S1). As anticipated co-exposure to 1 μM TCC and varying concentrations of DHT amplified the luciferase reporter activity by ∼40% ( Fig. 2A). Meanwhile the corresponding EC50 values remained unchanged at 8.0 × 10−11 M and 8.2 × 10−11 M for control and TCC treated cells, respectively. Nevertheless, the suggested stimulation of the AR is in line with earlier studies, which confirms the functionality of the reporter construct ( Christen et al., 2010). The next aim was to validate the AR-antagonistic function of TCC on a transcriptional level. This was

done by RT-PCR, targeting several transcripts known to be regulated by the AR (i.e. SARG, NDRG1 and SORD) ( Doane et al., 2006). Prior to RNA-extraction the cells this website were treated for 24 h with 1 or 10 nM DHT ± 1 μM TCC, respectively. Exposure to DHT led to an increased expression of all three transcripts. Yet, in the presence of TCC only SORD see more showed a slight but statistically significant decrease in gene expression (p = 0.02) ( Fig. 2D). It was thus not possible to confirm the initially observed androgenic effect of TCC on the level of the AR regulon. An unspecific off-target effect of TCC on luciferase should, however, be apparent independent of the receptor investigated. Hence an estrogenic luciferase reporter was used to investigate the effects of TCC in presence of estrogen

(E2). The corresponding results were then compared with those of a commonly used proliferation assay, namely the E-screen. The effect of TCC on an estrogenic luciferase reporter was studied using HeLa9903 cells, a cell line previously suggested for the detection Olopatadine of xenoestrogens by the OECD and EPA (OECD, 2009). These cells are stably transfected with human ERα and an ERE-driven luciferase reporter gene. The molecular phenotype was verified by quantitative RT-PCR, detecting transcripts of ESR1, GPR30 and AHR but not AR or ESR2 ( Fig. S1). As described previously ( Ahn et al., 2008) cellular co-exposure

to E2 and 1 μM TCC resulted in a 50% increase of luciferase signal intensity ( Fig. 3). Although signal amplification was consistent for all E2-concentrations tested (10−11 to 10−8 M), the maximal effect was seen at 1 nM E2 and 1 μM TCC ( Fig. 3). Higher concentrations of TCC quickly became cytotoxic ( Fig. S2). The suggested xenoestrogenic potential of TCC was further examined using the E-screen (Fig. 4). This assay uses the estrogen dependent proliferation of human mammary carcinoma MCF-7 cells as readout. Cellular exposure to E2 triggered a dose dependent increase of MCF-7 cell numbers. Addition of 1 μM TCC, however, failed to have any further proliferative effect. The E2 concentrations producing 50% of the maximal effect (EC50) were comparable between the two assays, ranging from 2.9 × 10−11 M for the luciferase assay to 3.0 × 10−11 M for the E-screen.