Screening of endocrine disrupting chemicals with MELN cells, an ER-transactivation assay combined with cytotoxicity assessment.
There is growing concern that some chemicals can cause endocrine disrupting effects to wild animals and humans. Therefore a rapid and reliable screening assay to assess the activity of endocrine disrupting chemicals (EDCs) is required. These EDCs can act at multiple sites. Most studied mechanism is direct interaction with the hormone receptors, e.g. estrogen receptor. In this study the luciferase reporter gene assay using transgenic human MELN cells was used. Since cytotoxicity of the chemicals can decrease the luminescent signal in the transactivation assays, a cytotoxicity assay must be implemented. Mostly the neutral red (NR) assay is performed in parallel with the estrogenicity assay. To increase the reliability and cost-efficiency of the test, a method to measure estrogenicity and cytotoxicity in the same cell culture plate instead of in parallel plates was developed and evaluated. Therefore the NR-assay was compared with the CytoTox-ONE homogeneous membrane integrity assay. The latter measures LDH (lactate dehydrogenase) leakage based on a fluorometric method. For all compounds tested, the CytoTox-ONE test showed comparable curves and EC50-values to those obtained by the NR-assay. So the CytoTox-ONE kit, which seemed more sensitive than measurements of LDH-leakage based on a colorimetric method, is recommended to test cytotoxicity to MELN cells, with the advantage to use the same cells for ER-transactivation measurements. The chemicals tested in the optimised MELN assay showed estrogenic potencies comparable to those reported for several other transactivation assays.
Berckmans
P
P
VITO, Flemish Institute for Technological Research, Department of Environmental Toxicology, Boeretang 200, 2400 Mol, Belgium. pascale.berckmans@vito.be
Leppens
H
H
Vangenechten
C
C
Witters
H
H
D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects; D052244:Endocrine Disruptors / Q000633:toxicity*; D005470:Fluorometry; D017930:Genes, Reporter / Q000187:drug effects; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000187:drug effects / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D009499:Neutral Red / Q000378:metabolism; D011960:Receptors, Estrogen / Q000187:drug effects* / Q000378:metabolism; D015203:Reproducibility of Results; D018675:Toxicity Tests / Q000379:methods*; D015533:Transcriptional Activation / Q000187:drug effects*
D052244:Endocrine Disruptors; D011960:Receptors, Estrogen; D009499:Neutral Red; D007770:L-Lactate Dehydrogenase; D008156:Luciferases