Antimutagenic effects of the mushroom Agaricus blazei Murrill extracts on V79 cells

Agaricus blazei Murrill, a native mushroom in Brazil, has been widely consumed in different parts of the world due to its medicinal power. Its anticarcinogenic activity has been shown in experimental animals, and antimutagenic activity has been demonstrated only in Salmonella. In this work, the multagenic and antimutagenic activities of mushroom teas of strains AB96/07, AB96/09 and AB97/11 were evaluated in Chinese hamster V79 cells, using the comet assay and the micronucleus test. The cells were treated with three different concentrations (0.05, 0.1 and 0.15) of teas prepared from a 2.5% aqueous solution, under three different temperatures: (1) room (20-25 degreesC); (2) ice-cold (2-8 degreesC); and (3) warm (60 degreesC). The teas were applied in co-, pre- and post-treatments in combination with the mutagen methyl methanesulfonate (MMS; 1.6 x 10(-4) and 4 x 10(-4) M). The duration of the treatment was 1 h in the comet assay and 2 h in the micronucleus test. The results showed that the mushroom was not mutagenic itself. Nevertheless, the mushroom is an efficient antimutagen against the induction of micronuclei by MMS in all concentrations and preparations tested. The observed reductions in the frequencies of micronuclei ranged from 61.5 (room temperature 0.1% tea in post-treatment) to 110.3% (co-treatment with warm and ice-cold 0.15% tea). In the comet assay, the antimutagenic activity was detected only when the cells were pre-treated with the following teas: warm 0.1 and 0.15%, room temperature 0.05% and ice-cold 0.1%. The results indicate that the mushroom A. blazei extracts are antimutagenic when tested in V79 cells. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Genotoxicity of antimicrobial endodontic compounds by single cell gel (comet) assay in Chinese hamster ovary (CHO) cells

Objective. In the current study, the potential DNA damage associated with exposure to a number of antimicrobial endodontic compounds was assessed by the single cell gel (comet) assay in vitro.Study design. Chinese hamster ovary (CHO) cells were exposed to formocresol, paramonochlorophenol, calcium hydroxide, or chlorhexidine at final concentration ranging from 0.01% to 1%.Results. Formocresol, paramonochlorophenol, and calcium hydroxide, as well as chlorhexidine in all concentrations tested did not contribute to the DNA damage.Conclusion. These findings are clinically relevant since they represent an important contribution to the correct evaluation of the potential health risk associated with exposure to dental agents.

The HUman MicroNucleus project on eXfoLiated buccal cells (HUMNXL): The role of life-style, host factors, occupational exposures, health status, and assay protocol

The human buccal micronucleus cytome assay (BMCyt) is one of the most widely used techniques to measure genetic damage in human population studies. Reducing protocol variability, assessing the role of confounders, and estimating a range of reference values are research priorities that will be addressed by the HUMNXL, collaborative study. The HUMNXL, project evaluates the impact of host factors, occupation, life-style, disease status, and protocol features on the occurrence of MN in exfoliated buccal cells. In addition, the study will provide a range of reference values for all cytome endpoints. A database of 5424 subjects with buccal MN values obtained from 30 laboratories worldwide was compiled and analyzed to investigate the influence of several conditions affecting MN frequency. Random effects models were mostly used to investigate MN predictors. The estimated spontaneous MN frequency was 0.74 parts per thousand (95% CI 0.52-1.05). Only staining among technical features influenced MN frequency, with an abnormal increase for non-DNA-specific stains. No effect of gender was evident, while the trend for age was highly significant (p < 0.001). Most occupational exposures and a diagnosis of cancer significantly increased MN and other endpoints frequencies. MN frequency increased in heavy smoking (>= 40 cig/day. FR = 1.37:95% CI 1.03-.82) and decreased with daily fruit consumption (FR = 0.68; 95% CI 0.50-0.91). The results of the HUMNXL, project identified priorities for validation studies, increased the basic knowledge of the assay, and contributed to the creation of a laboratory network which in perspective may allow the evaluation of disease risk associated with MN frequency. (C) 2011 Elsevier B.V. All rights reserved.

Genetic biomonitoring of an urban population exposed to mutagenic airborne pollutants

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation of Diuron (3-[3,4-dichlorophenyl]-1,1-dimethyl urea) in a Two-stage Mouse Skin Carcinogenesis Assay

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection of IgG in cerebrospinal fluid for diagnosis of neurocysticercosis: evaluation of saline and SDS extracts from Taenia solium and Taenia crassiceps metacestodes by ELISA and immunoblot assay

We compared saline (S) and sodium dodecyl sulphate (SDS) extracts from Taenia solium (homologous species - HO) and Taenia crassiceps (heterologous species - HE) metacestodes in order to detect Ige by ELISA and immunoblot assay (IBA) in cerebrospinal fluid (CSF) for the diagnosis of human neurocysticercosis (NC). CSF samples were obtained from 93 patients. of these, 40 had NC, five had a diagnosis of probable NC, nine had central nervous system schistosomiasis or strongyloidiasis and 39 had other neurological alterations. Samples were analysed by ELISA and the results were compared with IBA in all samples with confirmed and probable NC diagnosis, in all samples with other central nervous system parasitic infection, and in 10 of those with another neurological alterations. ELISA sensitivity was 100%, 85%, 95% and 87.5% for the S-HO, S-HE, SDS-HO and SDS-HE extracts, respectively, and ELISA specificity was 100% for S-HO, S-HE, SDS-HO extracts and 97.9% for SDS-HE antigen. Immunodominant peptides detected by IBA were, by decreasing percentage of recognition: 64-68 and 45 kDa for S-HO; 108-114, 92-95, 64-68, 83 and 88 kDa for S-HE; 64-68, 108-114, 77 and 86 kDa for SDS-HO; and 108-114, 88 and 92-95 kDa for SDS-HE. Overall the homologous antigenic extracts showed higher sensitivity than the heterologous extracts in the diagnosis of NC in CSF samples. The heterologous extracts contained most of the immunodominant peptides presented in the homologous extracts, which are recognized by Ige antibodies in CSF samples.

Hazards in non-pasteurized milk on retail sale in Brazil: prevalence of Salmonella spp, Listeria monocytogenes and chemical residues

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Single-cell gel (comet) assay detects primary DNA damage in nonneoplastic urothelial cells of smokers and ex-smokers

A protocol for DNA damage assessment by the single-cell gel (SCG)/comet assay in human urinary bladder washing cells was established. Modifications of the standard alkaline protocol included an increase to 2% of sodium sarcosinate in the lysis solution, a reduction in the glass-slide area for comet analysis, and a cutoff value for comet head diameter of at least 30 mum, to exclude contaminating leukocytes. Distinguishing cell populations is crucial, because significant differential migration was demonstrated for transitional and nontransitional cells, phenomena that may confound the results. When applying the modified protocol to urinary bladder cells from smokers without urinary bladder neoplasia, it was possible to detect a significant (P = 0.03) increase in DNA damage as depicted by the tail moment (6.39 +/- 3.23; mean 95% confidence interval; n = 18) when compared with nonsmokers (1.94 +/- 1.41; n = 12). No significant differences were observed between ex-smokers and current smokers regarding comet parameters. Inflammation was not a confounding factor, but DNA migration increased significantly with age in nonsmokers (r = 0.68; P = 0.014). Thus, age matching should be a concern when transitional cells are analyzed in the SCG assay. As it is well known, DNA damage may trigger genomic instability, a crucial step in carcinogenesis. Therefore, the present data directly support the classification of individuals with smoking history as patients at high risk for urinary bladder cancer.

Acute bacterial cystitis does not cause deoxyribonucleic acid damage detectable by the alkaline comet assay in urothelial cells of dogs

Considering the high incidence of dogs with acute bacterial cystitis (BC) and the relationship among inflammation, genotoxicity, and carcinogenesis, we conducted a case-control study comparing the frequency of deoxyribonucleic acid (DNA) lesions assessed by the comet assay between disease-free animals (13 males and 13 females) and cytology-confirmed cases of acute BC (12 males and 12 females), which was mainly caused by Staphylococcus sp. (40%) and Escherichia coli (35%). The results show no increase in DNA damage in cells obtained by bladder washings and no influence of age, sex, and breed due to acute BC. In conclusion, DNA damage was seemingly not associated with the infection by specific bacteria.

Reversion to virulence evaluation of a 9R vaccine strain of Salmonella enterica serovar gallinarum in commercial brown layers

The live vaccine Cevac S. Gallinarum, made from a rough strain of Salmonella enterica subspecies enterica serotype Gallinarum is used for preventing fowl typhoid, a disease that still causes considerable economic losses in countries with a developing poultry industry. The objective of this paper was to evaluate a possible reversion to virulence of the strain used in a vaccine in commercial brown layers. Only Salmonella-free chicks were utilized. One hundred twenty (120) 12-day-old Dekalb brown layers divided in two trials were used. The first trial had six groups of 15 birds each. Birds of group 1 were vaccinated with 10 doses of Cevac S. Gallinarum subcutaneously and 10 doses orally, in a total of 20 doses of vaccine. Then the birds of groups 2, 3, 4, and 5 received inocula that contained feces and a pool of organs with fragments of liver, heart, spleen, and cecal tonsils obtained from the immediately previous group. The second trial had three groups with 10 birds each. Birds in group 7 received inocula containing a pool of organs from birds of group 5 from trial 1, whilst the birds in group 8 were vaccinated subcutaneously with one dose of vaccine. Both trials included negative control groups (6 and 9). Throughout the experimental period, birds were monitored for reactions to the vaccination on the site of administration, clinical signs, and post-mortem lesions. In each passage, in addition to the birds euthanized to provide the inocula material, two birds from each group were euthanized for assessment of possible lesions, and their organs (liver, heart, spleen and cecal tonsils) were cultured in an attempt to isolate the vaccine strain. Except for one bird from group 1, that had a local reaction on the site of vaccination - a small vesicle with less that 0.5 mm that persisted until the third day post vaccination -, no other bird had any local reaction to the vaccine or any visible clinical alteration. Birds in group 8 did not present any reaction or clinical alteration because of the vaccine. We only managed to re-isolate the vaccine strain in the inocula made from organs of birds in group 1. We confirmed the isolation by means of biochemical tests, serology, and acriflavine agglutination test. All other cultures made from organs or feces, from all the other experimental groups did not show any growth of the vaccine strain or any other Salmonella serovar, suggesting that the vaccinated birds did not shed the SG9R vaccine strain. No bird presented any clinical symptoms or died during the trials, and no gross lesions were observed in the post-mortem examinations. Under the controlled conditions and time-frame of the present experiment, it was possible to conclude that the rough 9R strain of Salmonella Gallinarum present in the vaccine Cevac S. Gallinarum (Ceva Campinas Ltda. - Campinas, SP - Brazil) did not revert to virulence.

Uso de microbiota cecal congelada com crioprotetores em pintos infectados experimentalmente com Salmonella enterica sorovar Enteritidis

Pintos de corte com um dia de idade foram tratados com microbiota cecal cultivada em condição de aerobiose, nos tempos de congelamento de 90, 200, 290 e 360 dias, e associada aos crioprotetores sacarose, trealose, dimetilsulfóxido (DMSO) e glicerol. Posteriormente as aves foram desafiadas com Salmonella Enteritidis, visando determinar a eficácia dos tratamentos em relação à quantidade de bactérias viáveis da microbiota que foi maior aos 90 dias (10,58 Log10 UFC/ml), quando as aves foram tratadas com sacarose, e menor aos 290 dias, quando tratadas com glicerol (7,73 Log10 UFC/ml). No tempo zero, todas as aves apresentaram Salmonella (100%) quando tratadas com DMSO e glicerol, com colonização cecal de 4,9 e 5,2 Log10 UFC/g do conteúdo cecal, respectivamente; aos 360 dias nenhuma ave foi infectada, independente do tratamento. A microbiota cecal, independente de tratamento, sempre determinou menor quantidade de S. Enteritidis em qualquer um dos parâmetros pesquisados, quando comparada com a das aves não tratadas. O congelamento em nitrogênio líquido foi eficaz na manutenção da viabilidade da microbiota cecal até 360 dias.

Efeito protetor da microbiota cecal congelada e liofilizada sobre a infecção experimental de frangos de corte por Salmonella enterica sorovar Enteritidis

Estudaram-se os efeitos do tratamento de frangos de corte com microbiota cecal anaeróbia liofilizada (MCL) e congelada (MCC) sobre a infecção do trato digestivo das aves por Salmonella enterica sorovar Enteritidis. Foi usada microbiota intestinal sem prévia identificação bacteriana. A infecção foi persistente, em ordem, no ceco, inglúvio e duodeno. A infecção também foi autolimitante nos grupos tratados e no controle. Não ocorreu diferença entre o grupo-controle positivo e os tratados com MCL ou MCC. Houve redução da colonização do ceco no período de 12 dias após o desafio nos grupos tratados com MCL e MCC, o que não ocorreu no grupo-controle positivo. Não houve variação entre os tratamentos com MCL e MCC quanto às características pesquisadas. A S. Enteritidis reduziu o ganho de peso médio nas aves inoculadas. Os tratamentos com MCL e MCC minimizaram a redução de peso nos grupos infectados.

Efeito da vacina contra coccidiose sobre a colonização de Salmonella enteritidis em frangos de corte tratados com microbiota cecal anaeróbia

Avaliou-se o efeito da vacina contra coccidiose sobre a habilidade da microbiota cecal anaeróbia (MCA), administrada por diferentes vias (aves tratadas), em proteger pintos de corte da colonização por Salmonella enteritidis (Se). Utilizaram-se 120 aves assim divididas: grupo A, pulverização de MCA em aves vacinadas contra coccidiose e desafiadas com Se, grupo B, inoculação endoesofágica de MCA em aves vacinadas e desafiadas, grupo C, MCA na água de bebida de aves vacinadas e desafiadas, grupo D, aves não tratadas, vacinadas e desafiadas, grupo E, aves não tratadas, não vacinadas e desafiadas e, grupo F, aves não tratadas, não vacinadas e não desafiadas (controle negativo). Utilizaram-se como parâmetros a colonização do trato digestivo por Se e sua presença nas fezes, bem como o peso corporal das aves, avaliados aos dois, sete e 12 dias após o desafio. Nas aves tratadas com MCA, especialmente por meio de pulverização e inoculação endoesofágica, a colonização do ceco por Se e sua presença nas fezes foram menores, mostrando que a ação da MCA contra a colonização de ceco e excreção fecal não foi afetada pelo uso da vacina contra coccidiose em pintos de corte e que a associação MCA/vacina contra coccidiose pode ser utilizada sem que haja comprometimento na eficácia da MCA. Nos grupos que não receberam MCA, vacinados ou não contra coccidiose, houve aumento da colonização cecal, bem como excreção fecal da amostra desafio. O ceco foi o local de maior presença e persistência da Se. O resultado de administração de MCA pela água de bebida não foi tão eficiente, mas somente este tratamento resultou em peso corporal das aves significativamente superior ao das aves do grupo não tratado, não vacinado e desafiado, indicando que a presença de salmonelas paratifóides não interfere na produtividade de frangos de corte. Não se observou diferença no peso das aves dos grupos D (vacinados contra coccidiose) e E (não vacinadas) desafiadas com Se, demonstrando que a vacina não influenciou negativamente o ganho de peso das aves desafiadas com Salmonela enteriditis.

Efeito da microbiota cecal e do Lactobacillus salivarius inoculados in ovo em aves desafiadas com Salmonella enterica sorovar Enteritidis

Ovos embrionados provenientes de matrizes pesadas foram inoculados na câmara de ar com microbiota cecal total, microbiota cecal diluída e cultura de Lactobacillus salivarius, no 18º dia de incubação. Dois dias após o nascimento, as aves foram desafiadas com Salmonella enterica sorovar Enteritidis (SE) e, cinco dias após o desafio, avaliou-se a presença da bactéria no fígado e ceco. O efeito de exclusão competitiva, após o desafio com SE, somente foi observado pela ausência da bactéria no fígado das aves tratadas in ovo com L. salivarius. A inoculação in ovo de microbiota cecal indefinida ou diluída não reduziu a colonização de SE no fígado e no ceco das aves, incluindo, neste último, também o tratamento com L. salivarius. Nenhum dos tratamentos in ovo determinou índice de eclodibilidade superior a 65%.