In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters

doi:10.1016/j.ab.2005.02.032

Analytical Biochemistry

Volume 340, Issue 2, 15 May 2005, Pages 295-302

https://doi.org/10.1016/j.ab.2005.02.032 Get rights and content

Abstract

Reporter gene technology is widely used to measure activity of hormone analogs, and bioluminescent in vitro assays have allowed rapid screening of numerous chemicals either to identify new agonists or antagonists of hormones or to detect the presence of endocrine disrupters in the environment. Stable bioluminescent cell lines have been established and they provide reproducible dose–response curves and accurate determination of in vitro efficiencies of various chemicals. In vivo, however, these molecules can be metabolized, bound by proteins, or stored in fats and thus could display efficiencies different from those observed in vitro. In vivo assays, such as the uterotrophic bioassay, require numerous sacrificed animals, and responses not only are dependent on an estrogenic action but also imply other factors. For a faster assay and to avoid the use of numerous animals, we developed an in vivo biosensor constituted of stable bioluminescent cells implanted in nude mice. MCF-7 bioluminescent cell lines were chosen since their proliferation is low in the absence of estrogen and the xenograft size can thus be stable for several weeks. Luciferase gene expression was monitored noninvasively with a cooled charge-coupled device camera. Quantitative analysis allowed us to compare in vitro and in vivo actions of different estrogenic compounds (estradiol, estrone) and endocrine disruptors (ethynylestradiol, genistein, octylphenol, and 2,4′-dichlorodiphenyldichloroethylene) in the same cell lines and to follow hormone action on a living animal as a function of time. Different administration protocols have been used and good correlation was observed for most products. However, we found that ethynylestradiol was the most efficient chemical when orally administered.

Section snippets

Materials

Materials for cell culture were obtained from Invitrogen (Cergy-Pontoise, France). Luciferin was purchased from Promega (Charbonnières, France). Tribromoethanol (TBE), 17β estradiol (E₂), ethynylestradiol (EE₂), estrone (E₁), genistein (Gen), 2,4′-dichlorodiphenyldichloroethylene (2,4′-DDE), and 4-tert-octylphenol (OP) were purchased from Sigma Chemical (Saint Quentin-Fallavier, France). Effectors were dissolved in dimethyl sulfoxide at 10⁻² M.

Generation of stably transfected reporter cell lines

The stably transfected luciferase reporter MELN cell

MELN cell in vitro assay

A great number of compounds able to activate ERα were tested with the MELN cell in vitro assay. The typical dose–response curve of most estrogenic compounds in the MELN cell assay was a sigmoid curve leveling off at a value corresponding to the maximal inductive effect (Fig. 1). Maximum value, taken as 100, was obtained in presence of 10 nM E₂ in cell medium; basal activity was 15% of maximal activity and presence of antagonist reduced transactivation [15], [22]. Since antiestrogens were able to

Discussion

In this study, bioluminescence-based measurements were performed to evaluate estrogenic activities of several estrogens and endocrine disrupters using in vitro and in vivo luciferase-expressing tumor cells. The optical reporter gene that we used in our oncology models is a modified version of that isolated from firefly P. pyralis. Luciferase enzyme produces light in presence of luciferin substrate, oxygen, and ATP [25]. in vitro, luciferase uses intracellular oxygen and ATP, and adding 0.3 μM

Acknowledgments

This research was supported by INSERM (ATC), Montpellier 1 University (BQR), l’ARC and la Ligue de lutte contre le cancer (RA). The authors thank Michel Brissac for help in performing animal experiments.

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In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters

Abstract

Section snippets

Materials

Generation of stably transfected reporter cell lines

MELN cell in vitro assay

Discussion

Acknowledgments

Toxicol. Lett.

Sci. Total Environ.

Sci. Total Environ.

J. Steroid Biochem. Mol. Biol.

Toxicology

Am. J. Clin. Nutr.

Gene

Insights from the study of animals lacking functional estrogen receptor

Science

Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta

Endocrinology

In vitro bioassays for assessing estrogenic substances

Environ. Sci. Technol.

Identification of estrogenic chemicals in STW effluent. 1. chemical fractionation and in vitro biological screening

Environ. Sci. Technol.

Identification of environmental chemicals with estrogenic activity using a combination of in vitro assays

Environ. Health Perspect.

Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line

Toxicol. Sci.