EHP Toxicogenomics 112-4, 2004: In This Issue
Environews
Research Roundup
This month's Forum (p. A216) describes advances in gene silencing, in
which a team from The Wistar Institute may have discovered a long-awaited histone
demethylation protein. In other news, the U.S. Food and Drug Administration may
soon accept microarray data in drug applications, researchers find functional
nonexonic sequences in 13 vertebrate species, and Kullback-Leibler clustering
takes on a new role in deciphering microarray data.
Microarrays Express Themselves
The NCT Update (p. A222) looks at the Microarray Group, where scientists
analyze changing patterns of gene expression across the entire genome, studying
thousands of affected genes at a time. Microarrays allow researchers to screen
for large numbers of gene expression responses to toxic substances, determine
if toxic effects occur at low-dose exposures, highlight vulnerable tissue or
cell types, and begin to extrapolate effects from one species to another.
Enter RNAi
The discovery of RNA interference (RNAi) has revolutionized genetic research,
and is on the verge of spawning an entirely new class of drugs to treat human
diseases with a genetic component. The Focus (p. A224) investigates
both the vast potential and the daunting obstacles as scientists seek to leverage
RNAi into one of the first tangible and widespread benefits to be derived from
the sequencing of the human genome.
Toxicogenomics
Gene-Gene Interactions Regulated by Ahr Ligands![](gene.jpg)
One of the most important uses of new toxicogenomics technologies is in resolving
gene-gene, and gene-network interactions. Johnson et al. (p. 403) have
used co-expression of genes coupled to additive probabilistic relationships
to identify gene sets predictive of the complex biological interactions regulated
by the aryl hydrocarbon receptor (Ahr). Biologically relevant gene predictor
sets were calculated for Ahr, cytochrome P450 1B1, insulin-like growth
factor-binding protein-5, lysyl oxidase, and osteopontin. Results demonstrate
how predictor gene combinations can be effectively used to resolve gene-gene
interactions regulated by Ahr ligands. (Also see Science Selections,
p. A230)
New Biotechnologies and Cancer and Occupational Cancer
As high-throughput technologies in genomics, transcriptonomics, and proteomics
evolve, it becomes important to evaluate their utility for assessing occupational
cancers. Toraason et al. (p. 413) summarize the efforts of a workshop
designed to identify the best means for exploiting new technologies to enhance
methods for laboratory investigation, epidemiological evaluation, risk assessment,
and prevention of occupational cancer. The workshop focused on identifying
and interpreting markers for early biological effect and inherited modifiers
of risk.
An ILSI HESI Genomics Committee Overview![](ilsi.jpg)
The value of genomic approaches in hypothesis generation is being realized as
a tool for understanding toxicity and consequently, contributing to an assessment
of drug and chemical safety. The membership of the International Life Sciences
Institute (ILSI) Health and Environmental Sciences Institute (HESI) formed
a committee to develop a collaborative scientific program to address issues,
challenges and opportunities afforded by the emerging field of toxicogenomics. Pennie
et al. (p. 417) provide an overview of the efforts of the committee
to design, conduct and analyze numerous toxicogenomics experiments within the
broad fields of hepatotoxicity, nephrotoxicity and genotoxicity. (Also see
Science Selections, p. A231)
Characterizing Genotoxicity using DNA Microarrays
The opportunity afforded by micrarrays for analysis of thousands of genes may
provide mechanistic insights into the response of cells to DNA damage. Newton
et al. (p. 420) give an overview of ILSI HESI Genomics Committee studies
to evaluate gene expression changes caused by prototypical direct and indirect
acting genotoxins. The data indicate that the value of the technology is in
its potential to provide mechanistic insight for differentiating compounds
that interact directly with DNA from those compounds that are genotoxic via
a secondary mechanism.
Overview of Hepatic Gene Expression Collaboration
The Hepatotoxicity Working Group (ILSI-HESI) evaluated biological and gene
expression responses in rats exposed to two model hepatotoxins, clofibrate
and methapyrilene. Ulrich et al. (p. 423) report on the effort to evaluate
and compare multiple biological, genomic and toxicological parameters across
different laboratories and microarray platforms. Despite discrepancies there
was overall agreement in the affected biological pathways, demonstrating that
transcriptional profiling is reproducible between laboratories.
A Cross-laboratory cDNA Array Study of Clofibrate Hepatotoxicity
Baker et al. (p. 428) compared liver cDNA microarray data from multiple
clofibrate doses and treatment times in two laboratories. In general, there
was a high level of concordance between the gene expression profiles generated
from pooled and individual RNA samples. Though the results indicate some variability
in the quantitative nature of the microarray data, these appear largely due
to differences in experimental and data analysis procedures used within each
laboratory.
Toxicogenomics Analysis Across Multiple Laboratories
Although changes in gene expression provide information about mechanisms
of toxicity, the issue of whether gene expression profiles are reproducible
across different laboratories is uncertain. Waring et al. (p. 439) report
results of gene expression profile comparisons across 5 different laboratories
using a single sample/RNA preparation sample set. Preliminary evaluation uncovered
striking differences between sites. Additional data analysis demonstrated that
the differences affected absolute gene expression, but not the study outcome.
Results show that, despite some variability, robust gene expression changes
were consistent between sites.
Cross-Site Similarity of Gene Expression Data
Reliable comparison of gene expression data across multiple laboratories
depends on several factors such as platform (oligo, cDNA, etc.), condition
of lab and data quality. Chu et al. (p. 449) present methods for quantitatively
assessing differences in data with a principal goal being the generation of
lists of site-insensitive genes responsive to low and high doses of Methapyrilene.
A combination of numerical and graphical techniques reveals important patterns
and partitions of variability in the data, including the magnitude of the site
effects. Although the site effects are significantly large they appear to be
primarily additive and can be adjusted in a way that does not bias conclusions
about treatment differences.
Quantitative PCR Deconstruction of Microarray Discrepancies
Differences in hybridization platforms used in gene array analyses can lead
to significant differences in hybridization results. Goodsaid et al. (p.
456) applied quantitative RT-PCR to investigate discrepancies between cDNA
and oligo platforms used to evaluate hepatic gene expression changes in methapyrilene-exposed
rats. Results demonstrate the importance of using a "gold standard", such as
quantitative RT-PCR to confirm key hybridization results and understand the
sources of discrepancies.
Molecular Markers of Nephrotoxicity
Kramer et al. (p. 460) analyzed the experimental information derived
from analysis of the biological response to three mechanistically distinct
nephrotoxins: cisplatin, gentamicin, and puromycin aminonucleoside. The analyses
detailed in the manuscripts from the HESI Nephrotoxicity Working Group suggest
at least equal sensitivity of microarray technology compared to traditional
endpoints. Additionally, microarray analysis of these prototypical nephrotoxicants
provided an opportunity for the development of candidate bridging biomarkers
of nephrotoxicity.
Gene Based Markers of Renal Toxicity
Amin et al. (p. 465) examined the changes in the expression profile
of genes associated with the nephrotoxicants, cisplatin, gentamicin, and puromycin.
By combining classical toxicology endpoints with toxicogenomics, several gene-based
markers of renal toxicity were identified. Gene expression changes were indicative
of damage to specific portions of the nephron.
Dye Bias Correction in Dual-labeled cDNA Microarrays
A significant limitation to the analytical accuracy and precision of dual-labeled
spotted cDNA microarrays is the signal error due to dye bias. Rosenzweig
et al. (p. 480) report that split control microarrays within a set of concurrently
processed hybridizations to specifically measure dye bias can eliminate the
need for technical dye swap replicates, and reduce costs, while maintaining
experimental accuracy and technical precision.
Platform-Independent Markers of Cisplatin Nephrotoxicity
Thompson et al. (p. 488) compared gene expression results using RNA
prepared from cisplatin treated rats across a variety of microarray formats
at multiple sites. Results suggest that gene profiles that are linked to specific
types of tissue injury or mechanisms of toxicity, and identified in well-performed,
replicated microarray experiments may be extrapolated across platform technologies,
laboratories, and in-life studies.
Toxicogenomic Database Review
The challenges of combining toxicology and genomics are best addressed using
data collected into well-designed toxicogenomics databases. Mattes et al.
(p. 495) review issues common to such databases such as: minimal descriptors,
standardized data storage and exchange, nomenclature and ontologies, data upload,
and the necessity of data quality.
Cross-Indexing of Microarray Platforms
One problem with transcript profiling using microarrays is that different
short nucleic acid sequences mapped to the same genetic locus may be given
different names and descriptions when they are deposited in public sequence
databases. Mattes (p. 506) describes an algorithm that uses a hierarchical
approach to assign a single "best annotation" to elements in a given microarray,
in such a fashion that elements from one microarray platform may be cross-indexed
with those of another.
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Last Updated: March 8, 2004