Antiproliferative and photoprotective activities of the extracts and compounds from Calea fruticosa.

In this paper, we complement our previous study on the antiproliferative activity of Calea fruticosa (Asteraceae) by isolating the compounds apigenin-4',7-dimethyl ether (1), budlein A (2), quercetin (3), and cichoriin (4) from the plant's aerial parts. The antiproliferative activity of these compounds was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method against human tumor cell lines. Compound 3 displayed moderate antiproliferative activity in three cell lines (HCT-116, PC-3, and SF-295, with cell growth inhibition values of 72.97, 74.55, and 68.94%) and high antiproliferative activity (90.86%) in the HL-60 cell line. The in vitro sun protection factor (SPF) of the extracts and compound 4, with and without sunscreen, was determined by a spectrophotometric method. The ethanol extract exhibited the highest SPF (9.67) at a concentration of 0.100 mg/mL, while compound 4, isolated from this extract, showed a SPF of 13.79 at the same concentration. A relative increased efficacy of SPF was observed for the extracts and compound 4 when sunscreen was also used. Compound 4 has not been reported previously from any species within the genus Calea. Compounds 1-4 were obtained from this species for the first time.

Antiproliferative and photoprotective activities of the extracts and compounds from Calea fruticosa

In this paper, we complement our previous study on the antiproliferative activity of Calea fruticosa (Asteraceae) by isolating the compounds apigenin-4',7-dimethyl ether (1), budlein A (2), quercetin (3), and cichoriin (4) from the plant's aerial parts. The antiproliferative activity of these compounds was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method against human tumor cell lines. Compound 3 displayed moderate antiproliferative activity in three cell lines (HCT-116, PC-3, and SF-295, with cell growth inhibition values of 72.97, 74.55, and 68.94%) and high antiproliferative activity (90.86%) in the HL-60 cell line. The in vitro sun protection factor (SPF) of the extracts and compound 4, with and without sunscreen, was determined by a spectrophotometric method. The ethanol extract exhibited the highest SPF (9.67) at a concentration of 0.100 mg/mL, while compound 4, isolated from this extract, showed a SPF of 13.79 at the same concentration. A relative increased efficacy of SPF was observed for the extracts and compound 4 when sunscreen was also used. Compound 4 has not been reported previously from any species within the genus Calea. Compounds 1-4 were obtained from this species for the first time.

Esophageal pythiosis in an ostrich (Struthio camelus) / Ptiose Esofágica em avestrus (Struthio camelus)

Uma avestruz-do-pescoço-vermelho, com dois anos de idade, apresentava um nódulo no terço médio do esôfago e foi submetida a procedimento cirúrgico. Histologicamente, observou-se uma área focalmente extensa de necrose estendendo-se da túnica mucosa à muscular, e, em algumas secções, à túnica adventícia. Circundando a área de necrose, observou-se uma reação inflamatória composta principalmente por granulócitos e macrófagos, associada à fibroplasia e neovascularização. Em meio às áreas de necrose e inflamação, verificavam-se numerosas imagens negativas de hifas em seções longitudinais e transversais, melhor apreciadas pela coloração de metenamina nitrato de prata de Grocott. O diagnóstico definitivo de infecção por Pythium insidiosum foi confirmado por imuno-histoquímica. A avestruz recebia água para consumo de um lago localizado em uma área de pastagem, no qual alguns cavalos haviam desenvolvido pitiose cutânea anteriormente.(AU)

Assessment of the antibacterial, cytotoxic and antioxidant activities of Morus nigra L. (Moraceae) / Avaliação das atividades antibacteriana, citotóxica e antioxidante de Morus nigra L. (Moraceae)

Abstract This study was carried out to assess the antibacterial, cytotoxic and antioxidant activities of extracts of Morus nigra L. HPLC was used to determine the fingerprint chromatogram of the crude ethanolic extract (Mn-EtOH). The antibacterial effect was assessed through the method of microdilution. The cytotoxicity was tested against human tumour cell lines using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The total phenolic and flavonoid contents were also assessed through the Folin-Ciocalteu and aluminum chloride methods, respectively. Antioxidant activities of the extracts were evaluated by using 2,2-diphenyl-1-picrylhydrazil (DPPH) radical scavenging and β-carotene-linoleic acid bleaching methods. The presence of phenolic compounds in Mn-EtOH was confirmed using HPLC. The extracts showed activity against most microorganisms tested. The extracts did not show any expressive antiproliferative effect in the assessment of cytotoxicity. The most significant total phenolic content was 153.00 ± 11.34 mg of gallic acid equivalent/g to the ethyl acetate extract (AcOEt). The total flavonoid content was 292.50 ± 70.34 mg of catechin equivalent/g to the AcOEt extract, which presented the best antioxidant activity (IC50 50.40 ± 1.16 μg/mL) for DPPH scavenging. We can conclude that this species shows strong antibacterial and antioxidant activities, as well as weak cytotoxic effects.

Resumo Este estudo foi realizado para avaliar as atividades antibacteriana, citotóxica e antioxidante de extratos de Morus nigra L. HPLC foi utilizado para determinar o perfil de compostos fenólicos do extrato etanólico bruto (Mn-EtOH). O efeito antibacteriano foi avaliado através do método de microdiluição. A citotoxicidade foi testada contra linhagens celulares de tumores humanos utilizando o ensaio do brometo de 3-(4,5-dimetil-2-tiazolil)-2,5-difenil-2H-tetrazólio (MTT). O conteúdo total de compostos fenólicos e flavonoides também foi avaliado por meio dos métodos de Folin-Ciocalteu e cloreto de alumínio, respectivamente. A atividade antioxidante dos extratos foi avaliada por meio do sequestro do radical livre 2,2-difenil-1-picrilhidrazil (DPPH) e co-oxidação do sistema β-caroteno-ácido linoleico. A presença de compostos fenólicos em Mn-EtOH foi confirmada utilizando HPLC. Os extratos mostraram atividade contra a maioria dos microrganismos testados. Os extratos não mostraram qualquer efeito antiproliferativo expressivo na avaliação da citotoxicidade. O conteúdo fenólico total mais significativo foi de 153,00 ± 11,34 mg de equivalente de ácido gálico/g para o extrato acetato de etila (AcOEt). O conteúdo de flavonoides totais foi de 292,50 ± 70,34 mg de equivalente de catequina/g para o extrato AcOEt, que apresentou a melhor atividade antioxidante (IC50 50,40 ± 1,16 mg/mL) para o sequestro do DPPH. Podemos concluir que esta espécie apresenta forte atividade antibacteriana e antioxidante, bem como fraca atividade citotóxica.

Assessment of the antibacterial, cytotoxic and antioxidant activities of Morus nigra L. (Moraceae).

This study was carried out to assess the antibacterial, cytotoxic and antioxidant activities of extracts of Morus nigra L. HPLC was used to determine the fingerprint chromatogram of the crude ethanolic extract (Mn-EtOH). The antibacterial effect was assessed through the method of microdilution. The cytotoxicity was tested against human tumour cell lines using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The total phenolic and flavonoid contents were also assessed through the Folin-Ciocalteu and aluminum chloride methods, respectively. Antioxidant activities of the extracts were evaluated by using 2,2-diphenyl-1-picrylhydrazil (DPPH) radical scavenging and ß-carotene-linoleic acid bleaching methods. The presence of phenolic compounds in Mn-EtOH was confirmed using HPLC. The extracts showed activity against most microorganisms tested. The extracts did not show any expressive antiproliferative effect in the assessment of cytotoxicity. The most significant total phenolic content was 153.00 ± 11.34 mg of gallic acid equivalent/g to the ethyl acetate extract (AcOEt). The total flavonoid content was 292.50 ± 70.34 mg of catechin equivalent/g to the AcOEt extract, which presented the best antioxidant activity (IC50 50.40 ± 1.16 µg/mL) for DPPH scavenging. We can conclude that this species shows strong antibacterial and antioxidant activities, as well as weak cytotoxic effects.

In vitro antitumor effect of a lignan isolated from Combretum fruticosum, trachelogenin, in HCT-116 human colon cancer cells.

The use of natural products in therapeutics has been growing over the years. Lignans are compounds with large pharmaceutical use, which has aroused interest in the search for new drugs to treat diseases. The present study evaluated the cytotoxicity of (-)-trachelogenin, a dibenzylbutyrolactone type lignan isolated from Combretum fruticosum, against several tumor and non-tumor cell lines using the MTT assay and its possible mechanism of action. (-)-Trachelogenin showed IC50 values ranging of 0.8-32.4µM in SF-295 and HL-60 cell lines, respectively and IC50 values >64µM in non-tumor cell lines. (-)-trachelogenin persistently induced autophagic cell death, with cytoplasmic vacuolization and formation of autophagosomes mediated by increasing LC3 activation and altering the expression levels of Beclin-1.

Isolation and characterization of 2-pyridone alkaloids and alloxazines from Beauveria bassiana.

Two novel compounds bearing heterocyclic nitrogen, 2-pyridone alkaloid (1) and alloxazine derivative (2), along with the known pretenellin B (3), pyridovericin (4) and lumichrome (5) were isolated from a culture of the entomopathogenic fungal strain Beauveria bassiana. The chemical structures of 2-pyridone alkaloid and alloxazine derivative were established on the basis of the interpretation of spectroscopic data. The isolated compounds were evaluated in a panel of five cancer cell lines and pyridovericin exhibited cytotoxicity (IC50, µM) against cancer cell lines: HL-60 (25.9 ± 0.3), HCT8 (34.6 ± 3.6), MDA-MB435 (34.8 ± 3.8) and SF295 (31.1 ± 0.6). Considering that other pyridone compounds display good cytotoxic activity, it would be suggested to obtain new semi synthetic derivatives of pyridovericin, for the development of new cytotoxic chemical entities.

Assessment of the antibacterial, cytotoxic and antioxidant activities of Morus nigra L. (Moraceae)

Abstract This study was carried out to assess the antibacterial, cytotoxic and antioxidant activities of extracts of Morus nigra L. HPLC was used to determine the fingerprint chromatogram of the crude ethanolic extract (Mn-EtOH). The antibacterial effect was assessed through the method of microdilution. The cytotoxicity was tested against human tumour cell lines using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The total phenolic and flavonoid contents were also assessed through the Folin-Ciocalteu and aluminum chloride methods, respectively. Antioxidant activities of the extracts were evaluated by using 2,2-diphenyl-1-picrylhydrazil (DPPH) radical scavenging and -carotene-linoleic acid bleaching methods. The presence of phenolic compounds in Mn-EtOH was confirmed using HPLC. The extracts showed activity against most microorganisms tested. The extracts did not show any expressive antiproliferative effect in the assessment of cytotoxicity. The most significant total phenolic content was 153.00 ± 11.34 mg of gallic acid equivalent/g to the ethyl acetate extract (AcOEt). The total flavonoid content was 292.50 ± 70.34 mg of catechin equivalent/g to the AcOEt extract, which presented the best antioxidant activity (IC50 50.40 ± 1.16 g/mL) for DPPH scavenging. We can conclude that this species shows strong antibacterial and antioxidant activities, as well as weak cytotoxic effects.

Resumo Este estudo foi realizado para avaliar as atividades antibacteriana, citotóxica e antioxidante de extratos de Morus nigra L. HPLC foi utilizado para determinar o perfil de compostos fenólicos do extrato etanólico bruto (Mn-EtOH). O efeito antibacteriano foi avaliado através do método de microdiluição. A citotoxicidade foi testada contra linhagens celulares de tumores humanos utilizando o ensaio do brometo de 3-(4,5-dimetil-2-tiazolil)-2,5-difenil-2H-tetrazólio (MTT). O conteúdo total de compostos fenólicos e flavonoides também foi avaliado por meio dos métodos de Folin-Ciocalteu e cloreto de alumínio, respectivamente. A atividade antioxidante dos extratos foi avaliada por meio do sequestro do radical livre 2,2-difenil-1-picrilhidrazil (DPPH) e co-oxidação do sistema -caroteno-ácido linoleico. A presença de compostos fenólicos em Mn-EtOH foi confirmada utilizando HPLC. Os extratos mostraram atividade contra a maioria dos microrganismos testados. Os extratos não mostraram qualquer efeito antiproliferativo expressivo na avaliação da citotoxicidade. O conteúdo fenólico total mais significativo foi de 153,00 ± 11,34 mg de equivalente de ácido gálico/g para o extrato acetato de etila (AcOEt). O conteúdo de flavonoides totais foi de 292,50 ± 70,34 mg de equivalente de catequina/g para o extrato AcOEt, que apresentou a melhor atividade antioxidante (IC50 50,40 ± 1,16 mg/mL) para o sequestro do DPPH. Podemos concluir que esta espécie apresenta forte atividade antibacteriana e antioxidante, bem como fraca atividade citotóxica.

O-naphthoquinone isolated from Capraria biflora L. induces selective cytotoxicity in tumor cell lines.

Biflorin is an o-naphthoquinone isolated from the roots of the plant Capraria biflora L. (Scrophulariaceae). In this study, the cytotoxic effects of biflorin were verified, and late apoptosis was detected in various cancer cell lines by in situ analysis. The cytotoxicity was further evaluated exclusively for 48 h of treatment in different tumor and non-tumor cell lines (Hep-2, HeLa, HT-29, A-375, and A-549, and HEK-293, respectively). The results indicated that biflorin induced selective cytotoxicity in tumor cells. HeLa cells were more susceptible to biflorin, followed by HT-29, A-549, A-375, and Hep-2 at all concentrations (range 5-50 µg/mL), and the highest half-maximal inhibitory concentration IC50 (56.01 ± 1.17 µg/mL) was observed in HEK-293 cells. Late apoptotic/necrotic events, observed by in situ immunostaining with Annexin V, varied with each cell line; an increase in late apoptotic events was observed corresponding to the increase in biflorin dosage. Hep-2 cells showed a greater percentage of late apoptotic events among the tumor cell lines when treated with higher concentrations of biflorin (69.63 ± 2.28%). The non-tumor HEK-293 line showed greater resistance to late apoptotic events, as well as a lower level of cytotoxicity (77.69 ± 6.68%) than the tested tumor lines. The data presented indicate that biflorin showed an important, possibly selective, cytotoxicity against tumor cell lines, thereby revealing a promising novel substance with potential anticancer activity for tumor therapy.

Surgical approach and radioactive iodine therapy for small well-differentiated thyroid cancer.

BACKGROUND: Management of small well-differentiated thyroid cancer (DTC) has generated much debate regarding the surgical approach and radioactive iodine treatment (RAI). AIM: The aim of the study was to evaluate the impact of surgical extension and RAI on the outcome of DTC ≤2 cm. METHODS: A retrospective analysis of 176 cases of DTC ≤2 cm was performed. RESULTS: At diagnosis, tumor size was 1.38 ± 0.55 cm, age 40.2 ± 13.6 years. After a mean follow-up period of 14.1 ± 4.5 years, 15.9 % patients had recurrent/persistent structural disease, with cervical neck disease (thyroid gland area and/or cervical lymph nodes) in 11.9 % cases and distant metastasis in 5.1 %. Disease specific mortality was of 1.1 %. No difference in outcome was observed between patients submitted to total or subtotal thyroidectomy. After total and subtotal thyroidectomy, the rate of recurrent/persistent structural disease was 19.1 and 10.6 % (p = 1.00), respectively. Using the multivariate cox proportion hazards analysis, no difference in the clinical outcome was observed after total or subtotal thyroidectomy (p = 0.703) neither after RAI (p = 0.807). Similar results were observed after stratification by tumor size. Multifocal disease (p = 0.007), extra-thyroid extension (p = 0.007) and presence of lymph node metastasis (p = 0.000) were associated with unfavorable outcome. CONCLUSIONS: Total thyroidectomy and RAI did not improve clinical outcomes of DTC ≤2.0 cm when compared with less extensive surgery and no RAI in selected patients. Therefore, in carefully selected patients with DTC ≤2.0 cm and no unfavorable risk factors (multifocal disease, extra-thyroid extension, lymph node and/or distant metastasis), less extensive surgery and no RAI may be acceptable treatment options.

Growth inhibitory effects of 3'-nitro-3-phenylamino nor-beta-lapachone against HL-60: a redox-dependent mechanism.

In this study, the cytotoxicity, genotoxicity and early ROS generation of 2,2-dimethyl-(3H)-3-(N-3'-nitrophenylamino)naphtho[1,2-b]furan-4,5-dione (QPhNO(2)) were investigated and compared with those of its precursor, nor-beta-lapachone (nor-beta), with the main goal of proposing a mechanism of antitumor action. The results were correlated with those obtained from electrochemical experiments held in protic (acetate buffer pH 4.5) and aprotic (DMF/TBABF(4)) media in the presence and absence of oxygen and with those from dsDNA biosensors and ssDNA in solution, which provided evidence of a positive interaction with DNA in the case of QPhNO(2). QPhNO(2) caused DNA fragmentation and mitochondrial depolarization and induced apoptosis/necrosis in HL-60 cells. Pre-treatment with N-acetyl-l-cysteine partially abolished the observed effects related to the QPhNO(2) treatment, including those involving apoptosis induction, indicating a partially redox-dependent mechanism. These findings point to the potential use of the combination of pharmacology and electrochemistry in medicinal chemistry.

Structure-mutagenicity relationship of kaurenoic acid from Xylopia sericeae (Annonaceae).

Kaurane diterpenes are considered important compounds in the development of new highly effective anticancer chemotherapeutic agents. Genotoxic effects of anticancer drugs in non-tumour cells are of special significance due to the possibility that they induce secondary tumours in cancer patients. In this context, we evaluated the genotoxic and mutagenic potential of the natural diterpenoid kaurenoic acid (KA), i.e. (-)-kaur-16-en-19-oic acid, isolated from Xylopia sericeae St. Hill, using several standard in vitro and in vivo protocols (comet, chromosomal aberration, micronucleus and Saccharomyces cerevisiae assays). Also, an analysis of structure-activity relationships was performed with two natural diterpenoid compounds, 14-hydroxy-kaurane (1) and xylopic acid (2), isolated from X. sericeae, and three semi-synthetic derivatives of KA (3-5). In addition, considering the importance of the exocyclic double bond (C16) moiety as an active pharmacophore of KA cytotoxicity, we also evaluated the hydrogenated derivative of KA, (-)-kauran-19-oic acid (KAH), to determine the role of the exocyclic bond (C16) in the genotoxic activity of KA. In summary, the present study shows that KA is genotoxic and mutagenic in human peripheral blood leukocytes (PBLs), yeast (S. cerevisiae) and mice (bone marrow, liver and kidney) probably due to the generation of DNA double-strand breaks (DSB) and/or inhibition of topoisomerase I. Unlike KA, compounds 1-5 and KAH are completely devoid of genotoxic and mutagenic effects under the experimental conditions used in this study, suggesting that the exocyclic double bond (C16) moiety may be the active pharmacophore of the genetic toxicity of KA.

Pisosterol induces interphase arrest in HL60 cells with c-MYC amplification.

The leukaemia cell line HL60 is widely used in studies of the cell cycle, apoptosis and adhesion mechanisms in cancer cells. One marked characteristic of HL60 cells is the c-MYC proto-oncogene amplification, resulting in the formation of homogeneously staining regions (HSRs) at 8p24. We conducted a fluorescence in situ hybridization study in an HL60 cell line, using a locus-specific probe for c-MYC, before and after treatment with pisosterol (at 0.5, 1.0 and 1.8 microg/mL), a triterpene isolated from the fungus Pisolithus tinctorius. Before treatment, 87.5% of the cells showed HSRs. After treatment, no effects were detected at lower concentrations of pisosterol (0.5 and 1.0 microg/mL). However, at 1.8 microg/mL only 15% of the cells presented HSRs, and 39.5% presented few fluorescent signals (3 or 4 alleles), suggesting that pisosterol probably blocks the cells with HSRs at interphase. This result is particularly interesting because cells that do not show a high degree of c-MYC gene amplification have a less aggressive and invasive behaviour and are easy targets for chemotherapy. Therefore, further studies are needed to examine the use of pisosterol in combination with conventional anti-cancer therapy.

The antitumoral, trypanocidal and antileishmanial activities of extract and alkaloids isolated from Duguetia furfuracea.

The alkaloid extract and five alkaloids isolated from subterranean stem bark of Duguetia furfuracea (Annonaceae) were investigated for the following activities: antitumoral, trypanocidal and leishmanicidal. Dicentrinone showed weak cytotoxicity, but it had the strongest leishmanicidal activity (IC(50) 0.01 microM). Duguetine and duguetine beta-N-oxide caused considerable antitumoral activity in every cell lines evaluated, although duguetine was more active against trypomastigote forms (IC(50) 9.32 microM) than other alkaloids tested.

Cell cycle arrest induced by pisosterol in HL60 cells with gene amplification.

The leukemia cell line HL60 is widely used in studies of the cell cycle, apoptosis, and adhesion mechanisms in cancer cells. We conducted a focused cytogenetic study in an HL60 cell line, by analyzing GTG-banded chromosomes before and after treatment with pisosterol (at 0.5, 1.0, and 1.8 microg/ml), a triterpene isolated from Pisolithus tinctorius, a fungus collected in the Northeast of Brazil. Before treatment, 99% of the cells showed the homogeneously staining region (HSR) 8q24 aberration. After treatment with 1.8 microg/ml pisosterol, 90% of the analyzed cells lacked this aberration. We further performed a pulse test, in which the cells treated with pisosterol (0.5, 1.0, and 1.8 microg/ml) were washed and re-incubated in the absence of pisosterol. Only 30% of the analyzed cells lacked the HSR 8q24 aberration, suggesting that pisosterol probably blocks the cells with HSRs at interphase. No effects were detected at lower concentrations. At the highest concentration examined (1.8 microg/ml), pisosterol also inhibited cell growth, but this effect was not observed in the pulse test, reinforcing our hypothesis that, at the concentrations tested, pisosterol probably does not induce cell death in the HL60 line. The results found for pisosterol were compared with those for doxorubicin. Cells that do not show a high degree of gene amplification (HSRs and double-minute chromosomes) have a less aggressive and invasive behavior and are easy targets for chemotherapy. Therefore, further studies are needed to examine the use of pisosterol in combination with conventional anti-cancer therapy.

Característica histológica, ultra-estrutural e produção de nitrito de folículos pré-antrais caprinos cultivados in vitro na ausência ou presença de soro / Histological and ultrastructural feature and nitrite production of caprine preantral follicles in vitro cultured in the presence or absence of serum

Avaliou-se o efeito da adição de diferentes tipos e concentrações de soro sobre o desenvolvimento e a sobrevivência de folículos ovarianos pré-antrais (FOPA) caprinos in vitro. Além disso, verificou-se a relação entre as concentrações de nitrito presentes no meio de cultivo e a viabilidade folicular. Cada par ovariano foi dividido em 29 fragmentos, sendo um destinado ao controle. Os fragmentos foram cultivados por um ou sete dias em meio essencial mínimo suplementado (MEM+) ou MEM+ com diferentes concentrações (10 ou 20 por cento) de soro fetal bovino (SFB), soro de cabra em estro (SCE) ou soro de cabra em diestro (SCD). Na análise morfológica após sete dias, apenas o tratamento com 10 por cento de SFB apresentou percentual de FOPA normais similar ao MEM+ (P>0,05). A análise ultra-estrutural dos folículos cultivados por sete dias com MEM+ ou MEM+ com 10 por cento de SFB mostrou danos oocitários, porém células da granulosa normais. A análise do meio de cultivo revelou correlação positiva entre a viabilidade folicular e a produção de nitrito. A suplementação com soro não melhorou a viabilidade de FOPA e a concentração de nitrito no meio de cultivo funcionou como um indicador da viabilidade das células da granulosa de FOPA caprinos cultivados in vitro.

The effect of the addition of different types and concentrations of sera on the viability and development of caprine preantal follicles (PAF) in vitro cultured was analyzed. In addition, it was evaluated the correlation between nitrite concentrations in culture medium and folicular viability. Each ovarian pair was divided in 29 fragments and one was used as control. The fragments were cultured for one or seven days in minimal essential medium (MEM+) or MEM+ with different concentrations of (10 or 20 percent) bovine fetal serum (BFS), estrous goat serum (EGS), or diestrous goat serum (DGS). After seven days, the morphological analysis showed that only the treatment with 10 percent BFS maintained the percentage of normal PAF similar to MEM+ (P>0.05). The ultrastructural analysis of follicles cultured for seven days in MEM+ or MEM+ with 10 percent BFS showed some oocyte damage, although the granulosa cells were normal. Analysis of culture medium revealed a positive correlation between follicular viability and nitrite production. Supplementation with serum did not improve the viability of PAF and nitrite levels in culture medium served as an indicator of viability of granulose cells from caprine PAF in vitro cultured.

Genotoxic effect of Physalis angulata L. (Solanaceae) extract on human lymphocytes treated in vitro

Physalis angulata L (Solanaceae) is a medicinal plant from North of Brazil, whose different extracts and infusions are commonly used in the popular medicine for the treatment of malaria, asthma, hepatitis, dermatitis and rheumatism. However, the genotoxic effects of P. angulata on human cells is not well known. The main purpose of the present study was to evaluate the in vitro genotoxic effects of aqueous extract of P. angulata using the comet assay and the micronucleus assay in human lymphocytes provided from 6 healthy donors. Treatments with P. angulata extracts were performed in vitro in order to access the extent of DNA damage. The comet assay has shown that treatments with P. angulata at 0.5, 1.0, 2.0, 3.0 and 6.0 microg/mL in culture medium were genotoxic. Lymphocytes treated with P. angulata at the concentrations of 3.0 and 6.0 microg/mL in culture medium showed a statistically significant increase in the frequency of micronucleus (p<0.05), however, the cytokinesis blocked proliferation index (CBPI) was not decreased after P. angulata treatment. In conclusion, the present work demonstrated the genotoxic effects of P. angulata extract on human lymphocytes in vitro.

Genotoxic and cytotoxic effects of manganese chloride in cultured human lymphocytes treated in different phases of cell cycle.

Manganese (Mn) has a natural occurrence and is necessary during the initial periods of the development. However, in high concentrations, Mn can be related to neurodegenerative disorders. The aim of the present study was to evaluate the mutagenic potential of manganese chloride (MnCl2.4H2O). Comet assay and chromosome aberrations analysis were applied to determine the DNA-damaging and clastogenic effects of MnCl2.4H2O. Cultured human lymphocytes were treated with 15, 20 and 25 microM manganese chloride during the G1, G1/S, S (pulses of 1 and 6h), and G2 phases of the cell cycle. All tested concentrations were cytotoxic and reduced significantly the mitotic index in G1, G1/S and S (1 and 6h) treatments, while in G2 treatment only the higher concentrations (20 and 25 microM) showed cytotoxic effects. Clastogenicity and DNA damage were found only in treatments with the highest concentration (25 microM). Chromosome aberrations were found exclusively in the G2 phase of the cell cycle. The absence of polyploidy in mitosis, suggests that manganese does not affect the formation of the mitotic spindle with the concentrations tested. The genotoxicity found in G2 phase and in the comet assay can be related to the short time of treatment in both cases.

Genotoxic and cytotoxic effects of iron sulfate in cultured human lymphocytes treated in different phases of cell cycle.

Iron (Fe) is a common chemical element that is essential for organisms as a co-factor in oxygen transport, but that in high amounts presents a significant risk of neurodegenerative disorders. The objective of this study was to evaluate the mutagenic potential of iron sulfate. The comet assay and chromosome aberration (CA) analysis were applied to determine the DNA-damaging and clastogenic effects of iron sulfate. Human lymphocytes were treated in the quiescent phase for the comet assay and proliferative phase during the G1, G1/S, S (pulses of 1 and 6 h), and G2 phases of the cell cycle for CA analysis, with 1.25, 2.5 and 5 microg/mL concentrations of FeSO(4).7H2O. All tested concentrations were cytotoxic and reduced significantly the mitotic index (MI) in all phases of the cell cycle. They also induced CA in G1, G1/S and S (pulses of 1 and 6 h) phases. Iron sulfate also induced polyploidy in cells treated during G1. In the comet assay, this metal did not induce significant DNA damage. Our results show that Fe causes alteration and inhibition of DNA synthesis only in proliferative cells, which explain the concomitant occurrence of mutagenicity and cytotoxicity, respectively, in the lymphocytes studied.

Genotoxic effects of tanshinones from Hyptis martiusii in V79 cell line.

The genotoxic effect of two tanshinones isolated from roots of Hyptis martiussi Benth (Labiatae) was studied using V79 (Chinese hamster lung) cells by the alkaline comet assay and micronucleus test. Tanshinones were incubated with the cells at concentrations of 1, 3, 6 and 12 microg/mL for 3 h. Tanshinones were shown to be quite strongly genotoxic against V79 cells at all tested concentrations. The data obtained provide support to the view that tanshinones has DNA damaging activity in cultured V79 cells under the conditions of the assays.