Development of a cell-based screening assay for invertebrate molting disruption

Abstract

The use of transgenic cell lines for relatively rapid, sensitive and reproducible assays for the detection and semi-quantitative measurement of contaminants in environmental media has increased in recent years. For example, many accepted assays rely on the arylhydrocarbon receptor interaction to screen for dioxins and related compounds in environmental samples. However, these systems are poorly developed or absent in lower organisms, and since arylhydrocarbon receptor interaction is a necessary early step in the development of dioxin toxicity, dioxins are relatively non-toxic to invertebrates. As a result, assays based on this interaction have no relevance for assessing the risk of environmental contamination to these organisms at the base of all food chains. Alternatively, arthropods possess highly developed ecdysone receptor systems that can be used similarly to the arylhydrocarbon receptor system as the basis of an assay with high relevance for these important classes of invertebrates. A new transgenic cell line was developed in order to create a rapid assay for ecdysone interactions to detect and measure the activity of environmental contaminants that are chronically toxic to lower organisms, specifically the invertebrate phylum Arthropoda. The cell line is based on Invitrogen's^® Ecdysone-Inducible Mammalian Expression System, which consists of two plasmids, one of which expresses the heterodimeric ecdysteroid receptor while the other contains the receptor-ligand response element E/GRE. When ecdysone or another ligand having ecdysteroid activity is present, the binding of the receptor-ligand complex to the response element results in transcription of the reporter gene (β-galactosidase), the activity of which can be monitored colorimetrically. The plasmids were stably transfected into HepG2 human liver cells. Preliminary results show that Aroclor 1242, which is known to inhibit molting in invertebrates, also inhibited molting in juvenile crawfish (Procambarus clarkii) at 100 μg l⁻¹ and normal ecdysteroid response in this new transgenic cell line, HepG2-EcR, at similar concentrations indicating that this new assay shows promise for future use as a screening tool.