
the microarray chip, or essentially missing cDNA fea-
tures. Typical microarray data analysis software in-
cludes functions to log transform gene expression data
into a more linear state and to essentially stabilize the
variance in the data. In addition, cut-off values may be
applied to the pixel size of cDNA features as well as the
intensity range of the values to refine the data set for
further analysis. The use of these processing features is
a form of “filtering” to microarray gene expression data
and improves the reliability of results.
At the onset of the microarray era, simple statistical
methods facilitated the interpretation of complex and
large-scale microarray gene expression results. For ex-
ample, a set number of + and − standard deviations
from the mean intensity values were typically used to
identify changes in biologically relevant genes in most
published microarray-related studies. However, more
sophisticated methods have been developed in which
computed confidence intervals are used to determine
significantly changed genes from a ratio distribution of
gene expression data [8]. More importantly, differen-
tially expressed genes can be validated by comparing
replicate measurements of microarrays and perform-
ing biological assays to confirm the significance of al-
tered expression [9,10]. Hence, for a given confidence
level, the probability of chance occurrences in replicate
experiments can be determined using a binomial dis-
tribution model [11].
At this time, these methods do not control for
false positives and false negatives, low fold changes,

TABLE 1. Number of Genes Altered in a Statistically Significant Manner for an Experiment Replicated Three Times
Times Gene Flagged # of Genes Expected to be
# of Outliers at 99% Confidencea by Chance Flagged by Chanceb Probabilityc

1650 0 1650 0.5

215 1 50 0.25
88 2 1 0.01
48 3 0 0.001
a
# of genes that were altered in a statistically significant manner in replicate experiments.
b
Probability of genes flagged by chance based on a 1650 gene chip.
c
Probability calculations using a binomial distribution (5).
Volume 15, Number 5, 2001
variation of data across independent replicate experi-
ments, nor do they account for genes exhibiting a bias
for incorporation of a particular fluor, as detected from
microarray fluor-reverse experiments. Fortunately,
microarray data has attracted significant attention
and gained concerted interest from practitioners
in the statistics, mathematics, and applied physics
communities as well as expert bioinformatics compa-
nies committed to applying rigorous and innovative
computational error models to measurements of gene
expression data. For instance, a pioneering approach
to analysis of microarray data is currently underway
using various mixed models and general analysis
of variance models to assess and increase statistical
significance in the microarray-derived gene expression
data [12]. Similarly, a combination of robust additive
and multiplicative error models are being used to
account for the uncertainty and variation in measuring
gene expression data as well as to assign confidence
and probability values, weight, and error bars to
define statistical significance of individual genes
[13].
The final stage in microarray gene expression anal-
ysis is multivariate analysis, in which higher-order
computational tools and procedures are utilized to as-
sist with the interpretation and visualization of com-
plex multivariate microarray gene expression data. Al-
though there is no definitive answer as to which form
of multivariate analysis to use on microarray data, it
is critical to understand the implicit characteristics of
each algorithm with the intention of prudently exploit-
ing inherent advantages of each to ultimately assemble
the best insight and interpretation of analyzed results.
For example, hierarchical clustering methods are es-
sentially an iterative process to organize objects into
groups with similar attributes based on resemblance,
proximity, similarity, or dissimilarity measurements.
The four basic steps in conducting a cluster analysis
on microarray data are 1) data collection and selection
of variables (genes and experiments) for analysis, 2)
generation of a resemblance matrix using a particular
mathematical formula to measure the extent of similar-
ity, 3) selection and execution of an iterative clustering
method to produce a phylogenetic tree diagram (den-
drogram) with the branches representing the amount
of similarity between clusters, and 4) determination of
where to “cut” the tree into a select number of nodes.
Recently, robust computational tools and an interactive
graphical interface have been developed to facilitate
the compilation and visualization of clustered gene ex-
pression data [14] (Figure 3). Although cluster analysis
is a classic and rather simple partitioning technique, its
application is relatively new to the field of microarray
data analysis and is not supported by any comprehen-
sive body of statistical literature.
GENE ARRAY TECHNOLOGY IN TOXICOLOGY 235
Self-organizing maps (SOMs) is another useful
algorithm used to interpret and visualize large high-
dimensional data sets in two-dimensional space. Basi-
cally, SOMs are an array of interconnected cells that be-
come refined to various input signal patterns or classes
of patterns in an orderly fashion. These artificial neu-
ral networks undergo a competitive and unsupervised
iterative learning process. When data is used to train
SOMs, structure is imposed on the data with neigh-
boring nodes defining related clusters. In essence, the
SOM constitutes a new paradigm in artificial intelli-
gence and cognitive learning and only recently has been
implemented into a computer program that can be uti-
lized as a genetic neural network model for interpreting
and displaying various patterns of gene expression data
[15].
Finally, discriminant analysis is commonly used in
multivariate analysis to determine which variables best
discriminate between two or more groups. The general
concept underlying the use of discriminant analysis to
analyze microarray data is to determine whether classes
of gene expression measurements significantly differ
with regard to distinct and characteristic gene expres-
sion profiles. Microarray data is used as a set of training
observations to assess and rank the importance of par-
ticular genes for discrete sample classification. When
identified, hallmark gene markers are used to predict
the classifications of a collection of test microarray treat-
ments as well as apply robust pattern recognition pro-
cedures to large volumes of complex gene expression
data. Computationally, discriminant analysis is very
similar to the analysis of variance; however, a novel
approach that combines a genetic algorithm and the
k-nearest neighbor method has been described to posi-
tively identify genes that discriminate between classes
of tumor cells [16] and toxicant treatments [17].
Data derived from microarray analysis may be ap-
plied to the identification of complex genetic diseases,
drug targets, toxicant and pathogen analysis, detection
of differences over time between normal and diseased
states of tissues, and mutation/polymorphism analy-
sis. The field of genomics that deals with generating
high-output gene expression data will have a great im-
pact on preventive medicine where diseases will be
sub-typed prior to the onset of physiological symp-
toms or progressed stages and drugs will be designed
to target causes of disease rather than symptoms. There
has already been some interesting studies published
in these areas. For example, gene expression analysis
of serum-exposed human fibroblasts highlighted the
physiological role of these cells in wound repair [18].
This type of analysis was also used to monitor signal
transduction during yeast pheromone response [19],
define regulatory pathways in stem cells as well as
their global genetic program [20], and validate drug
236 HAMADEH ET AL. Volume 15, Number 5, 2001

FIGURE 3. Two-dimensional hierarchical clustering analysis of 28 animals and 457 genes that were altered in a statistically significant manner at
95% confidence level in at least two replicate hybridizations at 2 time points [13]. Clustering analysis was performed across genes but not animals.
Genes are represented on the vertical axis while animals are plotted on the horizontal axis. Red or green indicates upregulation or suppression
respectively while black indicates no change in levels of mRNA as a result of exposure. Amplified node II shows an example of genes that were
induced by the peroxisome proliferators (PP) collectively but suppressed with phenobarbital treatment while node I is an example of genes that
were upregulated by phenobarbital (PB) treatment at the 24 h and 2 week time points while levels mostly decreased or remained unchanged
with the pp exposures.

target and identify secondary drug target effects [21].
The technology has been used to discover potential hu-
man biomarkers of ionizing radiation by ex vivo irra-
diation of human peripheral blood lymphocytes [22].
This study suggests that monitoring the gene expres-
sion of these biomarkers may provide estimates of
environmental radiation exposures. The collective re-
sults of these studies provide tangible evidence that
this technology can be utilized to confront a variety of
important medical problems that have been intractable
with past technology.
cDNA MICROARRAYS AND DISCOVERY
IN TOXICOLOGY
Although the field of toxicology has yet to benefit
from this revolutionary technology, other disciplines,
including cancer biology, cell biology, physiology, and
pharmacology, have made great advances using DNA
microarrays [13,23–31].
Many reports describe the theoretical application of
high-density gene expression analysis to challenges in
toxicology [32–39]. However, researchers are just start-
ing to test whether the application of microarrays can
help answer key toxicological questions. The under-
lying basis of chemical effects stems from the com-
plex interaction between the environment and different
genes. By understanding the molecular details of this
interaction, we can identify toxicity-associated genes
and cellular response mechanisms. This would allow
for the characterization of these “response” genes and
the elucidation of the mechanisms by which they are
regulated.
Microarray technology promises to speed the an-
notation of uncharacterized or poorly characterized
genes. The belief is based on the premise that shared
expression often implies shared function. Thus, when
a gene with an unknown function is included in a
cluster of known genes sharing similar gene expres-
sion patterns, it is implied that the function of the un-
known gene bears similarity to its neighbors in that
cluster. This hypothesis was first tested by Brown and
colleagues in 1998 in a study of global gene expres-
sion in Saccharomyces cerevisiae, that demonstrated co-
regulation of genes that participated in shared cellular
Volume 15, Number 5, 2001
function [14]. Theoretically, clustering will aid in de-
termining the function of a substantial number of un-
known genes, even when sequence similarity has not
been established with characterized genes [40–42].
A demonstration of the assignment of putative
function to a number of unknown genes is derived
by studying gene expression patterns generated
in the livers of rats that were treated in vivo with
phenobarbital or one of three peroxisome proliferators
(clofibrate, Wyeth 14,643, gemfibrozil) [17]. Male
Sprague-Dawley rats were exposed to one of the
compounds, then livers were harvested 24 h post
single-dose or 2 weeks post daily-dosage. In this study,
clustering illustrated upregulation of genes in response
to phenobarbital treatment but not with peroxisome
proliferator exposures (Figure 3). Not only did these
genes have a high correlation (R > + 0.8) in their expres-
sion levels, but were also related biologically. Genes in
node II, which were upregulated by peroxisome prolif-
erators but suppressed by phenobarbital, have reported
involvement in mitochondrial b-oxidation. This corrob-
orates previously published findings demonstrating
the induction of b-oxidation by peroxisome prolifera-
tors [43]. Node I contains genes that are mostly involved
in the detoxification and metabolism of phenobarbital.
Consequently these genes were upregulated with
phenobarbital treatment, but not with peroxisome
proliferators. Interestingly, an EST is included in node I,
indicating similarity in the expression pattern between
this unknown gene and the rest of the genes in this node.
The classification of this EST in node I raises the ques-
tion of whether we can infer that this gene is involved
in the detoxification and metabolism of phenobarbital.
This study illustrates the feasibility of discovering the
function of unknown genes, that may lead to a better
understanding of mechanisms of action by using cDNA
microarrays containing significant numbers of ESTs to
study toxicant-exposed biological samples in vivo.
The study of mechanisms of toxicant action is vi-
tal to understanding their adverse effects on human
health. Mechanistic information is crucial to making
more informed regulatory decisions regarding expo-
sure levels. Because of the ability to monitor expression
of thousands of genes on a cDNA chip, researchers can
now study the effects of a chemical insult on multi-
ple molecular pathways. This investigative approach
allows for the accumulation of mechanistic data that
will provide an insight into vital cellular pathways that
are affected by the exposure to a chemical agent.
Given the large number of genes on a chip, high-
density gene expression data theoretically should sum-
marize the findings from previous reports as well as
identify unreported alterations of genes. Figure 4 de-
picts a portion of a toxicant effector pathway map where
gene expression data from phenobarbital-treated livers
GENE ARRAY TECHNOLOGY IN TOXICOLOGY 237
were mapped into functional pathways present in the
mitochondria. This demonstrates the potential of dis-
covering novel alterations in the expression levels of
genes associated with toxicant-specific molecular path-
ways [17]. Other studies that demonstrated the ability
of microarrays to discover new genes associated with
toxicant responses have been reviewed [44].
MICROARRAYS AND THE
EXPLORATION OF POLYMORPHISMS
DNA sequence polymorphisms are discovered
by comparing the base sequence of genes across a
population and observing interindividual variation.
In addition to traditional sequencing methodologies,
oligonucleotide-based arrays are also useful in geno-
typing. Once a target gene has been identified, oligo
arrays can be used for conducting mutation and poly-
morphism screening [2,45]. However, limitations of
oligonucleotide-based microarrays include the inabil-
ity to detect length polymorphisms or large deletions
and insertions [46,47].
DNA base (deletion or substitution) changes that
occur at a population frequency greater than 1% are
common variations that affect host susceptibility to
a variety of factors [48]. Single nucleotide polymor-
phisms (SNPs), which are a form of genetic variation,
occur approximately once every 1000 bases [49]. Al-
though interindividual similarity is more than 99.9% in
the human genetic makeup, it is subtle variation such
as polymorphisms that are responsible for differences
in resistance or sensitivity to certain drugs or toxicants
[50–54]. Polymorphisms in enzymes have been shown
to affect their inducibility, expression level, and activity
[55]. DNA microarrays offer a novel method of study-
ing variations in the response to toxicants or drugs by
monitoring genome-wide differences in gene expres-
sion of key metabolizing or detoxification enzymes in
a target cell type.
Under the premise that some polymorphisms lead
to wide fluctuations in gene expression across indi-
viduals, one could screen individuals and potentially
identify new polymorphisms. Changes in gene ex-
pression between resistant versus susceptible individ-
uals may indicate alterations in a signaling/metabolic
pathway, allowing further definition of the exact gene
that contains the critical polymorphism. The inclu-
sion of ESTs on cDNA chips generates the potential
to discover new members of gene families involved
in interindividual susceptibility to human disease. Un-
fortunately, polymorphisms do not always affect the
levels of mRNA expression, thus rendering them un-
detectable if cDNA arrays were the only tool used for
this purpose. Other challenges to using microarrays for
238 HAMADEH ET AL. Volume 15, Number 5, 2001

FIGURE 4. Map of Effector Pathways of Toxicant (MEPT) for the action of phenobarbital in the mitochondria. mRNA levels corresponding to
all enzymes on the diagram were found to be statistically altered by phenobarbital treatment of Sprague-Dawley rats when gene expression
data was analyzed at a 95% confidence level using Array Suite software. The diagram shows data from two time points: 24 h post single-dose
and two week post daily-dosage. Enzymes enclosed in blue boxes were altered in this study and were previously reported in the literature
to be modulated by phenobarbital treatment while those in green were not previously associated with phenobarbital exposure and are novel
observations. (o/o) denotes modulation of mRNA level at the two time points where the spots before and after the slash correspond to the 24 h
and 2 week time points respectively. Up- or downregulation is denoted by a red or green spot respectively, and a gray spot denotes no change.

FIGURE 5. Clustering output highlighting interanimal differences in gene alteration in response to chemical exposure. Validated gene expression
data from livers of phenobarbital treated animals (24 h post single-dose or 2 week post daily-dosage) were clustered and the output displayed
as a dendrogram. Three rats were represented from each time point. Nodes on the hierarchical tree, highlighting differences in gene expression
patterns between animals receiving similar exposures were magnified. Deviant animals are marked as indicated ∗.
Volume 15, Number 5, 2001
this purpose include tissue specificity of gene expres-
sion, limited availability of human target tissue, and
definition of reference samples.
In addition to comparison of normal versus dis-
eased, or sensitive versus resistant organisms or indi-
viduals, the identification of candidate genes that might
play a role in susceptibility to toxic agents can be con-
ducted using microarray analysis of samples from hu-
man derived cell lines. Hypotheses generated from in
vitro models can then be tested in the in vivo envi-
ronment. For example, interindividual variation in sus-
ceptibility to carcinogenic effects of arsenic has been
reported [56–59]. Since the mechanism of arsenic toxic-
ity is not fully understood, inclusion of ESTs on cDNA
microarrays may aid in the discovery of novel genes
that play a central role in sensitivity. Fresh or immortal-
ized cell populations (ex. keratinocytes) derived from
healthy human donors could be screened by biological
assays such as viability in order to measure the rela-
tive resistance or susceptibility of different cell pop-
ulations. This would identify cells sensitive and re-
sistant to the toxic effects of arsenic. Comparison of
the gene expression profiles of arsenic-resistant cells to
sensitive cells should indicate mechanistic differences
between the two responses to arsenic exposure. Addi-
tionally this analysis would potentially identify candi-
date genes that play a role in the increased sensitivity or
resistance to arsenic toxicity [60]. The hypotheses gen-
erated by these studies could be further investigated by
construction of cell or animal models that either over-
express or underexpress for those genes.
Although the field of toxicology is just beginning to
embrace the potential of DNA microarrays, investiga-
tors in other scientific disciplines have benefited from
the recent advancements in the field of pharmacoge-
nomics [61–63]. Pharmacogenomics is a recent term re-
ferring to the identification and elucidation of genetic
variations that could serve as targets for therapeutic
modalities or impact the efficacy of drugs [64–66]. The
field of pharmacogenomics has realized the benefits of
DNA microarrays and researchers in the field have al-
ready used this technology to improve their drug dis-
covery efforts. The future use of microarrays in the field
of toxicology will allow for a versatile approach to in-
vestigate interindividual susceptibility to toxicants due
to genetic factors.
CHALLENGES
Although the theoretical applications seem endless,
cDNA microarray technology has certain limitations.
One limitation is the number of samples that can be pro-
cessed efficiently at a time. Automation is being applied
to microarray technology and new equipment such as
GENE ARRAY TECHNOLOGY IN TOXICOLOGY 239
the automated hybridization stations and autoloaded
scanners are starting to emerge. Because techniques in-
volved in microarray studies are not yet standardized,
researchers face technical difficulties at different stages
when performing the assay. The success of hybridiza-
tion is highly correlated with superior RNA quality,
good signal-to-noise ratio, low cross-hybridization, and
exclusion of fluor incorporation bias during labeling re-
actions. Microarray technology monitors fluctuations
in mRNA levels, but it does not verify if the message
was translated into functional protein. Proteomics, a
field that deals with global protein expression and func-
tion, will complement and extend data obtained us-
ing genomic approaches [67–69]. Studies using paral-
lel analysis of samples with both high density gene
and protein expression tools will lead to verification
of the relevance and importance of gene expression
data. Challenges facing proteomics efforts include over-
coming labor intensive steps (i.e. protein fractiona-
tion, purification, separation) and the inability to match
the high output of data generated by genomics at the
present time.
Identification of environmental carcinogens and
other hazards constitutes a major challenge in the field
of toxicology. The risk assessment process associated
with these chemicals is limited because of the use of
a variety of assumptions, particularly relating to cross-
species extrapolation, dose response, and estimated ex-
posure levels of humans [70–73]. Thus, the need has
arisen for elucidation of transspecies, chemical-specific
biomarkers resulting from quantified exposures to
drugs or toxic chemicals. This will permit the extrapola-
tion of effects observed in surrogate systems to potential
effects in humans. At this time, there is a limited inven-
tory of gene expression patterns that reflect responses
to chemical effects in biological models. Other impor-
tant issues such as variations in gene expression due
to the animals’ status, diet, circadian rhythm, or meth-
ods of tissue collection are not well understood. Cells
in culture also undergo remarkable changes in gene ex-
pression solely due to culture conditions. Such informa-
tion would be equally beneficial to the toxicology and
pharmaceutical communities. Gene expression profiles
for compounds where toxicological, pharmacological,
and pathological information is abundant should be
generated in several biological models of interest. Dif-
ferent doses, time points, and organs should be in-
vestigated to generate a comprehensive gene expres-
sion response to the chemical. A database that is popu-
lated with this information could then be interrogated
for forming associations between gene expression and
development of various efficacious or toxicity-related
phenotypes.
An effort to distinguish classes of toxicants, based
solely upon cluster-type analysis of differentially
240 HAMADEH ET AL.
expressed genes in exposed HepG2 cells, was recently
reported [74] using an approximately 250-gene cDNA
microarray. Initial comparison of gene expression pro-
files for cytotoxic antiinflammatory drugs and DNA-
damaging agents to a database populated with gene
expression profiles for 100 toxicants failed to distin-
guish the two classes of toxicants. The main obstacle
encountered was the lack of replicates of the gene ex-
pression profiles populating their database leading to
low reproducibility in gene responses. This challenge
was partially overcome when a gene expression profile
was generated from 13 independent cisplatin-exposed
HepG2 samples, leading to a suite of genes that were
altered by toxicant exposure with relatively high repro-
ducibility [74]. This highlights the need for the gener-
ation of replicate gene expression profiles for a given
sample.
When designing toxicogenomic studies, the num-
ber of animals required for each time point or dose is
an important consideration. Because of economic lim-
itations when dealing with microarray studies, inves-
tigators strive to use the least number of animals that
produce statistically meaningful results. There are sev-
eral considerations that are of importance when choos-
ing the number of subjects per dose or time point for
a given study. A major consideration is the amount of
interanimal variation in the model system employed.
Figure 5 shows results of a hierarchical clustering tree
highlighting the differences between animals receiving
the same chemical treatments. Discrepancies in expres-
sion of the suites of genes shown were not unique to
one of the animals, rather different animals deviated
from the group of animals in the expression pattern of
different sets of genes (Figure 5). It is accepted that four
to six animals per dosage group would be adequate to
derive statistically and biologically significant data that
overcomes interanimals variability.
CONCLUSIONS
DNA microarrays have facilitated discovery in
many scientific disciplines because of the versatility of
this novel technology. We highlight the potential use
of this technology in discovering new aspects of mech-
anisms of action of toxicants, the discovery of toxico-
logical functions of unknown genes, and in investigat-
ing polymorphisms. The use of cDNA microarrays for
approaching polymorphisms in a novel fashion will
accelerate the identification of polymorphisms that con-
fer sensitivity to toxicants. Not only will these efforts
help advance the field of risk assessment by better
identifying susceptible populations, but will widen our
knowledge of the mechanisms of action for compounds
that we are exposed to on a continuous basis.
Volume 15, Number 5, 2001
ACKNOWLEDGMENTS
We gratefully acknowledge the helpful comments
and critical review of the manuscript by Drs. Doug
Bell, Mike Cunningham, Jack Taylor, Rupesh Amin,
Ray Tennant, and Edward Lobenhofer. We also thank
our collaborators at Boehringer-Ingelheim Pharmaceu-
ticals, Inc., Drs. Ray Stoll, Kerry Blanchard, and Supriya
Jayadev for their contribution to the studies described
in this review.
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