Evaluation of antimutagenic and cytotoxic activity of skin secretion extract of Rhinella marina and Rhaebo guttatus (Anura, Bufonidae) / Avaliação da atividade antimutagênica e citotóxica da secreção cutânea de Rhinella marina e Rhaebo guttatus (Anura, Bufonidae)

The skin secretion from toads of the Bufonidae family has great potential in the search for new active compounds to be used as drug candidates in treating some diseases, among them cancer. In this context, this study aimed to evaluate the cytotoxic and antimutagenic activity of the parotoid gland secretion extracts of Rhinella marina and Rhaebo guttatus, as well as biochemically analyze transaminases and serum creatinine for liver and renal damage, respectively. Cytotoxicity was performed by the colorimetric method based on MTT (3- [4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide) with different concentrations of the extracts in Walker or splenic tumor cell cultures from rats and mice. The micronucleus test was performed with male Swiss mice treated orally with the extracts for 15 days, and then intraperitoneally with N-ethyl-N-nitrosurea (50 mg kg-1). Micronucleated polychromatic erythrocytes (MNPCE) were evaluated in bone marrow. The extracts showed cytotoxic activity in the evaluated cells. There was a significant reduction in the frequency of MNPCE (R. marina = 56% and R. guttatus = 75%, p < 0.001), indicating antimutagenic potential of the extracts. The groups treated only with extract showed an increase in MNPCE frequency, evidencing mutagenic potential. Biochemical analyzes showed no significant difference between treatments. Thus, under our experimental conditions, the extracts of R. marina and R. guttatus skin secretions presented chemopreventive potential for cancer. (AU)

A secreção cutânea de anuros da família Bufonidae tem grande potencial na busca de novos compostos ativos para utilização como fármacos candidatos no tratamento de algumas doenças, entre elas o câncer. Neste contexto, este estudo teve como objetivo avaliar a atividade citotóxica e antimutagênica dos extratos da secreção da glândula parótida de Rhinella marina e Rhaebo guttatus, bem como a análise bioquímica de transaminases e creatinina séricas, para avaliar dano hepático e renal, respectivamente. A avaliação de citotoxicidade foi realizada pelo método colorimétrico baseado no MTT (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide), com diferentes concentrações dos extratos em culturas de células do Tumor de Walker ou células esplênicas de rato e camundongo. O teste do micronúcleo foi realizado com camundongos Swiss machos que receberam tratamento oral com os extratos durante 15 dias, seguido de tratamento intraperitoneal com N-etil-N-nitrosuréia (50 mg kg-1). A frequência de eritrócitos policromáticos micronucleados (PCEMN) foi determinada em medula óssea. Os extratos apresentaram ação citotóxica nas células avaliadas. Houve uma redução significativa na frequência de PCEMN (R. marina = 56% e R. guttatus = 75%, p < 0,001), observando-se um potencial antimutagênico dos extratos. Os grupos tratados somente com os extratos apresentaram um aumento na frequência de PCEMNs, evidenciando um potencial mutagênico. As análises bioquímicas não apresentaram diferença significativa entre os tratamentos. Assim, nas condições experimentais testadas, as secreções cutâneas de R. marina e R. guttatus apresentaram potencial quimiopreventivo para câncer.(AU)

Longevity and survival curves of Rhinella icterica (Anura, Bufonidae) under laboratory conditions / Longevidade e curva de sobrevivência de Rhinella icterica (Anura, Bufonidae) em condições de laboratório

Life tables and survival curves of tadpoles from Rhinella icterica species were studied in the laboratory, under abiotic conditions controlled by a purification filter, a timer and a chiller. The survival curve for larval stage confirms a great mortality trend in the initial stages, which decreases when reaching the mature morphological condition (r = –0.94). Stages 37, 38, 39, 40 and 41 showed gradual values for their age structures, while stages 42, 43 and 44 presented high variations. Based on the results under laboratory conditions, it can be concluded that the maturity of R. icterica tadpoles development between 37 and 44 stages has a negative correlation and their predicted life expectancy is a logarithmic growth curve (y=–761.96Ln(x)+5298.5).

Tabelas de vida e curvas de sobrevivência em estágios larvais de girinos da espécie Rhinella icterica foram estudadas em laboratório, sob condições abióticas controladas com uso de filtro de depuração, timer e chiller. A curva de sobrevivência por estágio larval confirma a tendência de maior mortalidade para os estágios iniciais e menor mortalidade para o avanço da condição morfológica madura (r = –0,94). Os estágios 37, 38, 39, 40 e 41 apresentaram valores gradativos para sua estrutura etária, enquanto nos estágios 42, 43 e 44 ocorreram variações acentuadas. Com base nos resultados obtidos, nas condições de laboratório, pode-se concluir que a maturidade de desenvolvimento de girinos de R. icterica entre os estágios 37 e 44, apresenta correlação negativa e a predição de esperança de vida a uma curva logarítmica de crescimento (y=–761,96Ln(x)+5298,5).

Water-balance response of Rhinella arenarum (Hensel, 1867) tadpoles to graduated increase in environmental osmolarity / Resposta de Rhinella arenarum larvae a meio electrolítico e não electrolítico com aumento escalonado da osmolaridade

The water balance and the upper limit of osmotic tolerance of premetamorphic Rhinella arenarum larvae (Gosner's stage 26) was evaluated after semistatic incubation in electrolyte (NaCl) and non-electrolyte (mannitol) media following a protocol of progressively increased osmotic pressure. Wet and dry weights were measured to calculate the water content as a derived variable indicative of the hydric balance. Statistical analysis was performed using univariate and integrated multivariate analysis. Tadpoles survived in electrolyte and non-electrolyte solutions up to 200 mOsm. The discriminant function was the best tool to describe the responses of the animals to external environmental stress under experimental conditions. The results were compared with those obtained in previous studies using a protocol of acute exposure to the same media used in this study. It was concluded that a) multivariate analysis is an appropriate approach to describe the responses of tadpoles to changes in the environmental physicochemical parameters, and b) progressive and acute acclimation to the experimental solutions induced similar responses.

Avaliou-se o balanço hídrico e o limite superior de tolerância osmótica em larvas pré-metamórficas do Rhinella arenarum (etapa 26 de Gosner) sob condições de incubação semiestáticas, num meio eletrolítico (NaCl) e não eletrolítico (manitol), seguindo um protocolo de aumento progressivo da pressão osmótica do meio. A quantificação das respostas se efetuou por meio da medição dos valores de peso úmido e seco e do cálculo, a partir destes, do conteúdo de água, como variável derivada indicativa do equilíbrio hídrico. A análise estatística foi realizada usando análise univariada e multivariada. As larvas conseguiram sobreviver em soluções eletrolíticas e não eletrolíticas até 200 mOsm. A função discriminante foi a melhor ferramenta para descrever as respostas dos animais ao estresse osmótico ambiental. Os resultados foram comparados com os obtidos em estudos anteriores, usando um protocolo de exposição aguda aos mesmos meios de incubação usados neste estudo. Concluiu-se: a) a análise multivariada é a aproximação adequada para descrever as respostas das larvas às mudanças nos parâmetros físico-químicos do seu meio; e b) tanto a aclimatação progressiva, como as exposições agudas às soluções experimentais induziram as respostas semelhantes.

Population traits of the burrowing toad Rhinella fernandezae (Gallardo, 1957) (Anura, Bufonidae)

Size distribution, sex ratio and use of burrows of the burrowing toad Rhinella fernandezae were studied in Buenos Aires province, Argentina. Two sites separated by approximately 300 m were studied: one was a road next to a swamp, and the other a garden of a country house located further from the swamp. We identified toad burrows, and individuals were sexed, measured and given an individual mark. Burrows were examined in subsequent months after the first sampling to assess the presence of toads. We found significant differences in the size distribution between areas, being the proportion of juveniles greater at the site next to the swamp where the reproduction of the species was observed. This result may suggest that the site located near to the swamp functions as a source habitat of individuals that migrate to the other site, where recruitment would be very scarce. Sex proportion of adults did not differ from 1:1 in neither the total population nor in each site, suggesting that there was not differential mortality by sex. Some toads changed burrows throughout the study period, but there were not differences in the frequency of change between adults and juveniles.

Distribuição de tamanho, razão sexual operacional, e uso de covas do sapo Rhinella fernandezae foram estudados na província de Buenos Aires, Argentina, em dois sítios afastados 300 m. Um deles é uma trilha próxima a um pântano, o outro, um jardim de uma casa rural, mais afastado do pântano. Identificamos as covas dos sapos, e os indivíduos dentro delas foram sexados, medidos e marcados individualmente. As covas foram monitoradas mensalmente, depois da primeira amostragem para avaliar a presença dos sapos. Achamos diferenças na distribuição de tamanhos entres os sítios, sendo a proporção de juvenis maior naquele próximo ao pântano, onde a reprodução da espécie foi observada. O resultado sugere que o sítio próximo ao pântano funciona como um habitat fonte de indivíduos que migram para aquele mais afastado, onde o recrutamento seria escasso. A taxa entre os sexos dos adultos não foi diferente de 1:1 em nenhum dos sítios amostrados, nem na população total, sugerindo que não existe mortalidade diferencial entre machos e fêmeas. Alguns sapos mudaram de cova durante o período do estudo, mas não houve diferenças na freqüência de mudança entre juvenis e adultos.

The importance of glucose for the freezing tolerence/intolerence of the anuran amphibians Rana catesbeiana and Bufo paracnemis

Several species of terrestrially hibernating frogs, turtles and inserts have developed mechanisms, such as increased plasma glucose, anti-freeze proteins and antioxidant enzymes that resist to freezing, for survival at subzero temperatures. In the present study, we assessed the importance of glucose to cryoresistance of two anuran amphibians: the frog Rana catesbeiana and the toad Bufo paracnemis. Both animals were exposed to -2 degrees Celsius for measurements of plasma glucose levels, liver and muscle glycogen content, haematocrit and red blood cell volume. Frogs survived cold exposure but toads did not. Blood glucose concentration increased from 40.35 + 7.25 to 131.87 + 20.72 mg/dl (P < 0.01) when the frogs were transferred from 20 to -2 degrees Celsius. Glucose accumulation in response to cold exposition in the frogs was accompanied by a decrease (P < 0.05) in liver glycogen content from 3.94 + 0.42 to 1.33 + 0.36 mg/100 mg tissue, indicating that liver carbohydrate reserves were probably the primary carbon source of glucose synthesis whereas muscle carbohydrate seems unimportant. In the toads, the cold-induced hyperglycaemia was less (P < 0.05) pronounced (from 27.25 + 1.14 to 73.72 + 13.50 mg/dl) and no significant change could be measured in liver or muscle glycogen. Cold exposition had no effect on the haematocrit of the frogs but significantly reduced (P < 0.01) the haematocrit of toads from 20.0 + 2.1 per cent to 5.8 + 1.7 per cent due to a decreased red blood cell volume (from 1532 + 63 70 728 + 87 mm3). When toads were injected with glucose, blood glucose increased to levels similar to those of frogs and haematocrit did not change, but this failed to make them cryoresistent. In conclusion, the lack of cold-induced glucose catabolism may not be the only mechanism responsible for the freeze intolerance of Bufo paracnemis, a freeze-intolerant species.

Hemoglobin affinity for oxygen in three subspecies of toads (Bufo sp.) living at different altitudes

Blood oxygen affinity and red blood cell properties were measured in three subspecies of genus Bufo: Bufo spinulosus limensis, collected at sea level and at an average day temperature of 20§C; Bufo spinulosus trifolium, from 3100 m, average day temperature of 15§C; and Bufo spinulosus flavolineatus, from 4100 m, average day temperature of 10§C. Electrophoresis of the hemoglobin showed the same component in each of the three subspecies. At 20§C the blood oxygen affinities (P50) showed small differences between Bufo spinulosus limensis and Bufo spinulosus trifolium, whereas the value for Bufo spinulosus flavolineatus was markedly lower. At 10§C, the ambient temperature of Bufo spinulosus flavolineatus, the P50 was extremely low compared with the other two subspecies at their corresponding ambient temperatures.

Influence of pituitary on histology of Bidder's organ in castrated toad Bufo melanostictus.

Weight, number of follicles and corpora lutea of Bidder's organ increased significantly after castration of adult toad in breeding season. Removal of pituitary along with testes decreased both weight and number of follicles and corpora lutea. Administration of luteinizing hormone (LH) in castrated and hypophysectomized toad showed more or less similar result as that of control indicating that LH has an effect on the histology of Bidder's organ in the toad.