EU Environmental Policy under Pressure: Chemicals Policy Change between Antagonistic Goals?

Until now, chemicals policy was unable to find a method to establish a sound balance between the benefits and risks of chemical substances. Policy learning within the European Union (EU) resulted in the formulation of an alternative approach to future chemicals policy in some quarters. The Commission's proposal for new chemicals legislation - presented as 'paradigmatic' policy change - led to a highly controversial lobbying debate. This paper deals in particular with the influence of structures, policy networks, multi-level governance and the influence of new modes of governance on chemicals policy-making. It argues that future policy will not represent the paradigmatic change announced by the Commission but only one incremental, cost-effective step towards such a reform. The final proposal is criticised as a neo-liberal interpretation of the Lisbon strategy which includes a dangerous shift in environmental policy making.

Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis.

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites -zearalenol (-ZOL) and -zearalenol (-ZOL). -ZOL exhibited the strongest estrogenic potency (EC50 0.022 ± 0.001 nM), slightly less potent than 17- estradiol (EC50 0.015 ± 0.002 nM). ZEN was ~70 times less potent than -ZOL and twice as potent as -ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, -ZOL or -ZOL. ZEN, -ZOL or -ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 M. At 100 M, cell viability decreased and levels of hormones were significantly reduced except for progesterone. -ZOL increased estradiol concentrations more than -ZOL or ZEN, with a maximum effect at 10 M, with -ZOL (562 ± 59 pg/ml) > -ZOL (494 ± 60 pg/ml) > ZEN (375 ± 43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.

The Type VI secretion system of Burkholderia cenocepacia affects multiple Rho family GTPases disrupting the actin cytoskeleton and the assembly of NADPH oxidase complex in macrophages

Burkholderia cenocepacia is a Gram-negative opportunistic pathogen of patients with cystic fibrosis and chronic granulomatous disease. The bacterium survives intracellularly in macrophages within a membrane-bound vacuole (BcCV) that precludes the fusion with lysosomes. The underlying cellular mechanisms and bacterial molecules mediating these phenotypes are unknown. Here, we show that intracellular B. cenocepacia expressing a type VI secretion system (T6SS) affects the activation of the Rac1 and Cdc42 RhoGTPase by reducing the cellular pool of GTP-bound Rac1 and Cdc42. The T6SS also increases the cellular pool of GTP-bound RhoA and decreases cofilin activity. These effects lead to abnormal actin polymerization causing collapse of lamellipodia and failure to retract the uropod. The T6SS also prevents the recruitment of soluble subunits of the NADPH oxidase complex including Rac1 to the BcCV membrane, but is not involved in the BcCV maturation arrest. Therefore, T6SS-mediated deregulation of Rho family GTPases is a common mechanism linking disruption of the actin cytoskeleton and delayed NADPH oxidase activation in macrophages infected with B. cenocepacia.

An in vitro investigation of endocrine disrupting effects of tricothecenes deoxynivalenol (DON), T-2, HT-2 toxins

Trichothecenes are a large family of chemically related mycotoxins. Deoxynivalenol (DON), T-2 and HT-2 toxins belong to this family and are produced by various species of Fusarium. The H295R steroidogenesis assay, regulation of steroidogenic gene expression and reporter gene assays (RGAs) for the detection of androgen, estrogen, progestagen and glucocorticoid (ant)agonist responses, have been used to assess the endocrine disrupting activity of DON, T-2 and HT-2 toxins.

H295R cells were used as a model for steroidogenesis and gene expression studies and exposed with either DON (0.1–1000 ng/ml), T-2 toxin (0.0005–5 ng/ml) or HT-2 toxin (0.005–50 ng/ml) for 48 h. We observed a reduction in hormone levels in media of exposed cells following radioimmunoassay. Cell viability was determined by four colorimetric assays and we observed reduced cell viability with increasing toxin concentrations partly explaining the significant reduction in hormone levels at the highest toxin concentration of all three trichothecenes.

Thirteen of the 16 steroidogenic genes analyzed by quantitative real time PCR (RT-qPCR) were significantly regulated (P < 0.05) by DON (100 ng/ml), T-2 toxin (0.5 ng/ml) and HT-2 toxin (5 ng/ml) compared to the control, with reference genes (B2M, ATP5B and ACTB). Whereas HMGR and CYP19 were down-regulated, CYP1A1 and CYP21 were up-regulated by all three trichothecenes. DON further up-regulated CYP17, HSD3B2, CYP11B2 and CYP11B1 and down-regulated NR5A1. T-2 toxin caused down-regulation of NR0B1 and NR5A1 whereas HT-2 toxin induced up-regulation of EPHX and HSD17B1 and down-regulation of CYP11A and CYP17. The expressions of MC2R, StAR and HSD17B4 genes were not significantly affected by any of the trichothecenes in the present study.

Although the results indicate that there is no evidence to suggest that DON, T-2 and HT-2 toxins directly interact with the steroid hormone receptors to cause endocrine disruption, the present findings indicate that exposure to DON, T-2 toxin and HT-2 toxin have effects on cell viability, steroidogenesis and alteration in gene expression indicating their potential as endocrine disruptors.

Endocrine disrupting effects of ochratoxin A at the level of nuclear receptor activation and steroidogenesis

Ochratoxin A (OTA) is a mycotoxin and extrolite of fungi which has been reported in a range of foods. This study uses mammalian reporter gene assays (RGAs) with natural steroid receptors and the H295R steroidogenesis assay to assess the endocrine disrupting activity of OTA.

At the receptor level, OTA (within a concentration range of 0.25–2500 ng/ml) did not induce an agonistic response in an oestrogen, androgen, progestagen or glucocorticoid RGA. An antagonistic effect was observed in all of the RGAs at the highest concentration tested (2500 ng/ml). However, while there was no significant cytotoxic effect observed in the MTT (thiazolyl blue tetrazolium bromide) cell viability assay at this concentration, there was a corresponding change in cell morphology which may be related to the resulting antagonistic effect.

At the hormone production level, H295R cells were used as a steroidogenesis model and exposed to OTA (within a concentration range of 0.1–1000 ng/ml). Treatment of the cells with 1000 ng/ml OTA increased the production of estradiol (117 ± 14 ng/ml) over 3 times that of the solvent control (36 ± 9 pg/ml). Western blotting confirmed an increase in aromatase protein.

Overall the results indicate that OTA does not appear to interact with steroid receptors but has the potential to cause endocrine disruption by interfering with steroidogenesis. This is the first study identifying the effect OTA may have on production of the steroid hormone estradiol.

An in vitro investigation of endocrine disrupting effects of the mycotoxin alternariol

Alternariol (AOH) is a mycotoxin commonly produced by Alternaria alternata on a wide range of foods. Few studies to date have been performed to evaluate the effects of AOH on endocrine activity. The present study makes use of in vitro mammalian cellular based assays and gene expression to investigate the ability of AOH to act as an endocrine disruptor by various modes of action. Reporter gene assays (RGAs), incorporating natural steroid hormone receptors for oestrogens, androgens, progestagens and glucocorticoids were used to identify endocrine disruption at the level of nuclear receptor transcriptional activity, and the H295R steroidogenesis assay was used to assess endocrine disruption at the level of gene expression and steroid hormone production. AOH exhibited a weak oestrogenic response when tested in the oestrogen responsive RGA and binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of AOH. H295R cells when exposed to 0.1-1000ng/ml AOH, did not cause a significant change in testosterone and cortisol hormones but exposure to 1000ng/ml (3.87µM) AOH resulted in a significant increase in estradiol and progesterone production. In the gene expression study following exposure to 1000ng/ml (3.87µM) AOH, only one gene NR0B1 was down-regulated, whereas expression of mRNA for CYP1A1, MC2R, HSD3B2, CYP17, CYP21, CYP11B2 and CYP19 was up-regulated. Expression of the other genes investigated did not change significantly. In conclusion AOH is a weak oestrogenic mycotoxin that also has the ability to interfere with the steroidogenesis pathway.