Urban effluents affect the early development stages of Brazilian fish species with implications for their population dynamics.

The pollution from urban effluents discharged into natural waters is a major cause of aquatic biodiversity loss. Ecotoxicological testing contributes significantly to understand the risk of exposure to the biota and to establish conservation policies. The objective of the current study was to assess the toxicity of a river highly influenced by urban effluents (Atuba River, Curitiba city, Southern Brazil) to the early stages of development in four South American native fish species, investigating the consequences at the population level through mathematical modelling. The species chosen were Salminus brasiliensis, Prochilodus lineatus, Rhamdia quelen, and Pseudoplatystoma corruscans, ecologically important species encompassing different conservation statuses and vulnerability. The embryos were exposed from 8 to 96 h post fertilization to the Atuba River water, collected downstream of the largest wastewater treatment plant in the Metropolitan Region of Curitiba, and their survival rates and deformities were registered. The species S. brasiliensis and P. lineatus presented the highest mortality rates, showing high sensitivity to the pollutants present in the water. According to the individual-based mathematical model, these species showed high vulnerability and risk of extinction under the tested experimental conditions, even when different sensitivity scenarios of juveniles and adults were considered. The other two species, R. quelen and P. corruscans, showed a more resistant condition to mortality, but also presented high frequency and severity of deformities. These results emphasize the importance of testing the sensitivity of different Brazilian native species for the conservation of biodiversity and the application of models to predict the effects of pollutants at the population level.

Low concentration of 2,4,6-tribromophenol (TBP) represents a risk to South American silver catfish Ramdia quelen (Quoy and Gaimard, 1824) population.

The 2,4,6-tribromophenol (TBP) is an environmental persistent pollutant widely used as flame retardant, antimicrobial and insecticide agent in wood preservation and plastic production. Currently, TBP is found in environmental compartments such as soil, freshwater, groundwater, sewage sludge and domestic dust, but the effects to biota and the risk of exposure to aquatic vertebrates are still scarce. In the present study, Rhamdia quelen fish embryos (8 h post-fertilization - hpf) were exposed to 0.3 and 3.0 µg L-1 of TBP until 96 hpf. Biochemical biomarkers, hatching, survival and larvae/embryo malformations were evaluated after exposure. Additionally, a mathematical model was proposed to evaluate the effects along further generations. The results showed that TBP decreased the survival level but did not cause significant difference in the hatching rates. After 72 and 96 hpf, individuals from the highest tested concentration group showed more severe malformations than individuals from control and the lower concentrations groups. The deformities were concentrated on the embryos facial region where the sensorial structures related to fish behavior are present. The biochemical biomarkers revealed both oxidative stress and neurotoxicity signs after exposure to the contaminant, while the application of the mathematical model showed a decrease of population in both tested TBP concentrations. In conclusion, the current results demonstrated that TBP is toxic to R. quelen embryos and represents a risk to population after early life stage exposure.

Malignancy and tumorigenicity of melanoma B16 cells are not affected by silver and gold nanoparticles.

Gold (AuNP) and silver (AgNP) nanoparticles have been incorporated into many therapeutic and diagnostic applications. However, previous studies revealed toxic properties as well as the hormesis phenomenon of many nanoparticles in different biological models. To evaluate the effects of low concentrations of AuNP and AgNP on murine melanoma cells B16F1 and B16F10 and relate them with phenotype changes, cells were exposed for 24 and 48 h. No cytotoxicity was observed for B16 cells through neutral red, MTT, trypan blue, and crystal violet assays at concentrations from 0.01 to 10 ng mL-1. Likewise, the nanoparticles did not interfere with drug-efflux activity, cell migration, cell cycle, and colony formation. Slight toxicity was observed for B16F10 exposed to 100 ng mL-1, with a decreased number of viable and attached cells, indicating differential sensitivity of B16F1 and B16F10 cells to the nanoparticles. Furthermore, colony size dispersion decreased for both B16 cell sub-lines. Therefore, there is no evidence that the tested concentrations of AuNP and AgNP can render B16 cells more aggressive and malignant, which is important since both nanoparticles are already largely used in nanotechnological products. Considering studies that have showed the hormesis effect of nanoparticles at low concentrations, which could protect cancer cells against chemotherapy, further investigation is advised.

Responses of human hepatoma HepG2 cells to silver nanoparticles and polycyclic aromatic hydrocarbons.

The nanotechnology has revolutionized the global market with silver nanoparticles (AgNP) occupying a prominent position due to their remarkable anti-bacterial properties. However, there is no data about the adverse and toxic effects of associations of AgNP and ubiquitous compounds, such as polycyclic aromatic hydrocarbons (PAH). In the current study, we investigated the responses of HepG2 cells to realistic concentrations of AgNP (0.09, 0.9, and 9 ng ml-1) and mixture of PAH (30 and 300 ng ml-1), separately and in association. Cell viability and cytotoxicity (neutral red retention and MTT production assays) and proliferation (crystal violet [CV] assay), xenobiotic efflux transporter activity (rhodamine B accumulation assay), ROS levels (dichlorodihydrofluorescein diacetate assay), and lipid peroxidation (pyrenylphosphine-1-diphenyl assay) were analyzed. There was no decreases of cell viability after exposure to AgNP, PAH and most of AgNP + PAH associations, but increases of cell viability/number (CV assay) occurred. Efflux transporter activity was not affected, with exception of one AgNP + PAH associations, ROS levels increased, but lipid peroxidation decreased. Some toxicological interactions occurred, particularly for the highest concentrations of AgNP and PAH, but there is no evidence that these interactions increased the toxicity of AgNP and PAH.

Glyphosate and aminomethylphosphonic acid metabolite (AMPA) modulate the phenotype of murine melanoma B16-F1 cells.

Pesticides are contaminants run-offs from agricultural areas with a global concern due to their toxicity for non-target organisms. The Brazilian Health Surveillance Agency reported about 63% of the food contain pesticide residues. Glyphosate is a herbicide used worldwide but its toxicity is not a consensus among specialists around the world. AMPA (aminomethylphosphonic acid) is a glyphosate metabolite that can be more toxic than the parental molecule. Melanoma murine B16-F1 cells were exposed to glyphosate and AMPA to investigate the cell profile and possible induction to a more malignant phenotype. Glyphosate modulated the multi-drug resistance mechanisms by ABCB5 gene expression, decreasing cell attachment, increasing cell migration and inducing extracellular vesicles production, and the cells exposed to AMPA revealed potential damages to DNA. The present study observed that AMPA exhibits high cytotoxicity, which suggests a potential impact on non-tumor cells, which are, in general, more susceptible to chemical exposure. Conversely, glyphosate favored a more metastatic and chemoresistant behavior in cancer cells, highlighting the importance of additional research in this area.

BDE-209 exposure in murine melanoma (B16-F1) cells modulates tumor malignancy and progression in vivo.

Melanoma is a type of skin cancer considered aggressive due to its high metastatic ability and rapid progression to other tissues and organs. BDE-209 (2,2',3,3',4,4',5,5',6,6'-decabromodiphenyl ether) is an additive used as a flame retardant and classified as a persistent organic pollutant that has a high bioaccumulation capacity due to its lipophilic nature. This substance has already been detected in rivers, air, soil, plants and even in different human biological samples, such as plasma, umbilical cord blood and breast milk, revealing a great concern to human populations. Thus, in the current study we investigated whether prior exposure of murine melanoma B16-F1 cells to BDE-209 modulates in vivo progression and malignancy of melanoma. B16-F1 cells were cultured and exposed in vitro to BDE-209 (0.01, 0.1 e 1 nM) for 15 days and then inoculated, via caudal vein, in C57BL/6 mice for experimental metastasis analysis after 20 days. Inoculation of BDE-209-exposed cells resulted in 82% increase of metastasis colonized area in the lungs of mice, downregulation of tumor suppressors genes, such as Timp3 and Reck, decrease of lipid peroxidation and increase of systemic and local inflammatory response. These findings are related to melanoma progression. Additionally, the histopathological analysis revealed greater number of focal points of metastases in the lungs and invasiveness of metastases to the mice brain (89%). The results showed that exposure to BDE-209 may alter the phenotype of B16-F1 cells, worsening their metastatic profile. Current data showed that BDE-209 may interfere with the prognosis of melanoma by modulating cells with less invasiveness capacity to a more aggressive profile.

Pollutant bioaccumulation in sentinel fish chronically exposed in Iguaçu river reservoirs (Southern Brazil) and human health risk of fish consumption.

Bioaccumulation studies in fish mark the initial phase of assessing the risk of chemical exposure to biota and human populations. The Iguaçu River boasting a diverse endemic ichthyofauna, is grappling with the repercussions of human activities. This study delved into the bioaccumulation of micropollutants, the early-warning effects on Rhamdia quelen and Oreochomis niloticus in the Segredo Reservoir (HRS) and the potential risk of human exposure. Two groups of caged fish in three sites of the reservoir were exposed during the autumn-winter and spring-summer, while a third group (O. niloticus) underwent a twelve-month exposure, and inorganic and organic chemicals analysis in water, sediment, and biota. Additionally, metallothionein expression and genotoxicity were employed as biomarkers. PAHs, PCBs, Al, Cu, Fe, and As in water and DDTs, Cu, Zn, and As in sediment surpassed the thresholds set by Brazilian regulations, where DDT exhibited bioaccumulation in muscle, alongside metals in liver, kidney, gills, and muscle tissues. R. quelen showed metallothionein expression whereas DNA damage and NMA frequencies were elevated in target organs and in brain and erythrocytes of O. niloticus during summer. In this species the DNA damage in liver was remarkable after twelve months. Target Hazard Quotients and Cancer Risk values shedding light on the vulnerability of both children and adults. The reservoir's conditions led to heightened sensitivity to micropollutants for R. quelen species. The data presented herein provides decision-makers with pertinent insights to facilitate effective management and conservation initiatives within the Iguaçu Basin.

Toxicity of glyphosate and aminomethylphosphonic acid (AMPA) to the early stages of development of Steindachneridion melanodermatum, an endangered endemic species of Southern Brazil.

This study aimed to evaluate glyphosate (GLY) and aminomethylphosphonic acid (AMPA) toxicity at 65, 650, and 6500 µg L-1 to the initial stages of development of Steindachneridion melanodermatum, an endangered endemic species from the Iguaçu River, assessing hatching, survival, total larval length, deformities, oxidative stress biochemical biomarkers, and neurotoxicity. Overall, looking at the sum of responses through the integrated biomarker response, the species was more sensitive to AMPA than GLY, especially at the lower concentration of 65 µg L-1, which induced mortality, deformities, underdevelopment, and oxidative stress. Considering the risk of exposure and the importance of conservation of the highly endemic ichthyofauna of this basin, it is urgent to investigate and regulate both GLY and AMPA levels at the Iguaçu River to protect not only this species, but the entire ecosystem.

Immunotoxicity of relevant mixtures of pesticides and metabolites on THP-1 cells.

Pesticides are used to combat agricultural pests but also trigger side effects on non-target organisms. Particularly, immune system dysregulation is a major concern due to the organism's increased vulnerability to diseases, including cancer development. Macrophages play essential roles in innate and adaptive immunity and can undergo classical (M1) or alternative (M2) activation. The M1 pro-inflammatory phenotype has an antitumor role, while M2 favors tumor promotion. Although previous studies have linked pesticide exposure to immune compromise, macrophage polarization is still poorly studied. Here, we investigated the effects of 72 h-long exposure to the mixture of four pesticides widely used in Brazil (glyphosate, 2,4-D, mancozeb, and atrazine), and their main metabolites (aminomethylphosphonic acid, 2,4-diclorophenol, ethylenethiourea, and desethylatrazine) on human leukemia monocytic THP-1 cell line at concentrations based on the Acceptable Daily Intake (ADI) value established in the country. The data revealed immunotoxicity related to impaired cell metabolism in all exposed groups, decreased cell attachment (Pes: 10-1; Met: 10-1; Mix: all concentrations), and disturbance in nitric oxide (NO) levels (Met: 10-1, 101; Mix: all concentrations). The polarization of macrophages towards a more pro-tumor M2-like phenotype was also supported by decreased secretion of the pro-inflammatory cytokine TNF-α (Pes 100, 101) and increased IL-8 (Pes 101). These outcomes alert about the risk of pesticide exposure in the Brazilian population.

Organic and inorganic pollutants in Jordão and Iguaçu rivers southern Brazil impact early phases of Rhamdia quelen and represent a risk for population.

The Iguaçu River basin presents high ecological importance due to its expressive endemic ichthyofauna rate, but chemical pollution may threat this biodiversity. Jordão River is one of the main tributaries of Iguaçu River and contribute to this pollution status, since it drains large agricultural areas receiving domestic and industrial effluents before flowing into the Iguaçu River. The objective of the current study was to evaluate the toxic effects of the Iguaçu, Jordão, and the combination of their waters to the embryo-larval phase of R. quelen, investigating the consequences to the population by means of mathematical modelling. R. quelen fertilized eggs were exposed for 96 h to water samples from Iguaçu River upstream (IR), Jordão River (JR), and downstream of both rivers (MR). The analysis of micropollutants in the water showed that JR presented the most complex mixture of substances and elements, followed by IR, while MR showed the lower number of micropollutants detected. Survival rate was not a sensitive endpoint, while the deformity indices were higher in individuals exposed to water from the three studied sites. Superoxide dismutase activity was increased in MR, while non-protein thiol levels were reduced in MR and JR showing the antioxidant mechanism activation. The mathematical modelling revealed that fish exposed to JR would lead to the greater population reduction (46.19%), followed by IR (40.48%) and MR (33.33%). Although the results showed toxicity in all studied sites, the JR site is the most impacted by micropollutants but decrease its toxicity after dilution with Iguaçu River.

BDE-209 and TCDD enhance metastatic characteristics of melanoma cells after chronic exposure.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and BDE-209 (decabromodiphenyl ether) are persistent organic pollutants (POPs) produced by industrial activities and associated with several diseases. TCDD is a known human carcinogen, but few studies investigated about the effects of exposure to both compounds, i.e., whether BDE-209 and TCDD can render tumor cells more aggressive and metastatic. In the current study we investigated if the exposure of B16-F1 and B16-F10 melanoma murine cells to environmental relevant concentrations of TCDD and BDE-209 at 24 h and 15-day exposure modulates the expression of genes related to metastasis, making the cells more aggressive. Both pollutants did not affect cell viability but lead to increase of cell proliferation, including the upregulation of vimentin, MMP2, MMP9, MMP14 and PGK1 gene expression and downregulation of E-cadherin, TIMP2, TIMP3 and RECK, strongly suggesting changes in cell phenotypes defined as epithelial to mesenchymal transition (EMT) in BDE-209 and TCDD-exposed cells. Foremost, increased expression of metalloproteinases and decreased expression of their inhibitors made B16-F1 cells similar the more aggressive B16-F10 cell line. Also, the higher secretion of extracellular vesicles by cells after acute exposure to BDE-209 could be related with the phenotype changes. These results are a strong indication of the potential of BDE-209 and TCDD to modulate cell phenotype, leading to a more aggressive profile.

2,4,6-Tribromophenol is toxic to Oreochromis niloticus (Linnaeus, 1758) after trophic and subchronic exposure.

The presence of 2,4,6-Tribromophenol (TBP) in the environment increased the risk of exposure to aquatic organisms affecting the animal development or metabolism. The current study investigated the low, subchronic and trophic effect of TBP in both, male and female adult of Oreochromis niloticus. The fish were exposed to 0.5 or 50 ng g-1 of TBP every ten days for 70 days. Then, hepatosomatic (HSI) and gonadosomatic (GSI) indexes, erythrocyte parameters (hemoglobin content, nuclear morphology and morphometrical abnormalities), biochemical endpoints (glutathione S-Transferase and catalase activities, non-protein thiols, lipid peroxidation and protein carbonylation levels in the liver; and acetylcholinesterase activity in the brain and muscle), histopathological analysis (liver) and vitellogenin levels (plasma) were considered. TBP affected the HSI in male and female fish, but not the GSI. Principal Component Analysis revealed that erythrocytes from males are more sensitive to TBP exposure. Likewise, TBP induced the expression of vitellogenin, CAT activity and liver lesion in male fish comparatively with control group, but GST and NPT were influenced only by sex. Finally, the results showed that the antioxidant mechanism and cholinesterase activity effects were more pronounced in male than in female. The current data shows evidences of estrogenic endocrine disruption and toxicity in O. niloticus exposed to TBP, revealing the risk of exposure to biota.

Morphological evidence of neurotoxic effects in chicken embryos after exposure to perfluorooctanoic acid (PFOA) and inorganic cadmium.

Perfluorooctanoic acid (PFOA) and cadmium (Cd) are persistent environmental pollutants, bioaccumulative, and capable of crossing the placental barrier. There is evidence of developmental toxicity induced by PFOA and Cd, but their effects on early development are still poorly understood. In the present study, chicken embryos were exposed to PFOA, Cd, and a mixture of PFOA + Cd from blastula stage up to 72 h of incubation. Embryo mortality and morphology were assessed. PFOA and Cd exposure increased mortality and induced malformations, mainly in the cephalic region, the neural tube, and the ventral body wall. After mixed exposure the effect was observed mostly in the neural tube and the cephalic region. However, the mechanism remains unknown. Because of the resemblance between human and chick development, it was possible to correlate the observed alterations in chickens with known human congenital malformations. Therefore, PFOA and Cd are neurotoxic to chicken embryos inducing morphological abnormalities and are possibly neurotoxic to human embryos.

Tribromophenol affects the metabolism, proliferation, migration and multidrug resistance transporters activity of murine melanoma cells B16F1.

Murine melanoma cells B16F1 were exposed to the flame retardant and wood preservative chemical 2,4,6-tribromophenol (TBP) during 24 and 48 h, at the concentrations found in human diet. TBP-exposed cells had increased MTT and Alamar blue® metabolism and ABCB5 mRNA levels (qPCR), but the cells had decreased proliferation (crystal violet assay), migration (scratch assay), and drug-effux transporters activity (rhodamine B efflux assay). Exposure to TBP did not affect the cell viability (neutral red and annexin V-PI assays), colony formation (colony number, clonogenic assay), and the levels of reactive oxygen species (DCF probe) or P53 mRNA (qPCR). The tested TBP concentrations had low toxicity to melanoma cells B16F1. However, dual effect on metastatic profile and chemoresistance suggests that the increase of ABCB5 positively modulates the cell chemoresistance, but decreases cell migration and proliferation. These findings may be explored in cancer therapy.

Metals and arsenic in fish from a Ramsar site under past and present human pressures: Consumption risk factors to the local population.

The risk of metals and As in seafood for traditional populations living in a Marine Protected Areas (MPA) is seldom assessed, although the risk of human exposure to contaminants is one of the indicators associated with the socioeconomic goals of MPAs. The current study aimed to estimate the potential risk of some metals (Cd, Pb, and Zn) and arsenic (As) for human health through the ingestion of fish locally harvested in a Ramsar site, the Cananéia-Iguape-Peruíbe Environmental Protected Area (APA-CIP). Previous studies showed environmental impacts in this area due to former mining activities and urbanization. Cathorops spixii, a catfish largely consumed by the local population, was collected along the estuary in three seasons with different rain regimes. Metals and As loads in muscle tissue were quantified and it was estimated (i) the target hazard quotient (THQ) and (ii) the daily intake (EDI) for metals and As, (iii) the cancer risk (CRisk) only for As, and (iv) the number of eligible meals per month. Cd, Pb, and As were found at concentrations above action levels for human consumption. Depending on the level of exposure of the local population, the consumption of C. spixii may pose risk to human health. Highest THQs were estimated for fish collected in sites closer to the main contamination sources in the APA-CIP, i.e. the mouth of Ribeira de Iguape River (P1) and the city of Cananéia (P4, P5, and P6). Arsenic showed high levels of cancer risk, although restricted to the area close to the city. The exposure of the local population to metal and As contaminated seafood cannot be disregarded in environmental studies and management of the APA-CIP.