Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies

Hepatic oxidative stress in an animal model of sleep apnoea: effects of different duration of exposure

Background: Repeated apnoea events cause intermittent hypoxia (IH), which alters the function of various systems and produces free radicals and oxidative stress. Methods: We investigated hepatic oxidative stress in adult mice subjected to intermittent hypoxia, simulating sleep apnoea. Three groups were submitted to 21 days of IH (IH-21), 35 days of IH (IH-35), or 35 days of sham IH. We assessed the oxidative damage to lipids by TBARS and to DNA by comet assay; hepatic tissue inflammation was assessed in HE-stained slides. Antioxidants were gauged by catalase, superoxide dismutase, glutathione peroxidase activity and by total glutathione. Results: After IH-21, no significant change was observed in hepatic oxidative stress. After IH-35, significant oxidative stress, lipid peroxidation, DNA damage and reduction of endogenous antioxidants were detected. Conclusions: In an animal model of sleep apnoea, intermittent hypoxia causes liver damage due to oxidative stress after 35 days, but not after 21 days

Megazol and its bioisostere 4H-1,2,4-triazole: comparing the trypanocidal, cytotoxic and genotoxic activities and their in vitro and in silico interactions with the Trypanosoma brucei nitroreductase enzyme

Megazol (7) is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8) in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR) enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7) for nitrogen (in the triazole in 8), the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7

An alkaline single-cell gel electrophoresis (comet) assay for environmental biomonitoring with native rodents

The main advantages of single-cell gel electrophoresis (SCG) are its applicability to any eukaryotic organism and cell type, its low cost and the short time required to obtain results. These properties make the SCG assay particularly useful in screening for environmental genotoxicity. The present study describes a modified version of this technique for use in field work with native rodents and examines some factors which influence the outcome of the assay. Wild rodents (Ctenomys torquatus, "tuco-tuco") from a region close to a strip coal mine and from a region with no coal mines were used. Animals from the coal mining region had significantly more DNA damage than those from the control area. The use of this SCG technique for direct sampling in the field should facilitate environmental genotoxicity studies with natural populations, without the need to remove the animals from their habitat or to sacrifice them.<br>A mais importante vantagem do Ensaio Cometa é a possibilidade do seu uso em qualquer organismo e tipo celular. Além de ser barato e rápido, se obtem resultados em poucas horas. Devido a isto é que ele vem sendo recomendado como teste inicial para monitoramento genotóxico ambiental. Neste trabalho investigaram-se adaptações na técnica para trabalhos de campo, com o uso de roedores endêmicos de região de mineração de carvão. O organismo utilizado foi o Ctenomys torquatus, capturado em duas diferentes áreas: Pelotas (região sem mineração de carvão-controle) e Candiota (zona de mineração de carvão). Foi coletado sangue periférico de 18 roedores, que após marcação foram liberados. O sangue foi protegido da luz e mantido sob refrigeração, e processado in loco. As concentrações das agaroses e as condições alcalinas de lise e eletroforese foram modificadas a partir das metodologias sugeridas pelas revisões existentes. As amostras restantes foram mantidas em RPMI 1640 (1:10) (4ºC) e pela nossa experiência podem ser utilizadas por até 4 dias. Foram analisadas 50 células por animal. As células foram classificadas de 0-4, segundo seu grau de lesão, além de serem medidas em unidades arbitrárias. Foi demonstrado aumento significante no nível de dano do DNA no grupo de Candiota (P < 0,05 - teste t), quando comparado com Pelotas. Ainda se demonstrou correlação entre o índice de danos e as medidas celulares. Estes resultados, neste estudo piloto, indicam que o carvão induz efeitos no DNA que são detectáveis através do Ensaio Cometa, o que indica a viabilidade deste teste para o biomonitoramento ambiental

Genotoxicity and antigenotoxicity assessment of shiitake (Lentinula edodes (Berkeley) Pegler) using the Comet assay

The mushroom shiitake (Lentinula edodes (Berkeley) Pegler) is been widely consumed in many countries, including Brazil, because of its pleasant flavor and reports of its therapeutic properties, although there is little available information on the genotoxicity and/or antigenotoxicity of this mushroom. We used the Comet assay and HEp-2 cells to evaluate the in vitro genotoxic and antigenotoxic activity of aqueous extracts of shiitake prepared in three different concentrations (0.5, 1.0 and 1.5 mg/mL) and three different temperatures (4, 22 and 60 °C), using methyl methanesulfonate (MMS) as a positive control and untreated cells as a negative control. Two concentrations (1.0 and 1.5 mg/mL) of extract prepared at 4 °C and all of the concentrations prepared at 22 ± 2 and 60 °C showed moderate genotoxic activity. To test the protective effect of the three concentrations of the extracts against the genotoxicity induced by methyl methanesulfonate, three protocols were used: pre-treatment, simultaneous-treatment and post-treatment. Treatments were repeated for all combinations of preparation temperature and concentration. Two extracts (22 ± 2 °C 1.0 mg/mL (simultaneous-treatment) and 4 °C 0.5 mg/mL (post-treatment)) showed antigenotoxic activity