Genotoxic and mutagenic effects of erythrosine B, a xanthene food dye, on HepG2 cells

Erythrosine (ErB) is a xanthene and an US Food and Drug Administration approved dye used in foods, drugs and cosmetics. Although its utilization is permitted, ErB is described as inhibitor of enzymes and protein-protein interactions and is toxic to pituitary and spermatogenesis processes. However, the genotoxicity and mutagenicity of ErB is inconclusive in the literature. This study aimed to analyze the genotoxicity of this dye using the alkaline comet assay and is the first investigation to evaluate ErB mutagenicity using the cytokinesis block micronucleus cytome (CBMN-Cyt) assay in HepG2 cells. These cells were chosen because they produce phase I and phase II enzymes that can mimic in vivo metabolism. The cells were treated with seven concentrations (0.1-70.0 mu g mL(-1)) of ErB, and the results showed genotoxicity at the two highest concentrations and mutagenicity at six concentrations. Furthermore, as micronuclei result from clastogenic and aneugenic processes, while comet assay is often considered more sensitive and detects DNA single strain breaks, we suggest that an aneugenic is responsible for the observed damage. Although ErB is approved for use in the food, cosmetic and pharmaceutical industries, it must be used carefully because it damages the DNA structure. (C) 2012 Elsevier Ltd. All rights reserved.

EROD activity and genotoxicity in the seabob shrimp Xiphopenaeus kroyeri exposed to benzo[a]pyrene (BaP) concentrations

Seabob shrimp Xiphopenaeus kroyeri is a marine species that lives in shallow waters of coastal environments, often impacted by polycyclic aromatic hydrocarbons (PAH) pollution. In the present study, seabob shrimp were exposed for 96 h to benzo[a]pyrene (BaP) at the nominal concentrations of 100, 200, 400 and 800 microg.L-1. Animals of the control groups were exposed either to clean water or to the BaP-carrier (DMSO). At the end of the exposures, muscle tissues were sampled for BaP uptake assessment and hepatopancreas and hemolymph for EROD enzyme activity and hemocytes DNA damage, respectively. EROD activity and DNA damage increased significantly as a function of BaP exposure concentrations. Significant correlations between BaP uptake and both EROD activity and DNA damage suggest that they can be used as suitable tools for integrated levels of study on the biomarkers of PAH exposure.

In Vivo Genotoxicity Assessment of Titanium Dioxide Nanoparticles by Allium cepa Root Tip Assay at High Exposure Concentrations

The industrial production and commercial applications of titanium dioxide nanoparticles have increased considerably in recent times, which has increased the probability of environmental contamination with these agents and their adverse effects on living systems. This study was designed to assess the genotoxicity potential of TiO2 NPs at high exposure concentrations, its bio-uptake, and the oxidative stress it generated, a recognised cause of genotoxicity. Allium cepa root tips were treated with TiO2 NP dispersions at four different concentrations (12.5, 25, 50, 100 mu g/mL). A dose dependant decrease in the mitotic index (69 to 21) and an increase in the number of distinctive chromosomal aberrations were observed. Optical, fluorescence and confocal laser scanning microscopy revealed chromosomal aberrations, including chromosomal breaks and sticky, multipolar, and laggard chromosomes, and micronucleus formation. The chromosomal aberrations and DNA damage were also validated by the comet assay. The bio-uptake of TiO2 in particulate form was the key cause of reactive oxygen species generation, which in turn was probably the cause of the DNA aberrations and genotoxicity observed in this study.

Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity

Objective: To investigate effects of cryopreservation on sperm motility and DNA integrity. Design: Pre-cryopreservation and post-cryopreservation analysis of motility and DNA integrity of semen and prepared sperm samples. Setting: A hospital andrology laboratory. Patient(s): Forty men attending the Regional Fertility Centre, Belfast, Northern Ireland. Intervention(s): Each sample was divided, and an aliquot was frozen unprepared. Remaining aliquots were prepared by Percoll density centrifugation (95.0:47.5) or direct swim-up procedure and divided into aliquots to allow direct comparison of fresh and frozen semen and prepared sperm (frozen with or without the addition of seminal plasma) from the same ejaculate. Samples were frozen by static-phase vapor cooling and being plunged into liquid nitrogen. Thawing was carried out at room temperature. Main Outcome Measure(s): Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay, and motility was determined using computer-assisted semen analysis. Result(s): Sperm frozen unprepared in seminal fluid appeared more resistant to freezing damage than frozen prepared sperm. Further improvements can be achieved by selecting out the subpopulation of sperm with best motility and DNA integrity and freezing these sperm in seminal plasma, making this the optimal procedure. Conclusion(s): Freezing sperm in seminal plasma improves postthaw motility and DNA integrity.

Assessment of DNA integrity and morphology of ejaculated sperm from fertile and infertile men before and after cryopreservation

Cryopreservation of human spermatozoa is extensively used in artifical insemination and IVF programmes. Despite various advances in cryopreservation methodology, the recovery rate of functional post thaw spermatozoa remains mediocre, with sperm motility being significantly decreased after freezing. The aim of this study was to investigate the effects of cryopreservation on both DNA integrity and morphology of spermatozoa from fertile and infertile men. Semen samples were obtained from 17 fertile men and 40 infertile men. All samples were prepared by discontinuous Percoll density centrifugation ( 95.0:47.5). Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. Aliquots for cryopreservation were mixed with a commercial cryoprotectant and frozen by static phase vapour cooling before plunging into liquid nitrogen. Thawing was carried out slowly at room temperature. Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis ( comet ) assay and sperm morphology analysed using the Tygerberg criteria. DNA of semen and prepared sperm from fertile men was found to be unaffected by cryopreservation. In marked contrast, spermatozoa from infertile men were significantly damaged by freeze- thawing. Cryopreservation had a detrimental effect on morphology of semen and prepared sperm from fertile and infertile men.

A comparison of mitochondrial and nuclear DNA status in testicular sperm from fertile men and those with obstructive azoospermia

Background: Mitochondria are vital to sperm as their motility powerhouses. They are also the only animal organelles with their own unique genome; encoding subunits for the complexes required for the electron transfer chain. Methods: A modified long PCR technique was used to study mitochondrial DNA (mtDNA) in ejaculated and testicular sperm samples from fertile men (n=11) and testicular sperm from men with obstructive azoospermia (n=25). Nuclear DNA fragmentation was measured by an alkaline single cell gel electrophoresis (COMET) assay. Results: Wild-type mtDNA was detected in only 60% of fertile mens�??�?�¢?? testicular sperm, 50% of their ejaculated sperm and 46% of testicular sperm from men with obstructive azoospermia. The incidence of mitochondrial deletions in testicular sperm of fertile and infertile men was not significantly different but the mean size of the deletions was significantly less in testicular sperm from fertile men compared with men with obstructive azoospermia (p<0.02). Nuclear DNA fragmentation in testicular sperm from fertile men and men with obstructive azoospermia was not significantly different. Conclusion: Multiple mtDNA deletions are common in testicular and ejaculated sperm from both fertile and infertile men. However, in males with obstructive azoospermia the mtDNA deletions in testicular sperm are of a larger scale.

Comet sensitivity in assessing DNA damage and repair in different cell cycle stages

The comet assay is a sensitive tool for estimation of DNA damage and repair at the cellular level, requiring only a very small number of cells. In comparing the levels of damage or repair in different cell samples, it is possible that small experimental effects could be confounded by different cell cycle states in the samples examined, if sensitivity to DNA damage, and repair capacity, varies with the cell cycle. We assessed this by arresting HeLa cells in various cell cycle stages and then exposing them to ionizing radiation. Unirradiated cells demonstrated significant differences in strand break levels measured by the comet assay (predominantly single-strand breaks) at different cell cycle stages, increasing from G1 into S and falling again in G2. Over and above this variation in endogenous strand break levels, a significant difference in susceptibility to breaks induced by 3.5 Gy ionizing radiation was also evident in different cell cycle phases. Levels of induced DNA damage fluctuate throughout the cycle, with cells in G1 showing slightly lower levels of damage than an asynchronous population. Damage increases as cells progress through S phase before falling again towards the end of S phase and reaching lowest levels in M phase. The results from repair experiments (where cells were allowed to repair for 10 min after exposure to ionizing radiation) also showed differences throughout the cell cycle with G1-phase cells apparently being the most efficient at repair and M-phase cells the least efficient. We suggest, therefore, that in experiments where small differences in DNA damage and repair are to be investigated with the comet assay, it may be desirable to arrest cells in a specific stage of the cell cycle or to allow for differential cycle distribution.

DNA damage in two bivalve species: Mytilus galloprovincialis and Ruditapes decussatus from the South coast of Portugal

Dissertação de mest., Estudos Marinhos e Costeiros, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2009

Suco de laranja e vitamina C : efeito sobre a estabilidade genômica

Existem evidências crescentes indicando a associação entre dietas ricas em frutas e vegetais e a diminuição da incidência de câncer. O suco de laranja (OJ) pode ser incluído entre os alimentos com potencial quimioprotetor e seu estudo é muito relevante pelo amplo consumo desta bebida. O OJ possui vários nutrientes e compostos bioativos com atividades antioxidante, antimutagênica, anticarcinogênica e antiaterogênica, entre outras. A vitamina C (Vit C) é um dos nutrientes mais abundantes no OJ, e o único nutriente que pode ser provido em quantidade superior à recomendação diária por uma única porção de 200 mL de OJ. A Vit C, a exemplo de outros componentes do OJ, pode ser tanto benéfica quanto maléfica para os sistemas biológicos, dependendo do contexto metabólico. Neste sentido, vários nutrientes presentes no OJ têm sido identificados como mutagênicos ou carcinogênicos, especialmente quando administrados de forma isolada. Este estudo utilizou o ensaio Cometa alcalino em sangue de camundongos (in vivo) para avaliar: 1) a genotoxicidade do OJ e da Vit C; 2) a genotoxicidade do FeSO4 e do CuSO4: 3) o efeito modulador do OJ e da Vit C sobre a genotoxicidade do FeSO4 e CuSO4, bem como do metilmetanosulfonato (MMS) e da ciclofosfamida (CP). A versão alcalina do ensaio Cometa foi utilizada para avaliar o dano no DNA em células brancas do sangue periférico de camundongos. Adicionalmente, os níveis de cobre e ferro no sangue e no fígado dos camundongos tratados com metais e OJ foram avaliados pela metodologia de PIXE (Particle-Induced X-ray Emission). Grupos com pelo menos 6 camundongos (metade de cada sexo) foram tratados por gavage com uma ou duas doses de água (controle), CP, MMS, FeSO4 ou CuSO4. OJ (0.1 mL/Kg) foi administrado tanto antes (pré-tratamento) quanto após a administração das substâncias-teste (pós-tratamento). A Vit C (1 e 30 mg/Kg) foi administrada apenas no pós-tratamento. O dano no DNA foi avaliado 24 e 48 h após o início do tratamento. Após 24 h, o OJ induziu um suave aumento no dano no DNA, enquanto a Vit C foi genotóxica (30 mg/Kg > 1 mg/Kg). O tratamento duplo com Vit C (a 0 e a 24 h) induziu uma resposta genotóxica cumulativa a 48 h, que foi mais intensa para a dose maior. O FeSO4 e o CuSO4 foram genotóxicos após 24 h, mas tiveram seu dano efetivamente reparado após 48 h do tratamento. O pré-tratamento com OJ reduziu a genotoxicidade do FeSO4 e do CuSO4 (efeito preventivo). O pós-tratamento com OJ também reduziu a genotoxicidade do CuSO4 (efeito reparador). O OJ mostrou tanto efeito preventivo quanto reparador sobre a genotoxicidade do MMS. O OJ teve apenas efeito reparador sobre a CP. Ambas doses de Vit C aumentaram os danos no DNA causados pelo FeSO4 e pelo CuSO4. Adicionalmente, os níveis de cobre e ferro no sangue e no fígado dos camundongos tratados com metais e OJ foram avaliados pela metodologia de PIXE (Particle-Induced X-ray Emission). Grupos com pelo menos 6 camundongos (metade de cada sexo) foram tratados por gavage com uma ou duas doses de água (controle), CP, MMS, FeSO4 ou CuSO4. OJ (0.1 mL/Kg) foi administrado tanto antes (pré-tratamento) quanto após a administração das substâncias-teste (pós-tratamento). A Vit C (1 e 30 mg/Kg) foi administrada apenas no pós-tratamento. O dano no DNA foi avaliado 24 e 48 h após o início do tratamento. Após 24 h, o OJ induziu um suave aumento no dano no DNA, enquanto a Vit C foi genotóxica (30 mg/Kg > 1 mg/Kg). O tratamento duplo com Vit C (a 0 e a 24 h) induziu uma resposta genotóxica cumulativa a 48 h, que foi mais intensa para a dose maior. O FeSO4 e o CuSO4 foram genotóxicos após 24 h, mas tiveram seu dano efetivamente reparado após 48 h do tratamento. O pré-tratamento com OJ reduziu a genotoxicidade do FeSO4 e do CuSO4 (efeito preventivo). O pós-tratamento com OJ também reduziu a genotoxicidade do CuSO4 (efeito reparador). O OJ mostrou tanto efeito preventivo quanto reparador sobre a genotoxicidade do MMS. O OJ teve apenas efeito reparador sobre a CP. Ambas doses de Vit C aumentaram os danos no DNA causados pelo FeSO4 e pelo CuSO4. processado e armazenado de forma a preservar o seu potencial biológico é um alimento sugerido como uma das porções de uma dieta equilibrada (contendo pelo menos 5 porções de frutas e vegetais), recomendada para uma vida saudável e longeva.

Estudo do efeito dos metabólitos do benzeno e tolueno sobre as mitocôndris cerebrais e hepáticas de ratos

Aim: The aim of this work was to investigate the hypothesis that catechol and 3MC inhibit FADH2-linked basal respiration in mitochondria isolated from rat liver and brain homogenates. Moreover, catechol ability to induce DNA damage in rat brain cells through the comet assay (alkaline single-cell gel electrophoresis assay) was also observed. Methods: Two different catechols were evaluated: pirocatechol (derived from benzene) and 3-methylcatechol (derived from toluene); rat liver and brain homogenates were incubated with 1mM catechol at pH 7.4 for up to 30 minutes. After that, mitochondrial fractions were isolated by differential centrifugation. Basal oxygen uptake was measured using a Clark-type electrode after the addition of 10 mM sodium succinate for a period of 12 minutes. In additional experiments, rat brain cells were treated with 1, 5 and 10mM pirocatechol for up to 20 minutes at 37º C, and submitted to electrophoresis. Results: Catechols (pirocatechol and 3methylcatechol) induced a time-dependent partial inhibition of FADH2-linked basal mitochondrial respiration. Indeed, pirocatechol was able to produce a dosedependent DNA oxidative damage in rat brain cells by 2 and 4 injury levels. These results suggest that reactive oxygen species generated by the oxidation of catechols, induced an impairment on mitochondrial respiration and a DNA damage, which might be related to their citotoxicity. Conclusion: Catechols produced an inhibition of basal respiration associated to FADH2 in isolated liver and brain mitochondria; 3-methylcatechol, at the same concentration, produced similar toxicity in the mitochondrial model. Indeed, pirocatechol induced a DNA damage in rat brain cells, mainly observed in comets formation and consequent DNA degradation

Mutagenicity and antimutagenicity of (-)-hinokinin a trypanosomicidal compound measured by Salmonella microsome and comet assays

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

Absence of mutagenicity effects of Psidium cattleyanum Sabine (Myrtaceae) extract on peripheral blood and bone marrow cells of mice

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Oxidative stress status of highly prolific sows during gestation and lactation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biocompatibility in vitro tests of mineral trioxide aggregate and regular and white Portland cements

Mineral trioxide aggregate (MTA) and Portland cement are being used in dentistry as root end-filling materials. However, biocompatibility data concerning genotoxicity and cytotoxicity are needed for complete risk assessment of these compounds. In the present study, genotoxic and cytotoxic effects of MTA and Portland cements were evaluated in vitro using the alkaline single cell gel (comet) assay and trypan blue exclusion test, respectively, on mouse lymphoma cells. The results demonstrated that the single cell gel (comet) assay failed to detect DNA damage after a treatment of cells by MTA and Portland cements for concentrations up to 1000 mu g/ml. Similarly, results showed that none of the compounds tested were cytotoxic. Taken together, these results seem to indicate that MTA and Portland cements are not genotoxins and do not induce cellular death.

Evaluation of genetic damage in human peripheral lymphocytes exposed to mineral trioxide aggregate and Portland cements

Mineral trioxide aggregate (MTA) and Portland cement are being used in dentistry as root-end-filling material for periapical surgery and for the sealing of communications between the root canal system and the surrounding tissues. However, genotoxicity tests for complete risk assessment of these compounds have not been conducted up to now. In the present study, the genotoxic effects of MTA and Portland cements were evaluated in peripheral lymphocytes from 10 volunteers by the alkaline single cell gel (comet) assay. The results pointed out that the single cell gel (comet) assay failed to detect the presence of DNA damage after a treatment of peripheral lymphocytes by MTA and Portland cements for concentrations up to 1000 mu g mL(-1). In summary, our results indicate that exposure to MTA or Portland cements may not be a factor that increases the level of DNA lesions in human peripheral lymphocytes as detected by single cell gel (comet) assay.