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.

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)

Mode of Action of Pulegone on the Urinary Bladder of F344 Rats

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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.

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.

Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Enrofloxacin assay validation and pharmacokinetics following a single oral dose in chickens

The pharmacokinetics of enrofloxacin (ENRO), a fluoroquinolone antimicrobial agent, was studied in male broiler chickens (Cobb) after single oral administration of 10 mg of ENRO/kg b.w. A high-performance liquid chromatography-photodiode array detector (DAD) (HPLC-DAD) method was developed and validated and used for quantitation of ENRO and its major metabolite ciprofloxacin in plasma. The HPLC analyses were carried out using a cationic-octadecyl mixed column and 0.05 mol/L phosphate buffer (pH 2.5)/acetonitrile as mobile phase. The sample preparation of plasma consisted of the precipitation of proteins followed by solid phase extraction on cationic-octadecyl mixed cartridges. The method was validated considering linear range, linearity, selectivity, sensitivity, limit of detection (LOD), limit of quantitation (LOQ), intra- and inter-day precisions and accuracy. The LOD and LOQ for both fluoroquinolones were 60 and 200 ng/mL for plasma. The plasma concentration vs. time graph was characteristic of a two-compartment open model. The maximal plasma concentration of 1.5 +/- 0.2 mg/mL was achieved at 9 +/- 2 h. The elimination half-life and the mean residence time of ENRO were 1.5 +/- 0.2 and 15.64 h, respectively. The area under the concentration-time curve was calculated as 35 +/- 4 mg(.)h/mL.

Evaluation of fertilizing potential of frozen-thawed dog spermatozoa diluted in ACP-106 (R) using an in vitro sperm-oocyte interaction assay

The aim of present study was to evaluate frozen canine semen with ACP-106 (R) (Powder Coconut Water) using an in vitro sperm-oocyte interaction assay (SOIA). Ten ejaculates from five stud dogs were diluted in ACP-106 (R) containing 20% egg yolk, submitted to cooling in a thermal box for 40 min and in a refrigerator for 30 min. After this period, a second dilution was performed using ACP-106 (R) containing 20% egg yolk and 12% glycerol. Samples were thawed at 38 degrees C for 1 min. Post-thaw motility was evaluated by light microscopy and by using a computer aided semen analysis (CASA). Plasma membrane integrity and sperm morphology/acrosomal status were evaluated by fluorescent probes (C-FDA/PI) and Bengal Rose respectively. Moreover, frozen-thawed semen was analysed by a SOIA. Subjective post-thaw motility was 52.0 +/- 14.8% and it was significant higher than the total motility estimated by CASA (23.0 +/- 14.8%) because this system considered the egg yolk debris as immotile spermatozoa. Although normal sperm rate and acrosomal integrity evaluated by Bengal Rose stain was 89.6 +/- 3.1 % and 94.3 +/- 3.1 %, respectively, post-thaw percentage of intact plasma membrane was only 35.1 +/- 14.3%. Regarding SOIA, the percentage of interacted oocytes (bound, penetrated and bound and/or penetrated) was 75.3%. Using regression analysis, it was found significant relations between some CASA patterns and data for SOIA. In conclusion, the freezing-thawing procedure using ACP-106 (R) was efficient for maintain the in vitro fertility potential of dog spermatozoa.

Tritrichomonas fetus extracellular products decrease progressive motility of bull sperm

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

Taenia saginata: Differential diagnosis of human taeniasis by polymerase chain reaction-restriction fragment length polymorphism assay

Speciation of Taenia in human stool is important because of their different clinical and epidemiological features. DNA analysis has recently become possible which overcomes the problems of differentiating human taeniid cestodes morphologically. In the present study, we evaluated PCR coupled to restriction fragment length polymorphism to differentiate Taenia solium from Taenia saginata eggs present in fecal samples from naturally infected patients. A different Dral-RFLP pattern: a two-band pattern (421 and 100 bp) for T saginata and a three-band pattern (234, 188, and 99 bp) for T solium was observed allowing the two species to be separated.. The lower detection limit of the PCR-RFLP using a non-infected fecal sample prepared with a given number of T saginata eggs was 34 eggs in 2 g stool sediment. The 521 bp mtDNA fragment was detected in 8 out of 12 Taenia sp. carriers (66.6%). of these, three showed a T solium pattern and five a T saginata pattern. (c) 2005 Elsevier B.V. All rights reserved.

Wild rabies virus detection by plaque assay from naturally infected brains in different species

A simple, sensitive and specific plaque assay protocol for the detection of wild type rabies virus in different species is described using confluent monolayers of chicken embryo cells in 6-well plates. Plaques are produced after application of either agarose or Sephadex G-100 overlay onto cell monolayers and incubation for 96 h after virus infection at 37 degreesC. The parameters affecting plaque appearance include cell seeding concentration, overlay composition and time of incubation after infection. Optimal conditions are seeding at a concentration of 4 x 10(6) cell/cm(3), incubation at 37 degreesC in 5% CO2 atmosphere during 96 h, using either 1% agarose or 2% Sephadex G-100 overlays. The described plaque assay would be a new valuable too] in conducting various quantitative investigations, since the chicken embryo cells are susceptible to rabies virus infection from all species studied. (C) 2004 Elsevier B.V. All rights reserved.