Current Status

ERDC TN-DOER-C19

December 2000

the gene products can be detected, it is possible to design the assays such that multiple endpoints can

be obtained on the same cell sample. Fluorescent endpoints can be run without addition of reagents,

and luminescent and colorimetric assays will not interfere with each other if the luminescent assay is

conducted first. Thus, it is possible to construct a cell line that can monitor for several classes of con-

taminants simultaneously.

Current Status. Current research is focused on developing an assay for the detection of com-

pounds that interrupt the hormonal cycle of arthropods (insects and crustaceans). Interference with

the interaction between ecdysone (a molting hormone) and its receptor can lead to the inability to

grow and reproduce properly, potentially leading to a decline in the population. Since these animals

are important components at the base of both the terrestrial and aquatic food chains, a significant de-

cline in population can affect the entire ecosystem. If compounds that adversely affect this hormonal

system are present in dredged sediments, they present a potential problem for both dredging and dis-

posal. At this time, no valid assays or tests are available to assess the effects of endocrine disrupters.

The current project will lead to the development of a screening assay for ecdysone-disrupting com-

pounds in sediment extracts. A reporter gene system has been obtained (Invitrogen's

ecdysone-inducible mammalian expression system), and the genes are being inserted into the hepG2

cell line (the same cell line used in the generation of the P450RGS cell line). At the time this techni-

cal note is being written, the first of two plasmids required for this system has been inserted into the

cells via electroporation. The gene insertion has been confirmed by both polymerase chain reaction

(PCR) of the ecdysone receptor gene and by the zeocin resistance conferred by the plasmid. The sec-

ond plasmid will be inserted once the transformed cells recover from the electroporation and pass

through several generations (to confirm stability of the initial gene). Once the second plasmid is in-

serted, the response of the cell-based assay will be tested with several compounds known to interact

with the system, as well as several environmental contaminants suspected of interfering with the

system. In a collaborative effort with other projects at the U.S. Army Engineer Research and Devel-

opment Center, the assay will also be validated against a whole organism assay (crawfish molting)

and a computer modeling program that is designed to determine the potential receptor-ligand inter-

action of a chemical.

DNA ARRAYS: An alternative approach for monitoring multiple toxicity endpoints simulta-

neously is the use of DNA arrays. The DNA array technology takes advantage of the recent explo-

sion in biotechnology. Both normal cell function and ability to adapt to changing conditions at a

cellular level by increasing or decreasing the production of the various proteins critical to cell sur-

vival are controlled at the DNA level. Basically, signals are sent to the DNA to increase or decrease

active transcription of genes into various mRNA, which then direct the cell to produce proteins. The-

oretically, if one could inventory the mRNA in a cell, a snapshot of all the proteins currently being

synthesized within the cell would be obtained. Briefly, a cDNA array consists of complementary

DNA (cDNA) of known genes bound to a substrate (the array). These can then be utilized to deter-

mine the type and concentrations of the expressed mRNA by essentially extracting the mRNA from

the cells and binding them to the array. Knowing what is being synthesized at a given moment would

enable the interpretion of how cells are responding to toxicant exposure. Conversely, if how cells

respond to different classes of toxicants can be characterized and quantified, then given an unknown

mixture such as a sediment extract, the toxicants present in the sample may be able to be identified

and quantified.