CONSTRUCTION OF THE HepG2-EcR CELL LINE

ERDC TN-DOER-C30

February 2003

These hormones function similarly to other steroidal endocrine hormones such as the estrogens or

corticoids in higher organisms. A cascade of events is initiated by binding of the hormones with

receptors within the cytoplasm of cells. When bound, the receptor-hormone complexes are translo-

cated across the nuclear membrane. Activation of DNA by these complexes results in the synthesis

of proteins that regulate molting. Disruption of the molting process by chemicals has been observed.

For example, the PCB mixture Aroclor 1242 (A1242), a common environmental contaminant, has

been shown to drastically inhibit the molting of fiddler crabs (Uca pugilator) (Fingerman and

Fingerman 1977). In the same study octachlorodibenzofuran-exposed crabs molted at about the

same rate as did the controls. Both A1242 and octachlorodibenzofuran bind the AhR and are

active in the P450RGS assay. Aroclor 1242 is a mixture of about 100 PCB congeners, seven

of which are coplanar and bind the AhR. It is these seven that apparently account for all of the

A1242 activity in the P450RGS assay. Considering the lack of molt inhibition observed for

octachlorodibenzofuran which is also coplanar and binds the AhR it appears that the molt-

inhibiting potency of A1242 must reside with the non-coplanar PCB congeners.

In a recent investigation, exposure to a single PCB congener, 2,4,6-trichlorobiphenyl (PCB30),

disrupted molting in crawfish (Jones et al. 2000). This particular congener was chosen because it

was demonstrated to have potent estrogenic effects in juvenile fish (Westerlund et al. 2000), and

there are structural and functional similarities between the estrogen receptor in fish and the

ecdysteroid receptor in arthropods. It was hypothesized that the probable toxic mode of action of

molt disruption by such chemicals involves mimicking ecdysteroids or blocking their normal

receptor binding. PCB30 is not coplanar and does not bind the AhR. Consequently, PCB30 shows

no activity in the P450RGS assay. The implication of this is that environmental PCBs likely have

multiple modes of toxicity, and represent different kinds of hazard to different taxa. For complete-

ness in ecological risk assessments, tests are required with endpoints specific for those different

taxa and hazards.

Sediments contaminated with complex mixtures high in PAHs and metals have been shown to have

severe effects on molting and survival of crustaceans. Peddicord and McFarland (1976) exposed

3- to 4-cm juvenile crabs (Cancer magister) to high concentrations of suspended Oakland Inner

Harbor sediments over a period of 25 days. High mortality resulted, and 92 percent of the deaths

occurred during the molting process. The molt period was abnormally extended, and crabs were

trapped within the old exoskeleton and died. Those that were not killed during molting were

severely deformed and would not likely have survived in the wild. Survivors that were removed

from exposure and maintained in clean water resumed normal molt activity. These results indicate

that environmental contaminants that are characteristic of ship channel sediments can affect the

molting process and by so doing can have serious consequences for survival, growth, and repro-

duction of invertebrate organisms.

CONSTRUCTION OF THE HepG2-EcR CELL LINE: Initial attempts to develop a cell-based

assay for molt-disrupting chemicals were based on a commercially available ecdysone receptor-

based system, the Ecdysone-Inducible Mammalian Expression System (Invitrogen). Functioning

of the system is described at the Invitrogen Tech-Online website (http://www.invitrogen.com/). The

system can be used to study any gene of interest and its products by transiently transfecting it into

isolated human embryonic kidney cells (EcR-293 cell line, Invitrogen) where gene expression can

be triggered by the addition of a foreign agonist, in this case an ecdysteroid. The system was derived