RESUMO
Vitellogenin (Vtg), a large multidomain protein precursor of egg-yolk proteins, is used as an endocrine disruption biomarker in fish, and in the last decades, its use has been extended to invertebrates like mollusks. However, it remains unclear whether invertebrate endocrine system produces Vtg in response to estrogens, like it occurs in oviparous vertebrates. In a previous study, no evidence of induction of Vtg expression at protein level was found in gonads of the marine mussel Mytilus galloprovincialis after exposure to the estrogenic chemical 17α-ethinylestradiol (EE2). In the present follow-up study, it was investigated whether there is any effect of EE2 on Vtg abundance at transcriptional level in M. galloprovincialis gonads. To this aim, RT-qPCR analysis targeting three different domains of Vtg transcript was performed on gonads of mussels that were exposed either 4 or 24 days to 100 ng/L EE2. In addition, several reference genes were analysed and a selection of these for potential use in further RT-qPCR analyses on mussel male and female gonads is provided. Results showed higher expression in females than in males for the three analysed Vtg domains, and no evidence of Vtg mRNA induction due to EE2 either in females or males. The present results, together with those obtained from previous analysis at protein level, support that Vtg is not an adequate biomarker for xenoestrogenicity in marine mussels. Additionally, nucleotide sequences of Vtg transcripts of three closely-related species from Mytilus edulis complex (M. galloprovincialis, M. edulis and M. trossulus) are provided and compared with Vtg sequences from other mollusk species to assess the level of conservation and evolutionary relationships among species.
Assuntos
Mytilus , Poluentes Químicos da Água , Animais , Etinilestradiol/toxicidade , Feminino , Seguimentos , Expressão Gênica , Masculino , Mytilus/genética , Vitelogeninas/genética , Poluentes Químicos da Água/toxicidadeRESUMO
Organophosphate flame retardants (OPFRs) are (re-)emergent environmental pollutants increasingly being used because of the restriction of other flame retardants. The chlorinated OPFR, tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is among those of highest environmental concern, but its potential effects in the marine environment have rarely been investigated. We exposed a widely used sentinel marine mussel species, Mytilus galloprovincialis, to 10 µg L-1 of TDCPP during 28 days and studied: (i) the kinetics of bioaccumulation and elimination of the compound, (ii) the effect on two molecular biomarkers, glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities, and (iii) proteomic alterations in the gills, following an isobaric labeling quantitative shotgun proteomic approach, at two exposure times (7 and 28 days). Uptake and elimination of TDCPP by mussels were very fast, and the bioconcentration factor of this compound in mussels was 147 L kgww-1, confirming that this compound is not very bioaccumulative, as predicted by its chemical properties. GST activity was not affected by TDCPP exposure, but AChE activity was inhibited by TDCPP at both 7 and 28 days of exposure. Proteomic analysis revealed subtle effects of TDCPP in mussel gills, since few proteins (less than 2 % of the analysed proteome) were significantly affected by TDCPP, and effect sizes were low. The most relevant effects detected were the up-regulation of epimerase family protein SDR39U1, an enzyme that could be involved in detoxification processes, at both exposure times, and the down-regulation of receptor-type tyrosine-protein phosphatase N2-like (PTPRN2) after 7 days of exposure, which is involved in neurotransmitter secretion and might be related to the neurotoxicity described for this compound. Exposure time rather than TDCPP exposure was the most important driver of protein abundance changes, with 33 % of the proteome being affected by this factor, suggesting that stress caused by laboratory conditions could be an important confounding factor that needs to be controlled in similar ecotoxicology studies. Proteomic data are available via ProteomeXchange with identifier PXD019720.
Assuntos
Bioacumulação/efeitos dos fármacos , Retardadores de Chama/toxicidade , Brânquias/metabolismo , Mytilus/efeitos dos fármacos , Compostos Organofosforados/toxicidade , Poluentes Químicos da Água/toxicidade , Acetilcolinesterase/metabolismo , Animais , Biomarcadores/metabolismo , Relação Dose-Resposta a Droga , Retardadores de Chama/metabolismo , Brânquias/efeitos dos fármacos , Glutationa Transferase/metabolismo , Cinética , Modelos Biológicos , Mytilus/metabolismo , Compostos Organofosforados/metabolismo , Proteômica , Poluentes Químicos da Água/metabolismoRESUMO
A wide variety of endocrine disrupting chemicals reach the marine environment and can cause harmful effects in different marine organisms. Vitellogenin (Vtg), the egg-yolk precursor, is a commonly used endocrine disruption biomarker in fish and more recently in marine invertebrates under the assumption of high expected similarities in the endocrine system of vertebrates and invertebrates. However, this assumption has been recently questioned. The results from previous studies focused on bivalve molluscs showed that Vtg induction could be misleading because of the use of either non-robust or indirect techniques to measure Vtg. In this study, mussels (Mytilus galloprovincialis) were exposed to either 10 or 100â¯ng/L of the synthetic hormone 17α-ethinylestradiol (EE2) at different exposure times (4 and 24â¯days) and under different feeding regimes (representing different energy balances), and Vtg levels in both male and female mussel gonads were quantified by label free shotgun LC-MS/MS proteomic analysis. Vtg protein was not detected in male gonads. In female gonads, Vtg levels were not significantly affected by EE2 at any exposure time or EE2 concentration tested, whereas a significant correlation was found between the degree of maturation of the gonad and Vtg levels in females. Results obtained in the present study critically question the use of Vtg as a biomarker of endocrine disruption in marine mussels, and show that the degree of maturation of the gonad can be an important confounding factor in the attempts to evaluate estrogenic effects through Vtg measurement in mussel gonads.