Thyroid disrupting effects of low-dose dibenzothiophene and cadmium in single or concurrent exposure: New evidence from a translational zebrafish model.

Thyroid hormones (THs) are major regulators of biological processes essential for correct development and energy homeostasis. Although thyroid disruptors can deeply affect human health, the impact of exogenous chemicals and in particular mixture of chemicals on different aspects of thyroid development and metabolism is not yet fully understood. In this study we have used the highly versatile zebrafish model to assess the thyroid axis disrupting effects of cadmium (Cd) and dibenzothiophene (DBT), two environmental endocrine disruptors found to be significantly correlated in epidemiological co-exposure studies. Zebrafish embryos (5hpf) were exposed to low concentrations of Cd (from 0.05 to 2 µM) and DBT (from 0.05 to 1 µM) and to mixtures of them. A multilevel assessment of the pollutant effects has been obtained by combining in vivo morphological analyses allowed by the use of transgenic fluorescent lines with liquid chromatography mass spectrometry determination of TH levels and quantification of the expression levels of key genes involved in the Hypothalamic-Pituitary-Thyroid Axis (HPTA) and TH metabolism. Our results underscore for the first time an important synergistic toxic effect of these pollutants on embryonic development and thyroid morphology highlighting differences in the mechanisms through which they can adversely impact on multiple physiological processes of the HPTA and TH disposal influencing also heart geometry and function.

Vitamin D status and cardiovascular outcome.

INTRODUCTION: Vitamin D is classically involved in maintaining bone and mineral health, but it has been shown to exert many extraskeletal functions, including pleiotropic effects on cardiovascular system. MATERIALS AND METHOD: This review aims to summarize evidences in literature about vitamin D and cardiovascular outcome. RESULTS AND CONCLUSIONS: Calcitriol or 1,25(OH)2D, the active hormone, binds to the specific nuclear receptor VDR, which is expressed in rat and human heart and vasculature and has effects on myocardiocytes, smooth cells, and endothelial cells. 25-Hydroxy-vitamin D (25OHD) represents the biomarker of vitamin D levels and reflects vitamin D status. There is consistent evidence that low serum 25OHD levels are associated with increased risk of cardiovascular diseases, including hypertension, coronary artery disease, ischemic heart disease, heart failure, stroke, and type 2 diabetes. Randomized-controlled trials and Mendelian randomization studies so far have not succeeded in proving a benefit of vitamin D supplementation. However, the latter investigations are affected by some methodological limitations, and therefore, it is still unclear if vitamin D deficiency has a causative role in cardiovascular diseases or is rather a marker of poor health in chronic disease.

Measurement of urinary free cortisol by LC-MS-MS: adoption of a literature reference range and comparison with our current immunometric method.

PURPOSE: One of the best indicators of adrenal gland dysfunction is the level of free cortisol measured in the 24-h urine (UFC) which faithfully reflects the level of biologically active serum cortisol not subjected to circadian variations. Liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) is a sensitive, accurate and precise method recently available in routine laboratories that could remedy interference problems of immunoassays. METHODS: In this study, a literature reference range for UFC measured by LC-MS-MS was verified, and UFC values measured by LC-MS-MS and immunoassay were compared. Immunometric UFC measurement was performed by ACCESS CORTISOL assay without preliminary extraction, using Beckman Coulter UniCel DxI 600 highly automated platform. Liquid chromatography-tandem mass spectrometry UFC measurement was performed by a home-made validated method using cortisol-D4 as internal standard with preliminary deproteinization of urinary samples by centrifugal filter and injection on reverse-phase column. Cortisol was analyzed in positive ion mode with an ESI interface. RESULTS: The reference interval from literature (11-70 µg/day) was confirmed by results obtained for healthy study group. Comparison study of the two methods highlighted a constant and proportional systematic error with a general tendency to overestimate results for the in-use method. CONCLUSIONS: In conclusion, the direct immunometric method overestimates UFC results with respect to liquid chromatography-tandem mass spectrometry which represents the reference method. The literature reference range 11-70 µg/day was confirmed and can be adopted by our lab that will shift all UFC tests performed in routine to the mass spectrometry-based method, satisfying clinicians' request.

Effect of Seasonality on Rates of Gregarine Infection in Workers of a Social Wasp Polybia Paulista (Hymenoptera, Vespidae).

Social insects face strong selection from parasites because the conditions of group living often favor the transmission of infection among nestmates. However, there is little detailed information on the effects of parasite infection in the host species. Workers of Polybia species, neotropical swarm-founding wasps, are commonly infected by gregarines, protozoans that are exclusively parasitic on invertebrates. Previous studies showed that high rates of gregarine infection in workers of Polybia occidentalis (Olivier) have negative effects on their colony performance. However, the effect of seasonality on infection rates throughout the year or between wet and dry seasons has not been examined. Host-parasite interactions cannot be understood without consideration of the overall population dynamic. We compared rates of gregarine infection in workers of Polybia paulista (Ihering) between wet and dry seasons and among months. The 35% rate was by far the highest of the four wet seasons sampled, but the rates declined in the mid-wet season and were very low during the dry season. Strong seasonal differences in infection rates were also observed between the dry and wet seasons. Several potential factors affecting the seasonal differences are discussed.

Fruit Fly (Diptera: Tephritidae) Diversity and Host Relationships in Diverse Environments Estimated with Two Sampling Methods.

Knowledge of the diversity of fruit flies is essential for understanding aspects of their community ecology. However, the effectiveness of sampling methods in representing species diversity and relationships with hosts in a diverse environment has not been adequately assessed. This study aimed to determine the optimum method to represent the diversity of fruit flies and assess their relationships with host fruits. Species of Anastrepha sampled with both traps and fruits in the same area were comprehensively analyzed. Data were analyzed by Hill's numbers (species diversity in both sampling methods), heat map graph (interaction of species with methods), and rank clocks (population fluctuations of the most abundant species). We also measured the interaction strength of the species. Our results showed that estimated parameters of species diversity in an area may differ when sampled with traps or fruits. However, the most abundant species appeared to interact similarly in both methods. Fruits of members of the families Myrtaceae and Anacardiaceae contributed highly to the presence of Anastrepha. The optimum strategy to represent Anastrepha diversity in an area is the combined use of both sampling methods.

Geostatistics and Geographic Information System to Analyze the Spatial Distribution of the Diversity of Anastrepha Species (Diptera: Tephritidae): the Effect of Forest Fragments in an Urban Area.

Fruit flies are among the most damaging insect pests of commercial fruit in Brazil. It is important to understand the landscape elements that may favor these flies. In the present study, spatial data from surveys of species of Anastrepha Schiner (Diptera: Tephritidae) in an urban area with forest fragments were analyzed, using geostatistics and Geographic Information System (GIS) to map the diversity of insects and evaluate how the forest fragments drive the spatial patterns. The results indicated a high diversity of species associated with large fragments, and a trend toward lower diversity in the more urbanized area, as the fragment sizes decreased. We concluded that the diversity of Anastrepha species is directly and positively related to large and continuous forest fragments in urbanized areas, and that combining geostatistics and GIS is a promising method for use in insect-pest management and sampling involving fruit flies.

Toward an Automated Identification of Anastrepha Fruit Flies in the fraterculus group (Diptera, Tephritidae).

In this study, we assess image analysis techniques as automatic identifiers of three Anastrepha species of quarantine importance, Anastrepha fraterculus (Wiedemann), Anastrepha obliqua (Macquart), and Anastrepha sororcula Zucchi, based on wing and aculeus images. The right wing and aculeus of 100 individuals of each species were mounted on microscope slides, and images were captured with a stereomicroscope and light microscope. For wing image analysis, we used the color descriptor Local Color Histogram; for aculei, we used the contour descriptor Edge Orientation Autocorrelogram. A Support Vector Machine classifier was used in the final stage of wing and aculeus classification. Very accurate species identifications were obtained based on wing and aculeus images, with average accuracies of 94 and 95%, respectively. These results are comparable to previous identification results based on morphometric techniques and to the results achieved by experienced entomologists. Wing and aculeus images produced equally accurate classifications, greatly facilitating the identification of these species. The proposed technique is therefore a promising option for separating these three closely related species in the fraterculus group.

Quantification of thyroxine and 3,5,3'-triiodo-thyronine in human and animal hearts by a novel liquid chromatography-tandem mass spectrometry method.

Assaying tissue T3 and T4 would provide important information in experimental and clinical investigations. A novel method to determine tissue T3 and T4 by HPLC coupled to mass spectrometry is described. The major difference vs. previously described methods lies in the addition of a derivatization step, that is, to convert T3 and T4 into the corresponding butyl esters. The yield of esterification was Ì´ 100% for T3 and 80% for T4. The assay was linear (r>0.99) in the range of 0.2-50 ng/ml, accuracy was in the order of 70-75%, and the minimum tissue amount needed was in the order of 50 mg, that is, about one order of magnitude lower than observed with the same equipment (AB Sciex API 4000 triple quadrupole mass spectrometer) if derivatization was omitted. The method allowed detection of T3 and T4 in human left ventricle biopsies yielding concentrations of 1.51±0.16 and 5.94±0.63 pmol/g, respectively. In rats treated with different dosages of exogenous T3 or T4, good correlations (r>0.90) between plasma and myocardial T3 and T4 concentrations were observed, although in specific subsets different plasma T4 concentrations were not associated with different tissue content in T4. We conclude that this method could provide a novel insight into the relationship between plasma and tissue thyroid hormone levels.

Two new species of Anastrepha Schiner (Diptera, Tephritidae) closely related to Anastrepha pickeli Lima.

Anastrepha entodonta n. sp. and Anastrepha hadropickeli n. sp. are described and illustrated. The new species belong to the spatulata group. Both species occur sympatrically with Anastrepha pickeli Lima in the semiarid region of the state of Minas Gerais, Brazil. Anastrepha hadropickeli occurs also in the semiarid of the state of Rio Grande do Norte, Brazil, where it was misidentified as A. pickeli.

Estimates of genetic relatedness among males in a polygynous wasp.

Cyclical oligogyny is considered to be the mechanism that is most likely to be responsible for stabilizing cooperation in polygynous, epiponine wasps, in which single-queen colonies produce new queens and multiple-queen colonies produce males. In contrast with the number of studies on relatedness among adult females, we know little about relatedness among males in polygynous epiponine wasps. We estimated worker and male relatedness in the Brazilian epiponine wasp Polybia paulista Ihering and found that colonies of P. paulista produced males when they contained multiple queens. Although average relatedness within males did not differ significantly from 0.5, the number of alleles observed suggests that there were more than one queen to produce males in each colony. Our data would be helpful to elucidate dynamics of the male production in a colony of epiponine wasps.

NMR-based metabolomics and breath studies show lipid and protein catabolism during low dose chronic T(1)AM treatment.

OBJECTIVE: 3-Iodothyronamine (T1 AM), an analog of thyroid hormone, is a recently discovered fast-acting endogenous metabolite. Single high-dose treatments of T1 AM have produced rapid short-term effects, including a reduction of body temperature, bradycardia, and hyperglycemia in mice. DESIGN AND METHODS: The effect of daily low doses of T1 AM (10 mg/kg) for 8 days on weight loss and metabolism in spontaneously overweight mice was monitored. The experiments were repeated twice (n = 4). Nuclear magnetic resonance (NMR) spectroscopy of plasma and real-time analysis of exhaled (13) CO2 in breath by cavity ring down spectroscopy (CRDS) were used to detect T1 AM-induced lipolysis. RESULTS: CRDS detected increased lipolysis in breath shortly after T1 AM administration that was associated with a significant weight loss but independent of food consumption. NMR spectroscopy revealed alterations in key metabolites in serum: valine, glycine, and 3-hydroxybutyrate, suggesting that the subchronic effects of T1 AM include both lipolysis and protein breakdown. After discontinuation of T1 AM treatment, mice regained only 1.8% of the lost weight in the following 2 weeks, indicating lasting effects of T1 AM on weight maintenance. CONCLUSIONS: CRDS in combination with NMR and (13) C-metabolic tracing constitute a powerful method of investigation in obesity studies for identifying in vivo biochemical pathway shifts and unanticipated debilitating side effects.

The Identity of Specimens of the Anastrepha fraterculus Complex (Diptera, Tephritidae) with Atypical Aculeus Tip.

Several specimens collected in Paraguay along with Anastrepha fraterculus (sensu lato) have an aculeus tip similar to species from the fraterculus complex, but the teeth of the aculeus of these specimens are poorly defined. As Anastrepha species identification is based mostly on subtle differences in the aculeus tip, we studied these specimens with atypical aculeus tips (with poorly defined teeth) that slightly differs from the aculeus tip of species of the fraterculus complex (with well-developed blunt teeth), to determine if this is due to intraspecific variation or if it can characterize a full species. The Paraguayan specimens were separated in six groups under stereomicroscope according to variation in their aculeus tip. Specimens within each group were studied by means of morphometrics (traditional and geometric) and gene sequence analysis (COI and ITS1). Morphometric analyses were significant, but no clear groups were formed by the discriminant analyses of the aculeus and wing, and the COI and ITS1 sequence analysis clustered specimens with all six aculeus variations. Therefore, the subtle morphological differences observed in the aculeus tip of Paraguayan specimens are intraspecific variations and the Paraguayan specimens were more genetically closely related to Anastrepha sp. 3 from the fraterculus complex.

Correlation between mandibular gland secretion and cuticular hydrocarbons in the stingless bee Melipona quadrifasciata.

We investigated whether Melipona quadrifasciata worker mandibular gland secretions contribute directly to their cuticular hydrocarbon profile. The mandibular gland secretion composition and cuticular surface compounds of newly emerged worker bees, nurse bees, and foragers were determined by gas chromatography and mass spectrometry and compared. Both the mandibular gland secretions and the cuticular surface compounds of all worker stages were found to be composed almost exclusively of hydrocarbons. Although the relative proportion of hydrocarbons from the cuticular surface and gland secretion was statistically different, there was a high similarity in the qualitative composition between these structures in all groups of bees.

Analysis of insect cuticular compounds by non-lethal solid phase micro extraction with styrene-divinylbenzene copolymers.

Insect cuticular hydrocarbons including relatively non-volatile chemicals play important roles in cuticle protection and chemical communication. The conventional procedures for extracting cuticular compounds from insects require toxic solvents, or non-destructive techniques that do not allow storage of subsequent samples, such as the use of SPME fibers. In this study, we describe and tested a non-lethal process for extracting cuticular hydrocarbons with styrene-divinylbenzene copolymers, and illustrate the method with two species of bees and one species of beetle. The results demonstrate that these compounds can be efficiently trapped by Chromosorb® (SUPELCO) and that this method can be used as an alternative to existing methods.

Hymenopteran parasitoids associated with frugivorous larvae in a Brazilian caatinga-cerrado ecotone.

The purpose of this study was to investigate native species of parasitoids of frugivorous larvae and their associations with host plants in commercial guava orchards and in typical native dry forests of a caatinga-cerrado ecotone in the State of Minas Gerais, Brazil. Nine species of parasitoids were associated with larvae of Anastrepha (Tephritidae) and Neosilba (Lonchaeidae) in fruit of Psidium guajava L. (Myrtaceae), Ziziphus joazeiro Mart. (Rhamnaceae), Spondias tuberosa Arruda (Anacardiaceae), Spondias dulcis Forst. (Anacardiaceae), Syzygium cumini (L.) Skeels (Myrtaceae), and Randia armata (Sw.) DC. (Rubiaceae). Doryctobracon areolatus was the most abundant species, obtained from puparia of Anastrepha zenildae, An. sororcula, An. fraterculus, An. obliqua, and An. turpiniae. This is the first report of Asobara obliqua in Brazil and of As. anastrephae and Tropideucoila weldi in dry forests of Minas Gerais State. The number of species of parasitoids was higher in areas with greater diversity of cultivated species and lower pesticide use. The forest fragments adjacent to the orchards served as shelter for parasitoids of frugivorous larvae.

Mitochondrial F(0) F(1) -ATP synthase is a molecular target of 3-iodothyronamine, an endogenous metabolite of thyroid hormone.

BACKGROUND AND PURPOSE: 3-iodothyronamine (T1AM) is a metabolite of thyroid hormone acting as a signalling molecule via non-genomic effectors and can reach intracellular targets. Because of the importance of mitochondrial F(0) F(1) -ATP synthase as a drug target, here we evaluated interactions of T1AM with this enzyme. EXPERIMENTAL APPROACH: Kinetic analyses were performed on F(0) F(1) -ATP synthase in sub-mitochondrial particles and soluble F(1) -ATPase. Activity assays and immunodetection of the inhibitor protein IF(1) were used and combined with molecular docking analyses. Effects of T1AM on H9c2 cardiomyocytes were measured by in situ respirometric analysis. KEY RESULTS: T1AM was a non-competitive inhibitor of F(0) F(1) -ATP synthase whose binding was mutually exclusive with that of the inhibitors IF(1) and aurovertin B. Both kinetic and docking analyses were consistent with two different binding sites for T1AM. At low nanomolar concentrations, T1AM bound to a high-affinity region most likely located within the IF(1) binding site, causing IF(1) release. At higher concentrations, T1AM bound to a low affinity-region probably located within the aurovertin binding cavity and inhibited enzyme activity. Low nanomolar concentrations of T1AM increased ADP-stimulated mitochondrial respiration in cardiomyocytes, indicating activation of F(0) F(1) -ATP synthase consistent with displacement of endogenous IF(1,) , reinforcing the in vitro results. CONCLUSIONS AND IMPLICATIONS: Effects of T1AM on F(0) F(1) -ATP synthase were twofold: IF(1) displacement and enzyme inhibition. By targeting F(0) F(1) -ATP synthase within mitochondria, T1AM might affect cell bioenergetics with a positive effect on mitochondrial energy production at low, endogenous, concentrations. T1AM putative binding locations overlapping with IF(1) and aurovertin binding sites are described.

Application of microsatellite primers developed for Polistes in the independent-founding wasp Polists satan Bequaert (Hymenoptera: Vespidae).

Microsatellite primers developed for a given species are sometimes useful for another in the same genus and in other genera within the same family, making possible to search for pre-existing suitable primers in the databanks such as GenBank. We examined whether existing primers developed for Polistes could be used for Polistes satan Bequaert. We tested 50 microsatellite primers from three Polistes species and found that six microsatellite loci show polymorphism in size in P. satan. These six loci were highly polymorphic, having four to 15 alleles in P. satan with an expected heterozygosity of 0.525-0.832. These loci can be used to study parameters concerning genetic relatedness such as social interactions in colonies and genetic conflicts of interest among nestmate individuals.

Effects of the thyroid hormone derivatives 3-iodothyronamine and thyronamine on rat liver oxidative capacity.

Thyronamines T(0)AM and T(1)AM are naturally occurring decarboxylated thyroid hormone derivatives. Their in vivo administration induces effects opposite to those induced by thyroid hormone, including lowering of body temperature. Since the mitochondrial energy-transduction apparatus is known to be a potential target of thyroid hormone and its derivatives, we investigated the in vitro effects of T(0)AM and T(1)AM on the rates of O(2) consumption and H(2)O(2) release by rat liver mitochondria. Hypothyroid animals were used because of the low levels of endogenous thyronamines. We found that both compounds are able to reduce mitochondrial O(2) consumption and increase H(2)O(2) release. The observed changes could be explained by a partial block, operated by thyronamines, at a site located near the site of action of antimycin A. This hypothesis was confirmed by the observation that thyronamines reduced the activity of Complex III where the site of antimycin action is located. Because thyronamines exerted their effects at concentrations comparable to those found in hepatic tissue, it is conceivable that they can affect in vivo mitochondrial O(2) consumption and H(2)O(2) production acting as modulators of thyroid hormone action.

First record of the fruit fly parasitoid Doryctobracon crawfordi (Viereck) (Hymenoptera: Braconidae) in Brazil.

Doryctobracon crawfordi (Viereck) was recorded in the states of Amapá and Roraima, Brazil. This is also the first time that this parasitoid is associated with Anastrepha atrigona Hendel from Geissospermum argenteum.

The TRbeta-selective agonist, GC-1, stimulates mitochondrial oxidative processes to a lesser extent than triiodothyronine.

Specific tissue responses to thyroid hormone are mediated by the hormone binding to two subtypes of nuclear receptors, TRalpha and TRbeta. We investigated the relationship between TRbeta activation and liver oxidative metabolism in hypothyroid rats treated with equimolar doses of triiodothyronine (T(3)) and GC-1, a TRbeta agonist. T(3) treatment produces increases in O(2) consumption and H(2)O(2) production higher than those elicited by GC-1. The greater effects of T(3) on oxidative processes are linked to the higher hormonal stimulation of the content of respiratory chain components including autoxidizable electron carriers as demonstrated by the measurement of activities of respiratory complexes and H(2)O(2) generation in the presence of respiratory inhibitors. It is conceivable that these differential effects are dependent on the inability of GC-1 to stimulate TRalpha receptors that are likely involved in the expression of some components of the respiratory chain. The greater increases in reactive oxygen species production and susceptibility to oxidants exhibited by mitochondria from T(3)-treated rats are consistent with their higher lipid and protein oxidative damage and lower resistance to Ca(2)(+) load. The T(3) and GC-1 effects on the expression levels of nuclear respiratory factor-1 and -2 and peroxisome proliferator-activated receptor-gamma coactivator-1alpha suggest the involvement of respiratory factors in the agonist-linked changes in mitochondrial respiratory capacities and H(2)O(2) production.