Exposure to xenoestrogens alters the expression of key morphoregulatory proteins of oviduct adenogenesis in the broad-snouted caiman (Caiman latirostris).

Endocrine disrupting compounds (EDCs) are contaminants ubiquitously found in the environment, which pose a potential threat to aquatic and wetland ecosystems. Caiman latirostris, a crocodilian species that inhabits South American wetlands, is highly sensitive to EDC exposure. Previously, we reported that early postnatal exposure to EDCs such as Bisphenol A (BPA) and 17ß-Estradiol (E2) alters C. latirostris oviduct differentiation. The aim of this work was to elucidate the molecular mechanisms behind this alteration. To accomplish this, we established the ontogenic changes in histological features and the expression of Wnt-7a, Wnt-5a, ß-catenin, FoxA2, desmin, and alpha smooth muscle actin (α-SMA) in the oviduct of C. latirostris. Then, we evaluated the effects of BPA and E2 exposure on these histological features and protein expressions. Our results showed that during the postnatal differentiation of the oviduct the presence of histological features related to adenogenesis is associated with the levels of expression of FoxA2, ß-catenin, Wnt-5a and Wnt-7a. Early postnatal exposure to BPA and E2 decreased the presence of histological features related to adenogenesis and altered the levels of expression of FoxA2, ß-catenin, Wnt-5a and Wnt-7a, as well as the desmin/α-SMA ratio. These findings suggest that altered levels of Wnt-7a, Wnt-5a, ß-catenin and FoxA2 could play a role in the BPA and E2-induced alteration in oviduct differentiation in C. latirostris. Thus, impaired adenogenesis and, probably, impaired reproduction in wildlife naturally exposed to BPA and other estrogenic agonists cannot be completely ruled out.

p38ß (MAPK11) mediates gemcitabine-associated radiosensitivity in sarcoma experimental models.

BACKGROUND AND PURPOSE: Gemcitabine is an antitumour agent currently used in the treatment of several types of cancer with known properties as a radiosensitizer. p38MAPK signalling pathway has been shown to be a major determinant in the cellular response to gemcitabine in different experimental models. However, the molecular mechanism implicated in gemcitabine-associated radiosensitivity remains unknown. MATERIALS AND METHODS: The human sarcoma cell lines A673 and HT1080, and a mouse cell line derived from a 3-methylcholanthrene induced sarcoma were used as experimental models. Modulation of p38MAPKs was performed by pharmacological approaches (SB203580) and genetic interference using lentiviral vectors coding for specific shRNAs. Viability was assessed by MTT. Gene expression was evaluated by western blot and RT-qPCR. Induction of apoptosis was monitored by caspase 3/7 activity. Response to ionizing radiation was evaluated by clonogenic assays. RESULTS: Our data demonstrate that chemical inhibition of p38MAPK signalling pathway blocks gemcitabine radiosensitizing potential. Genetic interference of MAPK14 (p38α), the most abundantly expressed and best characterized p38MAPK, despite promoting resistance to gemcitabine, it does not affect its radiosensitizing potential. Interestingly, specific knockdown of MAPK11 (p38ß) induces a total loss of the radiosensitivity associated to gemcitabine, as well as a marked increase in the resistance to the drug. CONCLUSION: The present work identifies p38ß as a major determinant of the radiosensitizing potential of gemcitabine without implication of p38α, suggesting that p38ß status should be analysed in those cases in which gemcitabine is combined with ionizing radiation.

Metabolic control of acclimation to nutrient deprivation dependent on polyphosphate synthesis.

Polyphosphate, an energy-rich polymer conserved in all kingdoms of life, is integral to many cellular stress responses, including nutrient deprivation, and yet, the mechanisms that underlie its biological roles are not well understood. In this work, we elucidate the physiological function of this polymer in the acclimation of the model alga Chlamydomonas reinhardtii to nutrient deprivation. Our data reveal that polyphosphate synthesis is vital to control cellular adenosine 5'-triphosphate homeostasis and maintain both respiratory and photosynthetic electron transport upon sulfur deprivation. Using both genetic and pharmacological approaches, we show that electron flow in the energy-generating organelles is essential to induce and sustain acclimation to sulfur deprivation at the transcriptional level. These previously unidentified links among polyphosphate synthesis, photosynthetic and respiratory electron flow, and the acclimation of cells to nutrient deprivation could unveil the mechanism by which polyphosphate helps organisms cope with a myriad of stress conditions in a fluctuating environment.

Effects of agricultural pesticides on the reproductive system of aquatic wildlife species, with crocodilians as sentinel species.

Agricultural pesticides represent a significant class of endocrine-disrupting chemicals (EDCs) to which non-target organisms around the world are constantly exposed. Laboratory studies have found strong evidence showing the endocrine-disruptive potential of these pesticides at environmentally relevant exposure levels. Since the field of endocrine disruption continues to grow in richness and complexity, this review aims to provide an update on the effects of two agricultural pesticides that act as EDCs: atrazine and endosulfan. We will focus mainly on the effects on crocodilians due to their worldwide occurrence in tropical and sub-tropical wetland ecosystems and their ecological and physiological features, which render them vulnerable to exposure to pesticides with endocrine-disrupting action at all life stages. The results here reviewed provide important insights into the effects of hormonally active agricultural pesticides at cellular, tissue, and organ levels in the reproductive system of crocodiles. A better understanding of the effects of exposure to environmentally relevant doses of EDCs on the reproductive system of crocodilians will contribute to protect and improve the health of both wildlife species and humans.

The protective effect of extra-virgin olive oil in the experimental model of multiple sclerosis in the rat.

This study has evaluated the effect of EVOO (Extra-Virgin olive oil), OA (oleic acid) and HT (hydroxytyrosol) in an induced model of MS through experimental autoimmune encephalomyelitis (EAE).Dark Agouti 2-month old rats (25 males) were divided into five groups: (i) control group, (ii) EAE group, (iii) EAE+EVOO, (iv) EAE+HT, and (v) EAE+OA. At 65 days, the animals were sacrificed and the glutathione redox system and bacterial lipopolysaccharide (LPS) and LPS-binding protein (LBP) products of the microbiota in brain, spinal cord, and blood were evaluated.Gastric administration of EVOO, OA, and HT reduced the degree of lipid and protein oxidation, and increased glutathione peroxidase, making it a diet-based mechanism for enhancing protection against oxidative damage. In addition, it reduced the levels of LPS and LBP, which appeared as being increased in the EAE correlated with the oxidative stress produced by the disease.

Automatic Staging of Cancer Tumors Using AIM Image Annotations and Ontologies.

A second opinion about cancer stage is crucial when clinicians assess patient treatment progress. Staging is a process that takes into account description, location, characteristics, and possible metastasis of tumors in a patient. It should follow standards, such as the TNM Classification of Malignant Tumors. However, in clinical practice, the implementation of this process can be tedious and error prone. In order to alleviate these problems, we intend to assist radiologists by providing a second opinion in the evaluation of cancer stage. For doing this, we developed a TNM classifier based on semantic annotations, made by radiologists, using the ePAD tool. It transforms the annotations (stored using the AIM format), using axioms and rules, into AIM4-O ontology instances. From then, it automatically calculates the liver TNM cancer stage. The AIM4-O ontology was developed, as part of this work, to represent annotations in the Web Ontology Language (OWL). A dataset of 51 liver radiology reports with staging data, from NCI's Genomic Data Commons (GDC), were used to evaluate our classifier. When compared with the stages attributed by physicians, the classifier stages had a precision of 85.7% and recall of 81.0%. In addition, 3 radiologists from 2 different institutions manually reviewed a random sample of 4 of the 51 records and agreed with the tool staging. AIM4-O was also evaluated with good results. Our classifier can be integrated into AIM aware imaging tools, such as ePAD, to offer a second opinion about staging as part of the cancer treatment workflow.

The external genitalia in juvenile Caiman latirostris differ in hormone sex determinate-female from temperature sex determinate-female.

The broad-snouted caiman (Caiman latirostris) is a crocodilian species that inhabits South American wetlands. As in all other crocodilians, the egg incubation temperature during a critical thermo-sensitive window (TSW) determines the sex of the hatchlings, a phenomenon known as temperature-dependent sex determination (TSD). In C. latirostris, we have shown that administration of 17-ß-estradiol (E2) during the TSW overrides the effect of the male-producing temperature, producing phenotypic females (E2SD-females). Moreover, the administration of E2 during TSW has been proposed as an alternative way to improve the recovery of endangered reptile species, by skewing the population sex ratio to one that favors females. However, the ovaries of E2SD-female caimans differ from those of TSD-females. In crocodilians, the external genitalia (i.e. clitero-penis structure or phallus) are sexually dimorphic and hormone-sensitive. Despite some morphological descriptions aimed to facilitate sexing, we found no available data on the C. latirostris phallus histoarchitecture or hormone dependence. Thus, the aims of this study were: (1) to establish the temporal growth pattern of the phallus in male and female caimans; (2) to evaluate histo-morphological features and the expression of estrogen receptor alpha (ERα) and androgen receptor (AR) in the phallus of male and female pre-pubertal juvenile caimans; and (3) to determine whether the phallus of TSD-females differs from the phallus of E2SD-females. Our results demonstrated sexually dimorphic differences in the size and growth dynamics of the caiman external genitalia, similarities in the shape and spatial distribution of general histo-morphological compartments, and sexually dimorphic differences in innervation, smooth muscle fiber distribution, collagen organization, and ERα and AR expressions. The external genitalia of E2SD-females differed from that of TSD-females in many histological features and in the expression of ERα and AR, resembling patterns described in males. Our results alert on the effects of estrogen agonist exposure during TSW and suggest that caution must be taken regarding the use of E2SD as a procedure for wildlife population management.

The role of intragestational ghrelin on postnatal development and reproductive programming in mice

The purpose of this study was to evaluate the intragestational role of ghrelin in offspring development and reproductive programming in a mouse model of ghrelin imbalance during pregnancy. Female mice were injected with ghrelin (supraphysiological levels: 4 nmol/animal/day), antagonist (endogenous ghrelin inhibition with (D-Lys3)GHRP-6, 6 nmol/animal/day) or vehicle (control = normal ghrelin levels) throughout the pregnancy. Parameters evaluated in litters were growth, physical, neurobiological and sexual development and, at adulthood, reproductive function. Litter size and initial weight did not vary between treatments. Male pups from dams treated with ghrelin showed higher body weight increase until adulthood (31.7 ± 0.8 vs control = 29.7 ± 0.7, n = 11­14 litters/treatment; P < 0.05). Postnatal physical and neurobiological development was not modified by treatments. The antagonist accelerated male puberty onset, evidenced as earlier testis descent and increased relative testicular weight (antagonist = 0.5 ± 0.0% vs ghrelin = 0.4 ± 0.0% and control = 0.4 ± 0.0%, n = 5­10 litters/treatment; P < 0.05). At adulthood, these males exhibited lower relative testicular weight and reduced sperm motility (63.9 ± 3.6% vs control = 70.9 ± 3.3 and ghrelin = 75.6 ± 3.0, n = 13­15 animals; P < 0.05), without changes in plasma testosterone or fertility. Female pups intragestationally exposed to the antagonist showed earlier vaginal opening (statistically significant only at Day 25) and higher ovarian volume (antagonist = 1085.7 ± 64.0 mm3 vs ghrelin = 663.3 ± 102.8 mm3 and control = 512.3 ± 116.4 mm3; n = 4­6 animals/treatment; P < 0.05), indicating earlier sexual maturation. At adulthood, these females and those exposed to ghrelin showed a tendency to higher percentages of embryo loss and/or foetal atrophy. In conclusion, ghrelin participates in reproductive foetal programming: alterations in ghrelin activity during pregnancy modified body weight increase and anticipated puberty onset, exerting (or tending to) negative effects on adult reproductive function.

Maternal and postnatal high-fat diets with high ω6 : ω3 ratios affect the reproductive performance of male offspring in the mouse.

High-fat diets (HFDs) are an acknowledged risk factor for male subfertility, but the underlying mechanisms remain unclear. In the present study we compared the effects of two HFDs with different ω6:ω3 ratios, one enriched with soy oil (SOD; ω6:ω3=9.62) and another enriched with sunflower oil (SFOD; ω6:ω3=51.55), with those of a commercial diet (CD; ω6:ω3=19.87), supplied from pregnancy to adulthood, on morphometric parameters and reproductive performance in adult male mice (recommended ω6:ω3 for rodents=1-6). Bodyweight was significantly higher in the SFOD than CD group, and relative testicular weight was significantly lower in the SFOD than the other two groups. SFOD altered sperm performance: it reduced sperm viability (mean±s.e.m.; 76.00±1.35% vs 82.50±1.45% and 80.63±1.00% in the SFOD vs CD and SOD groups respectively; P<0.05) and increased the percentage of immature spermatozoa (71.88±7.17% vs 51.38±5.87% and 48.00±5.72% in the SFOD vs CD and SOD groups respectively; P<0.05). The epididymal ω6:ω3 ratio was higher in the SFOD versus CD and SOD groups, whereas the unsaturation index was higher in the SOD and SFOD groups than in CD group. Sperm membrane integrity was diminished in both the SOD and SFOD groups, but there was no difference in sperm reactive oxygen species production in these two groups compared with the CD group. The fertilisation rate was lower in the SFOD compared with the CD and SOD groups. In conclusion, although both HFDs affected sperm quality, the fertilising ability was more altered by the excessive dietary ω6:ω3 ratio than by the net ω6 content.

Development of a quantitative immuno-polymerase chain reaction assay to detect and quantify low levels of human thyroid stimulating hormone.

In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and a highly sensitive immuno-polymerase chain reaction (IPCR) assay specific for detection of human thyroid stimulating hormone (hTSH). Several anti-hTSH monoclonal antibodies (MAbs) were generated using hybridoma technology. Two pairs of MAbs (B-4 and B-9) were rationally selected and the optimal assay conditions of sandwich ELISAs were established. The ELISA prototypes were evaluated with standards calibrated with WHO 2nd International Reference Preparation for hTSH and in comparison with a commercial ELISA Kit. Although the limit of detection (LOD) was 0.1 µIU/ml in all cases, B-9-ELISA showed an analytical performance similar to commercial ELISA Kit. Therefore, we selected the B-9 ELISA to develop a hTSH-IPCR assay applying an "Universal-IPCR" format in standard PCR tubes without pretreatment. The signal amplification was achieved through the interaction between the biotinylated detection MAb and mono-biotinylated DNA probe pre-self-assembled with neutravidin. The hTSH-IPCR assay showed a significant increase in terms of the slope definition of sensitivity in low levels range. Our results support the potential of IPCR technique for being applied in clinical diagnosis of thyroid states.

Dose-dependent S-allyl cysteine ameliorates multiple sclerosis disease-related pathology by reducing oxidative stress and biomarkers of dysbiosis in experimental autoimmune encephalomyelitis.

Garlic is a component of the Mediterranean diet. S-allyl cysteine (SAC), the most common organosulphur present in garlic, possesses neuroprotective properties. This investigation was performed to evaluate the dose-dependent protective action of SAC on oxidative damage, inflammation and gut microbiota alterations biomarkers. Experimental autoimmune encephalomyelitis (EAE) as a model of multiple sclerosis (MS) was induced by the myelin oligodendrocyte glycoprotein (MOG), whose effects were quantified by examining the changes in: clinical score, lipid peroxidation products, carbonylated proteins, glutathione system, tumor necrosis factor alpha (TNFα), and lipopolysaccharide membrane bacteria (LPS). Our results reveal that MOG induces paralysis, oxidative damage and increases in LPS binding protein (LBP) and LPS levels. In this work, two doses of SAC were compared with two dose of N-acetyl cysteine (NAC). SAC was more effective than NAC and it prevented the harmful effects induced by MOG more effectively at the dose of 50mg/kg than that of 18mg/kg. Surprisingly, NAC increases LBP levels while SAC had not such negative effect. In conclusion the data show the ability of SAC to modify EAE evolution.

Impaired ovarian response to exogenous gonadotropins in female rat offspring born to mothers perinatally exposed to Bisphenol A.

The ovary is sensitive to disruption by the environmental estrogen Bisphenol A (BPA). Our aim was to investigate whether perinatal exposure to BPA (50µg/kgday), orally administered, affects ovarian response to exogenous gonadotrophins (PMSG or PMSG+hCG) in prepubertal female offspring. An altered response to gonadotrophins was observed in BPA-exposed rats. Increased proportion of antral follicles, altered levels of ovarian steroidogenic enzymes, gonadotropin receptors, AR and ERß were observed in PMSG group. Besides that, in response to PMSG+hCG, a persistent high Fshr mRNA expression and a decreased number of follicles with high expression of PR before ovulation were observed. After ovulation, there was an increase in antral atretic follicles, reduced Lhcgr mRNA expression and high serum levels of E2. Therefore, an early exposure to a low dose of BPA during perinatal period induces ovarian changes leading to an altered response to exogenous gonadotropin treatment later in life.

Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase.

GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST). rGST was expressed in Escherichia coli JM109, using a pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridoma technology. Two of these were rationally selected as capture and detection antibodies, allowing the development of a sandwich ELISA with a limit of detection (LOD) of 0.01 µg/ml. To develop the rGST-IPCR assay, we selected "Universal-IPCR" format, comprising the biotin-avidin binding as the coupling system. In addition, the rGST-IPCR was developed in standard PCR tubes, and the surface adsorption of antibodies on PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentration effect were studied and determined. Under optimized assay conditions, the rGST-IPCR assay provided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGST-ELISA. The proposed method exhibited great potentiality for application in several fields in which measurement of very low levels of GST is necessary, and might provide a model for other IPCR assays.

Body mass index and human sperm quality: neither one extreme nor the other.

The aim of the present study was to investigate the still contentious association between body mass index (BMI) and seminal quality. To this end, 4860 male patients (aged 18-65 years; non-smokers and non-drinkers), were classified according to BMI as either underweight (UW; BMI <20kgm-2; n=45), normal weight (NW; BMI 20-24.9kgm-2; n=1330), overweight (OW; BMI 25-29.9kgm-2; n=2493), obese (OB; BMI 30-39.9kgm-2; n=926) or morbidly obese (MOB; BMI ≥40kgm-2; n=57). Conventional semen parameters and seminal concentrations of fructose, citric acid and neutral α-glucosidase (NAG) were evaluated. The four parameters that reflect epididymal maturation were significantly lower in the UW and MOB groups compared with NW, OW and OB groups: sperm concentration, total sperm count (103.3±11.4 and 121.5±20.6 and vs 157.9±3.6, 152.4±2.7 or 142.1±4.3 spermatozoa ejaculate-1 respectively, P<0.05), motility (41.8±2.5 and 42.6±2.6 vs 47.8±0.5, 48.0±0.4 or 46.3±0.6 % of motile spermatozoa respectively, P<0.05) and NAG (45.2±6.6 and 60.1±7.9 vs 71.5±1.9, 64.7±1.3 or 63.1±2.1 mU ejaculate-1 respectively, P<0.05). Moreover, the percentage of morphologically normal spermatozoa was decreased in the MOB group compared with the UW, NW, OW and OB groups (4.8±0.6% vs 6.0±0.8%, 6.9±0.1%, 6.8±0.1 and 6.4±0.2%, respectively; P<0.05). In addition, men in the MOB group had an increased risk (2.3- to 4.9-fold greater) of suffering oligospermia and teratospermia (P<0.05). Both morbid obesity and being underweight have a negative effect on sperm quality, particularly epididymal maturation. These results show the importance of an adequate or normal bodyweight as the natural best option for fertility, with both extremes of the BMI scale as negative prognostic factors.

Analysis of the interaction between transcription factor 7-like 2 genetic variants with nopal and wholegrain fibre intake: effects on anthropometric and metabolic characteristics in type 2 diabetes patients.

The transcription factor 7-like 2 (TCF7L2) genetic variants have shown differential effect on low-fat and high-fat diet in obese subjects. Nopal is a Mexican variety of cactus that is a traditional food and has been used in the treatment of diabetes. Its hypoglycaemic effect may be because of its soluble fibre (mucopolysaccharide) content. This study analysed the effects of the rs7903146 and rs12255372 TCF7L2 variants on anthropometric, metabolic and hormonal parameters in type 2 diabetes mellitus patients who consumed fibre from either nopal tortilla or wholegrain bread for 8 weeks. We followed-up seventy-four patients who consumed an individualised isoenergetic diet that included nopal tortilla (Diet 1) and sixty-three patients with a diet that included wholegrain bread (Diet 2). Anthropometric, metabolic and hormonal measures were collected at baseline and final intervention. The size effect and carry-over effect were estimated. To assess the interaction of genotype and diets, we used a general linear model repeated-measures analysis. Minor allele frequency of rs7903146T was 0·27 and for rs12255372T it was 0·13. At 8 weeks after Diet 1 intake, weight, BMI, waist and hip circumference decreased (P=0·00015) in rs7903146CC and rs12255372GG genotypes. In particular, patients carrying of the rs7903146CC and consuming Diet 1 showed a reduction in waist circumference of more than 2·5 cm compared with Diet 2 (P<0·001). No significant interaction between rs7903146 or rs12255372 and diet was seen in this study. In conclusion, in the carriers of the rs7903146CC and rs12255372GG wild types, significant changes in all anthropometric measures were observed, and had better response to both diets.

Postnatal development and histofunctional differentiation of the oviduct in the broad-snouted caiman (Caiman latirostris).

Caiman latirostris is a South American crocodilian species characterized as a sentinel of the presence of endocrine-disrupting compounds (EDCs). Evaluating developmental events in hormone-dependent organs, such as the oviduct, is crucial to understand physiological postnatal development, to identify putative periods of exposure sensitive to EDCs, and/or to identify biomarkers useful to evaluate the effects of EDC exposure. In this study, we describe the histomorphological features of C. latirostris oviducts by establishing the ontogeny of changes at cellular, tissue and molecular levels from the neonatal to the pre-pubertal juvenile stages. Since the histological diagnosis of the adenogenic oviduct lies on a group of features, here we defined a histofunctional score system and a cut-off value to distinguish between preadenogenic and adenogenic oviducts. Our results showed that the maturation of the C. latirostris oviduct is completed postnatally and characterized by changes that mimic the pattern of histological modifications described for the mammalian uterus. Ontogenic changes in the oviductal epithelium parallel changes at subepithelial level, and include collagen remodeling and characteristic spatial-temporal patterns of α-actin and desmin. The expression pattern of estrogen receptor alpha and progesterone receptor evidenced that, even at early postnatal developmental stages, the oviduct of C. latirostris is a target organ of endogenous and environmental hormones. Besides, oviductal adenogenesis seems to be an estrogen-dependent process. Results presented here provide not only insights into the histophysiological aspect of caiman female reproductive ducts but also new tools to better characterize caimans as sentinels of endocrine disruption.

The eggshell features and clutch viability of the broad-snouted caiman (Caiman latirostris) are associated with the egg burden of organochlorine compounds.

Organochlorine compounds (OCCs) are toxic and have been identified as endocrine-disrupting chemicals (EDCs). The broad-snouted Caiman (Caiman latirostris) is an oviparous species widely distributed in South America with potential to accumulate OCCs. The eggshell is formed during passage of the eggs through the oviduct. Since the oviduct is a target of hormone actions, exposure to OCCs could modify eggshell quality, thus affecting clutch viability. Eight clutches were collected from wetlands of Parana River tributaries, in north-eastern Argentina. Two to four eggs per clutch were used to establish the burden of OCCs, eggshell thickness and eggshell porosity. The remaining eggs were incubated in controlled conditions. Ten days after hatching, hatchling survival was assessed. Organochlorine pesticide residues (OCPs) were found in all clutches, while polychlorinated biphenyls (PCBs) were present in all but one clutch. The principal contributors to the OCP burden were members of the DDT family and oxychlordane. Eggshell thickness was 400.9±6.0 µm and, unexpectedly, no association between eggshell thickness and the OCC burden was found. The number of pores in the outer surface was 25.3±4.3 pores/cm². A significant inverse correlation between porosity and OCC burden was found (Pearson r= -0.81, p= 0.01). Furthermore, a decrease in caiman survival with decreased pore density was observed (Pearson r= 0.73, p= 0.04). Our findings highlight another potential negative impact of current and past use of OCCs on wildlife species.

Effects of copper sulfate on growth and physiological responses of Limoniastrum monopetalum.

A glasshouse study of the coastal shrub Limoniastrum monopetalum was carried out to evaluate its tolerance and capacity to accumulate copper. We investigate the effects of Cu from 0 to 60 mmol l(-1) on the growth, photosynthetic apparatus, and nutrient uptake of L. monopetalum, by measuring gas exchange, chlorophyll fluorescence parameters, photosynthetic pigments, and total copper, nitrogen, phosphorus, sulfur, calcium, and magnesium content in the plant tissues. Although L. monopetalum did not survive at 60 mmol l(-1) Cu, the species demonstrated a high tolerance to Cu-induced stress, since all plants survived external Cu concentrations of up to 35 mmol l(-1) and displayed similar growth in the Cu-enriched medium as in the control treatment of up to the external level of 15 mmol Cu l(-1) (1,000 mg Cu l(-1)). The reduced growth registered in plants exposed to 35 mmol Cu l(-1) can be attributed to reduced photosynthetic carbon assimilation associated with the adverse effect of the metal on the photochemical apparatus and a reduction in the absorption of essential nutrients. Copper tolerance was associated with the capacity of the plant to accumulate the metal in its roots and effectively prevent its translocation to photosynthetic tissues. L. monopetalum has the characteristics of a Cu-excluder plant and could be used in the revegetation of Cu-contaminated soils.

Reproductive performance and fertility in male and female adult mice chronically treated with hexarelin.

Hexarelin (HEXr), a synthetic ghrelin analogue, has been associated with modifications of reproductive physiology. In previous studies of adult mice, we detected that HEXr induced significantly reduced ovulation rate and significant correlation coefficients between sexual maturation and corporal weight in offspring. In this study, we investigated the effects of chronic HEXr administration on sperm concentration and functional activity, oestrous cyclicity and pregnancy index, in addition to the number of fetuses and its correlation with the number of corpora lutea. Adult Albino swiss mice were injected (sc) daily with HEXr: 100 µgkg(-1) day(-1) (HEXr D1) or 200 µgkg(-1) day(-1) (HEXr D2) for 53 days in males and 30 days in females. We detected a significantly decreased ratio in the number of fetuses per corpora lutea in females treated with HEXr D2 for 30 days before mating and during the first 6 days of pregnancy, in addition to a downward trend in the pregnancy index and percentage of females impregnated by each male treated with both doses of the analogue. Although we did not find any significant effect on additional parameters evaluated in both genders, we propose certain effects of HEXr on the implantation process and/or early development of embryos and over the in vivo reproductive capability of males.

Neuroprotective effects of extremely low-frequency electromagnetic fields on a Huntington's disease rat model: effects on neurotrophic factors and neuronal density.

There is evidence to suggest that the neuroprotective effect of exposure of extremely low-frequency electromagnetic fields (ELF-EMF) may be due, at least in part, to the effect of these fields on neurotrophic factors levels and cell survival, leading to an improvement in behavior. This study was undertaken to investigate the neuroprotective effects of ELFEF in a rat model of 3-nitropropionic acid (3NP)-induced Huntington's disease. Behavior patterns were evaluated, and changes in neurotrophic factor, cell damage, and oxidative stress biomarker levels were monitored in Wistar rats. Rats were given 3NP over four consecutive days (20 mg/kg body weight), whereas ELFEF (60 Hz and 0.7 mT) was applied over 21 days, starting after the last injection of 3NP. Rats treated with 3NP exhibited significantly different behavior in the open field test (OFT) and the forced swim test (FST), and displayed significant differences in neurotrophic factor levels and oxidative stress biomarkers levels, together with a neuronal damage and diminished neuronal density, with respect neuronal controls. ELFEF improved neurological scores, enhanced neurotrophic factor levels, and reduced both oxidative damage and neuronal loss in 3NP-treated rats. ELFEF alleviates 3NP-induced brain injury and prevents loss of neurons in rat striatum, thus showing considerable potential as a therapeutic tool.