Impacts of food contact chemicals on human health: a consensus statement

Food Packaging Forum Foundation (FPF) and the Plastics Solution Fund (PSF

Microplastic in angling baits as a cryptic source of contamination in European freshwaters.

High environmental microplastic pollution, and its largely unquantified impacts on organisms, are driving studies to assess their potential entry pathways into freshwaters. Recreational angling, where many anglers release manufactured baits into freshwater ecosystems, is a widespread activity with important socio-economic implications in Europe. It also represents a potential microplastic pathway into freshwaters that has yet to be quantified. Correspondingly, we analysed three different categories of industrially-produced baits ('groundbait', 'boilies' and 'pellets') for their microplastic contamination (particles 700 µm to 5 mm). From 160 samples, 28 microplastics were identified in groundbait and boilies, with a mean concentration of 17.4 (± 48.1 SD) MP kg-1 and 6.78 (± 29.8 SD) mg kg-1, yet no microplastics within this size range were recorded in the pellets. Microplastic concentrations significantly differed between bait categories and companies, but microplastic characteristics did not vary. There was no correlation between microplastic contamination and the number of bait ingredients, but it was positively correlated with C:N ratio, indicating a higher contamination in baits with higher proportion of plant-based ingredients. We thus reveal that bait microplastics introduced accidentally during manufacturing and/or those originating from contaminated raw ingredients might be transferred into freshwaters. However, further studies are needed to quantify the relative importance of this cryptic source of contamination and how it influences microplastic levels in wild fish

Metamorphic T3-response genes have specific co-regulator requirements.

Thyroid hormone receptors (TRs) have several regulatory functions in vertebrates. In the absence of thyroid hormone (T3; tri-iodothyronine), apo-TRs associate with co-repressors to repress transcription, whereas in the presence of T3, holo-TRs engage transcriptional coactivators. Although many studies have addressed the molecular mechanisms of T3 action, it is not known how specific physiological responses arise. We used T3-dependent amphibian metamorphosis to analyse how TRs interact with particular co-regulators to differentially regulate gene expression during development. Using chromatin immuno-precipitation to study tissue from pre-metamorphic tadpoles, we found that TRs are physically associated with T3-responsive promoters, whether or not T3 is present. Addition of T3 results in histone H4 acetylation specifically on T3-response genes. Most importantly, we show that individual T3-response genes have distinct co-regulator requirements, the T3-dependent co-repressor-to-coactivator switch being gene-specific for both co-regulator categories

[Transcriptional repression of the TRH gene]

The synthesis and secretion of thyroid hormones (TH: T3, T4) must be strictly regulated. TH act on their own production via a negative feedback system. The synthesis of thyrotropin-releasing hormone (TRH), produced in the hypothalamus, and thyrotropin (TSH) in the pituitary is inhibited at the transcriptional level by TH. TRH and TSH stimulate production of TH. An outstanding, still open, question is the molecular basis of T3-dependent transcription repression of TRH and TSH genes. However, some regulatory components have been identified, with the b-TH receptor (TRb) playing a specific regulatory role (versus TRa) in the negative feedback effects of T3 on production of TRH and TSH. Moreover, the N-terminus of TRb is known to be a key element in this regulation. A hypothesis to explain this isoform specificity could be that TRb and TRa interact differentially with transcriptional comodulators. Thus, it is critical to characterize these comodulators and to analyse their contribution to the transcription regulation of TRH

Implication of bax in Xenopus laevis tail regression at metamorphosis.

Apoptosis is fundamental to normal vertebrate development. A dramatic example of postembryonic development involving apoptosis is tail regression during amphibian metamorphosis. Earlier studies led us to propose a functional role for the pro-apoptotic protein Bax in tadpole tail regression. However, its physiological relevance has never been analyzed. We have now cloned a cDNA encoding Xenopus laevis bax (xlbax) and used in vivo gene transfer in tail muscle to analyze the effects of xlbax overexpression. Furthermore, by using an antisense strategy in a similar experimental paradigm, xlbax antisense mRNA was shown to block the apoptotic effects of xlbax and protect against apoptosis in metamorphosing tadpoles. Our results suggest that xlbax is a regulator of muscle fiber death in the regressing tail during metamorphosis. Copyright (c) 2004 Wiley-Liss, Inc

In vivo siRNA delivery to the mouse hypothalamus confirms distinct roles of TR beta isoforms in regulating TRH transcription.

RNA interference mediated by small interfering RNAs (siRNAs) is a powerful tool for evaluating gene function in vivo. In particular it should be able to provide tissue-specific and developmental stage-specific knock-down of target genes in physiological contexts. However, demonstrations of its use on neuronal specific genes in vivo are lacking. We examined whether a recently developed cationic lipid based approach was applicable to study the differential effects of the two beta thyroid hormone receptor (TR) isoforms, TRbeta1 and TRbeta2, on T(3)-transcriptional repression of the hypothalamic gene, TRH. The cationic lipid based technique used, JetSItrade mark/DOPE, was previously shown to efficiently knock-down reporter gene mRNA in vivo. Here we now show that its use to vectorise siRNA against TRbeta1 and TRbeta2 mRNA abrogates T(3)-mediated repression of hypothalamic TRH transcription. In particular, when using siRNA against either TRbeta1 or TRbeta2 differential effects are revealed. siRNA directed against TRbeta1 blocks both T(3) independent activation and T(3) dependent modulation of TRH transcription. In contrast, siRNA directed against TRbeta2 abrogates only T(3) repression of transcription. These results corroborate our previous findings obtained in mutant TRbeta(-/-) mice, showing that the TRbeta1 and TRbeta2 isoforms have differential effects on T(3)-TRH transcription. The data thus show that the cationic lipid-based siRNA strategy can effectively be used to reveal fine, tissue specific and isoform specific effects on neuronal gene transcription in vivo

A rapid, physiologic protocol for testing transcriptional effects of thyroid-disrupting agents in premetamorphic Xenopus tadpoles.

Increasing numbers of substances present in the environment are postulated to have endocrine-disrupting effects on vertebrate populations. However, data on disruption of thyroid signaling are fragmentary, particularly at the molecular level. Thyroid hormone (TH; triiodothyronine, T3) acts principally by modulating transcription from target genes; thus, thyroid signaling is particularly amenable to analysis with a transcriptional assay. Also, T3 orchestrates amphibian metamorphosis, thereby providing an exceptional model for identifying thyroid-disrupting chemicals. We combined these two advantages to develop a method for following and quantifying the transcriptional action of T3 in Xenopus laevis tadpoles. This technology provides a means of assessing thyroid activity at the molecular level in a physiologically relevant situation. Moreover, translucent tadpoles are amenable to "on-line" imaging with fluorescent reporter constructs that facilitate in vivo measurement of transcriptional activity. We adapted transgenesis with TH-responsive elements coupled to either luciferase or green fluorescent protein to follow T3-dependent transcription in vivo. To reduce time of exposure and to synchronize responses, we optimized a physiologic pretreatment protocol that induced competence to respond to T3 and thus to assess T3 effects and T3 disruption within 48 hr. This pretreatment protocol was based on a short (24 hr), weak (10(-12) M) pulse of T3 that induced TH receptors, facilitating and synchronizing the transcriptional responses. This protocol was successfully applied to somatic and germinal transgenesis with both reporter systems. Finally, we show that the transcriptional assay allows detection of the thyroid-disrupting activity of environmentally relevant concentrations (10(-8) M) of acetochlor, a persistent herbicide

Polyethylenimine-based intravenous delivery of transgenes to mouse lung

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