Hydrogel-Containing Biogenic Silver Nanoparticles: Antibacterial Action, Evaluation of Wound Healing, and Bioaccumulation in Wistar Rats.

Wound infections are feared complications due to their potential to increase healthcare costs and cause mortality since multidrug-resistant bacteria reduce treatment options. This study reports the development of a carbomer hydrogel containing biogenic silver nanoparticles (bioAgNPs) and its effectiveness in wound treatment. This hydrogel showed in vitro bactericidal activity after 2 h, according to the time-kill assay. It also reduced bacterial contamination in rat wounds without impairing their healing since the hydrogel hydrophilic groups provided hydration for the injured skin. The high number of inflammatory cells in the first days of the skin lesion and the greater degree of neovascularization one week after wound onset showed that the healing process occurred normally. Furthermore, the hydrogel-containing bioAgNPs did not cause toxic silver accumulation in the organs and blood of the rats. This study developed a bioAgNP hydrogel for the treatment of wounds; it has a potent antimicrobial action without interfering with cicatrization or causing silver bioaccumulation. This formulation is effective against bacteria that commonly cause wound infections, such as Pseudomonas aeruginosa and Staphylococcus aureus, and for which new antimicrobials are urgently needed, according to the World Health Organization's warning.

What happens to Hoplias malabaricus fed on live prey (Astyanax altiparanae) previously exposed to copper? A multiple biomarker approach.

Copper waterborne toxicity is well understood in aquatic organisms. However, the dietary copper effects are much less known, especially in tropical fish. The toxicity of copper via the trophic route could be influenced by the composition of the food, and diets naturally impregnated with copper seem to have greater toxicity at lower concentrations than artificially impregnated ones. Thus, our objective was to investigate the effects of copper on juveniles of the Neotropical fish Hoplias malabaricus fed on live prey (Astyanax altiparanae) previously exposed to the metal (20 µg L - 1) for 96 h. The prey fish were given to H. malabaricus every 96 h, totaling 10 doses at the end of the experiment. Thus, after 40 days fish were killed and tissues were sampled. Blood showed to be the only tissue in which copper accumulated. Anemia was found and there was damage to the DNA of erythrocytes. Furthermore, ionic imbalances were observed in plasma. There was an increase in the concentration of Na+ and Cl- and a decrease in Ca2+, which were associated with increased copper uptake in the gastrointestinal tract of fish fed on copper exposed prey. All the antioxidant enzymes evaluated in the gills showed decreased activity compared to the control group. Copper seems to have interfered in the energy metabolism of H. malabaricus, since a lower condition factor and feed conversion efficiency rate were observed in fish fed with copper diet. The present study confirms the trophic route as an important copper toxicity pathway for H. malabaricus and reinforces the idea that metal toxicity can be increased when it is naturally impregnated in the prey tissues, even if the prey has been exposed to the metal only for a short period of time.

Biogenic metallic nanoparticles (Ag, TiO2, Fe) as potential fungicides for agriculture: are they safe for the freshwater mussel Anodontites trapesialis?

Silver (Ag), titanium dioxide (TiO2), and iron (Fe) nanoparticles (NPs) synthesized using the fungus Trichoderma harzianum are effective against the agriculture pathogen Sclerotinia sclerotiorum. However, their effects should be evaluated in aquatic organisms, as agriculture practices can contaminate the aquatic environment. Thus, this work evaluated sublethal effects of acute exposure (24 h) to AgNP, TiO2NP and FeNP, synthesized with T. harzianum, on the Neotropical freshwater bivalve Anodontites trapesialis, considering the hypothesis that suspension-feeding bivalves are susceptible to NPs toxicity. Individuals of A. trapesialis were divided into four groups (n = 8/group): a control group, kept in water only; a group exposed to AgNP; a group exposed to TiO2NP; and a group exposed to FeNP. The bioaccumulation of Ag, Ti, and Fe was evaluated in the gills, hemolymph, mantle, digestive gland, and muscle (foot). Lipoperoxidation, activities of the glutathione S-transferase, catalase, and superoxide dismutase, and glycogen concentration were quantified in the gills, mantle, and digestive gland. Ions (Na+, K+, Cl-, Ca2+, and Mg+2) and glucose concentrations were quantified in the hemolymph. Na+/K+-ATPase, H+-ATPase, Ca2+-ATPase, and carbonic anhydrase activities were assessed in the gills and mantle. Acetylcholinesterase activity was determined in the foot and adductor muscle. The mussels exposed to AgNP accumulated Ag in the gills, hemolymph, and foot, and showed a decrease in hemolymph concentrations of Na+ and Cl-, which was associated with the action of Ag ion (Ag+). The exposures to TiO2NP and FeNP led to the accumulation of Ti and Fe in the hemolymph, respectively, but did not promote additional effects. Accordingly, A. trapesialis showed bioaccumulation potential and susceptibility to AgNP, but was not susceptible to TiO2NP and FeNP. Thus, the preferential agricultural use of TiO2NP and FeNP over AgNP is highlighted.

Can Lemna minor mitigate the effects of cadmium and nickel exposure in a Neotropical fish?

We aimed to evaluate if Lemna minor can mitigate the observed effects of cadmium (Cd) and nickel (Ni) exposure in Prochilodus lineatus. Fish were exposed for 96 h to 20 µg L-1 of Cd, 1.5 mg L-1 of Ni, or to a mixture of these two metals. In all tests, one group was exposed to the metals with duckweed on the water surface, and other group was exposed only to the metals, without plants. After each exposure, samples of P. lineatus tissues were collected to evaluate multiple biomarkers. Duckweed prevented bioaccumulation in some fish tissues and attenuated changes in acetylcholinesterase activity, increases in erythrocytic nuclear abnormality frequency, and hyperglycemia. However, the changes in plasma ion concentrations, reduction in activity of ion transport enzymes, and histological damage were not mitigated. Therefore, we concluded that L. minor partially attenuates the effects caused by Cd and Ni exposure.

Effects of cadmium on the female reproductive axis of a Neotropical fish.

Metals are one of the contaminants released from the increase of anthropic activities. They can be classified as endocrine disruptors once they can affect the reproductive parameters of different organisms. The aim of the study was to evaluate the potential effects of cadmium on regulatory reproduction axis (Hypothalamic-Pituitary-Gonadal-Liver, the HPGL axis) in females of Rhamdia quelen exposed to nominal concentrations of 0.1; 1; 10 and 100 µg.L-1 of cadmium. After 15 days, tissues were collected for hormonal quantification, brain aromatase (cyp19a1b), hepatic vitellogenin (vtg) gene expression, and biomarkers analysis. Cadmium was quantified in water, gonad and liver samples. The plasma levels of estradiol, testosterone and gonad and hepatosomatic indexes did not changed after Cd exposure. The cyp19a1b was not different among the groups. Cadmium was detected at higher concentrations in the liver compared to the gonads. No genotoxicity was observed, only erythrocytes nuclear alterations. Metallothionein was reduced at 10 µg.L-1 in the liver and 10 and 100 µg.L-1 in the gonad. Hepatic superoxide dismutase activity increased and this can lead to a hydrogen peroxide increase, one of reactive oxygen species. This increase without a compensation of other enzymes of the antioxidant system can lead to lipoperoxidation, as occurred at 100 µg.L-1. Hepatic vitellogenin gene expression increased as well as the injury index at 0,1 and 100 µg.L-1. The tested cadmium concentrations have been found in the freshwater ecosystems and can affect the female reproductive regulation axis HPGL of the Neotropical species R. quelen.

Effects of copper on an omnivorous (Astyanax altiparanae) and a carnivorous fish (Hoplias malabaricus): A comparative approach.

Copper is an essential metal for life. However, in excess, it can lead to osmoregulatory disorders and oxidative stress in fish and these effects appear to be species specific. In order to evaluate the effects of copper and to compare the sensitivity of two Neotropical fishes that co-occur in nature as prey (Astyaynax altiparanae) and predator (Hoplias malabaricus), the fish were exposed to three concentrations of Cu (5 µg L-1, 10 µg L-1, and 20 µg L-1) for 96 h. At the end of the experimental period, copper concentration in tissues, osmoregulatory parameters, oxidative stress biomarkers, plasma glucose, muscle glycogen and acetylcholinesterase activity were evaluated. Fish mortality (25%) was only observed for A. altiparanae exposed to Cu 20 µg L-1. The results revealed species-specific ionic disturbances. Despite hypocalcemia, H. malabaricus showed an increase in the main gill ATPases, which probably guaranteed the maintenance of plasma Na+.  In A. altiparanae, there was no change in ATPase activity in the gills and hyponatremia was observed at all copper concentrations, as well as a decrease in plasma Cl- in the Cu 20 µg L-1 group. The strategy adopted by H. malabaricus seems to have contributed to the absence of copper accumulation in the tissues, in addition to possibly being related to the absence of oxidative stress in this species. On the other hand, there was an increase in the concentration of copper in the gills, liver, and gastrointestinal tract of A. altiparanae, as well as oxidative stress evidenced by increased lipoperoxidation in the liver and damage to erythrocytes DNA. This work reinforces the idea that copper effects are species specific and that a given concentration may not be safe for different species which can coexist in the same environment.

Microplastics and copper effects on the neotropical teleost Prochilodus lineatus: Is there any interaction?

Microplastics (MP) are emerging contaminants widely found in aquatic ecosystems. In addition to MP toxicity itself, there is increasing concern about the MP adsorption capacity and the interactive effects with other contaminants, such as copper. The objective of this research was to investigate the effects of polyethylene microplastic and its association with copper (Cu) in genotoxic, biochemical, and physiological biomarkers of the neotropical teleost Prochilodus lineatus. Fish were exposed for 24 and 96 h to MP (20 µg L-1) and Cu (10 µg L-1) and MP + Cu. The results showed that MP and Cu, both isolated and in combination, promoted DNA damage in erythrocytes (96 h) and liver cells (24 and 96 h) indicating that MP and Cu are genotoxic. Fish exposed only to Cu (96 h) showed a decrease in lipid peroxidation in the liver despite of the decrease in glutathione content, indicating the efficiency of other antioxidant defenses. Brain acetylcholinesterase was inhibited in the animals from all the treatments. Although MP did not influence on Cu accumulation in tissues, decreased plasma Na+ and Ca2+ (24 h) occurred after the exposure to MP and Cu, isolated and combined. Exposure to MP and MP + Cu resulted in decreased activity of Ca2+-ATPase (24 h). Taken altogether, these results showed that MP and Cu depicted genotoxic, neurotoxic, and physiological effects on P. lineatus, both alone and combined. An interaction between Cu and MP was observed in plasma Ca2+, where the combination of both contaminants caused a greater effect than the contaminants alone.

Sublethal effects of waterborne copper and copper nanoparticles on the freshwater Neotropical teleost Prochilodus lineatus: A comparative approach.

Copper nanoparticles can contaminate the aquatic environment, but their effects on fish and how they may differ from copper salts is not understood. Thus, in this work we compare the sublethal effects of copper nanoparticles (nCu) and copper chloride (Cu) on the freshwater teleost Prochilodus lineatus, known for its sensitivity to copper. Juveniles (n = 8/group) were exposed to 20 µg L-1 of copper as CuCl2 (Cu), 40 µg L-1 of copper nanoparticles (nCu), or only water (control), for 96 h. These concentrations were chosen to achieve similar dissolved copper concentration in both treatments (Cu: 10.29 ± 0.94 µg L-1; nCu: 12.16 ± 1.77 µg L-1). After the exposure, the following biological parameters were evaluated: copper accumulation in the gills, liver, gastrointestinal tract, kidney, and muscle; hematocrit (Ht) and hemoglobin content (Hb); branchial activity of Na+-K+-ATPase (NaKATP), H+-ATPase (HATP), Ca2+-ATPase (CaATP), and carbonic anhydrase (CA); glutathione content (GSH) and lipid peroxidation (LPO) in the liver; acetylcholinesterase activity (AChE) in the brain and muscle; and histopathology of the gills and liver. The gills of Cu-exposed fish were adversely affected, with increased copper content, inhibition of H+-ATPase and Ca2+-ATPase, and histological damage, including proliferation of mitochondria rich cells and/or mucous cells. In addition, LPO levels increased in the liver of Cu-exposed fish, indicating the occurrence of oxidative stress. Exposure to nCu promoted a decrease in Ht and Hb, indicating anemia, and an increase in branchial Na+-K+-ATPase and H+-ATPase activities, which can be an adaptive response to metabolic acidosis. Within the chosen biomarkers and the conditions tested, copper nanoparticles were less toxic than copper. However, the effects promoted by the nanoparticles were different from those promoted by copper. These results emphasize the need for a better understanding of copper nanoparticles toxicity in order to establish safe concentrations and avoid environment impacts.

An integrated approach in subtropical agro-ecosystems: Active biomonitoring, environmental contaminants, bioaccumulation, and multiple biomarkers in fish.

Aquatic contamination in agricultural areas is a global problem, characterized by a complex mixture of organic and inorganic pollutants whose effects on biota are unpredictable and poorly investigated. In this context, in the present study, the Neotropical fish Prochilodus lineatus was confined in situ for 120 days in two sites with different levels of anthropic impact: 1) a fish hatchery station, within the State University of Londrina (reference site - REF) and 2) an agro-ecosystem area in one of the most productive regions of southern Brazil (experimental site - EXP). We evaluated multiple biomarkers at different levels of biological organization, such as biotransformation and antioxidant enzymes, oxidative damages, DNA damages and liver histopathology. We also evaluated the occurrence of 22 organochlorine pesticides (OCPs) and 6 trace metals in water and sediment; and 33 current-use pesticides (CUPs) in the water; besides the presence of OCPs in the liver and metals in different tissues of the confined fish. The chemical analysis confirmed that the two environments presented different levels of contamination. We verified a distribution gradient of data in the principal component analysis (PCA), separating the REF fish to one side and the fish at the agricultural area (EXP) to the other side. In general, the biomarker responses were more altered in fish from the EXP than fish from the hatchery station; and this fish presented a greater accumulation of endosulfan (an increase of 18× compared to basal value) and showed oxidative, genetic, and histological damage. Through the Biomarkers Response Index (BRI), we found that the EXP fish demonstrated a decrease in health status compared with the REF fish during the confinement time, due to their exposure to a higher concentration of contaminants. In conclusion, the use of multiple biomarkers at different response levels is an important tool for environmental monitoring.

Single and combined effects of Zn, Mn and Fe on the Neotropical freshwater bivalve Anodontites trapesialis: Bioaccumulation and biochemical biomarkers.

Important concentrations of Zn, Mn and Fe were detected in a stream near a coal mining area and promoted, in field, biomarkers alterations in the bivalve Anodontites trapesialis. In order to understand the isolated and mixed effects of these metals on these Neotropical bivalves, we run short-term experiments under laboratory controlled conditions. After 96 h-exposure, tissues (gills, mantle, digestive gland, muscle, hemolymph) were removed for metal bioaccumulation analysis, oxidative stress biomarkers (reactive oxygen species (ROS), total antioxidant capacity, lipoperoxidation (LPO), proteins carbonylation (PC), metallothionein (MT), activity of superoxide dismutase and glutathione S-transferase and hemocytes DNA damage) and cholinesterase (ChE versus ASCh activity) activity evaluation. We run three independent tests. In Zn test, clams were exposed to three concentrations of Zn (0.18 mg L-1, 1.0 mg L-1, 5.0 mg L-1); in Mn test, clams were exposed to three concentrations of Mn (0.1 mg L-1, 0.5 mg L-1, 5.0 mg L-1) and in Mix test, clams were exposed to the mixture Zn (1 mg L-1) + Mn (0.5 mg L-1), with and without Fe (5.0 mg L-1). After single exposure to 5.0 mg L-1, Zn bioaccumulated in all tissues, but only in mantle and hemolymph after exposure to 1.0 mg L-1. The increased MT in gills of A. trapesialis exposed to Zn appears to be sufficient to avoid damage, since LPO occurred only in digestive glands from animals exposed to 5.0 mg L-1. We suggested that A. trapesialis had a metabolic suppression in consequence of Mn presence, based on the following results: the decrease of ROS in gills, the decrease of the Zn and Mn concentrations in tissues and the decrease of ChE versus ASCh activity in muscle. Despite this, animals exposed to Mn suffer oxidative damages (LPO and PC) in the mantle and digestive gland and MT increased in the mantle. These results showed A. trapesialis responded differently to each metal and Mn caused more damage. When exposed to Fe, gills level of ROS was increased, despite no changes in metal accumulation occurred. On the other hand, after exposure to the mixtures, tissues bioaccumulated Zn and previously observed damages caused by Mn and Fe disappeared. Consequently, biomarkers were less affected under mixture treatments, demonstrating mixtures effects or responses were not simply a combination of single exposures to Zn, Mn and Fe, but depend on metals toxicokinetics.

Triple-mixture of Zn, Mn, and Fe increases bioaccumulation and causes oxidative stress in freshwater neotropical fish.

Metal bioaccumulation and oxidative stress biomarkers were determined in Prochilodus lineatus to understand the effects of short-term exposure to a triple-mixture of Zn, Mn, and Fe. Three independent tests were carried out, in which fish were exposed to 3 concentrations of Zn (0.18, 1.0, and 5.0 mg L-1 ), Mn (0.1, 0.5, and 5.0 mg L-1 ), and in the mix test to Fe (5.0 mg L-1 ) and a mixture of Zn (1.0 mg L-1 ) + Mn (0.5 mg L-1 ), with and without Fe. After exposure for 96 h, tissues were removed for metal bioaccumulation analysis and oxidative stress biomarkers were determined in liver, along with DNA damage in blood cells. Our results revealed that Zn and Mn were bioaccumulated in fish tissues after exposure to 5.0 mg L-1 , whereas Fe only bioaccumulated in muscle and gills after mixture exposure. Results indicated that 1 metal interfered with the other's bioaccumulation. In P. lineatus, 5 mg L-1 of both Mn and Fe were toxic, because damage was observed (lipid peroxidation [LPO] in liver and DNA damage in blood cells), whereas Zn induced liver responses (metallothionein [MT] and reduced glutathione [GSH] increases) to prevent damage. In terms of bioaccumulation and alterations of oxidative stress biomarkers, we showed that Zn, Mn, and Fe triple-mixture enhances individual metal toxicity in Neotropical fish P. lineatus. Environ Toxicol Chem 2018;37:1749-1756. © 2018 SETAC.

Lead accumulation and metallothionein content in female rats of different ages and generations after daily intake of Pb-contaminated food.

Female Wistar rats of different ages (45, 90 and 140 days) and generations (mothers and offspring) were fed a feed containing 2.0mg of Pb kg-1 daily from weaning and the Pb accumulation was determined in different organs and in maternal milk, in addition metallothioneins (MTs) content was determined in the liver and kidneys. The results showed that Pb accumulation exhibited the following pattern: bone>liver>kidney>gut>blood cells>muscle>brain>ovary. Bones accumulated the most Pb in all animals, with its concentration increasing with age and prenatal exposure. Pb accumulation in the liver, kidney and blood cells, did not follow a consistent pattern with increasing age and our data did not indicate a relationship between the presence of MTs in liver and kidney and metal accumulation in these organs. However, in the offspring and with increasing age, Pb accumulated in more organs. Mothers fed with Pb produced contaminated milk, exposing their offspring to the metal via nursing Thus, increasing age and prenatal exposure increases susceptibility to Pb toxicity-induced damage.

Metals bioaccumulation and biomarkers responses in the Neotropical freshwater clam Anodontites trapesialis: Implications for monitoring coal mining areas.

As one of the most impactful industries, coal mining can promote several alterations at surrounding environment. In surface water, elevated concentrations of metals like Mn, Zn, Fe and Al are often observed. Thus, the aim of this study was to investigate the bioaccumulation and the sub-lethal effects of these metals on various organs of the Neotropical bivalve Anodontites trapesialis confined along a stream located near a coal mine, in order to assess a set of biomarkers that could be used for effectively monitoring coal mining areas. Clams were caged, for 96h, at two sites located upstream (Up1 and Up2) and two sites downstream (Dw1 and Dw2) from the mine. Metals bioaccumulation was determined in gills, mantle, digestive gland, muscle and hemolymph and the following biomarkers were measured in A. trapesialis tissues: total antioxidant capacity against peroxyl radicals, metallothionein content, lipid peroxidation (LPO), proteins carbonylation, glutathione S-transferase activity, superoxide dismutase activity and acetylcholinesterase (AChE) activity. The results showed that Al and Fe bioaccumulation in the gills and hemolymph, Al bioaccumulation in the mantle and muscle, increased LPO in the gills (Dw1 and Dw2) and mantle (Dw1), as well as reduced AChE activity in the muscle (Dw1 and Dw2) should be considered effective biomarkers for monitoring coal mining areas. A. trapesialis proved to be an efficient biological model, considering that biomarkers responses were observed in the clams after only 96h of confinement at Dw sites, accordingly this species could be a good candidate for monitoring Neotropical freshwaters.

Multiple biomarker responses in Prochilodus lineatus subjected to short-term in situ exposure to streams from agricultural areas in Southern Brazil.

In order to assess the quality of streams susceptible to contamination by pesticides we apply biochemical and genotoxic biomarkers in the Neotropical fish Prochilodus lineatus submitted to in situ tests. Fish were caged, for 96 h, in two streams located in areas with intensive use of pesticides, the Apertados (AP) and the Jacutinga (JC), and in a small stream (Godoy stream ­ GD) found inside a forest fragment adjacent to a State Park. Biochemical parameters, such as biotransformation enzymes 7-ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST), non-protein thiols (NPSH), lipoperoxidation (LPO), protein carbonylation (PCO) and acetylcholinesterase (AChE) were evaluated in various fish organs, as well as genotoxic biomarkers (damage to DNA and occurrence of micronuclei and erythrocyte nuclear abnormalities). Samples of water and sediment were collected for analysis of metals (Cu, Cr, Pb, Ni, Mn, Cd and Zn), organochloride pesticides, and triazine and glyphosate herbicides. We observed an increase in liver GST activity in fish at AP and gill GST activity in fish at JC. An increase in liver LPO was also observed in fish exposed to AP and JC. The same animals also exhibited increased DNA damage and erythrocyte nuclear abnormalities (ENAs) compared to the fish kept in GD. A number of compounds showed concentrations higher than the permitted levels, in particular, dichlorodiphenyltrichloroethane (DDT), its metabolites dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD), hexachlorocyclohexanes (HCH), heptachloride, diclofluanid and aldrins. These pesticides were detected at higher concentrations in water and sediment samples from AP, followed by JC and GD. The Integrated Biomarker Response Index (IBR) indicated that AP and JC (AP: 21.7 > JC: 18.5 > GD: 12.6) have the worst environmental quality. Integrated biomarker analysis revealed that the alterations observed related well with the levels of environmental contaminants, demonstrating the effectiveness of this biomonitoring approach.

Lead accumulation and its effects on the branchial physiology of Prochilodus lineatus.

The purpose of this work was to determine the tissue accumulation of lead (Pb) and its effects on osmoregulatory processes of the freshwater fish Prochilodus lineatus. Juvenile fish were exposed to Pb (from 1.7 to 0.7 mg of dissolved Pb L(-1)) for 6, 24 and 96 h and Pb accumulation was analyzed in the gills, liver, kidneys, blood cells and muscle. The following parameters were also analyzed: hematologic (hemoglobin content, hematocrit and number of red blood cells), metabolic (blood glucose), endocrine (blood cortisol), osmo ionic (plasma osmolality and Na(+), K(+), Cl(-) and Ca(+2) concentrations), gill enzymes (Na(+)/K(+)-ATPase and carbonic anhydrase), chloride cell (CC) density and CC location in the gills. Pb accumulated in all the analyzed tissues, with the kidneys showing the highest concentration, followed by the gills and liver. The lowest Pb concentrations were found in blood cells and muscle. Pb promoted an increase in blood glucose after 6 and 24 h exposure. Gill Na(+)/K(+)-ATPase was inhibited after 24 h of exposure, but its activity was restored at 96 h, probably due to the increase in CC in gill lamellae. Plasma Na(+) was reduced after 6 and 96 h, while K(+) concentrations increased at all the experimental times. Fish exposed to Pb showed reduced plasma Ca(+2) at all experimental periods. Hematologic parameters remained unchanged. Overall, this study demonstrated that Pb interferes in osmoregulatory processes of P. lineatus and the proliferation of CC in the gills is a response in order to reestablish adequate ion concentrations.