Endocrine disruptors in soil: Effects of bisphenol A on gene expression of the earthworm Eisenia fetida.

Xenobiotics such as bisphenol A (BPA), are present in biosolids, which are applied as organic fertilizers in agricultural fields. Their effects on soil life have been poorly assessed, and this is particularly important in the case of earthworms, which represent the main animal biomass in this medium. In the present work we study the impacts of BPA on gene expression of Eisenia fetida, a widely used ecotoxicological model. Chronic soil tests and acute contact tests were performed, and gene expression was analyzed in total tissue and in masculine reproductive organs of the earthworms. The genes studied in this research played a role in endocrine pathways, detoxification mechanisms, stress response, epigenetics, and genotoxicity. Most of the genes were identified for the first time, providing potentially useful biomarkers for future assessments. For chronic exposures, no changes were detected in whole-body tissue; however, masculine reproductive organs showed changes in the expression of genes related to endocrine function (EcR, MAPR, AdipoR), epigenetic mechanisms (DNMTs), genotoxicity (PARP1), and stress responses (HSC70 4). For acute exposures, the expression of one epigenetic-related gene was altered for both whole-body tissues and male reproductive organs (Piwi2). Further changes were detected for whole-body tissues involved in detoxification (Metallothionein), stress (HSC70 4), and genotoxicity (PARP1) mechanisms. Acute exposure effects were also tested in whole-body tissues of juveniles, showing changes in the expression of Metallothionein and Piwi2. The molecular changes found in the analyzed earthworms indicate that exposure to BPA may have negative implications in their populations. Particularly interesting are the alterations related to epigenetic mechanisms, which suggest that future generations may be impacted. This study is the first to evaluate the molecular effects of BPA on soil organisms, and further assays will be necessary to better characterize the true environmental repercussions. CAPSULE: Levels of gene expression in total-body tissues and masculine reproductive organs were analyzed in earthworms after exposure to bisphenol A and we observed associated changes in detoxification, endocrine, epigenetic, genotoxic and stress pathways.

Telomeric transcriptome from Chironomus riparius (Diptera), a species with noncanonical telomeres.

Although there are alternative telomere structures, most telomeres contain DNA arrays of short repeats (6-26 bp) maintained by telomerase. Like other diptera, Chironomus riparius has noncanonical telomeres and three subfamilies, TsA, TsB and TsC, of longer sequences (176 bp) are found at their chromosomal ends. Reverse transcription PCR was used to show that different RNAs are transcribed from these sequences. Only one strand from TsA sequences seems to render a noncoding RNA (named CriTER-A); transcripts from both TsB strands were found (CriTER-B and αCriTER-B) but no TsC transcripts were detected. Interestingly, these sequences showed a differential transcriptional response upon heat shock, and they were also differentially affected by inhibitors of RNA polymerase II and RNA polymerase III. A computer search for transcription factor binding sites revealed putative regulatory cis-elements within the transcribed sequence, reinforcing the experimental evidence which suggests that the telomeric repeat might function as a promoter. This work describes the telomeric transcriptome of an insect with non-telomerase telomeres, confirming the evolutionary conservation of telomere transcription. Our data reveal differences in the regulation of telomeric transcripts between control and stressful environmental conditions, supporting the idea that telomeric RNAs could have a relevant role in cellular metabolism in insect cells.

Gene expression analysis of Chironomus riparius in response to acute exposure to tire rubber microparticles and leachates.

Tire rubber microparticles (TRPs) entering aquatic ecosystems through stormwater runoffs is a significant challenge. TRPs are formed by the abrasion of tires with the road surface and include chemical additives that are an additional cause for concern. Currently, information on the molecular effects of TRPs, or especially its additives, in freshwater organisms is scarce. To address this problem, an array covering different cellular processes has been designed for the freshwater midge Chironomus riparius. Fourth-instar larvae were exposed to two concentrations of TRPs (1 mg L-1, 10 mg L-1) and tire rubber leachates (TRLs) (0,0125 %, 5 %) to evaluate the transcriptional activity by Real-Time PCR. To assess acute toxicity, larvae were exposed for 24 h and genes related to the endocrine system, stress response, DNA repair mechanisms, immune system, oxidative stress, and detoxification mechanisms were evaluated. The activity of the enzymes: glutathione S-transferase (GST) and catalase was also examined. The main pathway affected was the stress response showing overexpression of HSPs (HSC70.3, HSC70.4, HSC70.5, HSP60). Moreover, there was a reduction of the GSTd3 and catalase disrupting the antioxidant system. The upregulation of InR indicates a potential disturbance in the insulin pathway and ABCB6 activation only in TRPs exposure suggests its potential implication in their transport. However, most of these alterations are caused by TRLs, showing higher toxicity than TRPs. The results obtained in this work provide the first approach at the molecular and cellular levels to elucidate the impact of TRLs in freshwater organisms. To perform a realistic evaluation of the TR effects, additional research is required to assess the TR's long-term effects at the molecular level.

Transcription and activation under environmental stress of the complex telomeric repeats of Chironomus thummi.

In contrast to their traditional role, telomeres seem to behave as transcriptionally active regions. RNAs complementary to the short DNA repeats characteristic of telomerase-maintained telomeres have recently been identified in various mammalian cell lines, representing a new and unexpected element in telomere architecture. Here, we report the existence of transcripts complementary to telomeric sequences characteristic of Chironomus thummi telomeres. As in other Diptera, the non-canonical telomeres of chironomids lack the simple telomerase repeats and have instead more complex repetitive sequences. Northern blots of total RNA hybridized with telomere probes and RT-PCR with telomere-specific tailed primers confirm the existence of small non-coding RNAs of around 200 bp, the size of the DNA repeated telomeric unit. Telomere transcripts are heterogeneous in length, and they appear as a ladder pattern that probably corresponds to multimers of the repeat. Moreover, telomeres are activated under conditions of environmental stress, such as heat shock, appearing highly decondensed and densely labelled with acetylated H4 histone, as well as with RNA polymerase II antibodies, both marks of transcriptional activity. Changes in the expression levels of telomeric RNA were detected after heat shock. These findings provide evidence that transcriptional activity of the repetitive telomere sequences is an evolutionarily conserved feature, not limited to telomerase telomeres. The functional significance of this non-coding RNA as a new additional element in the context of telomere biology remains to be explained.

Applying sunscreens on earthworms: Molecular response of Eisenia fetida after direct contact with an organic UV filter.

The use of organic Ultraviolet (UV) filters has increased in the last years, either in sunscreens, other cosmetics, or even food packaging. These filters may end up in soil and water since the Wastewater Treatment Plants may not successfully remove them. Among them, benzophenones are known to act as endocrine disruptors. However, most of the studies are directed towards vertebrates and aquatic invertebrates, while there is a lack of information on the molecular mechanisms affected by these compounds on soil dwelling invertebrates. Here, we study the impact of direct acute (48 h) contact of 4-hydroxybenzophenone (4-OHBP) at two sublethal concentrations (0.02 and 0.2 mg/mL) on gene expression of the earthworm Eisenia fetida. Investigated genes were involved in endocrine pathways, stress response, detoxification mechanisms, genotoxicity, energy metabolism and epigenetics. Three of them were identified for the first time in earthworms. Our results suggest that exposure to 4-OHBP affected endocrine pathways, causing an increase in the Ecdysone receptor gene (EcR) expression. Moreover, the UV filter induced changes in the CuZn superoxide dismutase gene (CuZn SOD), indicating an effect in the stress response. Finally, significant changes were detected for glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) expression, indicating that energy metabolism is influenced by the 4-OHBP and highlighting the risks of using GAPDH as an internal reference for Real Time PCR.

The endocrine disruptor bisphenol A increases the expression of HSP70 and ecdysone receptor genes in the aquatic larvae of Chironomus riparius.

Bisphenol A (BPA) is an endocrine disruptor that can mimic the action of estrogens by interacting with hormone receptors and is, therefore, potentially able to influence reproductive functions in vertebrates. Although information about the interaction with the endocrine systems in invertebrates is limited, it has also been shown its effect on reproductive and developmental parameters in these organisms. As little is known about its mechanism of action in aquatic invertebrates, we have examined the effects of BPA on the expression of some selected genes, including housekeeping, stress-induced and hormone-related genes in Chironomus riparius larvae, a widely used organism in aquatic ecotoxicology. The levels of different gene transcripts were measured by Northern blot or by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Exposure to BPA (3 mgl(-1), 12-24h) did not affect the levels of rRNA or those of mRNAs for both L11 or L13 ribosomal proteins, selected as examples of housekeeping genes involved in ribosome biogenesis. Nevertheless, BPA treatment induced the expression of the HSP70 gene. Interestingly, it was found that BPA significantly increases the mRNA level of the ecdysone receptor (EcR). These results show for the first time that exposure to endocrine disrupting chemicals, such as BPA, can selectively affect the expression of the ecdysone receptor gene suggesting a direct interaction with the insect endocrine system. Furthermore, this finding suggests a common way of BPA action, shared by vertebrates and invertebrates, through interaction with steroid hormone receptors. Our study adds a new element, the EcR, which may be a useful tool for the screening of environmental xenoestrogens in insects.

Bisphenol A in artificial soil: Effects on growth, reproduction and immunity in earthworms.

The application of biosolids in agricultural fields is increasing annually. They contain not only nutrients but also xenobiotics, such as Bisphenol A (BPA). These compounds are not regulated in the use of biosolids in agriculture, which highlights the need to assess their effects on soil life, of which earthworms are most abundant of the animal representatives. In this study the effect of BPA on life-history parameters, such as mortality, growth and reproduction, and on immunity, is evaluated for Dendrobaena veneta and Eisenia fetida. Sublethal concentrations were evaluated by a modified OECD artificial soil test. Decline in growth with increasing concentration of BPA was detected during the first two weeks and the opposite effect for the next two, although these differences were only significant at the highest concentration. Reproduction traits were only significantly different for E. fetida, for which the number of juveniles decreased at higher concentrations, thus showing different sensitivity in both species. By using a contact test, the potentially harmful effect of direct contact with BPA was shown to be much higher than in soil (resembling natural) conditions. Finally, results indicate that BPA may not affect the immune system of these animals, at least in terms of coelomocyte viability.

Characterization and expression during development and under environmental stress of the genes encoding ribosomal proteins L11 and L13 in Chironomus riparius.

The Chironomus riparius gene sequences encoding ribosomal proteins L11 and L13 were characterized and their expression analysed during development, and under different types of cellular stress. A comparative and phylogenetic study among different orders of insects was carried out by analysis of sequence databases. L11 is highly conserved, both at the level of DNA and protein, and it shares over 90% amino acid identity with homologous sequences from other insects. Interestingly, the changes are mainly concentrated in the C-terminal domain of the protein. Conversely, L13 shows a lower degree of homology, around 60% amino acid identity, and the changes were dispersed throughout the length of the polypeptide. Surprisingly, when comparing L13 nucleotide sequences, only a very low or no homology was found even among diptera. These results are helpful for defining the structural and, therefore, evolutionary constraints of these proteins. Studies of gene expression by RT-PCR showed that they are differentially expressed in distinct stages of development. Both L11 and L13 were significantly upregulated during embryogenesis. The expression profiles of the transcripts were also analysed after a general stress, such as heat shock, as well as after a specific stress, such as acute cadmium treatment. In both conditions, no significant differences to controls were detected in L11 and L13 transcripts, in spite of the drastic changes observed in the stress-induced gene HSP70, and the inhibitory effect on rRNA transcription. These data confirm that both genes are equally robust against harmful environmental conditions, suggesting that they could be used as a control for environmentally responsive genes in Chironomus. Overall, our results show a coordinated expression of both the L11 and the L13 genes, but not a coordinated regulation of rRNA and ribosomal protein production.

Ribosomal genes as early targets of cadmium-induced toxicity in Chironomus riparius larvae.

Cadmium is a widespread environmental pollutant that causes severe impacts in organisms. Although the effects of cadmium on aquatic insects have been studied in terms of their toxicity and changes in developmental parameters, little is known about its molecular and genetic effects. We have investigated the alterations in the pattern of gene expression provoked by acute exposure to cadmium in Chironomus riparius Mg. (Diptera, Chironomidae), a sentinel organism widely used in aquatic toxicity testing. The early cytotoxic effects were evaluated using immunocytochemistry and specific fluorescent probes in fourth instar larvae after 12 h of 10 mM cadmium treatments; under these conditions no significant effect on larvae mortality was detected until after 36 h of exposure. The changes in the pattern of gene expression were analysed by means of DNA/RNA hybrid antibodies in the polytene chromosomes from salivary gland cells. A decrease in the activity of the nucleolus is especially remarkable, accompanied by a significant reduction in size and the modification in nucleolar architecture, as shown by FISH. The inhibition of rDNA transcription was further evaluated by Northern blot analysis, which showed a marked decrease in the level of preribosomal rRNA (54% 45S 12 h). However, the BR genes, whose products are the giant polypeptides that constitute the silk-like secretion for constructing housing tubes, remain active. Simultaneously, decondensation and activation take place at some chromosomal regions, especially at the centromeres. The changes observed in the pattern of gene expression do not resemble those found after heat shock or other cell stressors. These data provide the first evidence that cadmium interacts with ribosomal genes and results in a drastic impairment of the functional activity of the nucleolus, an essential organelle for cellular survival. Therefore, the depletion of ribosomes would be a long-term effect of Cd-induced cellular damage. These findings may have important implications for understanding the adverse biological effects of cadmium and its toxic mechanism, as yet not clearly defined, and provide a sensitive biomarker of cadmium exposure.

Effect of acute exposure to cadmium on the expression of heat-shock and hormone-nuclear receptor genes in the aquatic midge Chironomus riparius.

Cadmium is a widespread and highly toxic pollutant of particular ecotoxicological relevance for aquatic ecosystems where it accumulates. To identify biomarkers for ecotoxicity monitoring, the effect of cadmium on the expression of different genes related to the stress response as well as to the ecdysone hormone-signalling pathway was studied in the aquatic larvae of Chironomus riparius (Diptera, Chironomidae), a standard test organism in aquatic toxicology testing. Reverse Transcription Polymerase Chain Reaction (RT-PCR) was used to evaluate the effects of acute and short-term cadmium exposures (10mM CdCl(2), 12h and 24h) on the expression of hsp70, hsc70, hsp90 and hsp40 genes, as well as on that of the ecdysone hormonal-receptor genes (EcR and usp). A significant 3-fold increase in the level of hsp70 gene transcripts was induced by the treatment, whereas neither the other stress genes tested (hsp90 and hsp40) nor the constitutive form of hsp70, hsc70, was affected in the larvae exposed to cadmium. These results show that hsp70 is differentially activated to other environmentally regulated heat-shock genes, and constitutes a biomarker of exposure to this toxic metal. In addition, we also found that cadmium is able to alter the expression of the ecdysone receptor gene (EcR), whose mRNA level is significantly increased whereas usp levels remained unaltered. This finding, evidenced for the first time in invertebrates, supports the view that cadmium has the ability to mimic the effect of the hormone by the activation of the ecdysone nuclear receptor, which may partly explain the endocrine disruption capability that has been previously suggested for this toxic metal. Our research adds to the growing evidence implicating heavy metals, and cadmium in particular, as potential endocrine disruptive agents and may have significant implications for ecological risk assessment of endocrine-disrupting compounds in invertebrates.

Molecular profiling related to poor prognosis in thyroid carcinoma. Combining gene expression data and biological information.

Undifferentiated and poorly differentiated thyroid tumors are responsible for more than half of thyroid cancer patient deaths in spite of their low incidence. Conventional treatments do not obtain substantial benefits, and the lack of alternative approaches limits patient survival. Additionally, the absence of prognostic markers for well-differentiated tumors complicates patient-specific treatments and favors the progression of recurrent forms. In order to recognize the molecular basis involved in tumor dedifferentiation and identify potential markers for thyroid cancer prognosis prediction, we analysed the expression profile of 44 thyroid primary tumors with different degrees of dedifferentiation and aggressiveness using cDNA microarrays. Transcriptome comparison of dedifferentiated and well-differentiated thyroid tumors identified 1031 genes with >2-fold difference in absolute values and false discovery rate of <0.15. According to known molecular interaction and reaction networks, the products of these genes were mainly clustered in the MAPkinase signaling pathway, the TGF-beta signaling pathway, focal adhesion and cell motility, activation of actin polymerization and cell cycle. An exhaustive search in several databases allowed us to identify various members of the matrix metalloproteinase, melanoma antigen A and collagen gene families within the upregulated gene set. We also identified a prognosis classifier comprising just 30 transcripts with an overall accuracy of 95%. These findings may clarify the molecular mechanisms involved in thyroid tumor dedifferentiation and provide a potential prognosis predictor as well as targets for new therapies.