Anesthesia of fish with benzocaine does not interfere with comet assay results

Fish blood erythrocytes are frequently used as sentinels in biomonitoring studies. Usually, fish blood is collected by painful cardiac or caudal vein punctures. Previous anesthesia could decrease animal suffering but it is not known at present whether anesthesia can cause confounding effects. Therefore, using the alkaline single cell gel (SCG)/comet assay with blood erythrocytes of the cichlid fish Nile tilapia, we tested for a possible modulation of induced DNA damage (methyl methanesulfonate; MMS) by the anesthetic benzocaine administered by bath exposure (80mg/l for ∼10min). Furthermore, benzocaine (80-600mg/l) was tested for its genotoxic potential on fish erythrocytes in vitro and for potential interactions with two known genotoxins (MMS and hydrogen peroxide). Our results did neither indicate a significant increase in the amount of DNA damage (even after a 48h follow-up), nor indicated interactions with MMS-induced DNA damage when fish were exposed to benzocaine in vivo. There was also no increase in DNA damage after in vitro exposure of fish erythrocytes to benzocaine. Clear concentration-related effects were observed for the two genotoxins in vitro, which were not significantly altered by the presence of benzocaine. These results suggest that anesthesia of fish does not confound comet assay results and the use of blood samples from anesthetized fish can be recommended with regard to animal welfare. © 2002 Elsevier Science B.V. All rights reserved.

Comparison of a multiplex-PCR assay with mycolic acids analysis and conventional methods for the identification of Mycobacteria

A fast, sensitive and cost-effective multiplex-PCR assay for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium (M. avium) identification for routine diagnosis was evaluated. A total of 158 isolates of mycobacteria from 448 clinical specimens from patients with symptoms of mycobacterial disease were analyzed. By conventional biochemical methods 151 isolates were identified as M. tuberculosis, five as M. avium and two as Mycobacterium chelonae (M. chelonae). Mycolic acid patterns confirmed these results. Multiplex-PCR detected only IS6110 in isolates identified as MTC, and IS1245 was found only in the M. avium isolates. The method applied to isolates from two patients, identified by conventional methods and mycolic acid analysis, one as M. avium and other as M. chelonae, resulted positive for IS6110, suggesting co-infection with M. tuberculosis. These patients were successfully submitted to tuberculosis treatment. The multiplex-PCR method may offer expeditious identification of MTC and M. avium, which may minimize risks for active transmission of these organisms and provide useful treatment information.

Investigation of the Genotoxic Potential of the Waters of a River Receiving Tannery Effluents by Means of the in vitro Comet Assay

The comet assay has been described as an efficient tool for the detection of changes in the DNA molecule of cells exposed to contaminating agents in vivo and in vitro. The possible environmental contamination due to the persistence of chromium residues from tannery effluents was determined in the waters of the Córrego dos Bagres stream, Municipal district of Franca/SP, by the comet assay on CHO-K1 cells. Water samples were collected during the four seasons of the year 2001 at three distinct stations along the river. The data suggest that the comet test showed good sensitivity for the environmental monitoring of these waters and indicated that this test can be efficient for the determination of the quality of waters contaminated with effluents containing heavy metal residues such as chromium.

Immobilization of lipases and assay in continuous fixed bed reactor

Lipases are versatile enzymes regarding the range of reactions they catalyse and substrates on which they act. They are as well important as catalyst in organic synthesis. Their immobilization on appropriate supports confer them greater stability besides the possibility of operating in continuous reactors. In order to explore these abilities, the reactions involving hydrolysis of p-nitrophenyl acetate (PNPA) and transesterification of PNPA with n-butanol were chosen. Lipases from two different sources were assayed, namely: microbial (Candida rugosa, CRL, Sigma Type VII) and pancreatic (PPL, Sigma, Type 11). Two immobilization methods were also used, namely: 1) adsorption, using as support the following silica derivatives (150-300μm e 450μ): phenyl, epoxy, amino and without derivation, and 2) covalent binding, using glutaraldehyde as binding agent and silica amino as support. This later method led to better results. Hydrolytic activity was 6.1 U/gsupport for CRL and 0.97U/gsupport for PPL, and of transesterification, 2,8U/gsupport for CRL and 1,9U/gsupport for PPL. Stability of the immobilized enzyme as a function of temperature was evaluated for CRL at 40°C and 50°C and for PPL at 32°C and 40°C. The assays were initially carried out batchwise, both for soluble and immobilized enzymes, aiming to the obtention of parameters for the continues reactor. Lipases immobilized by covalent binding were used in the assays of operacional stability in continuos reactors. For PPL in aqueous medium, at 32°C, and CRL in organic medium at 40°C, both operating continuously, no significant loss of activity was detected along the analysis period of 17 days. In the case of CRL in aqueous medium at 40°C there was a loss of activity around 40% after 18 days. For PPL in organic medium at 40°C the loss was 33% after 20 days. Compairing both sources with each other, very different results were obtained. Higher activitiy was found for CRL, both for hydrolysis and for transesterification reactions, with higher stability in organic medium. PPL showed lower activity as well as higher stability in aqueous medium. The immobilization method by covalent binding showed to be the most appropriate. Immobilized lipases are therefore relatively stable both in aqueous and organic medium.

Lack of genotoxicity of formocresol, paramonochlorophenol, and calcium hydroxide on mammalian cells by comet assay

Formocresol, paramonochlorophenol, and calcium hydroxide are widely used in dentistry because of their antibacterial activities in root canal disinfection. However, the results of genotoxicity studies using these materials are inconsistent in literature. The goal of this study was to examine the genotoxic potential of formocresol, paramonochlorophenol, and calcium hydroxide using mouse lymphoma cells and human fibroblasts cells in vitro by the comet assay. Data were assessed by Kruskal-Wallis nonparametric test. The results showed that all compounds tested did not cause DNA damage for the tail moment or tail intensity parameters. These findings suggest that formocresol, paramonochlorophenol, and calcium hydroxide do not promote DNA damage in mammalian cells and that the comet assay is a suitable tool to investigate genotoxicity.

A new neolignan and antioxidant phenols from Nectandra grandiflora

The EtOH crude extract from the leaves of Nectandra grandiflora collected in the Atlantic Forest, Brazil, showed antioxidant activity towards β-carotene in a TLC assay. The bioassay-guided fractionation led to the isolation of protocatechuic acid and two flavonol glycosides: afzelin and quercetrin, which showed free radical scavenging activity towards DPPH (ΔA 32, 16 and 73% at 50 μmol L-1) and were compared to commercial antioxidants rutin (81% at 50 μmol L-1) and BHT (9% at 50 μmol L-1), used as standard compounds. Additionally, three inactive neolignans, including the new bicycle[3.2.1]octane neolignan 2′-oxo-piperol B were obtained and characterized by spectrometric methods. ©2005 Sociedade Brasileira de Química.

Microbiological assay for the determination of azithromycin in ophthalmic solutions

The validation of a simple, sensitive and specific agar diffusion bioassay, applying cylinder-plate method, for the determination of the antibiotic azithromycin in ophthalmic solutions is described. Using a strain of Bacillus subtilis ATCC 9372 as the test organism, azithromycin at concentrations ranging from 50.0 to 200.0 μg·mL-1 could be measured in 1.666 7 mg·mL-1 ophthalmic solutions. A prospective validation of the method showed that the method was linear (r = 0.999 9) and precise (RSD = 0.70) and accurate (it measured the added quantities). The results obtained by bioassay method could be statistically calculated by linear parallel model and by means of regression analysis and verified using analysis of variance (ANOVA). We conclude that the microbiological assay is satisfactory for quantification of azithromycin in ophthalmic solutions.

Evaluation of the genotoxic potential due to the action of an effluent contaminated with chromium, by the comet assay in CHO-K1 cultures

The comet assay technique has been considered to be more efficient in the biomonitoring of aquatic environments that the micronucleus and sister chromatid exchange techniques. The comet assay has been used to determine breaks in the DNA strands of organisms exposed to pollutants with a genotoxic potential. The comet technique was applied to CHO-K1 cells in order to evaluate the genotoxic potential of the waters of the Sapucaizinho River (Municipality of Patrocínio Paulista, State of São Paulo, Brazil), which receive tannery effluents and therefore are contaminated with chromium. The results indicated high genotoxicity of the waters collected at sites located downstream from the emission of tannery effluents, where the concentration of chromium was found to be high.

In vitro metabolism effect on genotoxicity and antigenotoxicity of Agaricus blazei organics and aqueous extracts by the comet assay

There is high interest in the natural products properties due to their use in popular medicine. Agaricus blazei Murrill ss. Heinem. (Ab) is native to Brazil and has been widely disseminated because its medicinal properties. In the present study, the genotoxic and antigenotoxic potential of Ab extracts were investigated using the comet assay. The cells utilized were the non drug-metabolizing line CHO-k1 (Chinese hamster ovary) and the drug-metabolizing line HTC (rat hepatoma). Cells were treated for 3 h in the absence of fetal bovain serum (FBS) with methanolic, hexanic and n-butanolic extracts at 50 μg/ml and 0.75% aqueous extract to test for genotoxicity. Antigenotoxic effects of extracts were determined in cells exposed to the DNA damage inducing agent ethyl methanesulfonate under simultaneous or simultaneous with 1 h pre-incubation conditions. The extracts did not show genotoxicity in HTC, while they were genotoxic in CHO-k1. No antigenotoxic effect was observed with any extract under any condition. These results demonstrate that the metabolism in presence or in absence has a direct influence on the genotoxicity of these extracts. © 2006 The Japan Mendel Society.

Lack of DNA damage induced by fluoride on mouse lymphoma and human fibroblast cells by single cell gel (comet) assay

Fluoride has widely been used in Dentistry because it is a specific and effective caries prophylactic agent. However, excess fluoride may represent a hazard to human health, especially by causing injury on genetic apparatus. Genotoxicity tests constitute an important part of cancer research for risk assessment of potential carcinogens. In this study, the potential DNA damage associated with exposure to fluoride was assessed by the single cell gel (comet) assay in vitro. Mouse lymphoma and human fibroblast cells were exposed to sodium fluoride (NaF) at final concentration ranging from 7 to 100 μg/mL for 3 h at 37μC. The results pointed out that NaF in all tested concentrations did not contribute to DNA damage as depicted by the mean tail moment and tail intensity for both cellular types assessed. These findings are clinically important because they represent a valuable contribution for evaluation of the potential health risk associated with exposure to agents usually used in dental practice.

In vitro cytotoxic and non-genotoxic effects of gutta-percha solvents on mouse lymphoma cells by single cell gel (comet) assay

Chloroform and eucalyptol are widely used in clinical dentistry as gutta-percha solvents. However, these compounds may represent a hazard to human health, especially by causing injury to genetic apparatus and/or inducing cellular death. In this study, the genotoxic and cytotoxic potentials associated with exposure to chloroform and eucalyptol were assessed on mouse lymphoma cells in vitro by the single cell gel (comet) assay and trypan blue exclusion test, respectively. Both gutta-percha solvents proved to be cytotoxic at the same levels in concentrations of 2.5, 5 and 10 μL/mL (p<0.05). On the other hand, neither of the solvents induced DNA breakage. Taken together, these results suggest that although both tested compounds (chloroform and eucalyptol) are strong cytotoxicants, it seems that they are not likely to increase the level of DNA damage on mammalian cells.

In vivo clastogenicity assessment of the Austroplenckia populnea (Celastraceae) leaves extract using micronucleus and chromosome aberration assay

The clastogenic effect of the A. populnea leaves extract was tested in vivo on bone marrow cells of Wistar rats by evaluating the induction of chromosome aberrations and micronuclei induction on polychromatic erythrocytes. The extract was administered by gavage at doses of 300, 600 and 900mg/kg body weight. Experimental and control animals were submitted to euthanasia 24 h after the treatment. Under the conditions used, A. populnea leaves extract did not induce decrease in mitotic index and did not induce a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes or chromosome aberrations in the bone marrow cells of Wistar rats. © 2007 The Japan Mendel Society.

Paracoccidioidomycosis: No genetic damage in human peripheral blood cells of patients assessed by single-cell gel (comet) assay

Paracoccidioidomycosis is a systemic fungal infection caused by Paracoccidioides brasiliensis. As infectious diseases can cause DNA damage, the authors aimed at analyzing DNA breakage in peripheral blood cells of patients with paracoccidioidomycosis by using the comet assay. The results suggested that paracoccidioidomycosis does not cause genotoxicity.

Influence of endogenous and synthetic female sex hormones on human blood cells in vitro studied with comet assay

The comet assay has been conducted with numerous cell lines to assess in vitro genotoxicity. In order to use the comet assay as part of an in vitro test for evaluating genotoxicity, however, there are cell-specific factors that need to be better understood. In this present study we have evaluated some factors that may impact upon the DNA damage detected in whole blood (WB) cells and lymphocytes (ILs). Experiments were conducted comparing responses of both cells, and investigating the effects of the female hormonal cycle, and oral contraceptive (OC) use on DNA damage detection in the in vitro comet assay, at three sampling time. No significant differences were detected in the basal levels of DNA damage detected in ILs and WB cells from women OC users and non-users and from men. Basal DNA damage in ILs was unaffected by gender and stage of the menstrual cycle or the stage of the treatment schedule. Our results also indicated that the H2O2 induces DNA damage in human lymphocytes independently of gender, low-dose OC use and hormonal fluctuation. However, data showed that in 3rd sampling of menstrual cycle, lymphocytes were more resistant to H2O2-induced DNA damage than those from OC users and men. © 2007 Elsevier Ltd. All rights reserved.

Immunohistochemistry assay to detect Turkey coronavirus (TCoV) from experimentally infected poults

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