Characterization of the androgen‐sensitive MDA‐kb2 cell line for assessing complex environmental mixtures

Abstract

Synthetic and natural steroidal androgens and estrogens and many other non-steroidal endocrine-active compounds commonly occur as complex mixtures in aquatic environments. It is important to understand the potential interactive effects of these mixtures to properly assess their risk. Estrogen receptor agonists exhibit additivity in mixtures when tested in vivo and in vitro. Little is known, however, concerning possible mixture interactions of androgen receptor agonists. In these studies we used the MDA-kb2 cell line, a human breast cancer cell line with endogenous androgen receptors and a stably transfected luciferase reporter gene construct to quantify the androgenic activity of seven natural and synthetic androgens: 17beta-trenbolone, dihydrotestosterone, methyltestosterone, testosterone, trendione, 17alpha-trenbolone, and androstenedione. We tested combinations of these androgens and compared the observed activity to expected androgenic activity based on a concentration addition model. Our analyses support the hypothesis that androgen receptor agonists cause additive responses in a mixture. Binary mixtures of 17beta-trenbolone with 17beta-estradiol or triclocarban (an anti-microbial found in the environment) were also tested. 17beta-Estradiol induced androgenic activity, but only at concentrations 600-fold greater than those found in the environment. Triclocarban enhanced the activity of 17beta-trenbolone. Additionally, three anti-androgens were each paired with three androgens of varying potencies. The relative potencies of the antagonists were a vinclozolin metabolite (M2) > procymidone > prochloraz regardless of the androgen used. The results of our studies demonstrate the potential utility of the androgen-responsive MDA-kb2 cell line for quantifying the activity of mixtures of endocrine-active chemicals in complex wastes such as municipal effluents and feedlot discharges.

Environmental (anti-)androgenic chemicals affect germinal vesicle breakdown (GVBD) of Xenopus laevis oocytes in vitro

“…Given the high sensitivity of amphibian oocyte maturation to environmental androgens, we suggest that Xenopus GVBD can be used to test androgenic activity of suspicious environmental androgens. Several in vitro AR transactivation assays, such as yeast transactivation assay, are used to assay androgenic activity of endocrine disruptors (Blake et al, 2010;Christen et al, 2010;Gee et al, 2008;Sun et al, 2007). Effective concentrations of androgens as positive reference substances in these in vitro assays generally ranged 1-1000 nM.…”

Use of chemical mixtures to differentiate mechanisms of endocrine action in a small fish model

“…Triclocarban, an anti-microbial chemical commonly found in aquatic systems (Sapkota et al, 2007), recently was reported to enhance AR-mediated responses to testosterone both in mammalian cell cultures and rats in vivo Chen et al, 2008;Blake et al, 2010). The mechanism whereby this effect occurs is not known Chen et al, 2008), but our basic mixture study design provided a unique opportunity to assess whether TCC could enhance AR-mediated responses in fish.…”

“…Although tubercle formation in fathead minnows is clearly an AR-mediated phenomenon, it is possible that ER agonists could affect development of tubercles in the fathead minnow (Miles-Richardson et al, 1999). A comparatively recent HPG-related response of interest involves some common antimicrobial chemicals (triclocarban, triclosan) that seem to enhance responsiveness to AR agonists in rats and in vitro Chen et al, 2008;Blake et al, 2010). To assess cross-class generality of this phenomenon in vivo, we also conducted mixture studies with TB and triclocarban (TCC).…”

The potential anti-androgenic effect of agricultural pesticides used in the Western Cape: In vitro investigation of mixture effects

“…Several natural and man-made compounds have been shown to modulate endocrine activity in vertebrates (Heath and Claassen, 1999;Urbatzka et al, 2007a;Blake et al, 2010). Compounds acting in this way are collectively referred to as endocrine-disrupting contaminants (EDCs).…”

“…Compounds acting in this way are collectively referred to as endocrine-disrupting contaminants (EDCs). Although the endocrine modulation may be subtle in cases, several of these EDCs have been shown to adversely disrupt the gonadal endocrine system of non-target organisms, potentially resulting in altered reproductive fitness, and may eventually show effects at a population level (Blake et al, 2010). Chemicals having endocrine-disrupting (ED) effects on the reproductive system can in general be categorised in 1 of 4 mode-of-action (MOA) groups (responses), namely, oestrogenic, anti-oestrogenic, androgenic or anti-androgenic (Behrends et al, 2010;Hoffmann and Kloas, 2010).…”