A New Transgenic Cell Line for Detection of Invertebrate Endocrine Disrupters: Laboratory Protocol for its Use

ERDC TN-DOER-C30

February 2003

A New Transgenic Cell Line for Detection

of Invertebrate Endocrine Disrupters:

Laboratory Protocol for its Use

PURPOSE: The purpose of this technical note is to report the development of a cell-based assay

for the detection of endocrine disrupting activity related to crustacean and insect molting and to

provide a detailed laboratory protocol for its conduct.

BACKGROUND: The advent of transgenic technology and the ability to insert foreign reporter

genes into the genomes of standard culturable cells has led to the improvement of cell-based assay

techniques. For example, mammalian cell cultures have been used for many years in assays for

dioxins and related compounds. The basis for their use is the presence in these cells of the aryl

hydrocarbon receptor (AhR) system that binds certain planar uncharged organic chemicals, and

mediates their toxicities. The assays have been improved by inserting genes of bacterial or insect

origin into the nuclear DNA that act as "reporters." When upstream dioxin recognition sequences

are activated by binding of AhR-dioxin complexes, the reporter genes express gene products that

result in signals that can be measured instrumentally (Inouye and McFarland 2000).

The P450RGS assay for polychlorinated dibenzodioxins/dibenzofurans (PCDD/Fs), polychlorinated

biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) is based on the transgenic 101L cell

line, a modification of human liver cancer HepG2 cells developed at the University of California,

Berkeley. In 101L cells the human CYP1A1 gene promoter sequence has been fused with the insect

firefly luciferase gene downstream as a reporter. Hence, the designation "P450" for the cytochrome P450

family of monoxygenases of which the CYP1A1 gene encodes isozymes responsive to dioxin and related

compounds, and "RGS," which refers to Reporter Gene System. When 101L cells are exposed in the

assay to chemicals with dioxinlike activity, light is produced in proportion to the intensity of the exposure

and is measured in a luminometer. The assay has relevance to biota that possess the CYP1A1 gene or

genes that are homologous with it. These include the teleost fishes, birds, amphibians, reptiles, and

mammals; the CYP1A1 gene is absent or less inducible in lower organisms. Consequently, although

chemicals such as dioxins and related compounds are extremely toxic to higher organisms possessing

the CYP1A1 gene, they are either nontoxic to lower organisms, or if toxic, their effects are manifested

through mechanisms that do not involve CYP1A1. Presently there are no methods similar to the

P450RGS assay that are capable of discriminating the potential for chronic toxicity to invertebrate

organisms in environmental samples.

INTRODUCTION: This Technical Note describes a newly developed transgene system designed

to detect and measure the activity of environmental contaminants that are chronically toxic to lower

organisms lacking the CYP1A1 gene but which are susceptible to contaminant toxicity through

other mechanisms. The assay based on these new cells detects chemicals that disrupt the invertebrate

endocrine system responsible for ecdysis (molting). Molting is a multistage process of shedding

the exoskeleton and replacing it with a larger one, and its successful completion is essential for the

survival, growth, and reproduction of arthropods, the invertebrate phylum that includes crustaceans

and insects. In both classes of organisms molting is under the control of ecdysteroid hormones.