Technique / Molecular Biology / Transfection and infection / Reporter gene assay

Disposable gloves made of nitrile-butadiene rubber (NBR) are used for contact with foodstuffs rather than polyvinyl chloride gloves containing di(2-ethylhexyl)phthalate (DEHP), because endocrine-disruptive effects are suspected for phthalate diesters including DEHP. However, 4,4'-butylidenebis(6-t-butyl-m-cresol) (BBBC), 2,4-di-t-butylphenol, and 2,2,4-trimetyl-1,3-pentanediol diisobutyrate can be eluted from NBR gloves, and possibly also detected in food. In this study, we examined the endocrine-disrupting effects of these chemicals via androgen receptor (AR) and estrogen receptor (ER)-mediated pathways using stably transfected reporter gene cell lines expressing AR (AR-EcoScreen system) and ER (MVLN cells), respectively. We also examined the binding activities of these chemicals to AR and ER. The IC50 value of BBBC for antagonistic androgen was in the range of 10(-6)M. The strength of inhibition was about 5 times that of a known androgen antagonist, 1,1'-(2,2-dichloroethylidene)bis[4-chlorobenzene] (p,p'-DDE), and similar to that of bisphenol A. The IC50 value of BBBC for antagonistic estrogen was in the range of 10(-6)M. These results suggest that BBBC and its structural homologue, 4,4'-thiobis(6-t-butyl-m-cresol) are androgen and estrogen antagonists. It is therefore necessary to study these chemicals in vivo, and clarify their effect on the endocrine system.

Interferons (IFNs) are cytokines playing an important role in the immune response and defence against viruses. They are widely used as biopharmaceuticals. Currently, the anti-viral assay (AVA) is the most commonly used bioassay for determining interferon potency. In the search for rapid and robust but reliable methods, reporter gene assays (RGA) appear to be the most promising approach, therefore we have designed a new reporter cell line, CHO-ISRE-SEAP, suitable for determination of type I interferon potency. Chinese hamster ovary (CHO-K1) cells were stably transfected with secretory alkaline phosphatase (SEAP) gene under the control of interferon stimulated response element (ISRE) promoter. The amount of SEAP in the cell culture medium can be easily measured colorimetrically and has been found to correlate with the amount of IFN added. The new assay is widely applicable for determination of type I IFNs, such as IFN-alpha, IFN-beta and IFN-omega, in research, development of IFN biopharmaceuticals, in batch release, etc. Interestingly, in this assay, IFN-beta shows approximately 6 times higher response than IFN-alpha, which makes it especially appropriate for measuring low levels of IFN-beta. Compared to other known RGAs, the novel CHO-ISRE-SEAP cell line-based RGA appears to have certain advantages with respect to cost and performance.

Alkylphenols (APs) are widely used as important industrial materials and have attracted lots of attention because of their potential estrogenic activities. In this study, we developed human estrogen receptor alpha (hERalpha) and rat estrogen receptor alpha (rERalpha) mediated reporter gene assays and compared the estrogenic activity of APs and related chemicals based on the two ERalpha. Human breast cancer cell line MCF-7 was co-transfected with Gal4-fused hERalpha and corresponding reporter plasmid; African green monkey kidney cell line CV-1 was co-transfected with rERalpha and reporter gene. Both assays showed acceptable response to natural estrogen 17beta-estradiol (E2) with EC50 of 0.16 nM and 4.7 nM. Then the estrogenic activity of 4-APs, 4-phenylphenol and bisphenol-A were evaluated and compared with the effects of E2. The data suggested that test APs and related chemicals possessed weakly estrogenic activity and the activity of test APs increased with the increase of substituent size. This structure-activity relationship helped to infer the activity of chemicals with similar feature. Furthermore, test APs showed similar effect on the function of hERalpha and rERalpha. This consistency helped to extrapolate in vivo rodent data to human being when performing risk assessment of endocrine disruptors.