Cytotoxic and genotoxic potential of industrialized powdered milk for infants and young children

This study aimed to evaluate the cytotoxicity and genotoxicity and determine the LC50 concentration of powdered infant formulas widely marketed in South American countries. To this, milk samples, called as A, B, C and D, were analyzed in root meristem cells of Allium cepa, at concentrations of 0.075; 0.15 and 0.30 g mL-1, for 24 and 48 hours; and through cell viability in culture of normal line cells, via MTT test, for 24 hours, in the concentrations 0.018; 0.0375; 0.075 and 0.15 g mL-1. In A. cepa, all dairy products in the three concentrations caused significant inhibition of cell division in the meristems within the first 24 hours of exposure. In the in vitro evaluation, all milk formulas at 0.15 g mL-1, as well as milk A at a concentration of 0.037 g mL-1, C at 0.075 g mL-1 and D at 0.037 g mL-1, significantly reduced the cellular viability of the cell culture exposed to the foods studied, being potentially toxic. The milk A was considered the most toxic, with LC50 of 0.031 g mL-1, and B as the least toxic, with LC50 of 0.15 g mL-1. Therefore, the milk evaluated caused significant instability in cells of the test systems used and were characterized as cytotoxic.

Lethal and sublethal effects of essential oil of Lippia sidoides (Verbenaceae) and monoterpenes on Chagas´ disease vector Rhodnius prolixus

The aim of this study was to identify the composition of the essential oil from leaves of Lippia sidoides (EOLS), a typical shrub commonly found in the dry northeast of Brazil, popularly known as “alecrim-pimenta”. Additionally, we investigated the nymphicidal, ovicidal, phagoinhibitory and excretion effects of EOLS, its major constituent thymol and its isomer carvacrol, on fourth instar nymphs and eggs of Rhodnius prolixus, the Chagas’ disease vector. The nymphicidal and ovicidal activity of thymol, carvacrol, and EOLS was assessed by tests using impregnated Petri dishes. The lethal concentration values (LC50) for EOLS, carvacrol, and thymol were 54.48, 32.98, and 9.38 mg/cm2, respectively. The ovicidal test showed that both carvacrol and thymol (50 mg/cm2) inhibited hatching (50% and 23.3%, respectively), while treatments with 10 mg/cm2 or 50 mg/cm2 EOLS did not affect the hatching rate at all (80% and 90%, respectively). We observed an anti-feeding effect in insects fed with blood containing natural products at the higher concentrations (100 µg/mL). Finally, excretion rate was affected by EOLS and carvacrol, but not by thymol. These findings offer novel insights into basic physiological processes that make the tested natural compounds interesting candidates for new types of insecticides.

Efecto citotóxico de los compuestos de inclusión de paladio (II) en la beta-ciclodextrina / Cytotoxic effect of palladium (II) inclusion compounds in beta-cyclodextrin

Resumen Introducción. Las tiosemicarbazonas y sus complejos de paladio (II) poseen actividad antineoplásica con pocos efectos secundarios, por lo cual se las considera como una nueva alternativa terapéutica. Sin embargo, existen diferencias en los rangos de la concentración inhibitoria media (CI50) asociada a la divergencia estructural y la solubilidad de los complejos, así como a la sensibilidad de los blancos celulares. La inclusión de fármacos en la beta-ciclodextrina con fines terapéuticos ha mejorado su solubilidad y estabilidad, pero los efectos de su combinación con los complejos de paladio (II) y las tiosemicarbazonas no se han comprobado aún. Objetivo. Estudiar el efecto citotóxico de los complejos de paladio en la beta-ciclodextrina. Materiales y métodos. La actividad citotóxica de los complejos de paladio en la beta-ciclodextrina se evaluó en la línea celular de cáncer de mama (MCF-7), empleando el método de la sulforodamina B. Resultados. Los ligandos MePhPzTSC y Ph2PzTSC, sus complejos de paladio (II) libres e incluidos en la beta-ciclodextrina y el cisplatino mostraron actividad citotóxica en la línea celular MCF-7; sin embargo, la citotoxicidad fue mayor con la inclusión en la beta-ciclodextrina ([Pd(MePhPzTSC)2]•ß-CD y [Pd(Ph2PzTSC)2]•ß-CD). La concentración inhibitoria media (CI50) para estos complejos se obtuvo en concentraciones de 0,14 y 0,49 μM, y con dosis hasta cinco veces inferiores comparadas con las concentraciones de los ligandos libres (1,4 y 2,9 μM), de los complejos de paladio (II) libres (0,57 y 1,24 μM) y del cisplatino (6,87 μM). Conclusiones. El uso de la beta-ciclodextrina mejoró significativamente la actividad citotóxica de las tiosemicarbazonas y sus complejos de paladio (II), lo cual probablemente está asociado al incremento de la solubilidad y biodisponibilidad del compuesto, estrategia que se puede sugerir para el diseño de futuros fármacos antineoplásicos.

Abstract Introduction: Thiosemicarbazones and palladium (II) complexes have antineoplastic activities with mild side effects, for which they are considered new alternative antineoplastic drugs. However, the IC50 ranges of these complexes vary due to differences in their structure and solubility and their sensitivities for various cellular targets. Beta-cyclodextrin is an additive used to improve the solubility and stability of various drugs for therapeutic use, but the combination of beta-cyclodextrin with palladium (II) complexes and thiosemicarbazones has not been tested yet. Objective: To study the cytotoxic effect of palladium (II) inclusion complexes in beta-cyclodextrin. Materials and methods: We tested the cytotoxic activity of palladium complexes combined with betacyclodextrin in the breast cancer cell line MCF-7 using a sulforhodamine B assay. Results: We tested the antiproliferative activity of palladium (II) complexes with and without the ligands MePhPzTSC and Ph2PzTSC and with and without beta-cyclodextrin in MCF-7 cells and compared them to that of cisplatin. All combinations showed antiproliferative activity; however, the activity was greater for the combinations that included beta-cyclodextrin: ([Pd (MePhPzTSC) 2] • ß-CD and [Pd (Ph2PzTSC) 2] • ß-CD), at concentrations of 0.14 and 0.49 μM, respectively. The IC50 for this complex was 5-fold lower than that of the ligand-free combinations (1.4 and 2.9 μM, respectively). The IC50 for free palladium (II) complex was 0.571.24 μM and that for cisplatin was 6.87 μM. Conclusions: Beta-cyclodextrin significantly enhanced the cytotoxic activities of palladium (II) complexes and thiosemicarbazones probably by improving their solubility and bioavailability. The addition of betacyclodextrin is a possible strategy for designing new anticancer drugs.

In vitro anticancer activity of microbial isolates from diverse habitats / In vitro atividade anticancerígena de isolados microbianos de diversos habitats

Extracts from natural products, especially microorganisms, have served as a valuable source of diverse molecules in many drug discovery efforts and led to the discovery of several important drugs. Identification of microbial strains having promising biological activities and purifying the bio-molecules responsible for the activities, have led to the discovery of many bioactive molecules. Extracellular, as well as intracellular, extracts of the metabolites of thirty-six bacterial and twenty-four fungal isolates, grown under unusual conditions such as high temperature, high salt and low sugar concentrations, were in vitro tested for their cytotoxic potential on various cancer cell lines. The extracts were screened on HeLa and MCF-7 cell lines to study the cytotoxic potential. Nuclear staining and flow cytometric studies were carried out to assess the potential of the extracts in arresting the cell cycle. The crude ethylacetate extract of isolate F-21 showed promising results by MTT assay with IC50 as low as 20.37±0.36 µg/mL on HeLa, and 44.75±0.81 µg/mL on MCF-7 cells, comparable with Cisplatin. The isolate F-21 was identified as Aspergillus sp. Promising results were also obtained with B-2C and B-4E strains. Morphological studies, biochemical tests and preliminary chemical investigation of the extracts were also carried out.

Extratos de produtos naturais, especialmente de microrganismos, constituíram-se em fonte valiosa de diversas moléculas em muitas descobertas de fármacos e levaram à descoberta de fármacos importantes. A identificação de espécies microbianas que apresentam atividade biológica e a purificação de biomoléculas responsáveis pelas atividades levou à descoberta de muitas moléculas bioativas. Extratos extracelulares tanto quanto intracelulares de metabólitos de 36 isolados de bactérias e 24 isolados de fungos, que cresceram sob condições não usuais, como alta temperatura, alta concentração de sal e baixa concentração de açúcar, foram testados in vitro quanto ao seu potencial citotóxico em várias linhagens de câncer. Os extratos foram ensaiados em células HeLa e MCF-7 para o estudo do potencial citotóxico. A coloração nuclear e os estudos de citometria de fluxo foram realizados para avaliar o potencial dos extratos em bloquear o ciclo celular. O extrato bruto em acetato de etila do isolado F-21 mostrou resultados promissores no ensaio de MTT, com IC50 de 20,37±0,36 µg/mL em células HeLa e 44,.75±0,81 µg/mL em células MCF-7, comparativamente à cisplatina. O isolado F-21 foi identificado como Aspergillus sp. Resultados promissores foram obtidos com cepas B-2C e B-4E. Realizaram-se, também, estudos morfológicos, testes bioquímicos e investigação química preliminar dos extratos.

Effects of aqueous leaf extracts of Azadirachta indica A. Juss. (neem) and Melia azedarach L. (Santa Barbara or cinnamon) on the intracellular development of Toxoplasma gondii / Efeito de extrato aquoso de folhas de Azadirachta indica A. Juss. (nim) e Melia azedarach L. (canela ou Santa Bárbara) sobre o desenvolvimento intracelular de Toxoplasma gondii

Melia azedarach (cinnamon) and Azadirachta indica (neem) have a variety of biologically active ingredients against virus, bacteria and protozoan parasites; however, little is known about their action on Toxoplasma gondii intracellular development. Toxoplasma gondii infects all eukaryotic cells, where it establishes and multiplies inside a modified vacuole called the parasitophorous vacuole until the cell ruptures, re-infecting other cells and establishing the infection. There are no efficient chemotherapies for the elimination of T. gondii, minimizing side effects. In this study, we performed in vitro assays with neem and cinnamon aqueous extracts against the intracellular development of T. gondii tachyzoites. After treatment with neem and cinnamon for 24 h, the percentage of infected cells and the number of intracellular parasites drastically decreased. This effect was concentration-dependent. During the incubation of the extracts, progressive morphological and ultrastructure alterations led to intense vesiculation and complete elimination of the parasite from the intracellular medium. However, during the treatment with extracts, no morphological effects were observed in the structure of the host cell. These results suggest that the aqueous extracts of neem and cinnamon were capable of interfering with and eliminating the intracellular development of Toxoplasma gondii.

Melia azedarach (canela) e Azadirachta indica (nim) apresenta grande variedade de ingredientes biologicamente ativos contra vírus, bactérias e protozoários, mas nenhum efeito sobre o desenvolvimento intracelular do Toxoplasma gondii é conhecido. Toxoplasma gondii infecta todos os tipos de células Eucarióticas, onde se estabelece no meio intracelular em vacúolo modificado conhecido como vacúolo parasitóforo. Neste vacúolo ocorre a replicação levando a ruptura da célula hospedeira e reinfecção de novas células, perpetuando a infecção. A quimioterapia utilizada não é capaz de eliminar o parasita além de induzir fortes efeitos colaterais. Neste estudo, nós demonstramos o efeito in vitro de extratos aquosos da canela e nim sobre o desenvolvimento intracelular do taquizoíto do Toxoplasma gondii. Após tratamento de nim e canela por 24 h, a porcentagem de infecção e o número de taquizoítos intracelulares decaiu drasticamente. Este efeito foi concentração-dependente. Durante incubação dos extratos, uma progressiva desorganização morfológica e ultraestrutural levaram a formação de intensa vesiculação e completa destruição do parasita, que passou a uma estrutura amorfa, antes da completa eliminação do meio intracelular. No entanto durante o tratamento com os extratos, efeitos morfológicos não foram observados nas estruturas da célula hospedeira. Estes resultados sugerem que os extratos aquosos de nim e canela foram capazes de interferir e eliminar o desenvolvimento intracelular do Toxoplasma gondii.

Virulence factors of Escherichia coli isolated from calves with diarrhea in Brazil

Duzentas e cinco amostras de Escherichia coli isoladas de bezerros com diarréia da região centro oeste do Brazil foram examinados quanto a presenca de fatores de virulência associados à colibacilose bovina. Cento e duas amostras (49,8 per center) de E. coli produziram toxinas: toxina de Shiga do tipo 1 (9,7 per center) e 2 (6,3 per center), a-hemolisina (9,7 per center), enterohemolisina (6,8 per center), Fatores Citotóxicos Necrotisantes tipo 1 (0,5 per center) e 2 (4,4 per center), enterotoxinas LT-II (8,3 per center), e STa (3,9 per center). Nenhuma amostra produziu enterotoxina LT-I. Adesinas fimbriais F5 e F17 foram produzidas por 7,3 per center e 4,8 per center das cepas, respectivamente, e nenhuma expressou F41. Sete das amostras (3,4 per center) apresentaram o gene eae e pertenceram aos sorotipos O26:H-; O111:H- e O118:H16. Estes resultados sugerem que bezerros no Brasil podem ser uma importante fonte de E. coli patogênica para animais e humanos.

Relation between cytotoxin producing Helicobacter pylori and Helicobacter pylorielated upper gastrointestinal disease

Helicobacter pylori [H. pylori], a major aetiological agent in gastritis, peptic ulcer and gastric cancer, is estimated to infect more than 50% of the world's population. However, only about 10% will develop peptic ulcer disease and 1 - 2% gastric malignancy. Virulent strains carrying the cag A gene and vac A s1 genotype and capable of cytotoxin production have been proposed to be associating with the severer forms or disease, although this was not universal. We were interested in studying the relation between cytotoxin-producing H. pylori strains and the H. pylori-related upper gastrointestinal disease in our community. Sixty patients were allocated into 3 predefined groups according to their endoscopic picture: gastroesophageal reflux disease [GERD], peptic ulcer disease, and gastritis groups. Gastric biopsies from the patients were examined for the presence of H. pylori by urease test and culture. The isolated H. pylori strains were subjected to cytotoxic assay to detect cytotoxin-producing strains. Forty-one patients [68.3%] were H. pylori positive, of them 19 [46.3%] were positive for cytotoxin production. Cytotoxin-producing H. pylori strains significantly associated peptic ulcer disease where 73.3% of peptic ulcer patients were infected with cytotoxin-producing strains. GERD was significantly associated with absence of H. pylori infection [66.7% of GERD patients were free of H. pylori infection]. The presence of gastritis did not correlate with the H. pylori status, however, there was a significant association between cytotoxin-producing strains and atrophic gastritis. Cytotoxin-producing H. pylori strains are associated with severer H. pylori-related upper gastrointestinal diseases such as atrophic gastritis and peptic ulcer disease. Our findings support the hypothesis of cag A [+] H. pylori being protective against GERD. Determination of cag A status of H. pylori strains bears importance in clinical practice in detecting patients at increased risk for developing gastric cancer and in helping planning treatment strategies

Purification and characterization of Klebsiella pneumoniae cytotoxins.

Isolation, purification and characterization of 3 new cytotoxins of a K. pneumoniae strain isolated from ready to eat pork sausage are reported. Purification process involved extraction of cytotoxins with polymyxin B sulphate, salt precipitation, gel filtration and anion exchange chromatography. Klebsiella cytotoxin (KCT) I, a glycoprotein of about 65 kDa was verocytotoxic, enterotoxic and dermonerotic. KCT II was erythemogenic, verocytotoxic and enterotoxic protein of co 55 kDa, while KCT III was about double in MW (110 kDa) hadverocytotoxicity but neither enterotoxicity nor dermatotoxicity. KCT I and II caused granulation, conglomeration, shrinkage, detachment and lysis of MDBK and Vero cells, while KCT III induced enlargement, vacuolation, granulation, multinucleolation and syncytia formation in exposed cells. All the three cytotoxins induced specific neutralizing antibodies and cytotoxins were detectable in nanogram quantities with enzyme-linked immunosorbant assay using homologous antibodies. None of the anticytotoxin cross-reacted with either heterologous Klebsiella cytotoxins or with verocytotoxic preparations of Shigella dysenteriae.