Development of Gene Expression Fingerprints for Identification of Environmental Contaminants Using cDNA Arrays

ERDC TN-DOER-R4

September 2004

Development of Gene Expression

Fingerprints for Identification of

Environmental Contaminants Using

cDNA Arrays

PURPOSE: This technical note reports the current status of work being done at the U.S. Army

Engineer Research and Development Center (ERDC) to develop cDNA array-based assays that

map gene expression from contaminant exposures. Results substantiate that distinct gene

expression profiles exist for major contaminant classes such as PAHs, PCBs, and PCDD/Fs.

Results also indicate that identification of these contaminant mixtures in environmental media is

possible by examining their effects on gene expression in mammalian cells.

BACKGROUND: Research at ERDC directed at developing screening assays for contaminated

sediments has been applied to extracts of both sediments and the organisms living in them.

Several microbial and cell-based in vitro methods are now routinely used for this purpose. These

were described, and the possibilities of using cDNA arrays in a screening assay were explored, in

a preceding Technical Note (TN-DOER-C19, Inouye and McFarland (2001)). Briefly, cDNA

arrays allow quantification of gene expression profiles (genes that are "turned on or off" at a

selected time) providing a "fingerprint" of the sub-cellular responses of the test subject, e.g.,

cultured human liver cells, to a chemical challenge. While current in vitro tests such as the 101L

cell-based assay for dioxin equivalents (TN-DOER-C10, Ang et al. (2000)) offer inexpensive

and rapid screening of one endpoint, cDNA arrays offer the potential for simultaneous screening

of multiple endpoints and mixtures of contaminants. The activation of multiple genes related to

disruption of normal cell functions, such as apoptosis, tumor suppression, cell proliferation, cell

cycles, cytokines, oxidative stress, and more, can be measured in a single exposure using cDNA

arrays. The resulting information can provide valuable insights into the toxic modes of action

(MOAs) of mixtures of contaminants present in sediments. This is not possible with chemical

analysis or any other current methods. Mode-of-action information can be used in several ways:

the need for, and the type of, more time-consuming and expensive chronic sublethal bioassays

(e.g., survival, growth, reproduction, genotoxicity) as well as costly and expensive chemical

analysis can be identified -- or their necessity can be eliminated. A priori interpretations of the

presence of known chemicals in sediment/soil samples are more defendable if accompanied by

mechanistic information, i.e., MOA, and the decisions that result are more certain.

In this investigation a cultured cell line (human hepatoma, HepG2) was exposed to known

compounds/mixtures in order to develop genetic-response fingerprints for common contaminant

classes; future work will involve testing extracts of environmental samples (sediments, tissues)

to relate the responses seen with model chemicals to those of complex mixtures present in field

samples. Gene responses were monitored with Clontech's commercially available AtlasTM

Human Toxicology 1.2 cDNA array, which includes 1176 genes known to be involved in

toxicological responses, e.g., genes linked to DNA synthesis/repair, stress proteins, and tumor

suppression or induction. Results substantiate that distinct gene expression profiles exist for