, 2004, Hu and Mackenzie, 2009 and Harrington c-Met inhibitor et al., 2006). As before all transcripts were altered following estrogenic treatment. For PGR and ESR1 treatment with BPA, butylparaben and genistein had an effect similar to E2, unlike UGT2B15 where the two xenoestrogens had a less pronounced

effect. With regard to trefoil factor 1 the prolonged exposure with genistein led to a 10-fold upregulation, a level twice as high as with E2. Again, none of the tested transcripts was influenced either by TCC alone or by co-stimulation with TCC and estrogens. Altogether the experiments therefore did not confirm a potential xenoestrogenic effect of TCC, neither on the molecular level, nor in whole cells (E-screen). Meanwhile the conflicting results for TCC in the various test systems point to an unspecific effect on luciferase. Ligand triggered stabilisation of luciferase has previously been reported to cause false positive readouts (Thorne et al., 2012). We therefore Temsirolimus used thermal shift to assay the effects of TCC and ATP

on the enzymes heat stability (Fig. 5A). The results showed that TCC indeed directly interacts with firefly luciferase, stabilising the enzyme. The effect is particularly pronounced in the presence of ATP as enzymatic cofactor. Addition of the latter shifted the T  m of luciferase by 3.3 °C. However, with increasing concentrations of TCC this shift increased further to up to 7 °C at 10 μM TCC. No such strong Tyrosine-protein kinase BLK interaction could be seen with structural similar antimicrobials such as TCS and HCP ( Fig. 5B). The first did not to stabilise luciferase at all, while the latter only interacted weakly ( ΔTm5μMHCP = 2 °C). Tested in the HeLa9903 estrogen reporter assay both substances were negative ( Fig. S2). Altogether the data indicate that the previously reported effects of TCC as a xenohormone in vivo are not related to a direct interaction with the AR or ER. It is well established though that AhR and ERα are connected via a complex regulatory crosstalk mediated by several

mechanisms and that interference with this crosstalk can lead to adverse phenotypes ( Rataj et al., 2012). On molecular level interactions comprise competition for co-activators as well as AhR mediated protein degradation of ERα by ubiquitinylation ( Ohtake et al., 2011). Further on some AhR regulated genes such as CYP1B1 are also known to be ERE regulated ( Tsuchiya et al., 2004). Following treatment with estrogens and TCC we therefore also measured the expression of two classical target genes of the AhR, CYP1A1 and CYP1B1 ( Fig. 6). Used as single substance TCC induced a slight increase in CYP1A1 expression which was comparable to the treatment with genistein. None of the other estrogens had a comparable effect when used alone. However, in combination with TCC they acted as strong inducers, increasing transcription of CYP1A1 by up to 20-fold.