Effect of Fe3+ on Na,K-ATPase: Unexpected activation of ATP hydrolysis.

Iron is a key element in cell function; however, its excess in iron overload conditions can be harmful through the generation of reactive oxygen species (ROS) and cell oxidative stress. Activity of Na,K-ATPase has been shown to be implicated in cellular iron uptake and iron modulates the Na,K-ATPase function from different tissues. In this study, we determined the effect of iron overload on Na,K-ATPase activity and established the role that isoforms and conformational states of this enzyme has on this effect. Total blood and membrane preparations from erythrocytes (ghost cells), as well as pig kidney and rat brain cortex, and enterocytes cells (Caco-2) were used. In E1-related subconformations, an enzyme activation effect by iron was observed, and in the E2-related subconformations enzyme inhibition was observed. The enzyme's kinetic parameters were significantly changed only in the Na+ curve in ghost cells. In contrast to Na,K-ATPase α2 and α3 isoforms, activation was not observed for the α1 isoform. In Caco-2 cells, which only contain Na,K-ATPase α1 isoform, the FeCl3 increased the intracellular storage of iron, catalase activity, the production of H2O2 and the expression levels of the α1 isoform. In contrast, iron did not affect lipid peroxidation, GSH content, superoxide dismutase and Na,K-ATPase activities. These results suggest that iron itself modulates Na,K-ATPase and that one or more E1-related subconformations seems to be determinant for the sensitivity of iron modulation through a mechanism in which the involvement of the Na, K-ATPase α3 isoform needs to be further investigated.

Can the exposure system adopted influence the results of the atrazine toxicity in hepatic tissue of fish?

The widespread use of atrazine, a herbicide used to control weeds, has contributed to the increased contamination of aquatic environments. To assess the toxicological effects of a xenobiotic on a nontarget organism in the laboratory, different models of toxicological exposure systems have been widely used. Therefore, the aim of this study was to evaluate and compare the action of sublethal concentrations of atrazine on the hepatic histology of Oreochromis niloticus, considering two models of exposure: static (where atrazine was only added once) and semi-static (where atrazine was periodically renewed). Fish were exposed to a concentration of 2 ppm atrazine for 15 days, which was verified by high-performance liquid chromatography. The livers were stained with hematoxylin and eosin and histopathological data were collected. In addition, they were submitted to immunohistochemistry for inducible nitric oxide synthase (iNOS). A maximum variation of 45% (static) and 12.5% (semi-static) was observed between the observed and nominal atrazine concentration. Nuclear and cytoplasmic changes were observed in both experimental models. Hepatocytes from the livers of the static system showed a degenerative appearance, while in the semi-static system, intense cytoplasmic vacuolization and necrosis were observed. iNOS positive cells were identified only in macrophages in the hepatocytes of fish in the semi-static system. These results directly showed how the choice of exposure system can influence the results of toxicological tests. However, future analysis investigating the by-products and nitrogen products should be carried out since the histopathological findings revealed the possibility of these compounds serving as secondary contamination routes.

Evaluation of the potential environmental risk from the destination of medicines: an epidemiological and toxicological study.

BACKGROUND: The high consumption of medicines by the population and their storage at home might cause an increase in the number of pharmaceutical substances that may be inappropriately discarded in the sanitary sewage, reaching an environmental aquatic. Thus, the effects of these emerging contaminants need more studies. OBJECTIVES: To identify the profile of most medicines that are discarded by users of community pharmacy and evaluate the toxicity of the most disposed drugs. METHODS: This was a translational study. A descriptive observational study was carried out for convenience of community pharmacy users using a standardized questionnaire. Subsequently, the lethal concentration 50 (LC50) for medicine that is most frequently discarded was determined. After LC50, the embryos (n = 144) were exposed to sublethal concentrations for most discarded drug at 24, 48, and 72 h. Mortality, heartbeat, and embryo deformities were used as parameters of toxicity. RESULTS: Most respondents (96%) had a "home pharmacy." The primary forms of disposal were in the common household waste, kitchen sink, and/or bathroom. The medicines that were most incorrectly discarded by the interviewees were nimesulide (17.1%), dipyrone (10.7%), and paracetamol (5.2%). LC50 of nimesulide was calculated (0.92 µgmL-1). The toxicological test revealed that embryos exposed to nimesulide showed several abnormalities, such as defects in the spinal cord, tail, yolk sac, as well as pericardial edema. Furthermore, the heartbeat decreased by 30% at a concentration of 0.4 µgmL-1 as compared with control group. The yolk sac and pericardial areas increased to >100% in all treatment groups when compared with the control group. CONCLUSION: Respondents disposed medicines in an inappropriate manner primarily in household waste and in the toilet. Nimesulide was the most discarded drug according to study population. Moreover, teratogenic effects such as spinal cord defects, decreasing heartbeats, and increasing pericardial and yolk sac area in embryos were observed after exposure to nimesulide. These results show that nimesulide may promote risk to aquatic organisms and to human health if it is discarded in an unsafe manner.

Thermal stress induces heat shock protein 70 and apoptosis during embryo development in a Neotropical freshwater fish.

Fish embryos are particularly vulnerable to temperature changes, with the effects varying with developmental stage. The major aim of the present study was to analyse the relationship between apoptosis and heat shock protein (HSP) 70 during embryo development under thermal stress conditions. To this end, Prochilodus lineatus embryos at the blastopore closure stage were subjected to one of three thermal treatments for 1h (Group 1, 25°C (control); Group 2, 20°C; Group 3, 30°C) and then examined at 0, 4 and 8h posttreatment (h.p.t.). The viability of embryos was highest in Group 1 (81.33±16.65%), followed by Group 3 and Group 2 (75.33±12.10% and 68.67±16.86% respectively), with significant difference between Groups 1 and 2 (P<0.05). At 0h.p.t., embryos subjected to thermal stress (Group 3) had a significantly higher number of terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL)- and caspase-3-labelled cells, and a lower number of HSP70-positive cells than those in the control group. At 4h.p.t., there was a decrease in the TUNEL reaction and an increase in HSP70 in embryos in Group 3. At 8h.p.t., the size of Group 3 embryos was significantly smaller than that of Group 1 embryos. The results indicate a cytoprotective role for HSP70, regulating caspase-3-mediated apoptosis during embryo development of P. lineatus; however, this mechanism is not effective in controlling embryo viability and larval malformations.

Oogenesis is accompanied by cyclic morphological changes in hepatocytes of Neotropical freshwater fish Piabina argentea.

We determined for the first time the reproductive biology of Piabina argentea through macroscopic and microscopic analysis of ovaries and evaluated the morphological changes in hepatocytes. Two hundred and 46 specimens were collected, 204 females and 42 males, between March 2014 and February 2015. Biometrics data were obtained. From females, gonad and liver samples were conducted to histological, histochemical and immunohistochemical techniques. Mature ovaries were used to determine absolute and relative fecundity. Total length and body weight values indicated that females were larger than males. The estimated weight-length ratio showed negative allometric growth. The absolute fecundity average was 171.83 ± 59.89 oocytes per ovary. In addition, females spawning capable and regressing stages were found throughout the sampling period and the presence of all oocyte types in regressing stage ovaries indicated asynchronous oocyte development and multiple spawning. From regenerating to spawning capable stage the oocytes accumulated yolk in cytoplasm became bigger. While in the liver hepatocytes with a larger cell area during regenerating stage and proliferative activity in the spawning capable stage were observed. Thus, our results indicate that P. argentea had an opportunistic reproductive strategy and cyclic morphological changes of hepatocytes occurred during the oogenesis.

Comparative histology in the liver and spleen of three species of freshwater teleost

We describe and compare the histology of liver and spleen ofGeophagus brasiliensis (Perciformes), Hypostomus francisci (Siluriformes) and Hoplias aff. malabaricus (Characiformes), tropical freshwater fishes. InG. brasiliensisandH. aff. malabaricusthe hepatocytes were arranged in tubular form whereas in H. franciscithey cord-like. In all species, hepatocytes presented glycogen, but in G. brasiliensis and H. aff. malabaricus they showed strong stained for hemossiderin in the cytoplasm. InG. brasiliensis and H. aff. malabaricus, melanomacrophage centres (MMCs) were associated to hepatic structures and only in G. brasiliensis was observed intrahepatic exocrine pancreas. The spleen, in all species, was characterized by red and white pulp without boundary between the two regions, but only in H. francisci was recorded nodular organization in splenic parenchyma. The G. brasiliensisandH. aff. malabaricuspresented in the white pulp MMCs linked mainly to ellipsoids. Besides, we observed large MMCs in the spleen in relation to liver of G. brasiliensis and H. aff. malabaricus. In liver, highest values of reticular fibers and collagen were observed inG. brasiliensis. In spleen, highest values of reticular fibers and collagen were recorded inH. aff. malabaricusandH. francisci, respectively. Histological differences confirm the hypothesis that the phylogenetic distance is reflected in liver and spleen.(AU)

Nós descrevemos e comparamos a histologia do fígado e do baço de Geophagus brasiliensis (Perciformes), Hypostomus francisci (Siluriformes) e Hoplias aff. malabaricus (Characiformes), peixes neotropicais de água doce. Em G. brasiliensis e H. aff. malabaricus os hepatócitos organizaram-se na forma tubular enquanto que em H. francisci eles apresentaram-se como cordões celulares. Em todas as espécies, os hepatócitos apresentaram glicogênio, mas em G. brasiliensis e H. aff. malabaricus, eles mostraram forte marcação para hemossiderina no citoplasma. Em G. brasiliensis e H. aff. malabaricus, centros melanomacrofágicos (CMMs) foram associados a estruturas hepáticas e somente em G. brasiliensis foi observado pâncreas exócrino intrahepático. O baço, em todas as espécies, foi caracterizado pela polpa vermelha e branca sem limites entre as duas regiões, mas somente em H. francisci foi registrado uma organização nodular no parênquima esplênico. G. brasiliensiseH. aff. malabaricusapresentaram na polpa branca CMMs associados principalmente a elipsoides. Além disso, nós observamos CMMs grandes no baço em relação ao fígado de G. brasiliensis e de H. aff. malabaricus. No fígado, valores altos de fibras reticulares e colágeno foram observado em G. brasiliensis. No baço, valores altos de fibras reticulares e colágeno foram registrados em H. aff. malabaricuseH. francisci, respectivamente. Diferenças histológicas confirmam a hipótese que a distância filogenética está refletida no fígado e no baço.(AU)

Biological variables of Hypostomus francisci (Siluriformes: Loricariidae) from Itapecerica River, Minas Gerais State, Brazil.

Herein we determine for the first time the reproduction parameters and population structure of Hypostomus francisci in the Itapecerica River, São Francisco Basin. A total of 250 specimens was captured quarterly between March 2010 and February 2012. Body weight, total length and weight of the gonads were obtained in the laboratory. Gonad samples were submitted to histological and histochemical techniques. Females with spawning capable ovaries were used to determine the fecundity and relative fecundity. Sex ratio with 1:1.01 (female:male) was observed. Males were more numerous than females for individuals smaller than 170 mm, however the number of females was significantly greater for specimens larger than 330 mm. The length-weight relationship estimated for H. francisci indicates negative-allometric growth. Females spawning capable were observed mostly in November-December-January. Two cohorts of oocytes at a determined time evidencing the development type group-synchronic. The eggs reaching 3.4 mm and the fecundity ranged from 312-1,460 oocytes with an average of 585.81 ± 337.43 oocytes per female. The reproductive parameters and population structure of H. francisci from Itapecerica River suggested that this species showed singular reproductive tactics among congeners.

Relationship between bcl-2, bax, beclin-1, and cathepsin-D proteins during postovulatory follicular regression in fish ovary.

In fish ovaries, postovulatory follicles (POFs) are key biomarkers of breeding and provide an interesting model for studying the relationship between autophagy and apoptosis. In this study, we investigated the immunohistochemical expression of autophagic and apoptotic proteins to improve the knowledge on the mechanisms regulating ovarian remodeling after spawning. Females from three neotropical fish species kept in captivity were submitted to hormonal induction. After ova stripping, ovarian sections were sampled daily until 5 days postspawning (dps). Similar events of POF regression were detected by histology, terminal transferase-mediated dUTP nick-end labeling (TUNEL), and electron microscopy in the three species: follicular cells hypertrophy, progressive disintegration of the basement membrane, gradual closing of the follicular lumen, theca thickening, and formation of large autophagic vacuoles preceding apoptosis of the follicular cells. Autophagic and apoptotic proteins were assessed by immunohistochemistry. Morphometric analysis of the immunolabeling revealed a more intense reaction for bcl-2 and beclin-1 (BECN1) in POFs at 0 to 1 dps and for bax at 2 to 3 dps (P < 0.001), the later period being the peak of apoptosis of the follicular cells. The immunostaining for cathepsin-D was more elevated until 2 to 3 dps and decreased significantly at 4 to 5 dps, when the POFs were in late stage of regression. Double labeling for BECN1 and caspase-3 indicated a shift in the relationship between autophagy and apoptosis at 2 to 3 dps, a critical period in determining the fate of follicular cells in POFs. Together, these results indicate that the bcl-2 family, BECN1, and cathepsin-D can be involved in the regulation of ovarian remodeling in teleost fish.

Evaluation of different methods of cryopreservation of Ehrlich tumor cells.

BACKGROUND: The Ehrlich Ascitic Carcinoma (EAC) is an experimental transplantable neoplasm that develops in several species of mice. The maintenance of the tumor occurs in vivo. Thus, freezing the cells would reduce the number of passages between animals, ensuring genetic stability and storage for longs period of experimentation. OBJECTIVE: Search by EAC cryoprotectants. MATERIALS AND METHODS: The combinations of nutrient medium (Tris, hen egg yolk, and DEMEN) and cryoprotective agent (Glicerol, Trehalose and DMSO) on freezing EAC cells and the transplantability after defrosting were evaluated. The cooling was conducted at 2 C/min. until -180 degree C and the thawing by immersion in water at 37 degree C. The transplantability was evaluated from cell inoculation in mice for 14 days. RESULTS: The best results were the associations IA (Cryoprotective agent Glycerol 6 % and medium containing 3.0 % Tris w / v, 1.8 % Citric acid w / v, 1.3 % D-fructose w / v and 20 % hen egg yolk v / v) and IIB (Cryoprotective agent Trehalose 100mM and medium containing 50 % coconut water v / v, 25 % sodium citrate 5 % v / v and 20 % hen egg yolk v / v) with 85.2 % and 55.1 % viable cells, respectively. CONCLUSION: These transplantable cells were efficient for tumor development, therefore demonstrating that this method of cryopreservation is simple and affordable.

Role of HSP70 in the regulation of the testicular apoptosis in a seasonal breeding teleost Prochilodus argenteus from the São Francisco River, Brazil.

This study investigated the relationship among heat shock protein 70 (HSP70), proliferating cell nuclear antigen (PCNA), and testicular apoptosis during a breeding cycle of Prochilodus argenteus, a neotropical migratory characiform fish of importance in commercial fishery from the São Francisco River basin. A total of 48 (12 fish/sampling) adult males were caught using casting and drifting nets in four samplings from June 2008 to March 2009. Immunohistochemistry, Western blotting, terminal transferase-mediated dUTP nick-end labeling (TUNEL), enzyme-linked immunosorbent assay (ELISA), and caspase-3 colorimetric assay were assessed in different phases of spermatogenesis. Labeling for HSP70 occurred in spermatogonia (SPG(A) 18.0±1.5 and SPGB 27.9±1.0 in 100 mm(2), respectively) and Sertoli cells in all sampling periods, with higher values in June (resting period) while spermatocytes were labeled in September (maturation period) and December (ripe period). For PCNA, immunoreaction was predominant in spermatogonia in June and September, while primary spermatocytes were labeled mainly in December (18.7±2.0). TUNEL-positive reaction occurred throughout the sampling periods, and labeling was detected in the nucleus of germ cells in all developmental phases, except spermatozoa. By ELISA, total HSP70 in testis increased significantly from June to December, and decreased in March (regression period), P<0.05. Caspase-3 activity decreased from June to December and increased in March. Taken together, our results suggest that HSP70 may protect the germ cells from caspase-3-dependent apoptosis during testicular activity and, reduction of HSP70 and increase of apoptosis contribute for testicular remodeling after the breeding season in wild populations of P. argenteus in the São Francisco River.

Assessment of spermatogenesis and plasma sex steroids in a seasonal breeding teleost: a comparative study in an area of influence of a tributary, downstream from a hydroelectric power dam, Brazil.

River damming and building of hydroelectric power plants interrupt the reproductive migration routes and change the major physicochemical parameters of water quality, with drastic consequences for populations of migratory fishes. The goal of this study was to evaluate proliferation and cell death during spermatogenesis and serum profiles of sex steroids in Prochilodus argenteus, from the São Francisco River, downstream from the Três Marias Dam. A total of 257 adult males were caught quarterly during a reproductive cycle in two sites: the first 34 km of the river after the dam (site 1) and the second 34-54 km after the dam (site 2), after the confluence with a tributary, the Abaeté River. Seasonal changes in the testicular activity associated with morphometric analyses of germ cells as well as proliferation and testicular apoptosis support a more active spermatogenesis in fish from site 2, where higher levels of sex steroids and gonadosomatic index (GSI) were also found. In site 1, fish presented low serum levels of testosterone, 17ß-estradiol and 17α-hydroxyprogesterone and a low GSI during gonadal maturation. Spermatogonial proliferation (PCNA) and apoptosis (TUNEL) were more elevated in fish from site 1, but spermatocytes were mainly labelled in fish from site 2. Overall, these data demonstrate changes in testicular activity and plasma sex steroids in a neotropical teleost fish living downstream from a hydroelectric dam, supplying new data on fish reproduction in regulated rivers. Moreover, morphometric analyses associated with sex steroids profiles provide reliable tools to assess fish spermatogenesis under environmental stress conditions.

Dual roles for autophagy during follicular atresia in fish ovary.

Autophagy, a highly conserved catabolic program for degrading proteins and organelles, is essential for cell and tissue homeostasis. Primarily, this process has a cytoprotective role under nutrient deprivation, but several stress stimuli can induce autophagy and, thus, distinct programmed cell death (PCD) pathways can be actived when stress is not abolished. Fish ovaries are a suitable experimental model system for studying the mechanisms of PCD due to the presence of postovulatory and atretic (i.e., nonovulated) follicles, which follow different routes after spawning. Apoptosis of the follicular cells is the major mechanism responsible for the rapid resorption of the postovulatory follicles. Recently, we investigated the contribution of PCD during follicular atresia in two species of freshwater fish. In contrast to mammals, this study revealed that follicular apoptosis is not a major process for deletion of follicular cells in atretic follicles. Furthermore, we detected autophagic vacuoles containing degenerating organelles increasing through follicular atresia in both species. In this addendum, we propose a hypothesis for follicular cell removal during ovarian regression in oviparous fish. In this model, autophagy could have dual roles in follicular atresia. Thus, fish ovaries after breeding are suitable models for studying the interactions among the different cell death pathways.