Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya.

Isothiocyanates are electrophiles that are able to induce phase II biotransformation enzyme gene expression via an electrophile-responsive element (EpRE) in the gene regulatory region. To study the potency of different isothiocyanates to induce the expression of EpRE-regulated genes, a Hepa-1c1c7 luciferase reporter cell line was exposed to structurally different isothiocyanates. The reporter cell line, EpRE(mGST-Ya)-LUX, contains the EpRE from the regulatory region of the mouse glutathione S-transferase Ya gene. Isothiocyanates containing a methyl-sulfur side chain, e.g. sulforaphane, showed a lower EC(50) (0.8-3.2 microM) and a comparable induction factor (17-22.4) compared to the structurally different isothiocyanates containing an alkyl or aromatic side chain, e.g. allyl and phenylethyl isothiocyanate (EC(50) 3.9-6.5 microM, induction factor 17.5-23). After 24h of exposure, on average (+/-SD) 23+/-5% of the isothiocyanate was found in the cells and 77% in the cell medium. Isothiocyanates prove to be strong inducers of electrophile-responsive element-mediated gene expression at physiological concentrations. The here described luciferase reporter cell line is a suitable assay to measure the potency of compounds to induce EpRE-regulated gene expression.

Androgenic activity in surface water samples detected using the AR-LUX assay: indications for mixture effects.

This paper describes the screening of 22 extracts from 18 different aquatic environmental samples for androgenic activity, including indirect and interactive effects on androgen receptor (AR)-mediated signal transduction, using the AR-LUX bioassay. Four samples, originating from an industrial wastewater treatment plant (WTP) or the river Meuse, were shown to contain substantial androgenic activity. Moreover, the samples originating from the industrial WTP showed an enhancement of the maximal androgenic response relative to that elicited by the standard androgen methyltrienolone (R1881) in the AR-LUX assay. This indicates the involvement of cellular mechanisms other than receptor-ligand interaction influencing AR-regulated pathways. This also demonstrates the additional value of cell based assays featuring a more complete array of fully functional interacting pathways. Chemical analysis using GC-MS confirmed the presence of a number of androgens and also estrogens in these WTP samples. Subsequently, we showed that estrone and tributyltin hydride (TBT-H) enhance the response to androgens. This indicates that the presence of numerous compounds in addition to androgens in environmental mixtures might very well result in a more profound perturbation of the normal physiology of exposed organisms than estimated based on the androgen levels alone. Therefore, risk assessment of environmental samples should include an evaluation of the presence and the interactive effects of (ant)agonists of carefully selected relevant cellular receptors in order to provide a realistic estimate of the integrated ecotoxicological risk of the compounds present.

Detection of hormonal anabolic compounds in calf urine and unverified growth-promoting preparations: application of the AR-LUX bioassay for screening and determination of androgenic activity.

Despite a ban by the European Union, the use of anabolic steroids and repartitioning agents in cattle is still occasionally observed. Due to continuing improvements in analytical techniques, very low detection limits for individual compounds have been achieved. In response to these developments, cocktails composed of several steroids have been applied, thus hampering detection due to lower levels of the individual compounds. Bioassays capable of measuring the integrated effect of cocktails might therefore provide valuable additional tools in controlling the use of illegal anabolics. We investigated the feasibility of using the AR-LUX assay to detect the presence in cattle urine of growth promoters that exert their effects via androgen response elements (AREs). The AR-LUX assay is based on a human cell line featuring a luciferase reporter gene under transcriptional control of an authenticated ARE. Several column purification and liquid/liquid extraction methods were investigated to optimize the efficiency of anabolic compounds extraction and minimize cytotoxic effects of the urine matrix. The AR-LUX assay was found to be applicable to the detection of anabolic steroids excreted in urine samples with a discriminatory power similar to that of GC-MS analysis. Finally, some liquid products probably destined for growth-promoting purposes confiscated outside the Netherlands were analyzed. Although common chemical-analytical methods did not detect any anabolic steroids in these samples, the presence of compounds activating ARE-mediated gene expression was clearly established.