Tityus stigmurus venom causes genetic damage in blood and testicular cells and affects the number and morphology of gametogenic lineage cells in mice.

Scorpion envenomation represents an important health problem in many parts of the world, due to the high number and severity of accidents. Recent studies demonstrated that some species can produce venoms with genetic damage potential. Here, we evaluated whether Tityus stigmurus venom causes genetic damage in blood and testicular cells of Swiss mice. We also analyzed the effect of the venom on the number of spermatogenic lineage cells. Five groups of mice received 0.387 mg/kg of the venom, intraperitoneally; one group received saline solution (control group). Blood and testicular cells were collected for comet assay and histological analysis at different times after treatment (1, 2, 6, 12, and 48 h). Blood was also collected 48 h after treatment for the micronucleus test in erythrocytes. Histological analysis was performed by counting cells of the spermatogenic lineages; the nuclear area of elongated spermatocytes was also evaluated. Treatment with the venom induced DNA damage that endured from 1 h to 48 h, as confirmed by the comet assay. The micronucleus test demonstrated that the venom induced mutations in erythrocytes. The number of spermatogonia and rounded spermatids decreased in some groups; the number of elongated spermatids increased, and their nuclear size decreased 1 h after treatment. Genetic damage can be caused directly by the venom, but we suggested that reactive oxygen species that result from inflammatory process caused by the envenomation may have an important role in the DNA damage. Genetic damage and apoptosis may explain the changes in the number of spermatogenic cells. Furthermore, the decrease in nuclear area may result from chromatin loss. Genetic damage in testicular cells, associated with alterations in the number and morphology of spermatogenic cells, can result in reproduction disorders in animals, or humans, stung by T. stigmurus.

Seasonal influence on testicular morphophysiological parameters of bat Carollia perspicillata in fragments of the Atlantic Forest, northeastern Brazil / Influência sazonal sobre os parâmetros morfofisiológicos testiculares de Carollia perspicillata (Chiroptera: Phyllostomidae) em fragmentos florestais de Mata Atlântica, Nordeste do Brasil

Bats belong to the order Chiroptera, family Phyllostomidae, and present a wide diversity of reproductive strategies. However, information on the reproductive biology of male bats is scarce, mainly in the Northeast Region of Brazil. Thus, this study evaluated the seasonal testicular histomorphometry of the bat Carollia perspicillata in fragments of the Atlantic Forest in Pernambuco state. To this end, adult males were collected, euthanized for removal of the testicles, and later submitted to a routine histological technique. Histomorphometric analysis included assessment of the areas of tubular and intertubular compartment occupation, as well as quantification of spermatocytes, rounded spermatids, elongated spermatids, and Sertoli and Leydig cells. Results indicated that this bat species presents reproductive seasonality, because significantly higher averages of the testicular parameters were observed in the rainy season, which is a period of greater availability of food resources. Such inferences indicate that there is a synchrony between peak spermatogenesis and hormonal inversion in the months of high precipitation; furthermore, a higher carrying capacity of the Sertoli cells is noted. C. perspicillata males possibly present greater sperm and androgenic activity in the rainy season, associated with increased tubular area and number of spermatogenic cells, as well as with the intertubular area and number of Leydig cells, respectively.(AU)

Os morcegos pertencem a ordem Chiroptera, família Phyllostomidae, e apresentam ampla diversidade de estratégias reprodutivas. Entretanto, as informações relacionadas à biologia reprodutiva dos machos são escassas, principalmente no Nordeste do Brasil. Dessa forma, o trabalho avaliou a histomorfometria sazonal testicular de Carollia perspicillata em fragmentos de Mata Atlântica de Pernambuco. Para tanto, os machos adultos foram coletados, eutanasiados para a remoção dos testículos, e posteriormente submetidos à técnica histológica de rotina. As análises histomorfométricas avaliaram as áreas de ocupação do compartimento tubular e intertubular, assim como a quantificação dos espermatócitos, espermátides arredondadas, espermátides alongadas, células de Sertoli e de Leydig. Os resultados indicaram que a espécie apresenta sazonalidade reprodutiva, visto que maiores médias significativas dos parâmetros testiculares foram encontradas na estação chuvosa, que é um período de maior disponibilidade de recursos alimentares. Tais inferências indicam que existe uma sincronia entre o pico de espermatogênese e investimento hormonal nos meses de alta precipitação, atrelado a isso, nota-se ainda, uma maior capacidade de suporte das células de Sertoli. Os machos de C. perspicillata possivelmente apresentam uma maior atividade espermática e androgênica na estação chuvosa, associadas ao aumento da área tubular e do número de células espermatogênicas, assim como da área intertubular e do número das células de Leydig, respectivamente.(AU)

Hepatic toxicity caused by PLGA-microspheres containing usnic acid from the lichen C ladonia substellata (AHTI) during pregnancy in Wistar rats.

This study aimed to evaluate the teratogenic and hepatotoxic potential of the usnic acid encapsulated into PLGA-microspheres. In total, 12 female Wistar rats in pregnancy were randomly distributed in the control group (n= 6) that received 1.0 mL of physiological solution and treatment group (n= 6) that received 25 mg/kg of encapsulated usnic acid by oral administration. All females were euthanized at day 20 of pregnancy and their fetuses were removed and analyzed. During the pregnancy was observed a reduction in weight gain. There was no difference in serum transaminases levels analyzed as well as any difference in liver weight in both groups. The histomorphometric analysis of the liver from the treatment group revealed an increase in number of hepatocytes and a decrease in nuclear area of these cells. Moreover, no alteration was observed in cell area of hepatocytes or number of Kupffer cells. The fetuses had an increase in total number of hepatocytes and a reduction in the amount of megakaryocytes. These results show the hepatotoxic potential of usnic acid during pregnancy. However, its toxicity can be minimized by encapsulation in microspheres.

Hepatic toxicity caused by PLGA-microspheres containing usnic acid from the lichen C ladonia substellata (AHTI) during pregnancy in Wistar rats

ABSTRACT This study aimed to evaluate the teratogenic and hepatotoxic potential of the usnic acid encapsulated into PLGA-microspheres. In total, 12 female Wistar rats in pregnancy were randomly distributed in the control group (n= 6) that received 1.0 mL of physiological solution and treatment group (n= 6) that received 25 mg/kg of encapsulated usnic acid by oral administration. All females were euthanized at day 20 of pregnancy and their fetuses were removed and analyzed. During the pregnancy was observed a reduction in weight gain. There was no difference in serum transaminases levels analyzed as well as any difference in liver weight in both groups. The histomorphometric analysis of the liver from the treatment group revealed an increase in number of hepatocytes and a decrease in nuclear area of these cells. Moreover, no alteration was observed in cell area of hepatocytes or number of Kupffer cells. The fetuses had an increase in total number of hepatocytes and a reduction in the amount of megakaryocytes. These results show the hepatotoxic potential of usnic acid during pregnancy. However, its toxicity can be minimized by encapsulation in microspheres.

Ultrastructural aspects of melatonin cytotoxicity on Caco-2 cells in vitro.

Colon adenocarcinoma is a disease expanding worldwide. Cancer of colon and rectum are among the top ten most insidious types in Brazil. In vitro and in vivo studies have demonstrated the efficacy of the hormone melatonin to prevent and reduce tumor growth. However, there are only few studies addressing the action of melatonin on Caco-2 cells. Thus, the cytotoxic effect of melatonin on the ultrastructure of Caco-2 cells was investigated. The MTT colorimetric method was used to assess the cytotoxicity. A total of 2×10(6)cells/mL were seeded in microplates and incubated at 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0.0 (control) µg/mL of melatonin. For ultrastructural analysis concentrations with low, medium and high cytotoxicity plus the control were used for ultrastructural analysis. The concentrations 50, 1.56 and 0.78 µg/mL of melatonin showed low, medium and high cytotoxicity, respectively. Ultrastructurally, the control tumor cells were shown to be preserved. Caco-2 cells showed morphological changes at 50 µg/mL of melatonin, with numerous vacuoles, mitochondrial degeneration and reduced glycogen. However, Caco-2 cells also showed altered morphology in treatments at 1.56 and 0.78 µg/mL of melatonin with characteristics of cells in degeneration by the presence of numerous vacuoles, absence of microvilli, mitochondrial degeneration and nuclear fragmentation. Thus, one can infer that concentrations of 1.56 and 0.78 µg/mL of melatonin promote cytotoxicity in Caco-2 cells, which can probably be related to the generation of reactive oxygen species (ROS).

Melatonin effect on the ultrastructure of Ehrlich ascites tumor cells, lifetime and histopathology in Swiss mice.

AIMS: One of the models used for studying cancer is the Ehrlich ascites tumor (EAT) due to its ability to grow in liquid suspension, allowing a standard number of cells to be inoculated, growth quantification and regression of tumor mass. Among the oncostatic substances, melatonin has shown effectiveness in limiting the tumor cell proliferation. However, studies have shown contradictory effects of melatonin on the EAT. This study has investigated the melatonin effect on tumor growth, time and survival percentage, ultrastructure and metastasis of EAT cells in mice submitted or not to pinealectomy. MAIN METHODS: Animals were inoculated with 5×106 cells/mL and treated or not with exogenous melatonin with doses of at 150 and 300 µg/30 g animal weight for 12 days. Melatonin significantly reduced the abdominal circumference, volume of ascites liquid and EAT-cell viability, raising rates of time and mice survival percentage. KEY FINDINGS: Ultrastructurally, the melatonin treatment revealed changes in the shape of cells, the cell surface showed numerous projections, some bifurcated, cytoplasmic vacuolation, mitochondrial degeneration and nuclear fragmentation, peculiar characteristics of apoptosis. Histopathology revealed no metastasis in the liver, small intestine and large intestine in any of the animals in the experimental groups; however this process was evident in the lungs and kidneys, being inhibited by melatonin administration. SIGNIFICANCE: Thus,we can conclude that doses of 150 and 300µg/30g of melatonin for 12 consecutive days have a very effective oncostatic and cytotoxic activity on EAT cells in mice.

Podisus nigrispinus requer cópulas longas para o sucesso reprodutivo / Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae) requires long matings for successful reproduction

The occurrence of multiple and long matings seem to play an important role in the reproduction of the predatory stinkbugs such as Podisus nigrispinus (Dallas). However, the mechanisms underlying these behaviors remain unclear. In this study, the reproduction of P. nigrispinus was investigated as function of females' choice for their mating pair based on male body size and previous mating status, risk of predation and duration of mating. The female mating behavior was studied with female having multiple, partial or no partner choice based on male body size. Time to initiate a mating and its duration was observed after pairing males and females under risk of predation imposed by the presence of the predatory wasp Polistes versicolor Oliver. In addition, aiming to determine the reasons for long lasting matings, female had their mating interrupted at different intervals. Our data indicated that P. nigrispinus females do not select male partners as function of their body size and mating status. Duration of mating, mating partners' choice, and female fecundity and fertility were not influenced by the male size and risk of predation imposed by the predatory wasps. Mating interrupted after 30, 60, 120 and 240 min resulted in only 0, 3.1, 7.7 and 34 percent of egg hatching compared to 74.2 percent under uninterrupted mating (338 to 671 min long). Therefore, the reproductive success of P. nigrispinus females does not depend on male size and male mating status, but requires long-lasting mating as a condition for adequate spermatozoa transference to females instead.

A realização de múltiplas e longas cópulas parece ser importante para a reprodução de percevejos predadores como Podisus nigrispinus (Dallas), mas o comportamento governando essas características permanece desconhecido. Neste estudo foram investigados o comportamento de cópula e a reprodução de P. nigrispinus em função do tamanho do macho, seu status prévio de acasalamento, risco de predação e interrupção de cópulas. A escolha da fêmea para o acasalamento foi estudada sob múltipla escolha, parcial ou sem chance de escolha por machos grandes ou pequenos. O comportamento para o início do acasalamento e tempo de cópula sob risco de predação quando na presença de Polistes versicolor Oliver foi comparado àquele de casais sem risco de predação. Machos e fêmeas de P. nigrispinus acasalaram independentemente do tamanho e status prévio de acasalamento dos machos, além de não apresentarem preferência por parceiros. A duração da cópula, escolha do parceiro e fertilidade das fêmeas não foram influenciadas pelo tamanho do macho, nem a presença do predador alterou o comportamento de acasalamento e duração de cópula. Fêmeas com cópulas interrompidas após 30, 60, 120 e 240 min apresentaram baixa viabilidade de ovos (0; 3,1; 7,7 e 34 por cento, respectivamente), enquanto aquelas com tempo natural de cópula (338 a 671 min) obtiveram 74,2 por cento de viabilidade. Portanto, o sucesso reprodutivo de P. nigrispinus não depende do tamanho do macho ou de seu status de acasalamento, mas requer longas cópulas para a transferência de espermatozóides.

[Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae) requires long matings for successful reproduction]. / Podisus nigrispinus requer cópulas longas para o sucesso reprodutivo.

The occurrence of multiple and long matings seem to play an important role in the reproduction of the predatory stinkbugs such as Podisus nigrispinus (Dallas). However, the mechanisms underlying these behaviors remain unclear. In this study, the reproduction of P. nigrispinus was investigated as function of females' choice for their mating pair based on male body size and previous mating status, risk of predation and duration of mating. The female mating behavior was studied with female having multiple, partial or no partner choice based on male body size. Time to initiate a mating and its duration was observed after pairing males and females under risk of predation imposed by the presence of the predatory wasp Polistes versicolor Oliver. In addition, aiming to determine the reasons for long lasting matings, female had their mating interrupted at different intervals. Our data indicated that P. nigrispinus females do not select male partners as function of their body size and mating status. Duration of mating, mating partners' choice, and female fecundity and fertility were not influenced by the male size and risk of predation imposed by the predatory wasps. Mating interrupted after 30, 60, 120 and 240 min resulted in only 0, 3.1, 7.7 and 34% of egg hatching compared to 74.2% under uninterrupted mating (338 to 671 min long). Therefore, the reproductive success of P. nigrispinus females does not depend on male size and male mating status, but requires long-lasting mating as a condition for adequate spermatozoa transference to females instead.

Spermatogenesis, changes in reproductive structures, and time constraint associated with insemination in Podisus nigrispinus.

Males of the predatory stinkbug, Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae), accomplish long and multiple matings. We hypothesize that this behavior is due to time requirement for spermatozoa production and their transference to the females. Thus, this work investigated the effect of mating status of males and mating duration on spermatozoa transference to the females and the location of spermatozoa in the male reproductive tract during mating. On females, morphological alterations on female spermatheca and associated structures during a mating were investigated. Analyses of male reproductive tract showed presence of spermatozoa in the lumen of vas deferens was independent of mating status (ca. virgin, 0, 12 and 24 h after having a full mating), indicating continuous spermatogenesis which is supported by the absence of a seminal vesicle for spermatozoa storage. Female spermatheca had no changes associated with the duration of mating. However, females exhibited spermathecal elastic duct swelling by 30 min of mating duration. The success of males in filling the female spermatheca with spermatozoa depends on duration of mating. Thus, the results indicate that multiple mating is a requirement for reproductive success in the species by transference of spermatozoa and accessory substances stored in the female spermathecal duct. Likewise, the long mating is a male requirement to transfer materials in appropriate amount to the female but it is not dependent on spermatozoa alone.