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Tributyltin (TBT) is a widespread environmental contaminant in aquatic systems whose adverse effects in development and reproduction are related to its well-known endocrine-disrupting activity. In this work, the early molecular effects of TBT in Chironomus riparius (Diptera) were evaluated by analyzing its DNA damaging potential and the transcriptional response of different endocrine-related genes. Twenty-four-hour in vivo exposures of the aquatic larvae, at environmentally relevant doses of TBT, revealed genotoxic activity as shown by significant increases in DNA strand breaks quantified with the comet assay. TBT was also able to induce significant increases in transcripts from the ecdysone receptor gene (EcR), the ultraspiracle gene (usp) (insect ortholog of the retinoid X receptor), the estrogen-related receptor (ERR) gene and the E74 early ecdysone-inducible gene, as measured by real-time RT-PCR. In contrast, the expression of the vitellogenin (vg) gene remained unaltered, while the hsp70 gene appeared to be down-regulated. The ability of TBT to up-regulate hormonal target genes provides the first evidence, at genomic level, of its endocrine disruptive effects and also suggests a mechanism of action that mimics ecdysteroid hormones in insects. These data reveal for the first time the early genomic effects of TBT on an insect genome., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cytochrome P450 family members participate in xenobiotic transformation as a detoxification mechanism. We have characterized a CYP gene, assigned to the 4G family, in Chironomus riparius, a reference organism in aquatic toxicology. Due to the potential interest of CYP genes and P450 proteins for monitoring pollution effects at the molecular level, the alterations in the pattern of expression of this gene, induced by different xenobiotics, were analyzed. Different compounds, such as the biocide tributyltin (TBTO) and two other well-known endocrine disruptors, nonylphenol (NP) and bisphenol A (BPA), were tested at different concentrations and acute exposures. Upregulation of the CrCYP4G gene was found after exposures to TBTO (1 ng/L 24h-0.1 ng/L 96 h) and, as measured by RT-PCR mRNA quantification, its level was up to twofold that of controls. However, in contrast, NP (1, 10, 100 μg/L, 24h) and BPA (0.5mg/L 24h-3mg/L 96 h) downregulated the gene (by around a half of the control level) suggesting that this gene responds specifically to particular chemicals in the environment. Glutathione-S-transferase (GST) enzymatic activity was also evaluated for each condition. A fairly good correlation was found with CYP4G gene behavior, as it was activated by TBTO (96 h), but inhibited by NP and BPA (24h). Only the higher concentration of BPA tested activated GST, whereas it inhibited CYP4G activity. The results show that different xenobiotics can induce distinct responses in the detoxification pathway, suggesting multiple xenobiotic transduction mechanisms. This work confirms that specific P450 codifying genes, as well as GST enzyme activities, could be suitable biomarkers for ecotoxicological studies., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We characterized the Hsp70 cDNA in Chironomus riparius and evaluated its expression profile under different environmental stressors. It is highly conserved, at both DNA and protein levels, displaying many of the hallmarks of Hsps and sharing 80-96% of overall amino acid identities with homologous sequences from other diptera. The changes are mainly concentrated in the C-terminal domain of the protein. Phylogenetic analysis was consistent with the known classification of insects. The Hsp70 gene was located by in situ hybridization in region III-3A at the third polytene chromosome, a locus activated upon heat shock as shown by RNA pol II binding. As C. riparius is widely used in aquatic ecotoxicology testing, we studied Hsp70 gene induction in fourth instar aquatic larvae submitted to heat shock and selected environmental pollutants classified as potential endocrine disruptors. RT-PCR analysis showed that Hsp70 mRNA levels increased significantly (p<0.05) after short-term acute exposures to a temperature shift (HS), cadmium chloride (Cd), butyl benzyl phthalate (BBP), diethylhexyl phthalate (DEHP), bisphenol A (BPA), 4-nonylphenol (NP) and ethinylestradiol (EE). However, neither pentachlorophenol (PCP) nor tributyltin (TBTO) treatments were able to activate the Hsp70 gene. The cognate form, Hsc70, was also analysed and, unlike Hsp70, was not altered by any of the different treatments assayed. Moreover, at the times tested, there was no significant mortality of the larvae. The rapid upregulation of the Hsp70 gene suggests that it is sensitive and selective for different environmental pollutants, and could be used as an early molecular endpoint in ecotoxicological studies., (Copyright © 2010 Elsevier Inc. All rights reserved.)