Pyrethrum extract encapsulated in nanoparticles: Toxicity studies based on genotoxic and hematological effects in bullfrog tadpoles.

The environment receives about 2.7 kg.ha-1 annually of pesticides, used in crop production. Pesticides may have a negative impact on environmental biodiversity and potentially induce physiological effects on non-target species. Advances in technology and nanocarrier systems for agrochemicals led to new alternatives to minimize these impacts, such as nanopesticides, considered more efficient, safe and sustainable. However, it is important to evaluate the risk potential, action and toxicity of nanopesticides in aquatic and terrestrial organisms. This study aims to evaluate genotoxic and hematological biomarkers in bullfrog tadpoles (Lithobates catesbeianus) submitted to acute exposure (48 h) to pyrethrum extract (PYR) and solid lipid nanoparticles loaded with PYR. Results showed increased number of leukocytes during acute exposure, specifically eosinophils in nanoparticle-exposed groups, and basophil in PYR-exposed group. Hematological analysis showed that PYR encapsulated in nanoparticles significantly increased the erythrocyte number compared to the other exposed groups. Data from the comet assay indicated an increase in frequency of the classes that correspond to more severe DNA damages in exposed groups, being that the PYR-exposed group showed a high frequency of class-4 DNA damage. Moreover, erythrocyte nuclear abnormalities were triggered by short-time exposure in all treatments, which showed effects significantly higher than the control group. These results showed genotoxic responses in tadpoles, which could trigger cell death pathways. Concluding, these analyses are important for applications in assessment of contaminated aquatic environments and their biomonitoring, which will evaluate the potential toxicity of xenobiotics, for example, the nanoparticles and pyrethrum extract in frog species. However, further studies are needed to better understand the effects of nanopesticides and botanical insecticides on non-target organisms, in order to contribute to regulatory aspects of future uses for these systems.

Genotoxic effect and rat hepatocyte death occurred after oxidative stress induction and antioxidant gene downregulation caused by long term fluoride exposure.

Studies focusing on possible genotoxic effects of excess fluoride are contradictory and inconclusive. Currently, studies have reported a probable link to oxidative stress, DNA damage and apoptosis induced by fluoride in rat hepatocytes. We developed an in vivo study administering three doses of fluoride by gavage given to rats for 60 day. Micronucleus test was applied to investigate genotoxic potential of fluoride. The TUNEL method determined DNA fragmentation and apoptosis. Biochemical parameters to investigate mitochondrial swelling and oxidative stress. Semi-quantitative RT-PCR and immunostaining to determine mRNA and protein expression of antioxidant enzymes. Analyses of the hepatic function and morphology were performed. Our results revealed the genotoxic potential of fluoride but did not confirm mitochondrial swelling nor an increase of positive TUNEL labelling induced by fluoride, indicating absence of apoptosis. Oxidative stress induction was confirmed and is probably associated to DNA damage. Cell death events such as empty nuclear spaces, cytoplasm degeneration, nuclear pyknosis, karyorrhexis and karyorrhexis followed by karyolysis were observed. Hepatic function did not appear to be significantly modified makes no evidence of necrosis and suggesting other cell death pathway, the autophagic. In conclusion, prolonged fluoride intake at chosen concentrations caused imbalance of the cellular oxidative state, affected DNA and disrupted cellular homeostasis. It is recommended that fluoride supplementation requires a fresh consideration in light of the current study.

Effects of the organophosphorus pesticide Folisuper 600 (methyl parathion) on the heart function of bullfrog tadpoles, Lithobates catesbeianus (Shaw, 1802).

The aim of this work was to evaluate whether the heart function of bullfrog tadpoles (25 Gosner stage) is affected by their acute exposure (48 h) to a sub-lethal concentration (10 µg.L-1) of the active principle of the organophosphorus pesticide Folisuper 600R (methyl parathion - MP). Our results demonstrated that MP causes not only a reduction in tadpoles' cardiac ventricular mass, resulting in a marked reduction in their cardiac twitch force, but also impairs their swimming performance, irrespective of increasing their heart rate. Together, these findings indicate that low and realistic concentration of MP have a negative impact on tadpoles' performance, jeopardizing their survival.

Effects of the organophosphorus pesticide Folisuper 600 (methyl parathion) on the heart function of bullfrog tadpoles, Lithobates catesbeianus (Shaw, 1802)

Abstract The aim of this work was to evaluate whether the heart function of bullfrog tadpoles (25 Gosner stage) is affected by their acute exposure (48 h) to a sub-lethal concentration (10 µg.L1) of the active principle of the organophosphorus pesticide Folisuper 600R (methyl parathion - MP). Our results demonstrated that MP causes not only a reduction in tadpoles cardiac ventricular mass, resulting in a marked reduction in their cardiac twitch force, but also impairs their swimming performance, irrespective of increasing their heart rate. Together, these findings indicate that low and realistic concentration of MP have a negative impact on tadpoles performance, jeopardizing their survival.

Resumo O objetivo do presente estudo foi avaliar se a função cardíaca de girinos de rãs-touro (estágio 25 de Gosner) é afetada pela exposição aguda (48h) a uma concentração sub-letal (10 µg.L1) do princípio ativo do pesticida organofosforado Folisuper 600R (metil paration MP). Nossos resultados demonstraram que o MP ocasionou não apenas uma redução na massa ventricular cardíaca dos girinos, como também provocou uma redução na sua força de contração cardíaca e de seu desempenho natatório, a despeito de ter sido observado um aumento de sua freqüência cardíaca. Conjuntamente, os achados aqui obtidos indicam que uma baixa e realística concentração de MP exerce um impacto negativo sobre o desempenho dos girinos, ameaçando sua sobrevivência.

Effects of the organophosphorus pesticide Folisuper 600 (methyl parathion) on the heart function of bullfrog tadpoles, Lithobates catesbeianus (Shaw, 1802) / Efeitos do pesticida organofosforado Folisuper 600 (metil paration) sobre a função cardíaca de girinos de rã-touro, Lithobates catesbeianus (Shaw, 1802)

Abstract The aim of this work was to evaluate whether the heart function of bullfrog tadpoles (25 Gosner stage) is affected by their acute exposure (48 h) to a sub-lethal concentration (10 µg.L–1) of the active principle of the organophosphorus pesticide Folisuper 600R (methyl parathion - MP). Our results demonstrated that MP causes not only a reduction in tadpoles’ cardiac ventricular mass, resulting in a marked reduction in their cardiac twitch force, but also impairs their swimming performance, irrespective of increasing their heart rate. Together, these findings indicate that low and realistic concentration of MP have a negative impact on tadpoles’ performance, jeopardizing their survival.

Resumo O objetivo do presente estudo foi avaliar se a função cardíaca de girinos de rãs-touro (estágio 25 de Gosner) é afetada pela exposição aguda (48h) a uma concentração sub-letal (10 µg.L–1) do princípio ativo do pesticida organofosforado Folisuper 600R (metil paration – MP). Nossos resultados demonstraram que o MP ocasionou não apenas uma redução na massa ventricular cardíaca dos girinos, como também provocou uma redução na sua força de contração cardíaca e de seu desempenho natatório, a despeito de ter sido observado um aumento de sua freqüência cardíaca. Conjuntamente, os achados aqui obtidos indicam que uma baixa e realística concentração de MP exerce um impacto negativo sobre o desempenho dos girinos, ameaçando sua sobrevivência.

Negative impact of a cadmium concentration considered environmentally safe in Brazil on the cardiac performance of bullfrog tadpoles.

A drastic amphibian decline has been observed worldwide, which can be attributed (among other factors) to exposure to pollutants. Considering that cadmium corresponds to the most rapidly increasing trace metal in the environment, the aim of this work was to evaluate whether the exposure (2 and 16 days) of bullfrog tadpoles to this trace metal, at the concentration currently considered environmentally safe (at 1ppb) in class 1 and 2 waters by the Brazilian Environmental Council, can affect the cardiac performance of these animals. The acute exposure (2 days) of tadpoles to cadmium resulted in a marked bradycardic response, which was correlated with an incomplete cardiac relaxation, without any compensation by improved cardiac twitch force (Fc) or contraction velocity (TPT), nor even by cardiac hypertrophy. Indeed, after 16 days of exposure, the cardiac function of tadpoles became even more depressed due to a marked decrease in Fc, a prolongation of TPT, and also incomplete relaxation (i.e. increases in the ventricle resting tension), without changes in ventricle relative mass. Altogether, the cardiodepressive effects of cadmium (especially after more prolonged exposure periods) impose negative alterations on a tadpole׳s development and also impede adequate homeostatic adjustments to respond appropriately to the exposure to cadmium with increase in energetic demand to counteract the deleterious effects of the xenobiotic. These disturbances can impair tadpoles׳ growth, development and reproduction. It is a fact that allows us to strongly suggest that cadmium concentrations, which are currently considered environmentally safe in Brazil, should be revised.

Histochemistry of the mucus gland of Bombus morio Swedurus,1787 (Hymenoptera, Apidae)

The male mucus gland is present in all species of bees, exception for meliponines. In bees, the function of themucus glands is not certain so far and its contribution to form the female espermatecal fluid is not assured.With the aim to contribute to the knowledge of the mucus gland in bees we carried out a histochemical studyof the gland in mature, adult males of Bombus morio, focusing on the histochemical nature of the secretion.The males were collected around the forest fragments into UFSCar, Campus Sorocaba. The glands werefixed and analysed under routine microscopy and for histochemistry techniques: Periodic Acid Schiff (PAS),Bromophenol Blue (BB), Sudan Black (SB) and Critical Electrolyte Concentration (CEC) variant. The resultsshowed that the mucus gland is constituted by a pair of large, thick tubular structures, which presented theirdistal portion more dilated and corn shaped. The glandular cells are columnar and in the apical portion presentseveral apocrine vesicles being released into the gland lumen. The histochemistry showed that gland secretionis very complex and contains protein (BB), neutral polysaccharide (PAS), as well as lipid (SB). The luminalgland secretion presents a background of homogenous content, presenting little dark dots stained by alltechniques used for this work. The investigation of the nucleolar activity (CEC) showed that the mucus glandepithelium is evolved in protein synthesis, presenting nucleoli developed and much RNA in the cytoplasm.

Effects of atrazine on female Wistar rats: morphological alterations in ovarian follicles and immunocytochemical labeling of 90 kDa heat shock protein.

Fertility in female mammals may be affected by a variety of endocrine disrupters present in the environment. Herbicide atrazine is an example of endocrine disrupter employed in agriculture, which disrupts estrous cyclicity in rats. Aiming to characterize morphologically the effect of low and sublethal doses of atrazine on the ovaries of Wistar rats, in an effort to determine the possible intrafollicular target site through which this herbicide acts adult females were submitted to both subacute and subchronic treatments. Additionally, immunocytochemical labeling of 90 kDa heat shock protein (HSP90) was performed in order to evaluate the role played by this protein in the ovary, under stressed conditions induced by herbicide exposure. The results indicated that atrazine induced impaired folliculogenesis, increased follicular atresia and HSP90 depletion in female rats submitted to subacute treatment, while the subchronic treatment with low dose of atrazine could compromise the reproductive capacity reflected by the presence of multioocytic follicle and stress-inducible HSP90.

Nuclear alterations associated to programmed cell death in larval salivary glands of Apis mellifera (Hymenoptera: Apidae).

The silk glands of bees are a good model for the study of cell death in insects. With the objective to detect the nuclear features during glandular regression stage, larvae at the last instar and pre-pupae were collected and their silk glands were dissected and processed for ultrastructural analysis and histologically for cytochemical and imunocytochemical analysis. The results showed that the cellular nuclei exhibited characteristics of death by atypical apoptosis as well as autophagic cell death. Among the apoptosis characteristic were: nuclear strangulation with bleb formation in some nuclei, DNA fragmentation in most of the nuclei and nucleolar fragmentation. Centripetal chromatin compaction was observed in many nuclei, forming a perichromatin halo differing from typical apoptotic nuclei. With regards to the characteristics of autophagic-programmed cell death, most relevant was the delay in the collapse of many nuclei.

Programmed cell death in the larval salivary glands of Apis mellifera (Hymenoptera, Apidae).

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine,the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy.Tissue sections were subsequently stained with haematoxylin -eosin,bromophenol blue,silver,or a variant of the critical electrolyte concentration (CEC) method.Ultrathin sections were contrasted with uranyl acetate and lead citrate.Glands were processed for the histochemical and cytochemical localization of acid phosphatase,as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed.The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and,additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen.The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed.The nuclei were pyknotic,with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.