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.
Disruption of the molting process by anthropogenic chemicals has been observed in various arthropods. For example, the polychlorinated biphenyl (PCB) mixture Aroclor 1242, a common environmental contaminant, has been shown to inhibit the molting of fiddler crabs, Uca pugilator (Fingerman and Fingerman, 1977). In a recent investigation, exposure to a PCB congener (2,4,6-trichlorobiphenyl, PCB30) disrupted molting in crawfish (Jones et al., 2000). The hypothesized toxic mode of action of molt disruption by such chemicals involves the mimicking of ecdysteroids or the blocking of normal receptor binding. Contaminated sediments 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 suspended Oakland Inner Harbor sediments (highly contaminated with PCB's, hydrocarbons, and metals) over a period of 25 days. High mortality resulted and 92% 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. Subsequent molts of survivors that were removed from exposure and maintained in clean water were normal. Results such as these indicate that environmental contaminants that affect the molting process can have serious consequences for invertebrate organisms, potentially resulting in adverse effects on population dynamics. There is a need for assays that can detect this type of effect, therefore, the purpose of this investigation was to determine whether responses in a new cell line, HepG2-EcR, correlated with biological effects in arthropods. Since Aroclor 1242 has been previously shown to inhibit molting, it was selected as a test compound.
Methods and materials
Generation of a stably transfected cell line
Details of the generation of this cell line are described in McFarland et al. (2003). The cell line is based on Invitrogen's® Ecdysone-Inducible Mammalian Expression System, which consists of two plasmids (Figure 1). The first plasmid (pVgRXR) expresses the heterodimeric ecdysteroid receptor while the second plasmid (pIND) 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 encoding the enzyme, β-galactosidase, which then cleaves a substrate producing a yellow product that can be monitored colorimetrically. Briefly, optimized electroporation methods were used to transfect HepG2 cells (human hepatocellular carcinoma) with the pVgRXR plasmid. Cells were allowed to recover for 3 d before treatment with Zeocin to select for cells containing the plasmid. Colonies were generated from single cells and screened for activity by transiently inserting the second plasmid, pIND, using the cationic transfection reagent LipoFectamine® (Gibco) and testing for β-galactosidase activity after exposure to the EcR agonist, Ponasterone A. Cells from the colony with the highest activity were subsequently electroporated with the second plasmid (pIND) and allowed to recover for 3 d before treatment with Hygromycin B to select for cells carrying the pIND plasmid. Colonies were again generated from single cells and screened to isolate the one with the highest activity, which was then designated HepG2-EcR.
Details of the assay are described in McFarland et al. (2003). Briefly, cells were plated in 96-well plates at 40000 cells per well. After allowing the cells to attach overnight, cells were treated with Aroclor 1242 (1, 10, 100, and 500 μg l−1) in the presence of 0, 1, 3, 10, 30, or 100 μM Ponasterone A. After 48 h, cells were lysed in 100 μl of lysis buffer. For the β-galactosidase assay, 50 μl of the lysate were transferred to a 96-well plate. Buffer A (110 μl; 100 mM NaH2PO4, pH 7.5, 10 mM KCl, 1 mM MgSO4, 50 mM β-mercaptoethanol) was added and allowed to incubate at 37°C for 5 min before addition of 50 μl of substrate (4 mg ml−1 of o-nitrophenyl-p-galactopyranoside). Immediately after addition of the substrate, the absorbance was measured at 405 nm every min for 15 min in a temperature controlled (37°C) plate reader. Data from 5 separate experimental runs were combined by normalizing the activity (mOD min−1) to the maximal activity achieved with Ponasterone A alone. Data was plotted as log-dose versus response to determine if there were shifts in the EC50s or maximal activities in the presence of Aroclor 1242. Data from 10 μM Ponasterone A co-exposures were compared to determine whether this endpoint was a viable alternative to running a complete dose response curve.
Laboratory-reared juvenile crawfish (Procambarus clarkii, obtained from Dr. Robert Romaire, Louisiana State University at Baton Rouge) were exposed to aqueous solutions of Aroclor 1242 (UltraScientific) to provide a comparison of the cell-based assay with whole-organism effects. Crawfish were held individually in 22 ml borosilicate glass vials (containing 2.0 ml of solution), and checked for molting three times d−1 every 8 hr; each day, water was changed and crawfish were fed (tetramin granules). Experiments were conducted at 21 ± 1°C, with a photoperiod of 16:8 (light:dark). Crawfish were allowed to molt at least twice to provide pre-exposure molting periods, after which exposure to Aroclor 1242 began (within 8 h of molting). Aqueous solutions of Aroclor 1242 (nominal concentrations of 50, 100, 250, 500, and 1000 μg l−1; n = >10) were changed daily and monitored until the crawfish either molted or died. Solutions were prepared daily by spiking 20 ml of aged tap water with 10 μl of stock Aroclor 1242 solutions made in acetone (acetone stocks ranged from 0.1 to 2 mg ml−1). Solvent and non-solvent (water only) controls were conducted with the Aroclor exposures.
All statistical analyses were conducted using SigmaStat (SPSS Inc, version 6) software. Graphical analyses were conducted with SigmaPlot (SPSS Inc, version 5) software. The results from five repeated experiments with the HepG2-EcR assay were used to determine mean values and SEM after normalization to control values.
Results and discussion
Figure 2 represents data from co-exposure of Ponasterone A and Aroclor 1242 expressed as activity normalized to the maximal induction of Ponasterone A alone to allow comparison between experiments. Error bars represent standard deviation of the measurements for the five experiments used to generate the data. Maximal activity is significantly suppressed (Mann-Whitney Rank Sum Test, p = 0.007) in the 500 μg l−1 Aroclor 1242 dose compared to Ponasterone A alone, indicating that Aroclor was preventing the normal receptor-ligand interaction; however, no significant shift in the EC50 was observed (12.8 ± 1.1 and 11.7 ± 1.1 μM Ponasterone A, respectively). Figure 3 represents the 10 μM Ponasterone A data from five exposures, each normalized to their respective 10 μM Ponasterone A response to allow combining of the data. Activity is significantly suppressed in the 500 and 100 μg l−1 Aroclor 1242 dose compared to Ponasterone alone, and the suppression appears to be dose-related (log-dose vs. response regression gives r2 = 0.80).
Molt inhibition by Aroclor 1242 in juvenile crawfish
Aroclor was acutely toxic at 1000 μg l−1, with five out of ten of the crawfish dying within 10 d of exposure. At 500 μg l−1, only one of ten crawfish died. Feeding was noticeably reduced in the 1000 and 500 μg l−1 exposures, but no feeding reduction was observed below 250 μg l−1. Reported solubility of Aroclor 1242 ranges from 100 to 340 μg l−1 (ATSDR, 2000), so it appears that acute toxicity and feeding reduction is only observed at concentrations exceeding solubility. Paired t-test (pre-exposure intermolt period compared to post-exposure intermolt period for each individual) showed significant delays in molting for all doses above 50 μg l−1 (Figure 4). Although the concentrations of Aroclor 1242 used in the crawfish exposures are higher than aqueous concentrations found under environmentally relevant conditions, crawfish are sediment dwelling organisms and may accumulate high levels of total PCB's. Crawfish have been found under field conditions containing nearly 800 μg kg−1 on a wet tissue weight basis (Zaranko et al., 1997). We are currently analyzing crawfish from a parallel experiment to determine the actual body burdens of PCBs in order to correlate body burden with molting inhibition.
The present study indicates that the use of transgenic cell lines can provide rapid detection of sublethal effects. The cell-based assay and crawfish molting inhibition have similar sensitivities with Lowest Observed Effect Levels (LOEL) of 100 μg l−1 Aroclor 1242 for both assays. Although both the crawfish and the cell-based assays indicated that Aroclor 1242 is capable of inhibiting molting, a direct connection with the observed molting inhibition cannot definitively be linked to EcR receptor disruption without more studies since it may be a non-receptor mediated toxic response. In order to definitively link the assay to molting disruption, numerous chemicals should be tested in both the assay and invertebrate toxicity tests, and the results compared to that of a non-molting specific assay such as the AhR system.
Currently, testing of more compounds is underway. Additionally, comparisons of the assay response to environmental water samples with alterations in invertebrate community structure are being conducted in collaboration with the USGS National Water Quality Assessment Program's urban landuse gradient study. The results of this comparison are expected to gauge the potential utility of the assay for screening environmental samples.
This work was supported by the Dredging Operations Environmental Research Program (Robert M. Engler, Ph. D., Program Manager) of the USACE. Permission was granted by the Chief of Engineers to publish this information.