Toxicological Effects of Thimerosal and Aluminum in the Liver, Kidney, and Brain of Zebrafish (Danio rerio).

Vaccination programs in the first years of a child's life are effective and extremely important strategies for the successful eradication of diseases. However, as no intervention is without risks, the metal-based components of some vaccines, such as thimerosal (TMS), a preservative composed of ethylmercury, and aluminum (Al), have begun to generate distrust on the part of the population. Therefore, this study evaluated the effects of exposure to thimerosal and aluminum hydroxide (alone or in mixture) on Danio rerio (zebrafish) specimens. The fish were exposed to thimerosal and/or aluminum hydroxide intraperitoneally. The liver, kidney, and brain were removed for a biochemical biomarker analysis, histopathological analysis, and metal quantification. As a result, we observed changes in the activity of the analyzed enzymes (SOD, GST, GPx) in the kidney and brain of the zebrafish, a reduction in GSH levels in all analyzed tissues, and a reduction in MT levels in the kidney and liver as well as in the brain. Changes in AChE enzyme activity were observed. The biochemical results corroborate the changes observed in the lesion index and histomorphology sections. We emphasize the importance of joint research on these compounds to increase the population's safety against their possible toxic effects.

Distinct macrophage phenotypes and redox environment during the fin fold regenerative process in zebrafish.

In contrast to mammals, zebrafish (Danio rerio) has the ability to regenerate injured sites such as different tissues present in the fin. It is known that cells of the innate immune system play essential roles in regeneration; however, some aspects of the molecular mechanisms by which these cells orchestrate regeneration remain unknown. This study aimed to evaluate the infiltration dynamics of neutrophils and macrophages in the regenerative process of fin fold in regard to the influence of the redox environment and oxidative pathways. Fin fold amputation was performed on transgenic larvae for macrophage-expressed gene 1 (mpeg1), lysozyme (lyz), myeloperoxidase (mpo) and tumour necrosis factor alpha (TNFα) at 3 days post-fertilization, followed by confocal microscopy imaging and measurement of the activities of oxidant and antioxidant enzymes. We observed initially an increase in the number of neutrophils (lyz:DsRed+/mpx:GFP+) and then macrophages (mpeg1+) in the injury site followed by a decrease in neutrophils at 7 days post-amputation (dpa). Moreover, macrophages switch from a pro-inflammatory to an anti-inflammatory profile throughout the process, while the activity of superoxide dismutase (SOD) increased at 1 dpa and catalase (CAT) at 5 dpa. Higher levels of lipid peroxidation were also detected during regeneration. Despite oxidative stress, there is, therefore, an antioxidant response throughout the regeneration of the caudal fin. The present work can contribute to future studies on the development of cell therapies, achieving greater effectiveness in the treatment of diseases related to the formation of fibrotic tissue.

Tissue-specific genotoxicity and antioxidant imbalance of titanium dioxide nanoparticles (NPTiO2) and inorganic lead (PbII) in a neotropical fish species.

The aquatic environment is the major recipient of wastes containing nanoparticles and other contaminants. Titanium dioxide nanoparticles (NPTiO2) are one of the most produced and used nanoparticle worldwide. This study investigated the toxicity of NPTiO2, as well as the toxicity interaction between NPTiO2 and lead (Pb), in response to genetic and biochemical biomarkers using freshwater fish Rhamdia quelen, as an animal model. The results showed genotoxicity in blood and kidney tissues. No effect of NPTiO2 alone or in co-exposure with Pb on liver genotoxicity were observed. Alterations in the antioxidant hepatic enzymes activities, as well as alterations in glutathione levels indicated that NPTiO2 alone or in co-exposure with Pb can cause antioxidant imbalance. The lipid peroxidation was also raised after exposure to NPTiO2. In general, the results of this study indicated that both NPTiO2 alone and their co-exposure with Pb are capable of producing significant toxic effects in short-term exposure.

Toxin accumulation, detoxification and oxidative stress in bivalve (Anomalocardia flexuosa) exposed to the dinoflagellate Prorocentrum lima.

Prorocentrum lima is a cosmopolitan benthic dinoflagellate capable of producing the diarrhetic shellfish toxins (DSTs) okadaic acid (OA) and dinophysistoxin (DTX). These compounds may cause oxidative stress and accumulate in bivalve tissues, which become vectors of intoxication to human consumers. We investigated DST accumulation, detoxification and oxidative stress biomarkers in clams (Anomalocardia flexuosa) experimentally exposed to P. lima cells or their compounds. Experimental diets consisted of 6000 cells mL-1 of the non-toxic chlorophyte Tetraselmis sp. (C; control condition), and combinations of C with 10 P. lima cells mL-1 (T10), 100 P. lima cells mL-1 (T100), or to a toxin concentration of ∼4 µg OA L-1 and ∼0.65 µg DTX-1 L-1 (T100d). Clams were exposed to these diets for 7 days (uptake phase), followed by a 7-day depuration period. No DSTs were detected in clams exposed to treatments C (control) nor to T100d (dissolved compounds) during either uptake or detoxification phase. Conversely, clams exposed to T10 or T100 accumulated, on average, up to 2.5 and 35 µg DST kg-1 in their whole bodies at the end of the uptake phase. These concentrations are ∼64 and ∼4.5 times lower than the regulatory level of 160 µg OA kg-1, respectively. Accumulated OA quotas were 12-22 times higher in the digestive gland (DG) than in remaining tissues over the uptake phase. Quick toxin transformation was indicated by the early detection of conjugated compounds - DTX-1 and OA esters - in the DG after 6 h of exposure, with OA-ester representing the main compound (30 - 100 %) in that tissue over the experiment. During the depuration period, detoxification rates represented 0.024 h-1, 0.04 h-1 and 0.052 h-1 for OA, DTX-1 and OA-ester, respectively. The activities of catalase, glutathione S-transferase, glutathione peroxidase and the levels of oxidative stress by lipoperoxidation varied similarly in the DG of A. flexuosa individuals subjected to T100, T100d and the control condition. However, contrasting antioxidant responses were measured in those exposed to T10. These findings indicate that no oxidative stress was primarily induced by DST-producing dinoflagellates in this clam species under laboratory conditions representative of toxic bloom situations. Even though, possible interactions should be considered under multistressor scenarios.

Multiple biomarkers response in a Neotropical fish exposed to paralytic shellfish toxins (PSTs).

The Alagados Reservoir (Southern Brazil) is used as water supply, and since 2002 there have been reports with a presence of cyanobacterial blooms and cyanotoxins. In order to assess the water quality and the ecological integrity of the reservoir, we evaluated biochemical, genotoxic and osmoregulatory biomarkers in the freshwater cichlid fish (Geophagus brasiliensis) that were exposed to PSTs. The fish were sampled in the Alagados Reservoir in February 2016 (Summer) and were divided in three groups: 1) Reservoir group (RES): fish were collected immediately after sampling; 2) Depuration group (DEP): fish were submitted to the depuration experiment for 90 days in the laboratory; and 3) Reproduction group (REP): fish were kept in the laboratory until the fertilization and the chemical analyses were performed on the offspring (F1 generation). In the RES and DEP the blood, brain, muscle, liver and gills were collected for biochemical, genotoxic and osmoregulatory biomarkers analysis. Our results showed that the fish from the Alagados Reservoir (RES) presented oxidative stress and DNA damage; and after 90 days (DEP), the antioxidant system and DNA damage were recovered. Although PSTs were considered a risk to the ecological integrity of this water body; PSTs concentrations were not found in the tissues of the F1 generation. In addition, the biomarkers used were useful tools to evaluate the effects of environment contamination. Therefore, it is necessary to develop new technologies and monitoring programs in order to reduce cyanobaterial blooms, cyanotoxins and human activities that cause the contamination in aquatic environments.

Sublethal effects of microcystin-LR in the exposure and depuration time in a neotropical fish: Multibiomarker approach.

Eutrophication is an ecological process that results in cyanobacterial blooms. Microcystin-LR is the most toxic variant of microcystins and may cause toxic effects in the organisms, mainly in hepatic tissues. The aims of this study were to use multiple biomarkers in order to evaluate the sublethal effects of a low concentration of MC-LR (1 µg/L) in fish Geophagus brasiliensis by waterborne exposure; and evaluate the depuration of this toxin during 15 days. A group of 30 fish was exposed to 1 µg/L of MC-LR solution for 96 h in a static bioassay. After this time, blood, brain, muscle, liver, gonad and gills were collected from half of the exposed fish group in order to evaluate chemical, biochemical, histological and genotoxic biomarkers. The rest of the fish group was submitted to the depuration experiment with free MC-LR water for 15 days. After this time the same tissues were collected and evaluated using biomarkers analysis. Toxic effects were found mostly in the fish liver from depuration time as alterations on the antioxidant system and histopathologies. The results showed that even low concentrations can cause sublethal effects to aquatic organisms, and cyanotoxins monitoring and regulation tools are required.

The accumulation dynamics, elimination and risk assessment of paralytic shellfish toxins in fish from a water supply reservoir.

Paralytic shellfish Toxins (PSTs) or saxitoxins are neurotoxins that block the neural transmission by binding to the voltage-gated sodium channels in the nerve cells. There are >50 analogues described, which could be biotransformed into a molecular form of greater or lesser toxicity. The Alagados Reservoir is used for water supply, and persistent cyanobacterial blooms as well as PSTs concentrations have been found in this water body since 2002. The aims of this study were to quantify the concentrations of PSTs in the water and fish samples from the Alagados Reservoir. In addition, we evaluated the elimination of PSTs for 90 days in fish and estimated the potential risk to human health. Water and fish samples were collected from the reservoir. For the water samples the phytoplankton and chemical analyses were carried out. Fish were divided into two sample times: Field Samples (FS) and Elimination Experiment Samples (EES), which were maintained for 90 days in filtered and dechlorinated water. For chemical analysis, the muscles of FS were collected on the fish sampling day and the muscles and feces of EES were collected at 7, 15, 30, 45, 60, 75 and 90 days. PSTs concentrations were present in water and fish samples, and they were estimated as a potential risk to humans; mainly for children. In addition, toxins were accumulated, biotransformed to other analogues and excreted by the fish. However, after 90 days, the toxins were still present in the water and fish muscle. Therefore, PSTs can remain for a long period in water, and fish can be a carrier of these neurotoxins. New approaches of monitoring and management are necessary in the actual global context of cyanobacteria and cyanotoxins.

Depuration time and sublethal effects of microcystins in a freshwater fish from water supply reservoir.

Microcystins (MCs) are hepatotoxins that have been considered to be a worldwide problem due the effects that they can cause to environmental and human health systems. The Iraí Reservoir, located in the South of Brazil, is used as a water supply and MCs concentrations have been reported in this ecosystem. This study aimed to determine the MCs concentrations in the Iraí Reservoir and to evaluate the MCs depuration time and the health of Geophagus brasiliensis using biomarkers. Water and fish samples were collected in the Iraí Reservoir from August 2015 to May 2016. Phytoplankton and chemical analyses were conducted using water samples and the fish were divided into two groups; the Immediate Group (IMM) and the Depuration Group (DEP). In the IMM group, the blood, liver, muscle, brain and gills were collected, in order to evaluate the genotoxic, biochemical and chemical biomarkers. The DEP group was used in the depuration experiment for 90 days, and after this period the fish were submitted to the same procedure as the IMM group. Our results suggested that fish accumulated MCs and it may have caused oxidative stress, neurotoxicity and molecular damage. Furthermore, MCs concentrations increased during the depuration time and it resulted in molecular damage over the first 30 days. After 90 days, the recovery of the antioxidant system occurred. The depuration started on the 15th day, however, the toxins were still present in the samples. Therefore, the effects and the persistence of MCs are a risk to environmental systems and human health.

Neurotoxins in a water supply reservoir: An alert to environmental and human health.

Reservoirs are important source of power generation, recreation, and water supply. Nevertheless, human activities have favored the bloom of toxic cyanobacteria in many reservoirs, which has resulted in environmental, social, and economic problems. This study aims to evaluate the water quality of a reservoir in South Brazil through the analysis of cyanobacteria and cyanotoxins PSTs (Paralytic Shellfish Toxins) and biomarkers of environmental contamination in fish. For this purpose, water samples and fish (Geophagus brasiliensis) (Perciformes: Cichlidae) were collected from September 2013 to May 2014. The fish G. brasiliensis were separated in two groups. The first one "site group" was euthanized after the sampling and their weight and length were measured. The blood, brain, muscle and liver were collected for chemical, biochemical and genetics biomarkers analysis. The second group "depuration group" was submitted to depuration experiment for 40 days in clean water. After that, the same procedures as for the first group were carried out. Cylindrospermopsis raciborskii was the dominant cyanobacteria found in the reservoir, and it showed a density above the recommended limit by Brazilian legislation of 20,000 cells/mL. Results showed that the fish accumulate PSTs in the Reservoir and these were not eliminated after 40 days. The biochemical and genotoxic biomarkers showed a significant difference between "site groups" and "depuration groups", which suggests a recovery of the antioxidant system and a reduction of cellular damage after 40 days in clean water. In conjunction with results reported earlier by others, Alagados Reservoir, in South Brazil, appears to have a persistent contamination of cyanotoxins. Moreover, the mixture of contaminants which may be present in the water body can explain the seasonal differences in fish at the sampled points.