Human reporter gene system

In 2005, the Ministry of the Environment of Japan evaluated four novel simple bioassays for monitoring dioxin contamination in effluent gas, fly ash, and cinders. In the CALUX assay, recombinant mouse hepatoma cells (HlL6.1c2) with four dioxin-responsive elements upstream of the luciferase gene are treated with extracts from contaminated samples, and the luciferase activity is then measured [2]. Luciferase activity induced in the recombinant human hepatoma cell line lOlL and the recombinant mouse hepatoma cell line HeB5 is utilized in the P450 Human Reporter Gene System (HRGS) and Ah luciferase assays, respectively [3]. 

To test for the effects of chemopreventive agents on COX-2 and/or iNOS transcriptional activity, we constructed a P-galactosidase (p-gal) reporter gene system in human colon cancer DLD-1 cells. 

The test system was considerably less sensitive to endosulfan when mouse ER, rather than human ER, was used to mediate (3-gal activity (Ramamoorthy et al. 1997). In similar assays, endosulfan at 10 jM had no effect on (3-gal activity in yeast Saccharomyces) transfected with either the human or rainbow trout ER (Andersen et al. 1999). In addition, no effect was observed on transcriptional activation of HeLa cells transfected with plasmids containing an estrogen receptor as a responsive element (Shelby et al. 1996). Endosulfan also did not induce transient reporter gene expression in MCF-7 human breast cancer cells at an incubation concentration of 2.5 pM (Andersen et al. 1999). Maximum endosulfan-induced ER-mediated luciferase reporter gene expression occurred in vitro in a T47D human breast adenocarcinoma cell line at approximately 10 pM, while 50% expression of luciferase occurred at about 5.9 pM the maximum expression was approximately 59% of the effect from exposure to 0.03 nM estradiol (0.00003 pM) (Legler et al. 1999). Luciferase expression from combined treatment with endosulfan and dieldrin was additive over concentrations ranging from 3 to 8 pM. 

The yeast reporter gene assays not only assess for the interaction of the chemical with the hormone receptor, but also the ability of that receptor-chemical ligand interaction to activate the hormone DNA response element. It should be realized, however, that most of these systems have been developed with human and mammalian hormone receptors and differences in ligand potencies can occur between different animal species. A comprehensive review of in vitro assays for measuring estrogenic activity, and some of the issues of comparability, is provided by Zacharewski (1997). 

Persson, KP., Ekehed, S., Otter, C., Lutz, E.S., McPheaL J., Masimirembwa, C.M. and Andersson, T.B. (2006) Evaluation of human liver slices and reporter gene assays as systems for predicting the cytochrome p450 induction potential of drugs in vivo in humans. Pharmaceutical Research, 23, 56-69. 

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