A multiple endpoint approach reveals potential in vitro anticancer properties of thymoquinone in human renal carcinoma cells.

Thymoquinone (TQ) is a monoterpene isolated from the oil of Nigella sativa seeds. The aim of this work was to evaluate the cytotoxic effects induced by TQ and its impact on the migration and invasion potential of 786-O human renal cancer cells. These cells were exposed to TQ (1-100 µM) for 24 and 48 h and cell viability assessed using the Crystal Violet and MTS assays. TQ treatment clearly decreased cell viability in a concentration- and time-dependent manner. TQ exposure moderately increased intracellular ROS levels and co-incubation with reduced glutathione markedly increased cell viability. Moreover, the effect of TQ in the cell cycle distribution was evaluated using flow cytometry, and an increase in the sub-G1 population was observed, especially at 30 µM, along with an increase in the % of apoptotic cells. TQ did not show genotoxic effects at a non-cytotoxic concentration (1.0 µM). At this concentration level, TQ significantly decreased the collective migration of 786-O cells, whereas it had no effect in chemotactic migration. TQ also decreased the invasiveness potential of 786-O cells, as evaluated by the transwell invasion assay. Overall, these results suggest that TQ presents an anticancer potential in the context of renal cancer, warranting further investigation.

The role of epigenetic modifiers in extended cultures of functional hepatocyte-like cells derived from human neonatal mesenchymal stem cells.

The development of predictive in vitro stem cell-derived hepatic models for toxicological drug screening is an increasingly important topic. Herein, umbilical cord tissue-derived mesenchymal stem cells (hnMSCs) underwent hepatic differentiation using an optimized three-step core protocol of 24 days that mimicked liver embryogenesis with further exposure to epigenetic markers, namely the histone deacetylase inhibitor trichostatin A (TSA), the cytidine analogue 5-azacytidine (5-AZA) and dimethyl sulfoxide (DMSO). FGF-2 and FGF-4 were also tested to improve endoderm commitment and foregut induction during Step 1 of the differentiation protocol, being HHEX expression increased with FGF-2 (4 ng/mL). DMSO (1%, v/v) when added at day 10 enhanced cell morphology, glycogen storage ability, enzymatic activity and induction capacity. Moreover, the stability of the hepatic phenotype under the optimized differentiation conditions was examined up to day 34. Our findings showed that hepatocyte-like cells (HLCs) acquired the ability to metabolize glucose, produce albumin and detoxify ammonia. Global transcriptional analysis of the HLCs showed a partial hepatic differentiation degree. Global analysis of gene expression in the different cells revealed shared expression of gene groups between HLCs and human primary hepatocytes (hpHeps) that were not observed between HepG2 and hpHeps. In addition, bioinformatics analysis of gene expression data placed HLCs between the HepG2 cell line and hpHeps and distant from hnMSCs. The enhanced hepatic differentiation observed was supported by the presence of the hepatic drug transporters OATP-C and MRP-2 and gene expression of the hepatic markers CK18, TAT, AFP, ALB, HNF4A and CEBPA; and by their ability to display stable UGT-, EROD-, ECOD-, CYP1A1-, CYP2C9- and CYP3A4-dependent activities at levels either comparable with or even higher than those observed in primary hepatocytes and HepG2 cells. Overall, an improvement of the hepatocyte-like phenotype was achieved for an extended culture time suggesting a role of the epigenetic modifiers in hepatic differentiation and maturation and presenting hnMSC-HLCs as an advantageous alternative for drug discovery and in vitro toxicology testing.

Faunistic inventory of spheciformes wasps at three protected areas in Portugal.

The importance of considering insects in the protection of biodiversity has been recently recognized. However, despite the importance of Spheciformes wasps (Hymenoptera: Ampulicidae, Sphecidae and Crabronidae) in natural ecosystems and their potential as bioindicators, the Spheciformes communities in Portugal (part of the European biodiversity hotspot) have rarely been studied, and data for Portuguese protected areas are scarce. The Spheciformes wasp communities at 3 protected areas in Portugal, Douro International Natural Park, Serras de Aire e Candeeiros Natural Park, and Paúl do Boquilobo Nature Reserve, were studied in 2000 and 2001. During the study, 134 species of Spheciformes belonging to 3 families, Ampulicidae, Sphecidae, and Crabronidae, were identified. The species collected constituted nearly 1/3 of the species known in the Iberian Peninsula, 42 were new records for Portugal. Additionally, several specimens of 6 potentially new species were collected. Douro International Natural Park had the highest species richness, followed by Serras de Aire e Candeeiros Natural Park and Paúl do Boquilobo Nature Reserve. All the protected areas studied had species that were found exclusively at an individual protected area and species that were found to be new records for Portugal. Based on the literature review of the geographic distribution, nidification types, and prey orders, it was found that most species collected had a Euroasiatic or Mediterranean distribution, species with fossorial habits predominated, and the orders/suborders of insects preyed upon by most species were Diptera, Orthoptera, Sternorrhyncha, and Auchenorrhyncha. This study underscores the importance of including the protected areas studied in the conservation of Spheciformes diversity and also suggests that insect diversity should be studied separately, as it does not necessarily follow the same patterns as other, more studied, groups.

Genotoxic effects of doxorubicin in cultured human lymphocytes with different glutathione S-transferase genotypes.

Doxorubicin (Dox) is a widely used drug in oncology with a broad spectrum of interactions with various cellular components; therefore, it is likely to act through different mechanisms. In clinical practice there is inter-individual variability in cytotoxic drug response and in the occurrence of adverse reactions. Glutathione S-transferases (GSTM1, GSTT1 and GSTP1) are thought to be involved in the detoxification of endogenous and exogenous genotoxicants. The aim of this work is the assessment of a possible influence of polymorphisms in GSTs on the levels of genetic damage induced in vitro by Dox in cultured human lymphocytes. For this purpose, whole blood cultures from individuals with different genotypes for GSTM1, GSTT1 and GSTP1 were exposed to Dox and the cytokinesis-blocked micronucleus (CBMN) assay was used as the endpoint for chromosomal damage in the lymphocytes. Genotyping of GSTM1 and GSTT1 was carried out by multiplex PCR and the GSTP1-Ile105Val polymorphism was determined by PCR/RFLP. The total number of micronuclei present in 1000 binucleated cells and the frequency of micronucleated binucleated lymphocytes in the different individuals were analyzed considering the GSTM1, GSTT1 and GSTP1 genotypes. The results obtained suggest that GSTM1 and GSTT1 deletion polymorphisms do not modify significantly the genotoxic potential of Dox. However, the GSTP1 Ile105Val polymorphism was associated with an increase of micronucleated binucleated cells induced by Dox. Lymphocytes from homozygous individuals for the variant form (Val/Val) presented a significant increase in micronucleated binucleated cells (approximately 1.5-fold; p<0.05) when compared with individuals with at least one wild-type allele. These results suggest a possible role for GSTP1 on the modulation of the genotoxicity induced by Dox, which may be considered in cancer therapy.

Use of cytogenetic indicators in radiobiology.

The study of ionising radiation has systematically relied on cytogenetic indicators to evaluate the biological effects and has led to theoretical approaches to explain observations associated with radiation exposure. In many of the early studies on radiobiology, the induction of chromosomal aberrations was the method of choice to evaluate dose-response relationships. But progressively, this and other cytogenetic biomarkers were used to obtain mechanistic insight on the biological effects induced by radiation. This paper attempts to give a view on the use of cytogenetic indicators in the study of various radiation-related phenomena, including radiation dosimetry, mechanisms involved in the various cellular responses to radiation, such as bystander effects, chromosomal instability and adaptive response, as well as DNA repair pathways. One future direction may involve the use of cytogenetic indicators to evaluate various molecular determinants in individuals' susceptibility to radiation, using other techniques such as fluorescence in situ hybridisation (FISH) and linking them to specific gene functions and single nucleotide polymorphisms.

Wortmannin enhances the induction of micronuclei by low and high LET radiation.

In mammalian cells, the repair of DNA double-strand breaks (DSBs) is mainly mediated by DNA non-homologous end joining. DNA-dependent protein kinase (DNA-PK), a nuclear serine-threonine kinase and a member of the phosphaditylinositol-3 kinase-related kinase family that is activated by DSBs, is a key component of this pathway. Wortmannin (WM) is known to be an irreversible and potent inhibitor of DNA-PK and has thus been proposed as an effective sensitizer for ionizing radiation and for radiomimetic compounds. The present study, using the cytokinesis block micronucleus assay, reports on the differential effect of WM on the repair of the DNA damage induced by low LET ((60)Co gamma-radiation) and high LET radiation by the boron neutron capture reaction (alpha and Li particles) in V79 Chinese hamster cells. Significant increases in the number of micronuclei per binucleated cell as well as in the frequency of micronucleated binucleated cells were observed in the presence of different concentrations of WM for high LET radiation from the boron neutron capture reaction. The increases observed reached a maximum of approximately 2-fold in comparison with the respective controls. WM, however, had a more pronounced effect on (60)Co gamma-radiation-induced micronuclei, increasing the genotoxic damage from this radiation by approximately 3- to 4-fold. These results are in general in agreement with the concept that DSBs induced by high LET radiation are not a more suitable substrate for the end joining processes mediated by DNA-PK, yet they do not preclude a role for DNA-PK in high LET-induced damage repair.

Micronuclei and sister chromatid exchanges induced by capsaicin in human lymphocytes.

Capsaicin is the main pungent and irritating component of hot peppers (species Capsicum annuum and C. frutescens). Genotoxicity and carcinogenicity studies evaluating capsaicin effects are sparse and contradictory. In this study, we investigated the genotoxicity of capsaicin (10-200 microM) in human peripheral blood lymphocytes using the cytokinesis-block micronucleus (CBMN) assay and the sister chromatid exchange (SCE) assay in the presence or absence of external metabolic activation. Capsaicin induced the formation of micronuclei (MN) in a dose-dependent manner in the cytokinesis-blocked lymphocytes. This increase was more evident in the absence of metabolic activation, with a maximum of 3.4-fold increase above the background. Some inter-individual variability was observed. The results for the SCE assay also show that capsaicin is genotoxic and in this case with a more homogeneous response among donors. This end-point, however, has proven to be less sensitive than the CBMN assay for capsaicin.

Evaluation of the genotoxic effects of the boron neutron capture reaction in human melanoma cells using the cytokinesis block micronucleus assay.

The present work reports on the genotoxicity of the boron neutron capture (BNC) reaction in human metastatic melanoma cells (A2058) assessed by the cytokinesis block micronucleus assay (CBMN) using p-borono-L-phenylalanine (BPA) as the boron delivery agent. Different concentrations of BPA (0.48, 1.2 and 2.4 mM) and different fluences of thermal neutrons were studied. Substantial genotoxic potential of alpha and lithium particles generated inside or near the malignant cell by the BNC reaction was observed in a dose-response manner as measured by the frequency of micronucleated binucleated melanoma cells and by the number of micronuclei (MN) per binucleated cell. The distribution of the number of MN per micronucleated binucleated cell was also studied. The BNC reaction clearly modifies this distribution, increasing the frequency of micronucleated cells with 2 and, especially, > or =3 MN and conversely decreasing the frequency of micronucleated cells with 1 MN. A decrease in cell proliferation was also observed which correlated with MN formation. A discrete genotoxic and anti-proliferative contribution from both thermal neutron irradiation and BPA was observed and should be considered secondary. Additionally, V79 Chinese hamster cells (chromosomal aberrations assay) and human lymphocytes (CBMN assay) incubated with different concentrations of BPA alone did not show any evidence of genotoxicity. The presented results reinforce the usefulness of the CBMN assay as an alternative method for assessment of the deleterious effects induced by high LET radiation produced by the BNC reaction in human melanoma cells.

No evidence of increased chromosomal aberrations and micronuclei in lymphocytes from nonfamilial thyroid cancer patients prior to radiotherapy.

The relationship between the presence of high frequencies of chromosomal aberrations in peripheral lymphocytes and predisposition to cancer has been suggested for some cancer diseases. In nonfamilial thyroid cancer, the few reports available are equivocal. The aim of this study was to assess the possible chromosomal instability in peripheral blood lymphocytes from 22 patients suffering from nonfamilial thyroid cancer. For this purpose, 2 classic cytogenetic assays, the chromosomal aberrations assay and cytokinesis-blocked micronucleus assay, were chosen. The frequency of chromosomal aberrations excluding gaps (%) was 1.68 +/- 1.39 (mean value +/- SD) for the patients group versus 2.20 +/- 1.87 for the control group. The frequency of binucleated lymphocytes with micronuclei ( per thousand) was 5.41 +/- 3.51 (mean value +/- SD) for the patients group versus 5.37 +/- 3.21 for the control group. The results obtained revealed no significant differences between both groups. The present study reinforces the idea that constitutional chromosomal instability in peripheral blood lymphocytes is not visible in nonfamilial thyroid carcinomas.

Induction of micronuclei and chromosomal aberrations by the mycotoxin patulin in mammalian cells: role of ascorbic acid as a modulator of patulin clastogenicity.

Patulin is a mycotoxin produced by several species of Penicillium, Aspergillus and BYSSOCHLAMYS: Patulin is a common contaminant of ripe apples used for the production of apple juice concentrates and is also present in other fruits, vegetables and food products. Patulin has been reported to have mutagenic, carcinogenic and teratogenic properties. Nevertheless, these properties are still a matter of debate. In this report, we further investigated the genotoxicity of patulin in mammalian cells by two different approaches. Firstly, we evaluated the induction of micronuclei in cytokinesis-blocked human lymphocytes. This approach is important because available data concerning the genetic toxicity of patulin in human cells is sparse. Secondly, we chose an established model for patulin genotoxicity, i.e. the chromosomal aberration assay in V79 Chinese hamster cells, to clarify whether concomitant exposure to ascorbic acid with the mycotoxin modulates or not the clastogenicity of patulin. The results unequivocally show induction of DNA-damaged cells by patulin as assessed by both cytogenetic assays. In addition, an almost complete abolition of patulin (0.8 microM) clastogenicity was observed in the presence of 80 microM ascorbic acid (P < 0.05), showing that although a genetic risk is present, ascorbic acid could somehow partially modulate this problem.

Cytogenetic alterations and oxidative stress in thyroid cancer patients after iodine-131 therapy.

This study aimed to assess two end-points of DNA damage, namely chromosomal aberrations and micronuclei in peripheral lymphocytes, and their possible relationship with oxidative stress (which may be related to DNA damage and repair) in thyroid cancer patients receiving therapeutic doses of (131)I. Nineteen patients receiving 2590 MBq (70 mCi) were studied. Chromosomal aberrations were scored using standard cytogenetic methods and micronuclei scored in cytokinesis-blocked lymphocytes. Oxidative stress was assessed by determining thiobarbituric acid-reactive substances in blood, total plasma antioxidant status and serum uric acid levels. All parameters were assessed before treatment and 1 and 6 months after (131)I administration. The frequency of micronucleated cells per 1000 binucleated cells scored (mean +/- SEM) increased significantly from 5.21 +/- 0.80 to 9.68 +/- 1.22 1 month after treatment (P < 0.01) and to 8.42 +/- 1.28 6 months after treatment (P < 0.05). The frequency of cells with chromosomal aberrations, excluding gaps, per 100 cells, increased significantly from 1.68 +/- 0.41 to 3.47 +/- 0. 55 1 month after treatment (P < 0.01) and to 4.05 +/- 0.46 6 months after treatment (P < 0.01). Oxidative stress parameters showed slight modifications over the time period studied, but the differences were not significant except for a decrease in thiobarbituric acid-reactive products 6 months after therapy (P < 0. 05) and in serum uric acid concentration 1 and 6 months after therapy (P < 0.01). This report demonstrates slight but significant and persistent DNA damage in (131)I-treated patients as assessed by cytogenetic assays. There was no clear correlation between the cytogenetic findings and oxidative stress parameters studied.

Assessment of the adaptive response induced by quercetin using the MNCB peripheral blood human lymphocytes assay.

Over more than two decades the existence of an adaptive response (AR) has been reported in several cell types and extensively studied with low doses of radiation. Besides radiation, some chemicals [alkylating compounds, mitomycin C (MMC), bleomycin, hydrogen peroxide and metals] may also induce an adaptive response. We have recently reported that the food mutagen quercetin can also induce an adaptive response in V79 Chinese hamster cells. In this work we have studied the effect of low doses of quercetin on the genotoxicity of MMC and bleomycin assessed by the formation of micronuclei in cytokinesis-blocked (MNCB) human peripheral blood lymphocytes. Our results suggest the existence of an AR induced by quercetin in human lymphocytes. Seven of the nine donors studied showed in at least one independent experiment a significant decrease in the frequency of MNCB induced by MMC. The range of these decreases varied between 31 and 58%. In addition, we observed an AR induced by quercetin towards challenging doses of bleomycin. In accordance with other studies with ionizing radiation in which heterogeneity of the AR in the population has been extensively observed, the response here reported also showed some degree of variability between the different donors studied. In view of the results obtained one cannot rule out a possible protective effect of low doses of quercetin leading to adaptation to further exposure to mutagens or carcinogens.

Induction of an adaptive response to quercetin, mitomycin C and hydrogen peroxide by low doses of quercetin in V79 Chinese hamster cells.

The adaptive response is a phenomenon by which cells exposed to low, non-cytotoxic doses of a genotoxicant become significantly resistant to a subsequent higher dose of the same or another genotoxic agent. Induction of the adaptive response has been mainly studied using ionizing radiation and alkylating agents as genotoxic agents. However, other mutagenic agents may warrant further study, since the adaptive response as a whole may be an important general biological mechanism to maintain genetic integrity and thus could prevent carcinogenic initiation of cells. The exposure to mutagenic agents present, or formed, in the diet is considered an important factor in the etiology of human tumors and a considerable number of these agents have not yet been identified or characterized. Flavonoids are a large group of polyphenolic quinoids found in a wide variety of edible fruits and vegetables and a few, such as quercetin, present genotoxic activity in vitro. The mechanisms of mutagenicity of quercetin involve the production of oxygen radicals through an autoxidation process dependent on pH value and the presence of oxygen. Although there are few doubts regarding the mutagenicity of quercetin in vitro, carcinogenicity of flavonoid is still controversial. In view of these conflicting results and the radiomimetic nature of the mutagenicity of flavonoids, we addressed the question of cell exposure to quercetin at the low levels present in the diet leading to adaptation to further exposure to mutagens or carcinogens. The work reported here concerns induction of an adaptive response by low doses of quercetin to challenging doses of quercetin and other compounds, namely hydrogen peroxide and mitomycin C, using induction of chromosomal aberrations in V79 cells as the end point.

Avaliaçäo dos projetos de educaçäo sexual; relatório / Evaluation of sex education project; report

Apresenta os dados de avaliaçäo dos Projetos de Educaçäo Sexual apoiados pela Fundaçäo Odebrecht e desenvolvidos nas cidades de Salvador e Rio de Janeiro

Incidence of temporomandibular joint disorders in patients seen at a hospital emergency room.

A retrospective survey of a hospital emergency room population seen at an oral and maxillofacial surgery clinic during a 6-month period found 62 patients (2.7% of the total population) with temporomandibular joint disorders. The diagnoses were myofascial pain-dysfunction/temporomandibular joint dysfunction (MPD/TMJ) syndrome (70.9% of the cases) and dislocation (luxation) (22.5% of the cases). The chief complaint was well defined in relation to the diagnoses: facial pain in the MPD/TMJ syndrome cases, and displacement of the mandible in the dislocation cases.