Effects of primary leachates of conventional and alternative plastics in Cyprinodon variegatus fish larvae: Endocrine disruption and toxicological responses.

The inclusion of hazardous substances in the formulation of plastics raises significant concerns, particularly, if those substances are released as primary leachates during plastic degradation and/or fragmentation. In this sense, the production of degradable plastics holding deleterious additives can increase the release of harmful substances into the environment. Additionally, the effects of primary leachates of "eco-friendly" materials remain unexplored. To address this, we performed exposures to primary leachates of alternative polymers, and commercial bags to verify possible responses associated with endocrine disruption and/or activation of the detoxification pathway in larvae of the marine fish model Cyprinodon variegatus. The chemical characterization evidenced a great number of additives in the formulation of the materials analyzed in this study. Those include, except for the PLA sample, relevant levels of the hazardous phthalates DEHP and DiBP. Regarding the effects on marine fish larvae, exposure to leachates from alternative polymers (10 g/L) PHB and PHBV produced remarkable mortality (100%). While the exposure to bag leachates of all tested materials (1 and 10 g/L) produced alterations in biomarkers for steroidogenic and detoxification pathways. To a lesser extent (10 g/L), three materials produced significant alterations in estrogenic biomarkers (Home-compostable bag 1, LDPE and Recycled PE bags). Although the alterations in gene expression were not directly correlated to the amount of DEHP or DiBP, we can conclude that primary leachates of "eco-friendly" bags are harmful to marine vertebrates.

Evaluating the alterations of the estrogen-responsive genes in Cyprinodon variegatus larvae for biomonitoring the impacts of estrogenic endocrine disruptors (EEDs).

Currently, endocrine disruptors (EDs) can be found in all the environmental compartments. To understand the effects of estrogenic EDs (EEDs), adults of Cyprinodon variegatus have been classically used as a marine model. However, it is during development that exposure to contaminants may generate permanent consequences. Thus, the aim of this study was to verify the effects produced by acute exposure to 17α-ethinylestradiol (EE2) in C. variegatus larvae. Quantitative PCR (qPCR) results revealed the induction of vtg and zp gene expression on exposure to 1000 ng/L EE2 and the induction of vtgc, zp2, zp3 and cyp19a2, and inhibition of vtgab, wap and cyp1a1 on exposure to 100 ng/L EE2. Lower concentrations inhibited the gene expression of vtgab and wap (50 ng/L), cyp1a1 (25 ng/L) and zp2 (12.5 ng/L). These alterations in gene expression allow us to affirm that larvae of C. variegatus are an efficient and sensitive model for biomonitoring EEDs.

The bisphenol A metabolite MBP causes proteome alterations in male Cyprinodon variegatus fish characteristic of estrogenic endocrine disruption.

The toxicological status of bisphenol A (BPA) is under strong debate. Whereas in vitro it is an agonist of the estrogen receptor with a potency ca. 105-fold lower than the natural female hormone estradiol, in vivo exposure causes only mild effects at concentration thresholds environmentally not relevant and inconsistent among species. By using a proteomic approach, shotgun liver proteome analysis, we show that 7-d exposure to 10 µg/L of the BPA metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), and not the same exposure to the parental molecule BPA, alters the liver proteome of male Cyprinodon variegatus fish. Different physiological and environmental conditions leading to biotransformation of BPA to MBP may partly explain the conflicting results so far reported for in vivo BPA exposures. The pattern of alteration induced by MBP is similar to that caused by estradiol, and indicative of estrogenic endocrine disruption. MBP enhanced ribosomal activity, protein synthesis and transport, with upregulation of 91% of the ribosome-related proteins, and 12 proteins whose expression is regulated by estrogen-responsive elements, including vitellogenin and zona pellucida. Whey acidic protein (WAP) was the protein most affected by MBP exposure (FC = 68). This result points at WAP as novel biomarker for xenoestrogens.

Life-time exposure to waterborne copper III: Effects on the energy metabolism of the killifish Poecilia vivipara.

Copper ions (Cu) are essential to life maintenance, nonetheless, elevated concentrations can be hazardous. Acute and sub-chronic toxic effects of this metal are well known and are usually related to enzymatic inhibition, elevated ROS production and dysfunction of energy metabolism. Despite that, chronic studies are extremely rare. Therefore, the aim of this study was to assess the effects of chronic exposure to 5, 9 and 20 µg/L Cu (28 ad 345 days) on the energy metabolism and survival of the killifish Poecilia vivipara. To accomplish that, we evaluated the activity of enzymes related to aerobic (pyruvate kinase (PK); citrate synthase (CS)) and anaerobic metabolism (lactate dehydrogenase (LDH)) in whole-body (28 days) or in gills, liver and muscle (345 days) of exposed fish. Additionally, whole-body oxygen consumption was evaluated in fish exposed for 28 days and hepatic and muscular expression of genes involved in mitochondrial metabolism (cox I, II and III and atp5a1) was assessed in animals exposed for 345 days. Finally, final survival was evaluated. Following 28 days, Cu did not affect survival neither enzyme activities. However, increased whole-body oxygen consumption was observed in comparison to control condition. After 345 days, 76.8%, 63.9%, 60.9% and 0% survival were observed for control, 5, 9 and 20 µg/L groups, respectively. Animals exposed to 5 and 9 µg/L had a significant reduction in branchial and muscular LDH activity and in hepatic PK activity. Also, exposure to 9 µg/L significantly increased hepatic CS activity. For gene expression, Cu down-regulated muscular cox II (9 µg/L) and III (5 and 9 µg/L), and up-regulated hepatic atp5a1 (9 µg/L). Findings reported in the present study indicate that chronic exposure to Cu induces tissue-specific responses in key aspects of the energetic metabolism. In gills and muscle, Cu leads to reduced energy production through inhibition of anaerobic pathways and mitochondrial respiratory chain. This effect is paralleled by an increased ATP consumption in the liver, characterized by the augmented CS activity and atp5a1 expression. Finally, reduced PK activity indicate that oxidative stress may be involved with the observed outcomes.

Life-time exposure to waterborne copper II: Patterns of tissue accumulation and gene expression of the metal-transport proteins ctr1 and atp7b in the killifish Poecilia vivipara.

The involvement of transporting proteins on copper (Cu) bioaccumulation was evaluated in the killifish Poecilia vivipara chronically exposed to environmentally relevant concentrations of waterborne Cu. Fish (<24 h-old) were maintained under control condition or exposed to different waterborne Cu concentrations (5, 9 and 20 µg/L) for 28 and 345 days in saltwater. Following exposure periods, Cu accumulation and the expression of genes encoding for the high affinity Cu-transporter (ctr1) and the P-type Cu-ATPase (atp7b) were evaluated. Whole-body metal accumulation and gene expression were evaluated in fish exposed to 28 days. Similarly, in fish exposed to 345 days, liver, gills and gut were also evaluated. No fish survival was observed after exposure to 20 µg/L for 345 days. Whole-body Cu accumulation was significantly higher in fish exposed to 20 µg/L Cu for 28 days and in fish exposed to 9 µg/L for 345 days in comparison to control animals. Similarly, tissue Cu accumulation was significantly higher in fish exposed to 9 µg/L for 345 days in comparison to control animal. However, no significant accumulation was observed in fish muscle. Following exposure for 28 days, whole-body ctr1 expression was slightly induced in fish exposed to 9 µg/L. In turn, no significant change in ctr1 expression was observed following exposure to Cu for 345 days. Differently, whole-body atp7b expression was markedly up-regulated in the whole-body of fish exposed Cu for 28 days and in tissues of fish exposed to Cu for 345 days. These findings indicate the expression of atp7b is more responsive to Cu accumulation in P. vivipara than ctr1 expression and, therefore, more suitable to be used as a biomarker of exposure to this metal. Also, we argue that the expression of atp7b is sustained at elevated levels for as much time as fish are maintained in Cu contaminated water.

Metal accumulation and expression of genes encoding for metallothionein and copper transporters in a chronically exposed wild population of the fish Hyphessobrycon luetkenii.

In the present study, metal (As, Cd, Cu, Fe, Mn, Pb and Zn) accumulation and expression of genes involved in metal metabolism (metallothioneins, ATP7A and CTR1) were evaluated in gills and muscle of the fish Hyphessobrycon luetkenii living in the João Dias creek, a site historically (~1870-1996) contaminated with a metal mixture associated with copper mining (Minas do Camaquã, southern Brazil). Fish were collected in a metal impacted site of the João Dias creek and kept in a cage at this site (PP fish) or translocated to a non-metal impacted reach of this creek (PC fish). Gill metal concentrations and metallothionein gene expression were lower in PC fish than in PP fish at any experimental time (24, 48 and 72 h). In muscle, no significant changes were observed. These findings indicate that metal accumulated in gills of wild fish chronically exposed to the metal mixture are more easily excreted than those accumulated in the muscle. In this case, expression of gene encoding for metallothionein is shown to play a key role in the regulation of metal accumulation in gills of H. luetkenii living in an area historically contaminated with a metal mixture associated with copper mining.

Expression of genes related to metal metabolism in the freshwater fish Hyphessobrycon luetkenii living in a historically contaminated area associated with copper mining.

Copper (Cu) mining in Minas do Camaquã-Brazil, released significant amounts of metals into the João Dias creek, where Hyphessobrycon luetkenii inhabit. Because the involvement of Cu in biological processes its concentration and availability is regulated by molecules as the metal regulatory transcription factor (MTF-1), metallothionein (MT) and transporters (ATP7A and CTR1). These genes were whole sequenced and their expression (GE) evaluated in gills, liver and intestine. Were collected fish in non-contaminated and contaminated (Cu 3.4-fold higher) sites of the creek (CC and PP) and respectively translocated (CP and PC) for 96 h. The GE of the non-translocated groups evidenced that MT, MTF-1 and CTR1 have organ specific differences between both communities. Additionally the translocation allowed to identify organ specific changes associated with the activation/inactivation of protective mechanisms. These findings indicate that MTF-1, MT and CTR-1 GE play an important role in the tolerance of H. luetkenii to Cu contamination.

Effects of life-time exposure to waterborne copper on the somatotropic axis of the viviparous fish Poecilia vivipara.

Reduced fish growth following chronic exposure to dissolved copper (Cu) is well reported in the literature. Nevertheless, information on the mechanism(s) involved in this process is scarce. Therefore, we evaluated growth, gene expression and concentrations of proteins related to growth regulation in the viviparous guppy Poecilia vivipara chronically exposed to dissolved Cu. Newborn (<24 h after birth) fish were kept under control conditions or exposed to environmentally relevant concentrations of Cu (5 and 9 µg/L) in salt water (24 ppt) for 345 days. After exposure, fish growth was evaluated based on body weight and length. Also, growth hormone (gh) mRNA expression was evaluated in brain, while growth hormone receptor 1 (ghr1) and 2 (ghr2) mRNA expressions were analyzed in brain, skeletal muscle and liver. In turn, insulin-like growth factor 1 (igf1) and 2 (igf2) mRNA expressions were evaluated in skeletal muscle and liver. Additionally, Gh concentration was assessed in brain, while Ghr concentration was evaluated in skeletal muscle and liver. Exposure to 9 µg/L Cu reduced fish body weigh and length. Metal exposure affected mRNA expression only in skeletal muscle. Reduced ghr2 mRNA expression was observed in guppies exposed to 5 and 9 µg/L Cu. Additionally, reduced igf1 and igf2 mRNA expressions were observed in guppies exposed to 9 µg/L Cu. However, no significant change in Ghr concentration was observed. The reduced ghr2 mRNA expression suggests that chronic Cu exposure induced an insensitivity of the skeletal muscle to Gh, thus resulting in reduced igf1 and igf2 mRNA expression which lead to reduced fish growth. These findings indicate that chronic exposure to dissolved Cu disrupts the somatotropic axis regulation, thus helping to elucidate the mechanism underlying the Cu-dependent inhibition of growth observed in the viviparous fish P. vivipara.

Salinity-dependent copper accumulation in the guppy Poecilia vivipara is associated with CTR1 and ATP7B transcriptional regulation.

Copper (Cu) accumulation and regulation of key-genes involved in Cu homeostasis were evaluated in freshwater- and saltwater-acclimated guppies Poecilia vivipara. Fish were exposed (96h) to environmentally relevant concentrations of dissolved Cu (0, 5.0, 9.0 and 20.0µg/L). In freshwater guppies, gill and liver Cu accumulation was dependent on Cu concentration in the exposure medium. In saltwater guppies, this dependence was observed only in the gut. These findings indicate that Cu accumulation was salinity- and tissue-dependent. Key genes involved in Cu metabolism were sequenced for the first time in P. vivipara. Transcripts coding for the high-affinity copper transporter (CTR1) and copper-transporting ATPase (ATP7B) were identified using polymerase chain reaction (PCR) and gene sequencing. The full-length CTR1 open reading frame (1560bp) and a partial ATP7B (690bp) were discovered. Predicted amino acid sequences shared high identities with the CTR1 of Fundulus heteroclitus (81%) and the ATP7B of Sparus aurata (87%). Basal transcriptional levels addressed by RT-qPCR in control fish indicate that CTR1 and ATP7B was highly transcribed in liver of freshwater guppies while CTR1 was highly transcribed in gut of saltwater guppies. This could explain the higher Cu accumulation observed in liver of freshwater guppies and in gut of saltwater guppies, because CTR1 is involved in Cu uptake. Reduced gill mRNA expression of CTR1 was observed in freshwater guppies exposed to 20.0µg/L Cu and in saltwater guppies exposed to 5.0µg/L Cu. In turn, reduced mRNA expression of gut ATP7B was observed in freshwater and salt water guppies exposed to 9.0 and 20.0µg/L Cu. Liver CTR1 and ATP7B transcription were not affected by Cu exposure. These findings suggest that gill CTR1 and gut ATP7B are down-regulated to limit Cu absorption after exposure to dissolved Cu, while liver CTR1 and ATP7B levels are maintained to allow Cu storage and detoxification. In conclusion, findings reported here indicate that Cu accumulation in the euryhaline guppy P. vivipara is tissue specific and dependent on water salinity. They also suggest that Cu homeostasis involves a differential transcriptional regulation of the newly identified Cu transporters, CTR1 and ATP7B.

Gasdermin-B promotes invasion and metastasis in breast cancer cells.

Gasdermin B (GSDMB) belongs to the Gasdermin protein family that comprises four members (GSDMA-D). Gasdermin B expression has been detected in some tumor types such as hepatocarcinomas, gastric and cervix cancers; and its over-expression has been related to tumor progression. At least four splicing isoforms of GSDMB have been identified, which may play differential roles in cancer. However, the implication of GSDMB in carcinogenesis and tumor progression is not well understood. Here, we uncover for the first time the functional implication of GSDMB in breast cancer. Our data shows that high levels of GSDMB expression is correlated with reduced survival and increased metastasis in breast cancer patients included in an expression dataset (>1,000 cases). We demonstrate that GSDMB is upregulated in breast carcinomas compared to normal breast tissue, being the isoform 2 (GSDMB-2) the most differentially expressed. In order to evaluate the functional role of GSDMB in breast cancer two GSDMB isoforms were studied (GSDMB-1 and GSDMB-2). The overexpression of both isoforms in the MCF7 breast carcinoma cell line promotes cell motility and invasion, while its silencing in HCC1954 breast carcinoma cells decreases the migratory and invasive phenotype. Importantly, we demonstrate that both isoforms have a differential role on the activation of Rac-1 and Cdc-42 Rho-GTPases. Moreover, our data support that GSMDB-2 induces a pro-tumorigenic and pro-metastatic behavior in mouse xenograft models as compared to GSDMB-1. Finally, we observed that although both GSDMB isoforms interact in vitro with the chaperone Hsp90, only the GSDMB-2 isoform relies on this chaperone for its stability. Taken together, our results provide for the first time evidences that GSDMB-2 induces invasion, tumor progression and metastasis in MCF7 cells and that GSDMB can be considered as a new potential prognostic marker in breast cancer.