METHODS AND MATERIALS

ERDC TN-DOER-C30

February 2003

from the fruitfly (Drosophila melanogaster) but modified to function in mammalian cells that do

not ordinarily respond to ecdysone or other ecdysteroids. Two plasmids, pVgRXR and pIND, form

the basis of the system. The pVgRXR plasmid expresses the ecdysteroid receptor and pIND contains

the receptor-ligand response element E/GRE. When ecdysone or another ligand having ecdysteroid

activity is present, binding of the receptor-ligand complex to the response element results in

transcription of the protein of interest (Figure 1). The bacterial -galactosidase gene is contained

downstream from the response element and serves as a reporter. The enzyme -galactosidase is

expressed and cleaves a substrate in response to binding of the receptor-ligand complex with the

response element. The yellow product that results from the cleavage can be monitored colorimet-

rically. This mechanism provides the principle on which an assay for contaminants affecting

molting at the level of receptor binding can be based. In the assay, Ponasterone A, a high-potency

analog of the molting hormone, ecdysone, is added to the cells prepared on a microtiter plate. Used

alone, Ponasterone A will produce an ecdysteroid response in proportion to its molar concentration.

If xenobiotic chemicals are present that also bind the ecdysone receptor, the production of

-galactosidase and the resultant amount of colored product will be affected; i.e., either increased

or decreased. An increase signals the presence of a xenobiotic agonist of the receptor, and a decrease

signals the presence of an antagonist. Both types of effect indicate the potential for disruption of

the molting process due to ecdysone receptor-active foreign chemicals in the assay.

Several problems were encountered with using Invitrogen's human embryonic kidney EcR-293 cell

line during preliminary assays. The cells are semi-adherent, resulting in high variability between

replicates as cell numbers varied due to losses in transfection and rinsing steps. Additionally, the

transient transfection with the second plasmid was time and reagent consuming, and required the

use of a marker plasmid to correct for transfection efficiency. It was decided to address these

problems by developing a stably transfected cell line in which the Invitrogen ecdysone receptor

plasmids are inserted into the genome of the adherent HepG2 cell line (human liver hepatoma origin)

also used in the P450RGS assay. This transfection was successful and resulted in permanent cultures

of the new cell line, HepG2-EcR.

Studies are now ongoing to characterize the responses of HepG2-EcR to a series of model

ecdysteroids, environmental chemicals having the potential for interaction with ecdysteroid recep-

tors, and extracts of contaminated sediments and surface water. Since A1242 has been previously

shown to inhibit molting, it was selected as a model contaminant to compare the responses of the

new cell line with molting inhibition in an experimental organism, the crawfish (Procambarus

clarkii). The laboratory protocol for performance of the assay is included as Appendix I. HepG2-EcR

culture samples are available by request for research purposes only.

METHODS AND MATERIALS

Generation of the New Cell Line. Optimized electroporation methods were used to transfect

HepG2 cells with the pVgRXR plasmid. Cells were allowed to recover for 3 days before treatment

with Zeocin to select for cells containing the plasmid. Colonies were then generated from single

cells and screened for activity (the second plasmid, pIND, was transiently inserted using the cationic

transfection reagent, LipoFectamine, then tested for -galactosidase activity after exposure to the

EcR agonist, Ponasterone A). Cells from the colony with the highest activity were further

transfected with the second plasmid (pIND) and allowed to recover for 3 days before treatment with