Use of erythrocytes in cytotoxicity and toxicity assays of medicinal plant extracts: analysis of their application and bibliometric study / Utilización de eritrocitos en ensayos de citotoxicidad y toxidez de extractos de plantas medicinales: análisis de su aplicación y estudio bibliométrico

Plant species have been used for therapeutic purposes since ancient times and are still in use today since these products represent a source of raw material for the production of phytotherapeutic formulations. Screening and investigation of plants with pharmacological potential require the evaluation of characteristics related to their action, efficacy and safety in different steps. Among these steps, pre- clinical trials are used to evaluate the properties of the test product in in vitro experiments, such as cytotoxicity assays. Within this context, this study consists of a bibliometric analysis of some in vitro cytotoxicity and toxicity assays in erythrocytes used during bioprospecting of medicinal plants. The results demonstrated the wide application of erythrocytes to evaluate the biological effects of medicinal plant extracts. The methods were found to be valid and effective for the preliminary investigation of the in vitro cytotoxicity and toxicity of plant products.

El uso de especies vegetales para fines terapéuticos es una práctica histórica y todavía bastante actual, ya que estos productos pueden representar una fuente de materia prima para la producción de formulaciones fitoterápicas. En investigación de plantas con potencial farmacológico requiere la evaluación de su acción, eficacia y seguridad, a través de diferentes etapas. Entre estas, en los ensayos preclínicos se evalúan las propiedades del producto-prueba en experimentos in vitro, tales como ensayos de citotoxicidad, entre otros. En este aspecto, el presente estudio consiste en un análisis bibliométrico acerca de algunas pruebas de citotoxicidad y toxicidad in vitro en eritrocitos realizados en los ensayos de bioprospección de plantas medicinales. Los resultados evidencian la amplia utilización de eritrocitos para la evaluación de los efectos biológicos de extractos de plantas medicinales, apuntándolos como métodos válidos y eficaces para la investigación preliminar de la citotoxicidad y toxicidad in vitro de productos vegetales.

An early stage in T4-induced hyperthyroidism is related to systemic oxidative stress but does not influence the pentose cycle in erythrocytes and systemic inflammatory status

ABSTRACT Objective Hyperthyroidism causes many injuries in its target organs and the consequences are reflected systemically. As systemic alterations in hyperthyroidism at earlier stages have received partial attention, this study aimed to investigate systemic redox and inflammatory status at an early stage of T4-induced hyperthyroidism. Materials and methods Male Wistar rats were assigned to control and hyperthyroid groups (n = 7/group). The hyperthyroid group received L-thyroxine (12 mg/L) in their drinking water for 14 days whereas control group received only the vehicle. Body weight was measured on the 1st and 14th day of the protocol. On the 14th day, animals were anaesthetized. Blood was then collected from the retro-orbital venous plexus and then the animals were euthanised. The blood was separated into plasma and erythrocytes. Plasma was used to measure ROS levels, sulfhydryl compounds, IL-10, TNF-α and LDH levels; erythrocytes were used for the analysis of thioredoxin reductase activity, glutaredoxin content, and pentose cycle enzymes (total G6PD, G6PD and 6PGD). Results Hyperthyroid animals presented body weight gain and final body weight reduction, which was associated with increased ROS levels and decreased sulfhydryl content in plasma. Thioredoxin reductase activity, glutaredoxin content, and pentose cycle enzymes levels in erythrocytes, as well as IL-10, TNF-α and LDH plasma levels were unaltered. Conclusion Taken together, our results suggest an impairment in corporal mass associated with systemic oxidative stress at this stage of hyperthyroidism. Meanwhile, the pentose cycle was not influenced and systemic inflammation and tissue damage seem to be absent at this stage of hyperthyroidism.

Comparative study with dry crude extracts of Ananas comosus L. Merril using LC50 and erythrocyte osmotic fragility as toxicity parameters / Estudio comparativo con extractos crudos secos de Ananas comosus L. Merril utilizando CL50 y fragilidad osmótica eritrocitaria como parámetros de toxicidad

Ananas comosus, conocido popularmente como piña, tiene una anatomía de planta bien definida y numerosos fitoquí­micos farmacológicamente activos. Algunos de éstos son responsables del potencial antimicrobiano de la especie, que ha sido ampliamente estudiado dada la resistencia bacteriana a los antibióticos actualmente utilizados. Teniendo esto en cuenta, se define que la característica principal de un medicamento es la ausencia de efectos tóxicos, por lo tanto, es necesario buscar datos sobre la toxicidad de A. comosus, colaborando para su posible uso como fármaco. Comparando extractos hidroalcohólicos de las hojas de la corona, cáscara y pulpa de la infrutescencia, fue posible determinar que los tres extractos probados no son tóxicos, siendo el de menor toxicidad para Artemia salina Leach (extracto de hojas con CL50 igual a 994 μg/mL) y a sangre de cordero (extracto de pulpa con porcentaje de hemólisis igual a 0,83%). Los estudios sobre principios y metodologías similares a los utilizados aquí han encontrado resultados comparables que indican la baja toxicidad de la planta. Estos resultados aportan a las investigaciónes que promueven el uso de la piña como agente fitoterapéutico y reafirman su presencia en el Sistema Único de Salud de Brasil.

Ananas comosus, popularly known as pineapple, has well defined plant anatomy and numerous pharmacologically active phytochemicals. Some of these are responsible for the species antimicrobial potential, which has been widely studied given the bac­terial resistance to the currently used antibiotics. Considering this, it is defined that the main characteristic of a drug is the absence of toxic effects, thus, there is a need to seek data regarding the toxicity of A. comosus, collaborating for its possible use as a drug. Comparing hydroalcoholic extracts from the crown leaves, skin and pulp of the infructescence, it was possible to determine that the three tested extracts are non-toxic, being the one with the lowest toxicity to Artemia salina Leach (leaves extract with LC50 equal to 994 μg/mL) and to lamb's blood (pulp extract with percentage of haemolysis equal to 0.83%). Studies concerning principles and methodologies similar to those used here have found comparable results stating the low toxicity of the plant. These results contribute to the research that promotes the use of pineapple as a phytotherapeutic agent and reaffirms its presence in the Brazilian Unified Health System.

Black and white teas as potential agents to combine with amphotericin B and protect red blood cells from amphotericin B-mediated toxicity / Chá preto e branco de Camellia sinensis como potenciais agentes para associar com anfotericina B

Abstract Amphotericin B is a fungicidal substance that is treatment of choice for most systemic fungal infections affecting immunocompromised patients. However, severe side effects have limited the utility of this drug. The aim of this study was to evaluate the antifungal effect of the combination of amphotericin B with black tea or white tea and protective of citotoxic effect. The present study shows that white and black teas have additive effects with amphotericin B against some species Candida. In addition, the combination of white and black tea with amphotericin B may reduce the toxicity of amphotericin B to red blood cells. Our results suggest that white and black tea is a potential agent to combine with amphotericin for antifungal efficacy and to reduce the amphotericin dose to lessen side effects.

Resumo A anfotericina B é o tratamento de escolha para a maioria das infecções fúngicas sistémicas que afetam os doentes imunocomprometidos. No entanto, efeitos secundários graves têm limitado a utilidade desta droga. O objetivo deste estudo foi avaliar o efeito antifúngico da combinação de anfotericina B com chá preto ou chá branco, bem como o efeito citotóxico desta combinação sobre hemáceas. O presente estudo demonstra que o chá branco e preto de Camellia sinensis têm efeitos aditivos com anfotericina B contra algumas espécies de Candida sp. Além disso, a combinação de chá branco e preto com anfotericina B pode reduzir a toxicidade da anfotericina B em hemáceas. Nossos resultados sugerem que o chá branco e preto são agentes potenciais para associação com anfotericina B contribuindo para eficácia antifúngica, bem como redução de toxicidade.

Mentha pulegium crude extracts induce thiol oxidation and potentiate hemolysis when associated to t-butyl hydroperoxide in human's erythrocytes

ABSTRACT Mentha pulegium (Lamiaceae) tea has been used as a traditional medicine; however, the modulatory effect of M. pulegium extracts on damage to human erythrocytes associated to t-butyl hydroperoxide (t-BHP) exposure remains to be investigated. Accordingly, we perform this study in order to test the hypothesis that aqueous and ethanolic extracts of M. pulegium could modulate the hemolysis associated to t-BHP exposure, non-protein thiol (NPSH) oxidation and lipid peroxidation (measured as thiobarbituric acid reactive substances - TBARS) in human erythrocytes. Samples were co-incubated with t-BHP (4 mmol/L) and/or aqueous or ethanolic extracts (10-1000 mg/mL) during 120 min to further analysis. We found that both extracts, when associated to t-BHP, potentiate NPSH oxidation and hemolysis. Moreover, both extracts significantly prevents against t-BHP-induced TBARS production. A significant correlation among hemolysis and NPSH levels was found. Taking together, our data points that the association of M. pulegium extracts with t-BHP culminates in toxic effect to exposed erythrocytes, besides its protective effect against t-BHP-induced TBARS production. So, we infer that the use of this extract may exert negative effect during painful crisis in sickle cell anemia. However, more studies are still necessary to better investigate/understand the mechanism(s) involved in the toxic effect resultant from this association.

Evaluación genotóxica del agua del Río Grande (Antioquia, Colombia) mediante frecuencia de eritrocitos micronucleados de Brycon henni (Characiformes: Characidae) / Genotoxic evaluation of Río Grande (Antioquia, Colombia) water using micronucleus frequency in erythrocytes of Brycon henni (Characiformes: Characidae)

ResumenLa producción agrícola de monocultivo permanente de banano y plátano en la parte media del Río Grande (Turbo - Antioquia) requiere de la aplicación de diferentes plaguicidas. Prácticas inapropiadas en la producción y cultivos de plátano realizadas en esta región a menudo conllevan a contaminación con agroquímicos que llegan a este cuerpo de agua por procesos de lixiviación y escorrentía. Los peces son los vertebrados más utilizados como especies bioindicadoras de la calidad del agua porque son muy sensibles a la presencia de contaminantes en el ambiente. El objetivo de esta investigación fue comparar la frecuencia de eritrocitos micronucleados (EMN) en sangre periférica de ejemplares de peces sabaleta (Brycon henni) procedentes de dos estaciones del Río Grande que difieren en su grado de contaminación por agroquímicos. Se evaluó la frecuencia de EMN en sangre periférica de peces B. henni procedentes de cada estación durante dos épocas de lluvia de 2010 y dos época secas de 2011. Las muestras de sangre fueron obtenidas de la vena caudal, fijadas durante 24 horas y luego teñidas con Giemsa. La frecuencia de EMN fue significativamente mayor en el área impactada por agroquímicos. La frecuencia media de EMN fue mayor en la estación con impacto directo de agroquímicos (0.15±0.18) que en la estación poco alterada (0.06±0.08). Además, la frecuencia de EMN en B. henni fue significativamente mayor en la época seca. Los resultados de este estudio indican que el análisis de las EMN en B. henni puede ser recomendada como un método adecuado para la detección in situ de agentes genotóxicos en el ambiente.

Abstract:The permanent monoculture of bananas and plantains farming in the middle of Río Grande (Turbo - Antioquia) requires the application of a variety of pesticides. Inappropriate banana production practices in this region, have often led to waterbody pollution by agrochemicals from leachate and runoff processes. Currently, fish are the most common vertebrates used as bioindicators of water quality, because they are very sensitive to the presence of contaminants. Our main goal with this study was to compare the frequency of micronucleated erythrocytes (MNE) in peripheral blood specimens of fish Brycon henni, from two locations (polluted and unpolluted) in the Rio Grande. We evaluated the frequency of MNE in peripheral blood samples of fish B. henni from each location during two rainy seasons in 2010 and two dry seasons in 2011. Blood samples were collected, fixed for 24 h, and then were stained with Giemsa. Among results, we found that the median frequency of MNE was higher in the polluted site by agrochemical discharges (0.15±0.18), than in the unimpacted site (0.06±0.08). Furthermore, the frequency of MNE in B. henni during the dry season was highly significant for both locations. The results of this study indicated that the analysis of MNE in B. henni could be recommended as a suitable method for in situ detection of environmental genotoxins. Rev. Biol. Trop. 65 (1): 405-414. Epub 2017 March 01.

Patient age does not affect mefloquine concentrations in erythrocytes and plasma during the acute phase of falciparum malaria

Abstract Objective To evaluate whether patient age has a significant impact on mefloquine concentrations in the plasma and erythrocytes over the course of treatment for uncomplicated falciparum malaria. Methods A total of 20 children aged between 8 and 11 years and 20 adult males aged between 22 and 41 years with uncomplicated falciparum malaria were enrolled in the study. Mefloquine was administered to patients in both age groups at a dose of 20 mg kg−1. The steady-state drug concentrations were measured by reversed-phase high performance liquid chromatography. Results All patients had an undetectable mefloquine concentration on day 0. In adults, the plasma mefloquine concentrations ranged from 770 to 2930 ng mL−1 and the erythrocyte concentrations ranged from 2000 to 6030 ng mL−1. In children, plasma mefloquine concentrations ranged from 881 to 3300 ng mL−1 and erythrocyte concentrations ranged from 3000 to 4920 ng mL−1. There was no significant correlation between mefloquine concentrations in the plasma and erythrocytes in either adults or children. Conclusion In the present study, we observed no effect of patient age on the steady-state concentrations of mefloquine in the plasma and erythrocytes. We found that the mefloquine concentration in the erythrocytes was approximately 2.8-times higher than in the plasma. There were no significant correlations between mefloquine concentrations in the erythrocytes and plasma for either age group.

Lectin activity in mycelial extracts of Fusarium species

ABSTRACT Lectins are non-immunogenic carbohydrate-recognizing proteins that bind to glycoproteins, glycolipids, or polysaccharides with high affinity and exhibit remarkable ability to agglutinate erythrocytes and other cells. In the present study, ten Fusarium species previously not explored for lectins were screened for the presence of lectin activity. Mycelial extracts of F. fujikuroi, F. beomiformii, F. begoniae, F. nisikadoi, F. anthophilum, F. incarnatum, and F. tabacinum manifested agglutination of rabbit erythrocytes. Neuraminidase treatment of rabbit erythrocytes increased lectin titers of F. nisikadoi and F. tabacinum extracts, whereas the protease treatment resulted in a significant decline in agglutination by most of the lectins. Results of hapten inhibition studies demonstrated unique carbohydrate specificity of Fusarium lectins toward O-acetyl sialic acids. Activity of the majority of Fusarium lectins exhibited binding affinity to D-ribose, L-fucose, D-glucose, L-arabinose, D-mannitol, D-galactosamine hydrochloride, D-galacturonic acid, N-acetyl-d-galactosamine, N-acetyl-neuraminic acid, 2-deoxy-D-ribose, fetuin, asialofetuin, and bovine submaxillary mucin. Melibiose and N-glycolyl neuraminic acid did not inhibit the activity of any of the Fusarium lectins. Mycelial extracts of F. begoniae, F. nisikadoi, F. anthophilum, and F. incarnatum interacted with most of the carbohydrates tested. F. fujikuroi and F. anthophilum extracts displayed strong interaction with starch. The expression of lectin activity as a function of culture age was investigated. Most species displayed lectin activity on the 7th day of cultivation, and it varied with progressing of culture age.

Preservação da fertilidade e dos ovários em mulheres com tumores anexiais benignos / Preservation of the fertility and the ovaries in women with benign adnexal tumors

OBJETIVO: Avaliar a preservação da fertilidade e dos ovários em mulheres submetidas à cirurgia por tumor anexial benigno. MÉTODOS: Para este estudo observacional com coleta prospectiva foram incluídas 206 mulheres operadas no CAISM-Unicamp de fevereiro de 2010 a janeiro de 2014. A preservação da fertilidade foi definida como tumorectomia ou anexectomia unilateral sem histerectomia em mulheres na pré-menopausa. A preservação ovariana foi considerada quando pelo menos um ovário ou parte dele foi preservado. RESULTADOS: Das 206 mulheres com tumores anexiais benignos, 120 (58%) estavam na pré-menopausa e 86 (42%) na pós-menopausa. Na pré-menopausa, foram encontrados 36 (30%) tumores de células germinativas, 31 (26%) neoplasias epiteliais e 11 (9%) do cordão sexual e estroma. Na pós-menopausa foram identificados 35 (41%) neoplasias epiteliais, 27 (31%) do cordão sexual e estroma e 8 (9%) de células germinativas. Entre as 36 mulheres com tumores ovarianos não neoplásicos, 21 (58%) apresentavam endometriomas e 8 (22%) cistos funcionais. Das 22 mulheres com tumores extra ovarianos, o leiomioma uterino foi o achado mais frequente (50%). Entre as pacientes com ≤35 anos, 26 (57%) foram submetidas à tumorectomia e 18 (39%) a anexectomia unilateral com preservação do útero e anexo contralateral. Mulheres com ≤35 anos foram mais frequentemente operadas por laparoscopia que esteve associada a maior taxa de preservação de fertilidade quando comparada com a laparotomia (p<0,01). Observou-se que 26 das pacientes submetidas à histerectomia com anexectomia (28%) bilateral estavam na pré-menopausa. CONCLUSÕES: Embora se observe uma tendência em realizar apenas tumorectomia em mulheres com ≤35 anos, uma proporção significativa de mulheres jovens ainda é ...

PURPOSE: To evaluate the sparing of fertility and ovaries in women submitted to surgical treatment for benign adnexal tumors. METHODS: Between February 2010 and January 2014, 206 patients were included in this observational study as they were submitted to surgical treatment for benign ovarian tumors at CAISM, a tertiary hospital. Fertility sparing surgery was defined as tumorectomy or unilateral salpingoophorectomy without hysterectomy in premenopausal women. Preservation of the ovary occurred when at least one ovary or part of it was mantained. RESULTS: Of the 206 women with benign tumors, 120 (58%) were premenopausal and 86 (42%) were postmenopausal. There were 36 (30%) ovarian germ cell tumors, 31 (26%) epithelial neoplasms and 11 (9%) sex-cord stromal tumors among premenopausal women. In the group of postmenopausal women, 35 (41%) epithelial neoplasms, 27 (31%) sex-cord stromal tumors and 8 (9%) ovarian germ cell tumors were identified. Among 36 women with non-neoplastic ovarian tumors, 21 (58%) had endometriomas and 8 (22%) functional cysts. Among 22 women with extra-ovarian tumors, uterine leiomyomatosis was the most frequent finding (50%). In the group of women who were ≤35 years old, 26 (57%) were treated by tumorectomy and 18 (39%) were submitted to unilateral salpingoophorectomy with sparing of the uterus and the contralateral ovary. Women who were ≤35 years old were more frequently operated by laparoscopy which was associated with a higher number of fertility sparing procedures when compared to laparotomy (p<0.01). Twenty-six (28%) women submitted to hysterectomy with bilateral salpingoophorectomy were premenopausal. CONCLUSION: Although there is a trend to perform only tumorectomy in women who are ≤35 years old, a significant number of young women is still treated by salpingoophorectomy. Among 36- to 45-year-old women, only 70% had their fertility spared, while 20% had both ovaries removed. ...

Re-emergence of Chorioptes bovis (Acari: Psoroptidae) in cattle in the state of Rio Grande do Sul, Brazil / Reemergência de Chorioptes bovis (Acari: Psoroptidae) em bovinos no Estado do Rio Grande do Sul, Brasil

Here we describe an outbreak of chorioptic mange in cattle, 56 years after its first identification in Brazil. Between the months of June and July 2011, dermatitis characterized by alopecia and crusted and thickened skin at the insertion of the tail and in the ischiorectal fossa was recognized in 40 (35.7%) out of 112 Holstein cows on a farm in the northeastern mesoregion of the state of Rio Grande do Sul, Brazil. After diagnosing mange caused by Chorioptes bovis, the cows were weighed and treated with 0.5% ivermectin, as a pour-on single dose, and were separated into two groups: cows in early lactation and those in late lactation. The survival rate of C. bovis and the healing rate in the two groups of infested cows were monitored every seven days through skin scrapings. After 28 days of evaluation, the cure rate through treatment was greater among cows in early lactation (p <0.0001). The survival rate of C. bovis was higher in cows in late lactation.

O objetivo deste estudo foi descrever um surto de sarna corióptica em bovinos, 56 anos após a sua primeira identificação no Brasil. Entre os meses de junho a julho de 2011, a dermatite caracterizada por alopecia, com crosta e espessamento da pele na inserção da cauda e na fossa isquiorretal, foi observada em 40 (35,7%) de 112 vacas holandesas de uma propriedade rural pertencente à Mesorregião do Nordeste do Estado do Rio Grande do Sul, Brasil. Após o diagnóstico da sarna causada por Chorioptes bovis, as vacas foram pesadas, tratadas com 0,5% de ivermectina pour on em dose única e separadas em dois grupos: vacas no início da lactação e no final da lactação. A taxa de sobrevivência de C. bovis e a taxa de cura dos dois grupos de vacas infestadas foram monitoradas a cada sete dias por meio de raspas de pele. Após 28 dias do estudo, a taxa de cura com o tratamento foi maior em vacas no início da lactação (p <0,0001). A taxa de sobrevivência de C. bovis foi maior em vacas no final da lactação.

Carum copticum and Thymus vulgaris oils inhibit virulence in Trichophyton rubrum and Aspergillus spp

Emergence of drug-resistant strains has demanded for alternative means of combating fungal infections. Oils of Carum copticum and Thymus vulgaris have long been used in ethnomedicine for ailments of various fungal infections. Since their activity has not been reported in particular against drug-resistant fungi, this study was aimed to evaluate the effects of oils of C. copticum and T. vulgaris on the growth and virulence of drug-resistant strains of Aspergillus spp. and Trichophyton rubrum. The gas chromatography-mass spectrometry analysis revealed thymol constituting 44.71% and 22.82% of T. vulgaris and C. copticum, respectively. Inhibition of mycelial growth by essential oils was recorded in the order of thymol > T. vulgaris > C. copticum against the tested strains. RBC lysis assay showed no tested oils to be toxic even up to concentration two folds higher than their respective MFCs. Thymol exhibited highest synergy in combination with fluconazole against Aspergillus fumigatus MTCC2550 (FICI value 0.187) and T. rubrum IOA9 (0.156) as determined by checkerboard method. Thymol and T. vulgaris essential oil were equally effective against both the macro and arthroconidia growth (MIC 72 µg/mL). A > 80% reduction in elastase activity was recorded for A. fumigatus MTCC2550 by C. copticum, T. vulgaris oils and thymol. The effectiveness of these oils against arthroconidia and synergistic interaction of thymol and T. vulgaris with fluconazole can be exploited to potentiate the antifungal effects of fluconazole against drug-resistant strains of T. rubrum and Aspergillus spp.

Baseline micronuclei and nuclear abnormalities frequencies in native fishes from the Paraná River (Argentina) / Frequências basais de micronúcleos e anormalidades nucleares em peixes nativos do Rio Paraná (Argentina)

This work aims to establish baseline frequencies of micronuclei (MN) and nuclear abnormalities (NA) in native fish species collected in situ from the Paraná River. For this purpose, the micronucleus test was applied in peripheral blood erythrocytes from specimens obtained from samplings collected at two localities (Posadas and Candelaria, Misiones, Argentina) during the period 2007-2010. The results were statistically analyzed using the Kruskal Wallis test. Data from nine fish species were obtained, among which Steindachnerina brevipinna (Characiformes) revealed the highest baseline frequency of MN and NA, showing statistically significant differences with regard to the other analyzed species. These results are the first report of baseline MN and NA frequencies for native fish species studied and could be useful for future comparisons with data of fishes belonging to other environments.

O presente trabalho tem como objetivo estabelecer frequências basais de micronúcleos (MN) e anormalidades nucleares (AN) em espécies nativas de peixes obtidas in situ no Rio Paraná. Para este efeito, o teste do micronúcleo foi aplicado em eritrócitos de sangue periférico de espécimes provenientes de amostragens efetuadas em duas localidades (Posadas e Candelaria, Misiones, Argentina) durante o período 2007-2010. Os resultados foram analisados estatisticamente empregando o teste de Kruskal Wallis. Foram coletados dados de nove espécies e dentre estas Steindachnerina brevipinna (Characiformes) revelou a maior frequência basal de MN e AN, mostrando diferenças estatisticamente significativas com respeito às outras espécies analisadas. Estes resultados são o primeiro relatório de frequências basais de MN e AN para espécies nativas de peixes estudadas e poderiam ser úteis para futuras comparações com dados de peixes pertencentes a outros ambientes.

Ultrastructural changes of erythrocytes in whole blood after exposure to prospective in silico-designed anticancer agents: a qualitative case study

BACKGROUND: Novel, in silico-designed anticancer compounds were synthesized in our laboratory namely, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). These compounds were designed to have improved bioavailability when compared to their source compound, 2-methoxyestradiol. This theoretically would be due to their increased binding affinity to carbonic anhydrase II, present in erythrocytes. Since the novel compounds under investigation are proposed to be transported within erythrocytes bound to carbonic anhydrase II, the morphological effect which they may exert on whole blood and erythrocytes is of great significance. A secondary outcome included revision of previously reported procedures for the handling of the whole blood sample. The purpose of this study was twofold. Firstly, the ultrastructural morphology of a healthy female's erythrocytes was examined via scanning electron microscopy (SEM) after exposure to the newly in silico-designed compounds. Morphology of erythrocytes following exposure to ESE-15-ol and ESE-16 for 3 minutes and 24 hours at 22°C were described with the use of SEM. The haemolytic activity of the compounds after 24 hours exposure were also determined with the ex vivo haemolysis assay. Secondly, storage conditions of the whole blood sample were investigated by determining morphological changes after a 24 hour storage period at 22°C and 37°C. RESULTS: No significant morphological changes were observed in the erythrocyte morphology after exposure to the novel anticancer compounds. Storage of the whole blood samples at 37°C for 24 hours resulted in visible morphological stress in the erythrocytes. Erythrocytes incubated at 22°C for 24 hours showed no structural deformity or distress. CONCLUSIONS: From this research the optimal temperature for ex vivo exposure of whole blood samples to ESE-15-ol and ESE-16 for 24 hours was determined to be 22°C. Data from this study revealed the potential of these compounds to be applied to ex vivo study techniques, since no damage occurred to erythrocytes ultrastructure under these conditions. As no structural changes were observed in erythrocytes exposed to ESE-15-ol and ESE-16, further ex vivo experiments will be conducted into the potential effects of these compounds on whole blood. Optimal incubation conditions up to 24 hours for whole blood were established as a secondary outcome.

Evaluación del efecto citotóxico y del daño genético de extractos estandarizados de Solanum nudumcon actividad anti- Plasmodium / Evaluation of cytotoxic effect and genetic damage of standardized extracts of Solanum nudum with antiplasmodial activity

Introducción. La planta Solanum nudum es ampliamente usada en la medicina tradicional del Pacífico colombiano para tratar las fiebres y la malaria, o paludismo, y se ha convertido en una fuente de nuevas moléculas promisorias. Objetivo. Evaluar el efecto citotóxico y daño genético de extractos estandarizados de S. nudum en diferentes modelos celulares. Materiales y métodos. A 66 extractos estandarizados de S. nudum se les evaluó la actividad anti- Plasmodiumin vitro en dos cepas de Plasmodium falciparum, una sensible (NF54) y otra resistente (FCB2) a la cloroquina, y la citotoxicidad en células U937 y HepG2. Se seleccionaron los extractos que presentaron actividad anti- Plasmodium y baja toxicidad, y se les estimó su efecto hemolítico en eritrocitos sanos O + , el efecto mutagénico en las cepas TA98 y TA100 de Salmonella Typhimurium y el efecto genotóxico en células U937. Resultados. Se seleccionaron cinco extractos como promisorios (28MA1, 29MA1, 51MA1, 55MA1 y 61MA1), los cuales fueron activos en las cepas de P. falciparum con concentración inhibitoria 50 (CI 50 ) entre 9,8 y 54,8 µg/ml. El extracto 29MA1 fue el más selectivo para Plasmodium, con índice de selectividad de 4,4 y 14,5 para las células U937 y HepG2, respectivamente. En ningún extracto se observó efecto hemolítico a 250 µg/ml, no causaron mutaciones en las cepas TA98 y TA100 de S.Typhimurium, ni generaron efectos genotóxicos en células U937. Conclusiones. La utilización de extractos estandarizados de S. nudum contribuye con los trabajos encaminados al desarrollo de una nueva formulación farmacéutica para tratar la malaria a partir de productos naturales.

Introduction. The plant Solanum nudum (Solanaceae) is extensively used for the treatment of malaria-related symptoms in traditional medicine practices in the Colombian Pacific. Recently, it has become a significant source of promising new molecules for developing a pharmaceutical malaria treatment. Objective. This research aimed to evaluate the cytotoxic effect and the genetic damage of standardized extracts of S. nudumon different cells. Materials and methods. Sixty six standardized S. nudum extracts were used, evaluating cytotoxicity in U937 and HepG2 cells and the antiplasmodial activity using both a chloroquine-sensitive (NF54) and a chloroquine-resistant (FCB2) strain. The hemolytic effect on healthy O + erythrocytes, the mutagenic effect on S.Typhimurium TA98 and TA100 strains and the genotoxic effect on U937 cells were evaluated. The extracts that displayed both antiplasmodial activity and low toxicity were selected. Results. Five extracts were selected: 28MA1, 29MA1, 51MA1, 55MA1 and 61MA1. These extracts were active against P. falciparum with IC 50 between 9.8 and 54.8 µg/ml and selectivity indexes were calculated between 0.9 and 4.4, the latter for 29MA1. Also, no hemolytic effects in healthy O + erythrocytes were shown at a concentration of 250 µg/ml, nor did they cause mutations in the TA98 and TA100 strains or generate genotoxic effects in U937cells. Conclusion. The use of standardized extracts of S. nudum could contribute to the body of work aimed at developing a new pharmaceutical treatment for malaria using natural products.

Assessment of methyl methacrylate genotoxicity by the micronucleus test

The aim of this study was to evaluate the genotoxic potential of methyl methacrylate (MMA) vapor by simulating standard occupational exposure of 8 hours per day and using the micronucleus test. We used 32 adult male Wistar rats divided into three groups: A - 16 rats exposed to MMA for 8 hours a day, B - Eight rats receiving single subcutaneous doses of cyclophosphamide on the first day of the experiment (positive control), C - Eight rats receiving only water and food ad libitum (negative control). Eight rats from group A and all of the rats from groups B and C were sacrificed 24 hours after beginning the experiment (acute exposure in group A). The remaining animals in group A were sacrificed 5 days after the experiment began (repeated exposure assessment in group A, simulating occupational exposure 40 hours/week). Femoral bone marrow was collected from each rat at the time of sacrifice for use in the micronucleus test. Two slides were completed per animal and were stained with Giemsa staining. Two thousand polychromatic erythrocytes were counted per animal. The Kruskal-Wallis test followed by a multiple comparisons test (Dunn test) was used for statistical analysis. The median number of micronuclei was 7.00 in the group exposed to MMA for 1 day, 2.00 in the group exposed to MMA for 5 days, 9.00 in the group exposed to cyclophosphamide (positive control) and 0.756 in the negative control group (p < 0.0001). MMA was genotoxic when measured after 1 day of exposure but was not evidently genotoxic after 5 days.

Protective role of vitamins C and E in diclorvos-induced oxidative stress in human erythrocytes in vitro

Organophosphate (OP) pesticides such as dichlorvos (DDVP) intoxication has been shown to produce oxidative stress due to the generation of free radicals, which alter the antioxidant defense system in erythrocytes. In this study, the effects of DDVP (1, 10, 100 µM) or DDVP + vitamin C (VC; 10 µM) or vitamin E (VE; 30 µM), on the levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities in human erythrocytes were examined in vitro. There were no statistical differences between all groups for 1 µM concentration of DDVP. Treatment with DDVP alone produced an increase in the level of MDA and decreased activities of antioxidant enzymes (P < 0.05). Groups treated with vitamins and DDVP showed protective effects of vitamins against DDVP-induced changes in antioxidant enzyme activity and lipid peroxidation (LPO) (10 µM). At 100 µM concentration of DDVP vitamins had no effect on DDVP-induced toxicity. The results show that administration of DDVP resulted in the induction of erythrocyte LPO and alterations in antioxidant enzyme activities, suggesting that reactive oxygen species (ROS) may be involved in the toxic effects of DDVP. Also the data show that the plasma level of VC and VE may ameliorate OP-induced oxidative stress by decreasing LPO in erythrocytes at certain doses of OP pesicides.

Calcium dobesilate may improve hemorheology in patients undergoing coronary artery bypass grafting / Dobesilato de cálcio pode melhorar hemorreologia em pacientes submetidos à cirurgia de revascularização miocárdica

BACKGROUND: Calcium dobesilate is an angioprotective agent that has positive effects on hemorheological parameters. It is an antioxidant that increases endothelial-derived vasodilator substance secretion, there are none that analyze its effects during the postoperative period of patients undergoing myocardial revascularization. OBJECTIVE: We aimed to determine the effects of calcium dobesilate on hemorheological parameters, such as reduced glutathione and malondialdehyde in patients with ischemic heart disease undergoing myocardial revascularization in the postoperative period. METHODS: One hundred and thirty-four patients operated for coronary heart disease were included in this study. Hemorheological, oxidant and antioxidant parameters were measured two days after surgery and after a period of treatment with calcium dobesilate. Then, 500 mg of calcium dobesilate was given twice a day to one group of 68 patients for three months. The control group was composed of 66 patients who did not receive this medication. RESULTS: The increase in the erythrocyte deformability index was found to be significant compared with both the pretreatment values and with the 1st and 2nd values of the control group after calcium dobesilate administration, whereas there were no significant changes in blood viscosity, glutathione (GSH) or malondialdehyde (MDA) values after the calcium dobesilate administration. The same improvement in the CCS class was observed in patients regardless of they received the calcium dobesilate treatment. CONCLUSION: In the present investigation, the same improvement in the CCS class was observed in patients regardless of they received the calcium dobesilate treatment. Improvements with calcium dobesilate were statistically significant only in the increase in erythrocyte flexibility.

ANTECEDENTES: O dobesilato de cálcio é um agente angioprotetor que tem efeitos positivos sobre os parâmetros hemorreológicos. É um antioxidante que aumenta a secreção endotelial derivada da substância vasodilatadora, não há nada que analisar os seus efeitos durante o período pósoperatório de pacientes submetidos a revascularização do miocárdio. OBJETIVO: Nosso objetivo foi determinar os efeitos de dobesilato de cálcio sobre os parâmetros hemorreológicos, tais como glutationa reduzida e malondialdeído em pacientes com doença cardíaca isquêmica submetidos a revascularização do miocárdio no pós-operatório. MÉTODOS: Cento e trinta e quatro pacientes operados por doença cardíaca coronária foram incluídos neste estudo. Parâmetros de oxidante, hemorreológicos e de antioxidantes foram medidos dois dias após a cirurgia e após um período de tratamento com o dobesilato de cálcio. Em seguida, 500 mg de dobesilato de cálcio foi administrado duas vezes por dia para um grupo de 68 pacientes durante três meses. O grupo controle foi composto por 66 pacientes que não receberam essa medicação. RESULTADOS: O aumento do índice de deformabilidade dos eritrócitos foi considerado significativo comparado com ambos os valores pré-tratamento e com os 1º e 2º valores do grupo controle após a administração dobesilato de cálcio, enquanto que não houve alterações significativas na viscosidade do sangue, na glutationa (GSH) ou malondialdeído (MDA) após a administração dobesilato de cálcio. A mesma melhoria na classe CCS foi observada em pacientes independentemente de terem recebido tratamento com dobesilato de cálcio. CONCLUSÃO: Na presente investigação, a mesma melhora na classe CCS foi observada em pacientes independentemente de terem recebido o tratamento com dobesilato de cálcio.

Avaliação de parâmetros antioxidantes em ratos tratados com sevoflurano / Evaluation of antioxidant parameters in eats treated with sevoflurane / Evaluación de parámetros antioxidantes en ratones tratados con sevoflurano

JUSTIFICATIVA E OBJETIVOS: O sevoflurano é um éter halogenado com flúor que sofre biotransformação hepática através do citocromo P450 2E1. Éteres halogenados que sofrem biotransformação pelo P450 2E1 podem produzir espécies reativas do oxigênio (ERO) e promover enfraquecimento do sistema de defesa antioxidante. O objetivo deste trabalho foi investigar a relação entre a atividade das enzimas antioxidantes eritrocitárias e o sevoflurano. MÉTODO: Os animais foram distribuídos em quatro grupos: Grupo 1 controle: apenas oxigênio a 100 por cento (1 L.min-1 por 60 minutos durante 5 dias consecutivos); Grupo 2 - sevoflurane 4,0 por cento em oxigênio a 100 por cento (1 L.min-1 por 60 minutos durante 5 dias consecutivos); Grupo 3 - isoniazida (i.p.), 50 mg.kg-1 de peso corporal /dia, durante 4 dias e em seguida tratados apenas com oxigênio a 100 por cento (1 L.min-1 por 60 minutos durante 5 dias consecutivos); Grupo 4 - isoniazida por via intraperitoneal na dose de 50 mg.kg-1 de peso corporal, diariamente durante 4 dias, seguido da administração do sevoflurane a 4,0 por cento em oxigênio a 100 por cento (1 L.min-1 por 60 minutos durante 5 dias). Após 12 horas da última exposição ao sevoflurane, os animais foram sacrificados e o sangue foi coletado através da veia porta para análise da atividade das enzimas antioxidantes. RESULTADOS: Aumento da atividade específica da glicose-6-fosfato desidrogenase, diminuição da atividade específica da catalase, principalmente no grupo de animais pré-tratados com isoniazida e, em seguida, tratados com sevoflurano. A glutationa peroxidase não apresentou alteração na sua atividade. CONCLUSÕES: A interação do sevoflurano com indutores enzimáticos do citocromo P450 2E1 pode propiciar a instalação do estresse oxidativo caso a exposição se torne prolongada e repetitiva.

BACKGROUND AND OBJECTIVES: Sevoflurane is a halogenated fluorinated ether that undergoes hepatic biotransformation through cytochrome P4502E1. Halogenated ethers undergoing biotransformation by P4502E1 can produce reactive oxygen species (ROS), weakening the antioxidant defense mechanism. The objective of this study was to investigate the relationship between the activity of erythrocyte antioxidant enzymes and sevoflurane. METHODS: Animals were divided in four groups: Group 1 - control: 100 percent oxygen (1 L.min-1 for 60 min during five consecutive days); Group 2 - 4.0 percent sevoflurane in 100 percent oxygen (1 L.min-1 for 60 minutes during five consecutive days); Group 3 - isoniazid (i.p.), 50 mg.kg-1/ day for four consecutive days, followed by 100 percent oxygen (1 L.min-1 for 60 minutes during four consecutive days); Group 4 - intraperitoneal isoniazid, 50 mg.kg-1 daily for four days, followed by 4.0 percent sevoflurane in 100 percent oxygen (1 L.min-1 for 60 minutes during five days). Twelve hours after the last exposure to sevoflurane, animals were sacrificed and their blood was collected through the portal vein for analysis of antioxidant enzymes. RESULTS: An increase in the activity of glucose-6-phosphate dehydrogenase and a decrease in the activity of catalase were observed, especially in the group of animals pre-treated with isoniazid. Changes in the activity of glutathione peroxidase were not observed. CONCLUSIONS: The interaction between sevoflurane and cytochrome P450 2E1 with enzymatic inducers can lead to oxidative stress with prolonged and repetitive exposure.

JUSTIFICATIVA Y OBJETIVOS: El sevoflurano es un éter halogenado con flúor que sufre una biotransformación hepática a través del citocromo P450 2E1. Los éteres halogenados que sufren biotransformación por el P450 2E1, pueden generar especies reactivas del oxígeno (ERO) y promover el debilitamiento del sistema de defensa antioxidante. El objetivo de este trabajo fue investigar la relación entre la actividad de las enzimas antioxidantes eritrocitarias y el sevoflurano. MÉTODO: Los animales fueron distribuidos en cuatro grupos: Grupo 1 control: apenas oxígeno a 100 por ciento (1 L.min-1 por 60 minutos durante 5 días consecutivos); Grupo 2 - sevoflurano 4,0 por ciento en oxígeno a 100 por ciento (1 L.min-1 por 60 minutos durante 5 días consecutivos); Grupo 3 - isoniazida (i.p.), 50 mg.kg-1 de peso corporal /día, durante 4 días y enseguida tratados apenas con oxígeno a 100 por ciento (1 L.min-1 por 60 minutos durante 5 días consecutivos); Grupo 4 - isoniazido por vía intraperitoneal en dosis de 50 mg.kg-1 de peso corporal, diariamente durante 4 días, seguido de la administración del sevoflurano a 4,0 por ciento en oxígeno a 100 por ciento (1 L.min-1 por 60 minutos durante 5 días). Después de 12 horas de la última exposición al sevoflurano, los animales se sacrificaron y la sangre se recolectó a través de la vena porta para el análisis de la actividad de las enzimas antioxidantes. RESULTADOS: Aumento de la actividad específica de la glucosa- 6-fosfato deshidrogenasa, reducción de la actividad específica de la catalasis, principalmente en el grupo de animales pretratados con isoniazida y enseguida, tratados con sevoflurano. El glutatión peroxidasa no presentó ninguna alteración en su actividad. CONCLUSIONES: La interacción del sevoflurano con inductores enzimáticos del citocromo P450 2E1 puede propiciar la instalación del estrés oxidativo en el caso que la exposición se prolongue y sea repetitiva.