Thiazolidine derivatives: In vitro toxicity assessment against promastigote and amastigote forms of Leishmania infantum and ultrastructural study.

Neglected diseases, such as Leishmaniasis, constitute a group of communicable diseases that occur mainly in tropical countries. Considered a public health problem with limited treatment. Therefore, there is a need for new therapies. In this sense, our proposal was to evaluate in vitro two series of thiazolidine compounds (7a-7e and 8a-8e) against Leishmania infantum. We performed in vitro evaluations through macrophage cytotoxicity assays (J774) and nitric oxide production, activity against promastigotes and amastigotes, as well as ultrastructural analyzes in promastigotes. In the evaluation of cytotoxicity, the thiazolidine compounds presented CC50 values between 8.52 and 126.83 µM. Regarding the evaluation against the promastigote forms, the IC50 values ranged between 0.42 and 142.43 µM. Compound 7a was the most promising, as it had the lowest IC50. The parasites treated with compound 7a showed several changes, such as cell body shrinkage, shortening and loss of the flagellum, intense mitochondrial edema and cytoplasmic vacuolization, leading the parasite to cell inviability. In assays against the amastigote forms, the compound showed a low IC50 (0.65 µM). These results indicate that compound 7a was efficient for both evolutionary forms of the parasite. In silico studies suggest that the compound has good oral bioavailability. These results show that compound 7a is a potential drug candidate for the treatment of Leishmaniasis.

Ultrastructural analysis of Leishmania infantum chagasi promastigotes forms treated in vitro with usnic acid.

Leishmaniasis is considered by the World Health Organization as one of the infectious parasitic diseases endemic of great relevance and a global public health problem. Pentavalent antimonials used for treatment of this disease are limited and new phytochemicals emerge as an alternative to existing treatments, due to the low toxicity and cost reduction. Usnic acid is uniquely found in lichens and is especially abundant in genera such as Alectoria, Cladonia, Evernia, Lecanora, Ramalina, and Usnea. Usnic acid has been shown to exhibit antiviral, antiprotozoal, antiproliferative, anti-inflammatory, and analgesic activity. The aim of this study was to evaluate the antileishmanial activity of usnic acid on Leishmania infantum chagasi promastigotes and the occurrence of drug-induced ultrastructural damage in the parasite. Usnic acid was effective against the promastigote forms (IC50=18.30±2.00 µg/mL). Structural and ultrastructural aspects of parasite were analyzed. Morphological alterations were observed as blebs in cell membrane and shapes given off, increasing the number of cytoplasmic vacuoles, and cellular and mitochondrial swelling, with loss of cell polarity. We concluded that the usnic acid presented antileishmanial activity against promastigote forms of Leishmania infantum chagasi and structural and ultrastructural analysis reinforces its cytotoxicity. Further, in vitro studies are warranted to further evaluate this potential.

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.

Histopathology and ultrastructure of midgut of Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) fed Bt-cotton.

The interaction of Cry toxins from Bacillus thuringiensis in the midgut of some insect larvae determines their efficacies as insecticides, due to the expression and availability of sites of action of the toxin in the midgut. Researches point out cases of resistance to Cry toxin due to alterations in the binding sites in columnar cell membrane. We analyzed the effects of Cry1Ac toxin expressed by Bt-cotton plants on Alabama argillacea midgut morphophysiology clarifying in levels of morphological and ultrastructural. Larvae in the 4th instar of A. argillacea after 20 min from ingesting Bt-cotton leaves expressing 0.183 ng of Cry1Ac exhibited ultrastructural and morphological modifications in the columnar cells with significant changes in the mitochondrial polymorphism, cytoplasmic vacuolization, microvillus and basal labyrinth. Expressive morphological alterations were also observed in the goblet cells indicating that the columnar cells are not the only target of the Cry1Ac toxin. The regenerative cells did not modify their structures and exhibited decrease in regeneration capacity. In conclusion, the ingestion of 0.183 ± 0.077 ng of Cry1Ac was enough to promote alterations in the columnar and goblet cells, besides reducing significantly the number of regenerative cells, which may have contributed to larval death. Nevertheless, further studies are necessary to determine the true cause of death.

Ultrastructure of the Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) midgut.

The insect midgut has ultimately been the focus of researches tempting to control insect pests because alterations in the insect gut may affect not only its development, but also physiological events such as nutrient absorption and transformation. The objective of the present work was to describe morphologically, histochemically, and ultrastructurally the larva midgut of Alabama argillacea (Hübner) (Lepidoptera: Noctuidae), a cotton key pest in Brazil. Light and electronic transmission microscopy was used to obtain images from midgut sections of late fourth-instar larvae of A. argillacea. In general, the morphology, histochemistry, and ultrastructure characteristics of A. argillacea midgut follow that described in the literature for other lepidopteran species. However, the results showed a mitochondrial polymorphism and branched microvilli, which suggest an ultrastrucutural and physiological modification possibly associated with a high absorption and secretion activity by the columnar cells of this species. This intense activity may favor a faster response related to the action of ingested microbial agents and/or toxins, and can explain the high susceptibility of A. argillacea to the agents of control such as the toxin of Bacillus thuringiensis.

[Ultrastructure of the ovarioles of Tropidacris collaris (Stoll) (Orthoptera: Romaleidae) submitted to three photoperiods]. / Ultra-estrutura dos ovaríolos de Tropidacris collaris (Stoll) (Orthoptera: Romaleidae) submetido a três fotoperíodos.

The research evaluated the ultrastructure of the ovarioles of Tropidacris collaris (Stoll), submitted to photoperiods 10L:14D, 12L:12D and 14L:10D. Sixty nymphs (30 males and 30 females) in the last stage of development were paired in ten couples in each treatment. Thirty days after adult emergence, the females were immobilized with ethylic ether and dissected under stereomicroscope. The ovarioles were transferred to Karnovsky fixative (2.5% glutaraldehyde, 4% paraformaldehyde and 0.1 M sodium cacodylate buffer) and analyzed in transmission and scanning electron microscopes. The different photoperiods had no effect on the ovarioles' ultrastructure. Each ovariole is covered by a thick sheath constituted by a homogeneous and filamentous material. In the terminal filament, there are cells with large nuclei, some with scarce cytoplasm and projections cytoplasmatic, besides filamentous structures assuming characteristic of conjunctive tissue. In the germarium, the germ cells are big with large nuclei, scarce cytoplasm and plasma membrane containing interdigitations. The follicular cells are small with a small nucleus, yet presenting cytoplasmatic projections. In the vitellarium the follicular cells suffer modifications in their morphology varying from cubic to flat.

Ultra-estrutura dos ovaríolos de Tropidacris collaris (Stoll) (Orthoptera: Romaleidae) submetido a três fotoperíodos / Ultrastructure of the ovarioles of Tropidacris collaris (Stoll) (Orthoptera: Romaleidae) submitted to three photoperiods

A pesquisa teve o objetivo de avaliar a ultra-estrutura dos ovaríolos de Tropidacris collaris (Stoll), submetido aos fotoperíodos de 10L:14E, 12L:12E e 14L:10E. Foram utilizadas 60 ninfas (30 machos e 30 fêmeas) no último estágio de desenvolvimento, sendo colocados 10 casais em cada tratamento. Trinta dias após atingirem o estágio adulto, as fêmeas foram sedadas com éter etílico e dissecadas sob estereomicroscópio. Os ovaríolos foram fixados em Karnovsky (glutaraldeido 2,5 por cento, paraformaldeído 4 por cento e tampão cacodilato de sódio 0,1 M) e analisados em microscopia eletrônica de transmissão e varredura. Os resultados mostraram não haver influência dos fotoperíodos sobre a ultra-estrutura dos ovaríolos, onde estes apresentaram-se revestidos por uma bainha espessa constituída por um material homogêneo e filamentoso. Na região do filamento terminal observaram-se células com núcleos volumosos, algumas com citoplasma escasso, além de estruturas filamentosas assumindo característica de tecido conjuntivo. No germário, as células germinativas são maiores, com núcleos volumosos, escassos citoplasma e membrana celular com interdigitações. As células foliculares são menores com núcleo pequeno, apresentando ainda projeções citoplasmáticas. No vitelário as células foliculares sofrem modificações na sua morfologia, variando de cúbica a achatada.

The research evaluated the ultrastructure of the ovarioles of Tropidacris collaris (Stoll), submitted to photoperiods 10L:14D, 12L:12D and 14L:10D. Sixty nymphs (30 males and 30 females) in the last stage of development were paired in ten couples in each treatment. Thirty days after adult emergence, the females were immobilized with ethylic ether and dissected under stereomicroscope. The ovarioles were transferred to Karnovsky fixative (2.5 percent glutaraldehyde, 4 percent paraformaldehyde and 0.1 M sodium cacodylate buffer) and analyzed in transmission and scanning electron microscopes. The different photoperiods had no effect on the ovarioles' ultrastructure. Each ovariole is covered by a thick sheath constituted by a homogeneous and filamentous material. In the terminal filament, there are cells with large nuclei, some with scarce cytoplasm and projections cytoplasmatic, besides filamentous structures assuming characteristic of conjunctive tissue. In the germarium, the germ cells are big with large nuclei, scarce cytoplasm and plasma membrane containing interdigitations. The follicular cells are small with a small nucleus, yet presenting cytoplasmatic projections. In the vitellarium the follicular cells suffer modifications in their morphology varying from cubic to flat.