Estrogen contamination increases vulnerability of amphibians to the deadly chytrid fungus.

Aquatic contaminants and infectious diseases are among the major drivers of global amphibian declines. However, the interaction of these factors is poorly explored and could better explain the amphibian crisis. We exposed males and females of the Brazilian Cururu Toad, Rhinella icterica, to an environmentally relevant concentration of the estrogen 17-alpha-ethinylestradiol (an emerging contaminant) and to the chytrid infection (Batrachochytrium dendrobatidis), in their combined and isolated forms, and the ecotoxicity was determined by multiple biomarkers: cutaneous, hematological, cardiac, hepatic, and gonadal analysis. Our results showed that Cururu toads had many physiological alterations in response to the chytrid infection, including the appearance of cutaneous Langerhans's cells, increased blood leukocytes, increased heart contraction force and tachycardia, increased hepatic melanomacrophage cells, which in turn led to gonadal atrophy. The estrogen, in turn, increased the susceptibility of the toads to the chytrid infection (higher Bd loads) and maximized the deleterious effects of the pathogen: reducing leukocytes, decreasing the contraction force, and causing greater tachycardia, increasing hepatic melanomacrophage cells, and leading to greater gonadal atrophy, which were more extreme in females. The exposure to estrogen also revealed important toxicodynamic pathways of this toxicant, as shown by the immunosuppression of exposed animals, and the induction of the first stages of feminization in males, which corroborates that the synthetic estrogen acts as an endocrine disruptor. Such an intricate relationship is unprecedented and reinforces the importance of studying the serious consequences that multiple environmental stressors can cause to aquatic populations.

Immunodetection of heat shock protein 70 and cell death in liver of a neotropical fish acutely and chronically exposed to acetaminophen and propranolol.

The use of pharmaceutical drugs ends frequently in their inappropriate disposal and treatment at waste water treatment plants, which is the cause of their widespread presence in the environment. Yet, there is limited understanding or knowledge of their effects to non-target aquatic organisms. The drugs acetaminophen (analgesic and antipyretic) and propranolol (ß-blocker) are widely found in the aquatic environment, where they can interact with non-target exposed organisms, causing adverse effects. Heat shock proteins (namely HSP70) are molecular chaperones which help to refold misfolded cellular proteins, and the increase in their in vivo levels indicates a change in the cell to counteract the proteotoxic effects of the triggered stress, namely which is consequent to exposure to toxicants. The objective of this study was to quantify the levels of liver HSP70 proteins in individuals of the neotropical fish species Phalloceros harpagos, acutely and chronically exposed to concentrations of acetaminophen and propranolol, in the range of those already determined to occur in the wild. Fish acutely exposed to acetaminophen (concentrations of 8, 80, 800, and 8000 µg L-1) and to propranolol (levels of 1, 10, and 1000 µg L-1) evidenced increased intensity of HSP70 immunolabeling in liver cells. Similarly, animals chronically exposed to propranolol, at concentrations of 0.0625, 0.125, 0.25, and 0.5 µg L-1, showed a comparable trend. This finding suggests the triggering of a cytoprotective effect that was effective in preventing cell death in exposed groups in relation to the control group. In contrast, chronic exposure to acetaminophen caused a decrease in HSP70 labeling intensity in fish hepatocytes (animals exposed to 5, 10, 20, 40, and 80 µg L-1), with no induction of DNA fragmentation in the nuclei of hepatocytes of these fish. Some of the hepatic HSP70 responses observed in this study were obtained at levels already reported to occur in the wild. Finally, this study showed how levels of acetaminophen at microliter concentration can exert side effects on non-target organisms after chronic exposure, suggesting that environmentally exposed organisms may be subjected to adverse conditions that modify the typical response pattern of HSP70 levels.

Biomarkers of the oxidative stress and neurotoxicity in tissues of the bullfrog, Lithobates catesbeianus to assess exposure to metals.

Lithobates catesbeianus tadpoles were exposed to 1 µg L-1 of zinc (Zn), copper (Cu) and cadmium (Cd) alone or combined (1:1 and 1:1:1) for 2 and 16 days. Results showed a significant increase in the superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities in the liver, kidney and muscle (except for GPx) in the groups exposed to metal either alone or co-exposed after 2 days compared to the control. After 16 days, SOD, CAT and GST activities decreased significantly in the liver and kidney and GPx activity increased in the liver. Reduced glutathione (GSH) increased in the liver and kidney following combined exposure and decreased after 2 days of metal exposure in the muscle. There were significant increases in lipid hydroperoxide (LPO) levels in the liver, kidney and muscle (2 and 16 days), with the highest levels after metal co-exposure. Cholinesterase (ChE) activity increased significantly in the brain after 2 days of exposure but decreased in the brain (16 days) and muscle (2 days) after exposure to metals, alone and combined. The current study highlighted that the antioxidant system of L. catesbeianus was sensitive to metals and specially to the co-exposure of the three metals, despite presenting differences in the response among tissues. In addition, tadpoles were sensitive at both periods of exposure, but in different modes with stress response (activation, up-regulation) at 2 days and oppression (down-regulation) at 16 days.

Neurotoxic and respiratory effects of human use drugs on a Neotropical fish species, Phalloceros harpagos.

Pharmaceutical drugs are usually and continuously carried to the aquatic environment in different ways. Thus, they are pseudo-persistent in the environment, and they may exert deleterious effects on aquatic organisms. The objective of the present study was to investigate the acute and chronic effects of two widely used pharmaceutical drugs, paracetamol (analgesic and antipyretic) and propranolol (ß-blocker) on the activity of specific biomarkers (namely cholinesterase enzymes and lactate dehydrogenase) of the neotropical fish Phalloceros harpagos. The obtained results indicate an inhibition of the activity of the enzyme lactate dehydrogenase (LDH) after acute exposure to paracetamol, and an increase in cholinesterase activity in acutely propranolol-exposed fish. Chronic exposure to both drugs did not modify the enzymatic activities. Such short-term changes in enzymatic activities may be harmful to organisms, altering the preferential pathway of energy metabolism, and may induce behavioral changes that may compromise prey capture and predator escape, and in the longer term may induce population declines.

Cholinesterases characterization of three tropical fish species, and their sensitivity towards specific contaminants.

Cholinesterases are frequent targets for toxic effects, namely by insecticides derived from phosphoric and carbamic acids. This effects allows the use of cholinesterase inhibition as a biomarker for contamination of aquatic environments by these specific chemical agents. However, cholinesterases are differently responsive to environmental contaminants, according to their different forms and locations. In addition, cholinesterases seem also to be inhibited by metals, so their use as an environmental criterion requires the prior characterization of their specific forms in each species and tissues, and the study of their sensitivity. The objective of this study was to characterize the cholinesterase isoenzymes present in the brain and dorsal muscle of three tropical fish species, namely Phalloceros harpagos (Lucinda, 2008), Pterygoplichthys pardalis (Castelnau, 1855) and Astyanax altiparanae (Garutti and Britski, 2000). In vitro assays were conducted to quantify the effect of pesticides (dimethoate and carbaryl) and metals (lead and copper) on cholinesterases activity. Although acetylcholinesterase seems to be the most prevalent and abundant form, as commonly described in vertebrates, the here-obtained results showed that three cholinesterase isoenzymes occur in tissues of the three fish species. In addition, the pesticide carbaryl caused a stronger inhibition than dimethoate. Copper caused a significantly higher cholinesterasic inhibition than lead, which is also in line with most results concerning the anticholinesterasic effects by these metals. The here obtained results allowed to conclude that acetylcholinesterase is the predominant form in all tissues from the three analyzed species. In addition, cholinesterases of these three fish were responsive to common environmental contaminants, namely metals and pesticides, similarly to what was already described for fish of temperate areas. This allows using the here proposed fish species in environmental studies for the assessment of the presence of neurotoxicants under neotropical conditions.

Behavior and histopathology as biomarkers for evaluation of the effects of paracetamol and propranolol in the neotropical fish species Phalloceros harpagos.

Pharmaceutical drugs in the aquatic environment can induce adverse effects on nontarget organisms. This study aimed to assess the short-term effects of sublethal concentrations of both paracetamol and propranolol on the fish Phalloceros harpagos, specifically light/dark preference, swimming patterns, skin pigmentation, histopathology, and liver glycogen levels. Fish were acutely exposed to sublethal concentrations of both paracetamol (0.008, 0.08, 0.8, 8, 80 mg L-1) and propranolol (0.0001, 0.001, 0.01, 0.1, 1 mg L-1) under controlled conditions. For scototaxis, a significant preference for the dark compartment was observed for the group exposed to the highest concentration of paracetamol (80 mg L-1). Propranolol exposure significantly altered the swimming pattern, especially in fish exposed to the 0.001 mg L-1 concentration. Pigmentation was reduced in propranolol-exposed fish (0.1, 1 mg L-1). The lowest concentration of propranolol (0.0001 mg L-1) induced a decrease of histochemical reaction for hepatic glycogen. These data demonstrate that pharmaceuticals can induce sublethal effects in nontarget organisms, even at low concentrations, compromising specific functions of the individual with ecological relevance, such as energy balance and behavior.

Assessment of biochemical alterations in the neotropical fish species Phalloceros harpagos after acute and chronic exposure to the drugs paracetamol and propranolol.

Over time, many pollutants of anthropogenic origin have caused the contamination of aquatic ecosystems. Among several characteristics, these compounds can reach the trophic chain, causing deleterious interactions with the biota. Pharmaceutical substances can be included in this scenario as emerging contaminants that reach the aquatic environment because of direct human and veterinary usage, and release by industrial effluents, as well as through domestic dumping of surplus drugs. The effects of these compounds on exposed organisms have been studied since the 1990s, but ecotoxicological data for such chemicals are still scarce especially concerning aquatic organisms from tropical regions. Paracetamol and propranolol were selected for this study since they are frequently found in surface waters. Paracetamol is a drug used as analgesic and antipyretic, while propranolol, a ß-blocker, is used in the treatment of hypertension. The objective of this study was to assess the toxic effects of these substances on the neotropical freshwater fish Phalloceros harpagos after acute (96 h) and chronic (28 days) exposures. In order to understand the effects of these drugs on P. harpagos, biochemical markers were selected, including the enzymes involved in oxidative stress, xenobiotic metabolism, and neurotransmission (catalase, glutathione-S-transferase, and cholinesterase activities, respectively). After acute exposure, no significant alterations were observed for catalase activity, suggesting the absence of oxidative stress. On the contrary, significant alterations in glutathione-S-transferases activity were described for the higher concentrations of both pharmaceuticals after acute exposure. In addition, acute exposure to paracetamol caused a significant increase of cholinesterase activity. None of the tested pharmaceuticals caused significant changes in catalase or cholinesterase activities after chronic exposure. Glutathione S-transferases activity was significantly increased for propranolol following chronic exposure, indicating the potential involvement of phase II detoxification pathway.

Thiamethoxam and picoxystrobin reduce the survival and overload the hepato-nephrocitic system of the Africanized honeybee.

Apis mellifera perform important pollination roles in agroecosystems. However, there is often intensive use of systemic pesticides in crops, which can be carried to the colony by forage bees through the collection of contaminated pollen and nectar. Inside the colony, pollen loads are stored by bees that add honey and several enzymes to this pollen. Nevertheless, intra-colonial chronic exposure could induce sublethal effects in young bees exposed to a wide range of pesticides present in these pollen loads. This study was aimed to both determine the survival rate and evaluate the sublethal effects on the hepato-nephrocitic system in response to continuous oral exposure to lower concentrations of neonicotinoid thiamethoxam (TXT) and picoxystrobin fungicide (PXT). Exposure to a single chemical and co-exposure to both pesticides were performed in newly emerged honeybee workers. A significant decrease in the bee survival rates was observed following exposure to TXT (0.001 ng a.i./µL) and PXT (0.018 ng a.i./µL), as well as following co-exposure to TXT+PXT/2. After five days of continuous exposure, TXT induced sub-lethal effects in the organs involved in the detoxification of xenobiotics, such as the fat body and pericardial cells, and it also induced a significant increase in the hemocyte number. Thus, the hepato-nephrocitic system (HNS) reached the greatest level of activity of pericardial cells as an attempt to eliminate this toxic compound from hemolymph. The HNS was activated at low levels by PXT without an increase in the hemocyte number; however, the mobilization of neutral glycoconjugates from the trophocytes of the fat body was prominent only in this group. TXT and PXT co-exposure induced intermediary morphological effects in trophocytes and pericardial cells, but oenocytes from the fat body presented with atypical cytoplasm granulation only in this group. These data showed that the realistic concentrations of these pesticides are harmful to newly emerged Africanized honeybees, indicating that intra-colonial chronic exposure drastically reduces the longevity of bees exposed to neonicotinoid insecticide (TXT) and the fungicide strobilurin (PXT) as in single and co-exposure. Additionally, the sublethal effects observed in the organs constituting the HNS suggest that the activation of this system, even during exposure to low concentrations of theses pesticides, is an attempt to maintain homeostasis of the bees. These data together are alarming because these pesticides can affect the performance of the entire colony.

Effects of glyphosate and the glyphosate based herbicides Roundup Original(®) and Roundup Transorb(®) on respiratory morphophysiology of bullfrog tadpoles.

Glyphosate-based herbicides are widely used in agriculture and are commonly found in water bodies. Roundup Original(®) (RO) contains an isopropylamine glyphosate (GLY) salt containing the surfactant POEA, while Roundup Transorb R(®) (RTR) contains a potassium salt of GLY with unknown surfactants. Both contain different compositions of so-called "inert" ingredients, more toxic than glyphosate. Amphibian tadpoles often experience variations in O2 availability in their aquatic habitats; an ability to tolerate hypoxia can condition their survival and fitness. We evaluated the impacts of sublethal concentrations of GLY (1 mg L(-1)), RO (1 mg L(-1) GLY a.e) and RTR (1 mg L(-1) GLY a.e) on metabolic rate (V·O2 - mLO2 Kg1 h(-1)) of bullfrog tadpoles during normoxia and graded hypoxia, and related this to morphology of their skin, their major site of gas exchange. In control (CT) V·O2 remained unaltered from normoxia until 40 mmHg, indicating a critical O2 tension between 40 and 20 mmHg. GLY significantly reduced V·O2, possibly due to epidermal hypertrophy, which increased O2 diffusion distance to O2 uptake. In contrast, RTR increased V·O2 during hypoxia, indicating an influence of "inert" compounds and surfactants. V·O2 of RO did not differ from CT, suggesting that any increase in V·O2 caused by exposure was antagonized by epidermal hypertrophy. Indeed, all herbicides caused marked alterations in skin morphology, with cell and epithelium wall presenting hyperplasia or hypertrophy and chromatid rupture. In summary, GLY, RO and RTR exert different effects in bullfrog tadpoles, in particular the surfactants and inert compounds appear to influence oxygen uptake.

Hepatic effects of the clomazone herbicide in both its free form and associated with chitosan-alginate nanoparticles in bullfrog tadpoles.

The use of agrochemicals in agriculture is intense and most of them could be carried out to aquatic environment. Nevertheless, there are only few studies that assess the effects of these xenobiotics on amphibians. Clomazone is an herbicide widely used in rice fields, where amphibian species live. Thus, those species may be threatened by non-target exposure. However, nanoparticles are being developed to be used as a carrier system for the agrochemicals. Such nanoparticles release the herbicide in a modified way, and are considered to be more efficient and less harmful to the environment. The aim of this study was to comparatively evaluate the effect of clomazone in its free form and associated with nanoparticles, in the liver of bullfrog tadpoles (Lithobates catesbeianus) when submitted to acute exposure for 96 h. According to semi-quantitative analysis, there was an increase in the frequency of melanomacrophage centres, in the accumulation of eosinophils and in lipidosis in the liver of experimental groups exposed to clomazone - in its free form and associated with nanoparticles - in comparison with the control group, and the nanotoxicity of chitosan-alginate nanoparticles. The increase of melanomacrophage centres in all exposed groups was significant (P < 0.0001) in comparison to control group. Therefore, the results of this research have shown that exposure to sublethal doses of the herbicide and nanoparticles triggered hepatic responses. Moreover, these results provided important data about the effect of the clomazone herbicide and organic nanoparticles, which act as carriers of agrochemicals, on the bullfrog tadpole liver.

Cardiac adaptations of bullfrog tadpoles in response to chytrid infection.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) can result in heart failure in Bd-susceptible species. Since Bd infection generally does not cause mortality in North American bullfrogs, the aim of this work was to verify whether this species presents any cardiac adaptation that could improve the tolerance to the fungus. Thus, we analyzed tadpoles' activity level, relative ventricular mass, ventricle morphology, in loco heart frequency, and in vitro cardiac function. The results indicate that infected animals present an increase in both ventricular relative mass and in myofibrils' incidence, which accompanied the increase in myocytes' diameter. Such morphological alterations enabled an increase in the in vitro twitch force that, in vivo, would result in elevation of the cardiac stroke volume. This response requires much less energy expenditure than an elevation in heart frequency, but still enables the heart to pump a higher volume of blood per minute (i.e., an increase in cardiac output). As a consequence, the energy saved in the regulation of the cardiac function of Bd-infected tadpoles can be employed in other homeostatic adjustments to avoid the lethal effect of the fungus. Whether other species present this ability, and to what extent, remains uncertain, but such possible interspecific variability might explain different mortality rates among different species of Bd-infected amphibians.

Differences in Ca2+-management between the ventricle of two species of neotropical teleosts: the jeju, Hoplerythrinus unitaeniatus (Spix & Agassiz, 1829), and the acara, Geophagus brasiliensis (Quoy & Gaimard, 1824)

This study analyzed the physiological role of the cardiac sarcoplasmic reticulum (SR) of two neotropical teleosts, the jeju, Hoplerythrinus unitaeniatus (Erythrinidae), and the acara, Geophagus brasiliensis (Cichlidae). While the in vivo heart frequency (fH - bpm) of acara (79.6 ± 6.6) was higher than that of the jeju (50.3 ± 2.7), the opposite was observed for the ventricular inotropism (Fc - mN/mm²) at 12 bpm (acara = 28.66 ± 1.86 vs. jeju = 36.09 ± 1.67). A 5 min diastolic pause resulted in a strong potentiation of Fc (≅ 90 percent) of strips from jeju, which was completely abolished by ryanodine. Ryanodine also resulted in a ≅ 20 percent decrease in the Fc developed by strips from jeju at both subphysiological (12 bpm) and physiological (in vivo) frequencies. However, this effect of ryanodine reducing the Fc from jeju was completely compensated by adrenaline increments (10-9 and 10-6 M). In contrast, strips from acara were irresponsive to ryanodine, irrespective of the stimulation frequency, and increases in adrenaline concentration (to 10-9 and 10-6 M) further increased Fc. These results reinforce the hypothesis of the functionality of the SR as a common trait in neotropical ostariophysian (as jeju), while in acanthopterygians (as acara) it seems to be functional mainly in 'athletic' species.(AU)

O presente estudo analisou o papel fisiológico desempenhado pelo retículo sarcoplasmático (RS) de duas espécies de teleósteos neotropicais, o jeju, Hoplerythrinus unitaeniatus (Erythrinidae), e o acará, Geophagus brasiliensis (Cichlidae). Enquanto a frequência cardíaca registrada in vivo (fH - bpm) para o acará (79.6 ± 6.6) foi superior àquela observada para o jeju (50.3 ± 2.7), resposta inversa foi verificada para o inotropismo ventricular (Fc - mN/mm²) na frequência de estimulação de 12 bpm (acará = 28.66 ± 1.86 vs. jeju = 36.09 ± 1.67). Uma pausa diastólica de 5 min resultou em uma expressiva potenciação da Fc (≅ 90 por cento) das tiras de jeju, a qual foi completamente abolida pela rianodina. A rianodina também resultou em um decréscimo de ≅ 20 por cento na Fc desenvolvida pelas tiras de jeju tanto a frequências sub-fisiológicas (12 bpm) quanto fisiológicas (in vivo). No entanto, o decréscimo da Fc promovido pela rianodina foi completamente compensado pela adição de adrenalina (10-9 e 10-6 M). Em contraste, as tiras de acará foram irresponsivas à rianodina, independentemente da frequência de estimulação utilizada, fazendo com que a adição de adrenalina (10-9 e 10-6 M) resultasse em incrementos ainda maiores da Fc. Esses resultados reforçam a hipótese de que a funcionalidade do RS seja uma característica comum aos ostariofíseos neotropicais (como o jeju), enquanto nos acantopterígios (como o acará) esta organela parece ser funcional principalmente em espécies ativas.(AU)