Alfonso Blanco

Dose-dependent antioxidant responses and pathological changes in tenca (Tinca tinca) after acute oral exposure to Microcystis under laboratory conditions

The effects of cyanobacterial cells containing microcystins (MCs), toxins from cyanobacteria, on oxidative stress biomarkers from liver and kidney of Tenca fish (Tinca tinca) were investigated under laboratory conditions. Moreover, a histopathological study of liver, kidney, heart and intestine tissues was performed. Fish were orally exposed to cyanobacterial cells dosing 0, 5, 11, 25 and 55 microg MC-LR/fish mixed with the food. Results showed a dose-dependent decrease of superoxide dismutase (SOD) activity, and also of catalase (CAT) in the liver. Glutathione levels and protein oxidation, however, were not altered by the exposure to the cyanobacterial material. The microscopic study revealed tissue alterations even at the lower cyanobacterial cells doses. Onion-like hepatocytes in the liver, glomerulopathy in the kidney, loss of myofibrils in the heart and vacuolated enterocytes in the gastrointestinal tract were the main changes observed. These findings suggest that this fresh water fish can be adversely affected by cyanobacterial blooms in their natural habitats.

Biological Role of Pituitary Estrogen Receptors ERα and ERβ on Progesterone Receptor Expression and Action and on Gonadotropin and Prolactin Secretion in the Rat

Estrogen (E) is a key regulator of the synthesis and secretion of pituitary reproductive hormones [luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL)]. Until recently, it was thought that all biological actions of E at the pituitary were manifested through a single E receptor (R). The pituitary, like many other reproductive tissues, expresses two isoforms of ER, α and β, both activated by E. The relative contribution of α and β forms in E regulatory actions is largely unknown. To this end, 2-week-old ovariectomized (OVX) rats were injected over 3 days with 25 µg estradiol benzoate (EB), 1.5 mg of propylpyrazole triol (PPT), a selective ERα agonist, 1.5 mg of the selective ERβ agonist diarylpropionitrile (DPN) or a combination of PPT and DPN. Controls were injected with 0.2 ml oil. At 10:00 h on the day after treatment, trunk blood was collected to determine serum concentration of LH, FSH and PRL, and pituitaries were processed for RT-PCR analysis of total (A+B) progesterone receptor (PR) mRNA, immunocytochemistry of PR and incubation. Pituitaries from each of the five groups were incubated in DMEM, with or without 20 nM of the antiprogestin at the receptor ZK299, for 3 h with: 10–8M 17β-estradiol, 10–6M PPT, 10–6M DPN, PPT+DPN or medium alone, respectively, to determine LH, FSH and PRL secretion, and, when challenged with two pulses of 15 min 1 h apart of 10–8M gonadotropin-releasing hormone (GnRH) (GnRH self-priming). EB, PPT and PPT+DPN treatments increased PR mRNA and the number and intensity of nuclei immunoreactive (IR) for PR in gonadotropes, and reduced the number of gonadectomy cells. Like E, PPT alone or in combination with DPN stimulated PRL secretion, increased basal and GnRH-stimulated LH and FSH secretion and induced GnRH self-priming in the absence of ZK299 in the incubation medium. DPN alone had only a significant E-like effect on gonadectomy cells and IR-PR, but not on GnRH self-priming. In addition, while DPN lacked an agonistic action on peripheral tissue and serum pituitary reproductive hormones concentration, EB, PPT and PPT+DPN induced similar uterine ballooning and vaginal cornification, and increased and decreased, respectively, serum concentrations of PRL and gonadotropins. Overall, these results indicate that most of these E actions on the pituitary are exerted through the ERα isoform. The finding that activation of ERβ with its selective DPN agonist had an estrogenic effect on IR-PR nuclei, but not on GnRH self-priming, a characteristic ERα-mediated effect of E, suggests that the biological action of E at the pituitary may involve both isoforms of ER.

Effects of dietary selenium on the oxidative stress and pathological changes in tilapia (Oreochromis niloticus) exposed to a microcystin-producing cyanobacterial water bloom

The present study investigates the role of selenium (Se) supplementation (as sodium selenite) on the oxidative stress and histopathological changes induced by cyanobacterial cells containing microcystins (MCs) in tilapia fish (Oreochromis niloticus). Variation in lipid peroxidation (LPO) levels and carbonyl groups content, reduced glutathione/oxidized glutathione (GSH/GSSG) ratio, and catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities in liver and kidney of tilapia fish exposed to a single oral dose of 120 microg MC-LR/fish and sacrificed in 24 h, were investigated in the absence and presence of 1.5, 3.0 and 6.0 microg Se/g diet. Results showed a protective role of Se depending on the dose and the biomarker considered. Thus, the lower Se dose made CAT, liver GR and kidney SOD converged to basal values, whereas LPO and liver SOD and GST needed the higher dose. Kidney GR, however, was not protected at any Se dose. Moreover, Se has also shown to have a pro-oxidant effect with increased kidney LPO values and liver and kidney GPx activities in MC-free fish. The microscopic study revealed tissue alterations induced by cyanobacterial cells in the liver, kidney, heart and gastrointestinal tract that were ameliorated by the highest Se dose assayed. The level of Se supplementation must be therefore carefully selected to provide beneficial effects and to avoid potential negative consequences.

Quantitative changes in the testicular structure in mice exposed to low doses of cadmium.

The purpose of this study was to quantify the consequences of a long-term exposure to low doses of cadmium on the testicular structure. Sexually mature male mice were orally exposed to cadmium (0.015g/1 of CdCl2 in drinking water) for 1, 3, 6 and 12 months and then sacrificed; cadmium withdrawal was also considered in two groups raised with cadmium for 3 and 6 months, and without cadmium for 3 and 6 months before sacrifice, respectively. Morphometrical and stereological estimations were applied to quantify the structural constituents of the testes. The morphological parameters (testicular mass and size) were significantly decreased at 6 and 12 months of cadmium exposure. Interstitium was the testicular constituent most sensitive to cadmium so that significant decreases in the volume fractions of interstitium and Leydig cells were recorded as from 3 months of cadmium exposure. Cadmium-exposed seminipherous tubules showed increased diameters and lumens together with decreased tubular densities and epithelial percentages.

Time-dependent histopathological changes induced in Tilapia (Oreochromis niloticus) after acute exposure to pure cylindrospermopsin by oral and intraperitoneal route

Although fish and aquatic organisms can be in contact with the cyanotoxin cylindrospermopsin (CYN), toxicological studies are practically nonexistent. CYN has a late and progressive acute toxicity in rodents, but no data have been reported in fish. In this work, tilapia (Oreochromis niloticus) were exposed for the first time to an acute dose of CYN (200 μg/kg fish) by intraperitoneal (i.p.) injection, and the effects were compared with the oral route (gavage). In both cases, fish were sacrificed after 24 h or 5 days of the toxin administration. CYN induced multiorganic damage, being the liver and kidney the main targets of toxicity. The histological findings were more pronounced after i.p. administration (in the liver, kidney, heart, gills) with the exception of the gastrointestinal tract. The time of sacrifice influenced the degree of histological damage in all organs studied, and was more severe after 5 d in comparison to 24 h. Moreover, CYN induced an increase in the average nuclear diameter of hepatocytes in the liver, and decreased cross sections of proximal and distal convoluted tubules in the kidney. The changes in these parameters were also more severe by i.p. route, and with the time of sacrifice, supporting the histopathological results obtained in these organs. Thus, both parameters could be useful for quantifying the extent of the damage in fish after CYN exposure.

Acute exposure to pure cylindrospermopsin results in oxidative stress and pathological alterations in tilapia (Oreochromis niloticus)

Cylindrospermopsin (CYN) is increasingly recognized as a potential threat to drinking water safety, due to its ubiquity. This cyanotoxin has been found to cause toxic effects in mammals, and although fish could be in contact with this toxin, acute toxicity studies on fish are nonexistent. This is the first study showing that single doses of CYN pure standard (200 or 400 μg CYN/kg fish bw) by oral route (gavage) generate histopathological effects in fish (Tilapia—Oreochromis niloticus) exposed to the toxin under laboratory condition. Among the morphological changes, disorganized parenchymal architecture in the liver, dilated Bowmans space in the kidney, fibrolysis in the heart, necrotic enteritis in the intestines, and hemorrhages in the gills, were observed. Moreover, some oxidative stress biomarkers in the liver and kidney of tilapias were altered. Thus, CYN exposure induced increased protein oxidation products in both organs, NADPH oxidase activity was significantly increased with the kidney being the most affected organ, and decreased GSH contents were also detected in both organs, at the higher dose assayed.

Time-dependent protective efficacy of Trolox (vitamin E analog) against microcystin-induced toxicity in tilapia (Oreochromis niloticus).

Microcystins (MCs), hepatotoxins from cyanobacteria, induce oxidative stress and pathological changes in fish that can be ameliorated with chemoprotectants such as vitamin E (vit E). This study investigated the time period after MCs exposure in which Trolox, a vitamin E analog, is effective against oxidative and histological damage in different organs of tilapia (Oreochromis niloticus). Fish were fed Trolox supplement (700 mg/kg diet) for 7 days, or received only commercial fish food, and then were exposed to a single oral dose of 120 μg/fish microcystin‐LR, and sacrificed in 24, 48, or 72 h. The Trolox protective efficacy was evaluated based on lipid peroxidation (LPO), protein oxidation, enzymatic and non‐enzymatic antioxidants, and a morphologic study. Regarding the oxidative stress biomarkers altered by MCs, the higher protective action of Trolox was observed 24 h post toxin exposure, although it extends also until 48 h in gills (superoxide dismutase (SOD), catalase (CAT)), and liver, where glutathione reductase (GR) backed to control values 48 and 72 h after the toxin application. Glutathione‐S‐ transferase (GST) activity in the liver was ameliorated by the chemoprotectant after 24 and 48 h, although control values were not recovered. Trolox modulation of these biomarkers and its ability to quench free radicals explain the recovery of LPO values in all organs at 24 h and also in gills at 48 h. Histopathologically, Trolox efficacy was more evident after 72 h.

Endocrine-active compound evaluation: qualitative and quantitative histomorphological assessment of zebrafish gonads after bisphenol-A exposure.

There is great social concern about the risk involved from exposure to BPA as an endocrine disrupter in humans, as well as the possible repercussion of this chemical on the environment. In this study, the short-term effects of BPA at a gonadal level were assessed by means of different biomarkers in a model animal organism in vogue, the zebrafish (Danio rerio). For this purpose, 60 female zebrafish aged 16 weeks were used. These were exposed for 14 days in aquariums (following OECD Directive no.204) to BPA concentrations of 1, 10, 100 and 1000 μg/L, in addition to a control batch. After the exposure period, the zebrafish were sacrificed and samples taken for a histopathological study by light and electron microscopy and morphometric analysis. During the fourteen days of exposure, water samples were taken from the aquariums to analyze the BPA levels. The BPA concentration in the fish and the water was determined by LC-MS/MS. The gonads of the zebrafish exposed to the BPA had a normal external appearance and there were no variations in their size or body weight. An accumulation of BPA was produced in the zebrafish tissues, and this increased as the BPA concentration to which the fish were exposed did. In the histopathological and morphometric studies, multiple alterations were observed in the zebrafish ovaries, particularly highlighting the vacuolization of the follicular cytoplasm, a great degeneration of all the cell components, and an important increase in the percentage of atretic follicles as from concentrations of 100 and 1000 μg/L of BPA, verified by morphometry. These data indicate that morphological endpoints are sufficiently sensitive to individuate early effects of environmental concentration of BPA on gonads after two weeks of exposure.

Regulation of corticosterone secretion is modified by PFOS exposure at different levels of the hypothalamic–pituitary–adrenal axis in adult male rats

Perfluorooctane sulfonate (PFOS) is a fluorinated compound and a Persistent Organic Pollutant which can disrupt the endocrine system. This work was undertaken to evaluate the possible effects of PFOS exposure on the regulation of corticosterone secretion in adrenal and pituitary glands and at hypothalamic level in adult male rat, and to evaluate the possible morphological alterations induced by PFOS in this endocrine tissue. Adult male rats were orally treated with 0.5, 1.0, 3.0 and 6.0 mg of PFOS/kg/day for 28 days. Corticosterone, adrenocorticotropic hormone (ACTH) and corticotrophin-releasing hormone (CRH) secretion decreased in PFOS-treated rats. After PFOS exposure, relative expression of adrenocorticotropic hormone receptor (ACTHr) and proopiomelanocortin (POMC) genes was increased in adrenal and in pituitary glands, respectively; while relative expression of ACTHr and CRH genes decreased in hypothalamus with the doses of 0.5 and 1.0 mg/kg/day. PFOS treatment increased relative nitric oxide synthase 1 and 2 (NOS1 and NOS2) gene expression in the adrenal gland, and incremented superoxide dismutase activity. PFOS exposure induces a global inhibition of the hypothalamic-pituitary-adrenal (HPA) axis activity, and small morphological changes were observed in adrenal zona fasciculata cells.

Multigenerational study of the hepatic effects exerted by the consumption of nonylphenol- and 4-octylphenol-contaminated drinking water in Sprague–Dawley rats

Our multigenerational study evaluates the hepatic effects of the xenoestrogens nonylphenol (NP), and 4-octylphenol (4OP) on male and female rats when they are exposed uninterruptedly, from conception to adult age, to tap water containing 25ppm of NP or 4OP. Our results showed that these compounds did not induce any change in liver/body weight ratio (relative liver weight, RLW). In the morphological analysis we did not find evident signs of cytotoxicity. The most relevant findings were the presence of both an increase in the apoptotic index and in the percentage of binuclear hepatocytes in livers from exposed animals. Additionally, our study revealed the presence of hepatocellular glycogenosis (mainly in 4OP-exposed rats): the type of glycogen accumulated was in aggregates (gamma-glycogen), a non-functional form of glycogen. This study demonstrates that, at levels close to those described in the environment, NP and 4OP are capable of inducing a number of hepatic effects, potentially related with adaptive, and/or metabolic alterations of liver tissue.