Common phytochemicals are ecdysteroid agonists and antagonists: a possible evolutionary link between vertebrate and invertebrate steroid hormones.

Many plant compounds are able to modulate growth and reproduction of herbivores by directly interacting with steroid hormone systems. In insects, several classes of phytochemicals, including the phytoestrogens, interfere with molting and reproduction. We investigated whether the anti-ecdysone activity may be due to interaction with the ecdysone receptor (EcR) using a reporter-gene assay and a cell differentiation assay of an ecdysone-responsive cell line, Cl.8+. We tested rutin (delays molt in insects); four flavones: luteolin and quercetin (metabolites of rutin), and apigenin and chrysin; and three non-flavones, coumestrol and genistein (both estrogenic) and tomatine (alters molt in insects). None of the phytochemicals tested were ecdysone agonists in the reporter-gene assay, but the flavones were able to significantly inhibit EcR-dependent gene transcription. In the Cl.8+ cells, quercetin and coumestrol were mixed agonists/antagonists, while genistein, tomatine and apigenin showed a synergistic effect with ecdysteroid in the reduction of cell growth. We suggest that the rutin effects on molting in insects are most likely due to the metabolites, luteolin or quercetin, while tomatine acts via a non-EcR pathway. Flavones not only interact with EcR and estrogen receptor (ER), but also signal nitrogen-fixing bacteria to form root nodules. The NodD protein which regulates this symbiosis has two ligand-binding domains similar to human ERalpha. The evolutionary significance of these findings are discussed.

Adhesion of Drosophila imaginal disc cells in vitro.

We have used our imaginal disc cell lines to carry out in vitro studies on the cell-cell and cell-substrate adhesion of Drosophila leg and wing disc cells. Single cells were allowed to reaggregate in roller culture, and this process was found to be partially dependent on the presence of magnesium and calcium ions in the suspension medium. Varying rates of reaggregation were observed in cells from different stages of a passage, correlating with the pattern of morphogenesis which occurs during the passage. We have demonstrated that cloned cell lines can be produced showing certain selected characteristics, such as reduced cell adhesiveness.

Cell-cell and cell-substrate adhesion in cultured Drosophila imaginal disc cells.

Drosophila imaginal disc cell lines were used to investigate various aspects of cellular adhesion in vitro. The distribution of PS integrins and their involvement in cell-cell and cell-substrate adhesion were assessed with the monoclonal antibody aBG-1 against the betaPS subunit, and both forms of adhesion were found to be impeded by the presence of the antibody. Adhesion to a number of extracellular matrix components was investigated, and the cells were found to adhere to human fibronectin. This adhesion was inhibited by aBG-1. The adhesion molecule fasciclin III was also found in these cells. Given that the cells are competent to perform cell-cell and cell-substrate adhesion, it was thought that apical basal polarity might be restored when other suitable conditions were provided, i.e., an artificial basement layer with feeder cells to provide nutrients basally to the cells, and some features of apical-basal morphology were seen in cells cultured under these conditions.

Interaction of PAHs and PCBs with ecdysone-dependent gene expression and cell proliferation.

This study was done to determine whether PAHs and PCBs can interact with the arthropod steroid hormone system. Ecdysteroid molting hormones control growth, molting, and reproduction in arthropods. A spike in 20-OH ecdysone (20 HE) triggers the molt cycle in crustaceans, and earlier studies have shown that PAHs can affect this molt cycle in several crab species. However, the mechanism of this molt cycle interaction is unknown. Both PAHs and PCBs interact with other nuclear receptors; however, nothing is known about their ability to interact with the invertebrate ecdysone receptor (EcR). Four PAHs, benzo[a]pyrene, benzo[b]fluoranthene, pyrene, and chrysene, and the commercial PCB mixture, Aroclor 1254, were used to determine the ability of these classes of compounds to induce ecdysone-dependent reporter gene expression and to modify the proliferation and differentiation response of the ecdysteroid-responsive Cl.8+ cell line. The four PAHs were each able to enhance the ecdysteroid response in both the reporter gene and the cell proliferation assays only when given in conjunction with ecdysteroids. Aroclor 1254 had no effect in either system, either alone or in conjunction with ecdysteroids. These studies show that although the PAHs alone do not activate ecdysteroid-dependent gene expression or cell differentiation, they are able to enhance the effect of ecdysteroids, presumably through a non-receptor-mediated process. This mechanism may explain the effects on molting which have been reported after low-level crude oil exposures in crustaceans.

The effect of juvenile hormone on the response of the Drosophila imaginal disc cell line Cl 8+ to moulting hormone.

The Drosophila wing imaginal disc cell line Cl 8+ was used to investigate the interaction between juvenile hormone III (JH) and 20-hydroxyecdysone (20HE). Cell cultures were exposed to either or both hormones at a range of concentrations and cell growth was observed. JH was found to ameliorate the effects of 20HE on cell growth, even when added after the cells had been exposed to 20HE for 4 or 24h.

The effects of several ecdysteroids and ecdysteroid agonists on two Drosophila imaginal disc cell lines.

Two Drosophila imaginal disc cell lines, C18+ (sensitive to 20-hydroxyecdysone, 20HE) and C18R (resistant to 20HE) were exposed to the ecdysteroid agonists RH5849 and RH5992 and the ecdysteroids inokosterone, makisterone A and muristerone A. All compounds tested were found to have similar effects on the cells, comparable to the effects of 20HE, although at different concentrations. C18R showed resistance to all compounds, again at varying concentrations. We conclude that it is likely that all the compounds tested use the same receptors as 20HE, but show maximum effectiveness at different concentrations.

Effect of age on the growth and response of a Drosophila cell line to moulting hormone.

Insect cell lines in culture are used for a variety of studies. In this laboratory imaginal disc cell lines have been established from primary cultures from third instar larvae, and used for a number of experiments. The effect of ageing on the morphology and physiology of Drosophila cell lines has received very little attention, although problems of genotypic or phenotypic changes in cell lines with age are recognized in other areas of animal cell culture. We tested our cell line Cl8+ for any difference in growth, morphology and response to 20-hydroxyecdysone (20HE) at different ages (passage numbers). The cells were found to multiply faster, adhere less firmly to the substrate and to lose the tendency to aggregate at higher passages. The response to 20HE in terms of cell numbers and induction of beta-galactosidase was similar at all passage numbers but morphological changes in hormone-treated cells were less obvious in the higher passages. Cell lines are likely to vary in the extent of ageing effects but workers are advised to be aware of the possibilities. We suggest the effects of age on cell lines should be established, and passage numbers noted in experimental reports.