Evaluation of the genotoxicity of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes

In the present study, experiments were carried out to evaluate the mutagenic potential and genotoxic effects of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes, using the micronucleus and comet assays. Significant damage to DNA was observed for crotoxin and crotapotin (CA). Basic phospholipase A(2) (CB) and crotamine did not present any mutagenic potential when evaluated by the micronucleus test. C. d. terrificus crude venom was able to induce the formation of micronuclei, similarly to the mutagenic drug used as a positive control. In the comet assay, all the toxins tested (crotamine, crotoxin, CB and CA) and C. d. terrificus venom presented genotoxic activity. Studies on the cytogenetic toxicology of animal venoms and their isolated proteins are still very scarce in the literature, which emphasizes the importance of the present work for the identification and characterization of potential therapeutic agents, as well as for the better understanding of the mechanisms of action of toxins on the human body. (C) 2011 Elsevier B.V. All rights reserved.

Metal embryotoxicity from urban particles in Sao Paulo city: An experimental study in chicken embryos

Chicken eggs were inoculated with suspensions of ambient air particles (<= 10 mu m, PM(10)) from Sao Paulo city in 3, 0.3 or 0.03 mu g doses on one of the four early days of embryo development. On the eleventh day of development alterations were observed on embryos inoculated with PM(10) 3 mu g on the third day. Particles analysis showed high content of metals. Hence, embryos were also inoculated with PM(10) (3 mu g) combined with metal chelating EDTA. PM(10) (3 mu g) embryos presented underdevelopment (stage 29.44 +/- 11.4) compared to vehicle and positive controls (stage 36.44 +/- 0.51 Saline and stage 31.20 +/- 9.7 Cyclophosphamide, p <= 0.05); higher (47%) mortality rate (23% Saline and 42% Cyclophosphamide) and low (68%) viability (100% Saline and 70% Cyclophosphamide, p = 0.04). Effects were attenuated when embryos received PM(10) + EDTA (stage 33.63 +/- 0.94, 18.9% mortality rate and 82% viability). PM(10) from Sao Paulo city is embryotoxic and metal may be implicated in the toxic mechanism. (C) 2010 Elsevier Inc. All rights reserved.

Determinants of beta-lactam resistance in meningitis-causing Enterobacteriaceae in Brazil

This study analyzed resistance determinants in extended-spectrum beta-lactamase (ESBL)-producing enterobacteria and the epidemiology of 11 Escherichia coli isolates obtained from meningitis patients in a region of Brazil from 2000 to 2005. ESBL-encoding genes and their genetic environment were investigated by PCR and sequencing. The gene bla(CTX-M-2) was identified in 3 different enterobacteria (E. coli. Serratia marcescens, and Proteus mirabilis) downstream of the insertion sequence ISCR1 (localized in class 1 integrons), hut not as part of the resistance cassettes region. Multi locus sequence typing (MLST) was used to investigate genetic relationships between the 11 E. coil isolates in this study and strains associated with meningitis in the E. coil MLST database. MLST analysis indicated high genetic diversity among isolates, and no significant genetic relationship was identified with meningitis-causing E. coil in the database. The results in this report reinforce the need to be attentive to meningitis suspected to be due to ESBL-producing enterobacterial isolates, especially where ESBL epidemiology is well known.

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirao Preto, Sao Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTET (TM) QPCR SYBR (R) Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fist and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method. (C) 2009 Elsevier Ltd. All rights reserved.

Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N-acetyl-beta-D-glucosaminidase activity

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa. which is purified by affinity, with immobilized N-acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels or TNF alpha. and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the beta-1,4-homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin-affinity purification or paracoccin. This procedure provided higher yields than those achieved by means of the technique based oil the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS-PAGE and Western blot analysis revealed similarities between the N-acetylglucosamine- and chitin-bound fractions, confirmed by MALDI-TOF-MS of trypsinic peptides. Western blot of two-dimensional gel electrophoresis of the yeast extract showed a major spot with M(r) 70000 and pl approximately 5.63. Moreover, an N-acetyl-beta-D-glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization. Copyright (C) 2009 John Wiley & Sons. Ltd.

Antitumor effects of snake venom chemically modified Lys49 phospholipase A(2)-like BthTX-I and a synthetic peptide derived from its C-terminal region

The present work evaluates both in vitro and in vivo antitumor activity of BPB-modified BthTX-I and its cationic synthetic peptide derived from the 115-129 C-terminal region. BPB-BthTX-1 presented cytotoxicity of 10-40% on different tumor cell lines, which were also susceptible to the lytic action of the synthetic peptide. Injection of the modified protein or the peptide in mice, 5 days after transplantation of S 180 tumor cells, reduced 30 and 36% of the tumor size on day 14th and 76 and 79% on day 60th, respectively, when compared to the untreated control group. Thus, these antitumor properties might be of interest in the development of therapeutic strategies against cancer. (C) 2009 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

Clonal relationships determined by multilocus sequence typing among enteropathogenic Escherichia coli isolated in Brazil

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infantile diarrhea in developing nations. Multilocus sequence typing (MLST) characterizes bacterial strains based on the sequences of internal fragments in housekeeping genes. Little is known about strains of EPEC analyzed by MLST from Brazil. In this study, a diverse collection of 29 EPEC strains isolated from patients with diarrhea, admitted to the University Hospital of Ribeirao Preto, was characterized by MLST. Strain analysis demonstrated 22 different sequence types (STs), of which almost half (48%) were new, indicating a high genotype diversity. The 22 STs were divided by eBURST into 12 clonal complexes. It was not possible to correlate typical and atypical EPEC with other strains in the MLST database. This is the first study that analyzed EPEC strains from South America that are included in the E. coli MLST database. Nine (31%) out of 29 strains are part of the CC10 clonal complex, the major clonal complex in the database, which comprises 174 strains and 86 different STs, suggesting that these strains might be the most important intestinal pathogenic E. coli worldwide. Genetic relationships between typical and atypical EPEC, enterohemorrhagic E. coli, and enteroaggregative E. coli strains were not established by MLST.

The azo dyes Disperse Red 1 and Disperse Orange 1 increase the micronuclei frequencies in human lymphocytes and in HepG2 cells

The use of azo dyes by different industries can cause direct and/or indirect effects oil human and environmental health due to the discharge of industrial effluents that contain these toxic compounds. Several studies have demonstrated the genotoxic effects of various azo dyes, but information on the DNA damage caused by Disperse Red 1 and Disperse Orange 1 is unavailable, although these dyes are used in dyeing processes in many countries. The aim of the present study was to evaluate the mutagenic activity of Disperse Red 1 and Disperse Orange 1 using the micronucleus (MN) assay in human lymphocytes and in HepG2 cells. In the lymphocyte assay. it was found that the number of MN induced by the lowest concentration of each dye (0.2 mu g/mL) was similar to that of the negative control. At the other concentrations, a dose response MN formation was observed up to 1.0 mu g/mL. At higher dose levels, the number of MN decreased. For the HepG2 cells the results were similar. With both dyes a dose dependent increase in the frequency of MN was detected. However for the HepG2, the threshold for this increase was 2.0 mu g/mL, while at higher doses a reduction in the MN number was observed. The proliferation index was also calculated in order to evaluate acute toxicity during the test. No differences were detected between the different concentrations tested and the negative control. (C) 2009 Elsevier B.V. All rights reserved.

Intrinsically bent DNA sites in the Drosophila melanogaster third chromosome amplified domain

Bent DNA sites promote the curvature of DNA in both eukaryotic and prokaryotic chromosomes. Here, we investigate the localization and structure of intrinsically bent DNA sites in the extensively characterized Drosophila melanogaster third chromosome DAFC-66D segment (Drosophila amplicon in the follicle cells). This region contains the amplification control element ACE3, which is a replication enhancer that acts in cis to activate the major replication origin ori-beta. Through both electrophoretic and in silico analysis, we have identified three major bent DNA sites in DAFC-66D. The bent DNA site (b1) is localized in the ACE3 element, whereas the other two bent DNA sites (b2 and b3) are localized in the ori-beta region. Four additional bent DNA sites were identified in the intron of the S18 gene and near the TATA box of the S15, S19, and S16 genes. The identification of DNA bent sites in genomic regions previously characterized as functionally relevant for DNA amplification further supports a function for DNA bent sites in DNA replication in eukaryotes.

Biotransformation using Mucor rouxii for the production of oleanolic acid derivatives and their antimicrobial activity against oral pathogens

The goal of this study is to produce oleanolic acid derivatives by biotransformation process using Mucor rouxii and evaluate their antimicrobial activity against oral pathogens. The microbial transformation was carried out in shake flasks at 30A degrees C for 216 h with shaking at 120 rpm. Three new derivatives, 7 beta-hydroxy-3-oxo-olean-12-en-28-oic acid, 7 beta,21 beta-dihydroxy-3-oxo-olean-12-en-28-oic acid, and 3 beta,7 beta,21 beta-trihydroxyolean-12-en-28-oic acid, and one know compound, 21 beta-hydroxy-3-oxo-olean-12-en-28-oic acid, were isolated, and the structures were elucidated on the basis of spectroscopic analyses. The antimicrobial activity of the substrate and its transformed products was evaluated against five oral pathogens. Among these compounds, the derivative 21 beta-hydroxy-3-oxo-olean-12-en-28-oic acid displayed the strongest activity against Porphyromonas gingivalis, which is a primary etiological agent of periodontal disease. In an attempt to improve the antimicrobial activity of the derivative 21 beta-hydroxy-3-oxo-olean-12-en-28-oic acid, its sodium salt was prepared, and the minimum inhibitory concentration against P. gingivalis was reduced by one-half. The biotransformation process using M. rouxii has potential to be applied to the production of oleanolic acid derivatives. Research and antimicrobial activity evaluation of new oleanolic acid derivatives may provide an important contribution to the discovery of new adjunct agents for treatment of dental diseases such as dental caries, gingivitis, and periodontitis.

Chlorination treatment of aqueous samples reduces, but does not eliminate, the mutagenic effect of the azo dyes Disperse Red 1, Disperse Red 13 and Disperse Orange 1

The treatment of textile effluents by the conventional method based on activated sludge followed by a chlorination step is not usually an effective method to remove azo dyes, and can generate products more mutagenic than the untreated dyes. The present work evaluated the efficiency of conventional chlorination to remove the genotoxicity/mutagenicity of the azo dyes Disperse Red 1, Disperse Orange 1, and Disperse Red 13 from aqueous solutions. The comet and micronucleus assays with HepG2 cells and the Salmonella mutagenicity assay were used. The degradation of the dye molecules after the same treatment was also evaluated, using ultraviolet and visible absorption spectrum measurements (UV-vis), high performance liquid chromatography coupled to a diode-array detector (HPLC-DAD), and total organic carbon removal (TOC) analysis. The comet assay showed that the three dyes studied induced damage in the DNA of the HepG2 cells in a dose-dependent manner. After chlorination, these dyes remained genotoxic, although with a lower damage index (DI). The micronucleus test showed that the mutagenic activity of the dyes investigated was completely removed by chlorination, under the conditions tested. The Salmonella assay showed that chlorination reduced the mutagenicity of all three dyes in strain YG1041, but increased the mutagenicity of Disperse Red 1 and Disperse Orange 1 in strain TA98. With respect to chemical analysis, all the solutions showed rapid discoloration and a reduction in the absorbance bands characteristic of the chromophore group of each dye. However, the TOC was not completely removed, showing that chlorination of these dyes is not efficient in mineralizing them. It was concluded that conventional chlorination should be used with caution for the treatment of aqueous samples contaminated with azo dyes. (C) 2010 Elsevier B.V. All rights reserved.

Evaluation of the genotoxic and antigenotoxic effects after acute and subacute treatments with acai pulp (Euterpe oleracea Mart.) on mice using the erythrocytes micronucleus test and the comet assay

Acai, the fruit of a palm native to the Amazonian basin, is widely distributed in northern South America, where it has considerable economic importance. Whereas individual polyphenolics compounds in Acai have been extensively evaluated, studies of the intact fruit and its biological properties are lacking. Therefore, the present study was undertaken to investigate the in vivo genotoxicity of Acai and its possible antigenotoxicity on doxorubicin (DXR)-induced DNA damage. The Acai pulp doses selected were 3.33, 10.0 and 16.67 g/kg b.w. administered by gavage alone or prior to DXR (16 mg/kg b.w.) administered by intraperitoneal injection. Swiss albino mice were distributed in eight groups for acute treatment with acai pulp (24 h) and eight groups for subacute treatment (daily for 14 consecutive days) before euthanasia. The negative control groups were treated in a similar way. The results of chemical analysis suggested the presence of carotenoids, anthocyanins, phenolic. and flavonoids in Acai pulp. The endpoints analyzed were micronucleus induction in bone marrow and peripheral blood cells polychromatic erythrocytes, and DNA damage in peripheral blood, liver and kidney cells assessed using the alkaline (pH > 13) comet assay. There were no statistically significant differences (p > 0.05) between the negative control and the groups treated with the three doses of Acai pulp alone in all endpoints analyzed, demonstrating the absence of genotoxic effects. The protective effects of Acai pulp were observed in both acute and subacute treatments, when administered prior to DXR. In general, subacute treatment provided greater efficiency in protecting against DXR-induced DNA damage in liver and kidney cells. These protective effects can be explained as the result of the phytochemicals present in Acai pulp. These results will be applied to the developmental of food with functional characteristics, as well as to explore the characteristics of Acai as a health promoter. (C) 2009 Elsevier B.V. All rights reserved.

Genotoxic studies in hypertensive and normotensive rats treated with amiodarone

Amiodarone, a benzofuran derivative. is a very effective antiarrhythmic medication, but has potential to cause side effects. Although its cytotoxicity potential is very well-known, there are few reports about its genotoxicity effects. Since amiodarone has not been investigated in genotoxicity studies, and the spontaneously hypertensive rat (SHR) is a well-characterized model for hypertension, the aim of the present study was to perform cytogenetic analysis on chromosome aberrations in bone marrow cells of SHRs and normotensive Wistar-Kyoto rats (WKYs) that received oral amiodarone treatment for 4 weeks. Amiodarone activity was also monitored using electrocardiograms. The presence of bradycardia in amiodarone-treated rats confirmed that this drug was really active. Metaphase analysis on bone marrow cells showed that there were significant differences in total chromosomal damage and percentage abnormal metaphase between WKY and SHR negative controls. In the SHR negative control, the frequencies of basal chromosomal aberrations and abnormal metaphases were significantly higher (p < 0.05). There were high numbers of chromosomal aberrations in all amiodarone-treated groups, compared with negative controls. In amiodarone-treated groups, the most frequent chromosomal aberration was chromatid breaks. More chromosomal aberrations were found in WKYs that received amiodarone, with a statistically significant difference in comparison with negative controls (p < 0.05). However, in SHR rats there was no significant difference between the amiodarone and negative groups regarding chromosomal damage induction. These results showed that treatment with amiodarone was genotoxic in WKYs, but not in SHRs. Further studies are needed to confirm whether amiodarone is genotoxic or efficient and harmless, among humans undergoing therapy. (c) 2008 Published by Elsevier B.V.

The food business environment and the role of China and Brazil building a ""food bridge""

Purpose - The purpose of this paper is to discuss the economic crisis of 2008/2009 and the major impacts on developing nations and food-producing countries Within this macro-environment of food chains, there is concern that food inflation might come back sooner than expected The role of China as one of the major food consumers in the future, and Brazil, as the major food producer, is described as the food bridge, and an agenda of common development of these countries suggested. Design/methodology/approach - This paper reviews literature on muses of food inflation, production shortages, and investigation of programs to solve the problem in the future, it is also based on author`s personal insights and experience of working on this field in the last 15 years, and recent discussions in forums and interviews Findings - The major factors that jointly caused food prices increase in 2007/2008 were population growth, Income distribution, urbanization, dollar devaluations, commodity funds, social programs, production shortages, and bionic`s A list of ten policies is suggested. horizontal expansion of food production, vertical expansion, reduction in transaction costs, in protectionism and other taxes, investment in logistics, technology and better coordination, contracts, new generation of fertilizers and to use the best sources of biofuels. Originality/value - Two major outputs from this paper are the ""food demand model"" that inserts in one model the trends and muses of food inflation and the solutions, and the ""food bridge concept"" that also aligns in one box the imminent major food chain cooperation between China and Brazil