Juliana da Silva

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, it s low cost and the short time required to obtain results. These properties make the SCG assay particularly useful in screening for environ mental genotoxicity. The present study describes a modified version of this technique for use in field work with native rodents and ex amines 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 genotoxic ity studies with natural populations, without the need to remove the animals from their habitat or to sacrifice them.

Genotoxicity biomonitoring in coal regions using wild rodent Ctenomys torquatus by Comet assay and micronucleus test

Coal is a mixture of a variety of chemicals, especially hydrocarbons, which may give rise to polycyclic aromatic hydrocarbons (PAH). Many PAH compounds produce mutagenic and carcinogenic effects. The quality of mineral coal in Rio Grande do Sul (RS) is low and it is typically obtained by stripping operations; it represents approximately 87% of the Brazil reserves. This report concerns the application of the Comet assay to Ctenomys torquatus to detect the effects of coal, comparing the results with a micronucleus (MN) assay, both using peripheral blood. This study was performed over a 2‐year period in an attempt to evaluate seasonal patterns. The wild rodent is fossorial, and its geographic distribution in RS coincides with the distribution of coal reserves. Three localitions were studied: two coal fields, Butiá (in a strip coal mine region) and Candiota (near a strip coal mine), and one control region, Pelotas (no coal). At the end of 2 years, 240 rodents had been analyzed. Our results showed that coal and derivatives induced DNA and chromosomal lesions in rodent cells that were demonstrated by Comet and MN assays. These tests also demonstrated quantitative differences between field exposures (Candiota > Butiá). The Comet assay was more sensitive and also showed a direct relationship between age and damage, and an inverse relationship between temperature and damage index. Environ. Mol. Mutagen. 35:270–278, 2000

Effects of chronic exposure to coal in wild rodents (Ctenomys torquatus) evaluated by multiple methods and tissues

Rio Grande do Sul (RS) coal is low quality and typically obtained by strip mining. In a recent study concerning 2 years of biomonitoring in coal regions, we demonstrated the genotoxicity of coal and related products on blood cells of native rodents, from RS, Brazil. With the goal of studying the variations in the effects of RS coal on different tissues of the same rodent, we utilized, besides the single cell gel (SCG) and micronucleus (MN) assay on blood, histological analyses and SCG assay of bone marrow, spleen, kidney, liver and lung cells, and MN assay of bone marrow and spleen cells. In addition, to identify agents that can potentially influence the results, concentrations of several heavy metals were analyzed in livers and in soil, and the total concentration of hydrocarbons in the soil was determined. Rodents exposed to coal were captured at two different sites, Butiá and Candiota, in RS. Reference animals were obtained from Pelotas, where there is no coal mining. This report provides chemical and biological data from coal regions, indicating the possible association between Zn, Ni, Pb and hydrocarbons in the induction of DNA damage (e.g. single strand-breaks and alkali-labile sites) determined by the alkaline SCG assay in cells from Ctenomys torquatus. The results of the present SCG study indicate that coal and by-products not only induce DNA damage in blood cells, but also in other tissue cells, mainly liver, kidney and lung. Neither the MN assay nor histopathological observations showed significant differences; these analyses may be useful under circumstances where genotoxicity is higher. In conclusion we believe that the in vivo genotoxicity of coal can be biomonitored by the SCG assay, and our studies suggest that wild rodents, such as C. torquatus are useful for monitoring genotoxic damage by both methods, the SCG assay and the MN test.

Effects of dermal exposure to Nicotiana tabacum (Jean Nicot, 1560) leaves in mouse evaluated by multiple methods and tissues.

Tobacco farmers are routinely exposed to complex mixtures of the compounds present in tobacco leaves, including organic and inorganic pesticides. Penetration through skin is the most significant route of uptake in occupational exposure to chemicals, including dust and liquids containing toxic and carcinogenic substances. This study evaluates the genotoxic effect of tobacco leaves with and without dermal exposure to flumetralin in Mus musculus, determining cell damage by the micronucleus test and the Comet assay as well as antioxidant enzyme activities and hematologic parameters. Nicotine was used as positive control. Blood samples were collected for 0, 3, 24 and 48 h exposure periods, and DNA damage by Comet assay and micronucleus test was evaluated for all these periods. Bone marrow and liver cells were also evaluated for the 48 h exposure period. Significant differences between Comet assay results in blood cells from animals exposed to tobacco leaves with and without pesticide were found in 24 and 48 h exposure periods in relation to negative control. Bone marrow cells from the group exposed to leaves with pesticide (48 h) also demonstrated significant increase in DNA damage. Concerning the micronucleus test, only animals exposed to tobacco leaves without pesticide (24 h) showed increase in frequency of micronuclei when compared to the negative control. Oxidative stress activities also were demonstrated for different groups. The results demonstrate the injury effect caused by tobacco leaves in different Mus musculus tissues, suggesting that the effects of dermal exposure to tobacco leaves are caused by complex mixtures present in the plant, but mainly by nicotine.

DNA damage and epigenetic alteration in soybean farmers exposed to complex mixture of pesticides

Exposure to pesticides can trigger genotoxic and mutagenic processes through different pathways. However, epidemiological studies are scarce, and further work is needed to find biomarkers sensitive to the health of exposed populations. Considering that there are few evaluations of soybean farmers, the aim of this study was to assess the effects of human exposure to complex mixtures of pesticides. The alkaline comet assay modified with restriction enzyme (hOGG1: human 8-oxoguanine DNA glycosylase) was used to detect oxidised guanine, and compared with the buccal micronucleus cytome assay, global methylation, haematological parameters, biochemical analyses (serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, gamma-glutamyl-transferase and butyrylcholinesterase), and particle-induced X-ray emission (PIXE) for the analysis of inorganic elements. Farm workers (n = 137) exposed to different types of pesticides were compared with a non-exposed reference group (control; n = 83). Results of the enzyme-modified comet assay suggest oxidation of guanine in DNA generated by pesticides exposure. It was observed that DNA damage (comet assay and micronucleus test) was significantly increased in exposed individuals compared to the unexposed group. The micronucleus test demonstrated elimination of nuclear material by budding, defective cytokinesis and dead cells. Occupationally exposed individuals also showed genomic hypermethylation of DNA, which correlated with micronucleus frequency. No differences were detected regarding the haematological and biochemical parameters. Finally, significantly higher concentrations of Al and P were observed in the urine of the soybean farmers. DNA damage could be a consequence of the ability of the complex mixture, including Al and P, to cause oxidative damage. These data indicate that persistent genetic instability associated with hypermethylation of DNA in soybean workers after long-term exposure to a low-level to pesticides mixtures may be critical for the development of adverse health effects such as cancer.

Unexpected chromosomal alterations in Tayassu tajacu (Artiodactyla: Tayassuidae) in captivity

Os animais silvestres tem sido utilizados como bioindicadores quando o ambiente e exposto a estressores quimicos. Em geral, os agentes quimicos podem induzir as alteracoes cromossomicas dos tipos falhas e quebras. Tayassu tajacu, e uma especie aparentada dos porcos verdadeiro e apresenta uma grande estabilidade cariotipica. As unicas alteracoes descritas sao em relacao a forma do cromossomo X. Foram observadas falhas e quebras cromossomicas durante as analise citogenetica. Estas alteracoes foram detectadas em cromossomos autossomicos. Levantamentos realizados na literatura associados as dados observados nos exemplares estudados, indicam um vermifugo, a base de ivermectina, como o possivel causador dessas alteracoes cromossomicas.

Genotoxic and chemopreventive assessment of Cynara scolymus L. aqueous extract in a human-derived liver cell line

Abstract Cynara scolymus L., popularly known as artichoke, is consumed as food and used as tea infusions for pharmacological purposes to treat liver dysfunctions and other conditions. Scientific data on the safety and protective effect of artichoke in human-derived liver cells is missing. This study investigated the genotoxic and modulatory effect of a liophilized extract suspended in water of C. scolymus L. leaves. Four extract concentrations (0.62, 1.25, 2.5 and 5.0 mg/mL) were evaluated using the comet assay on human hepatocyte cultures, HepG2 cells. Genotoxicity was assessed after two treatment periods, 1 and 24 h. Antigenotoxicity was evaluated against oxidative lesions induced by hydrogen peroxide in pre-, simultaneous and post-treatment protocols. Artichoke leaves aqueous extract induced genotoxic effects in HepG2 cells after 1- and 24-h treatments. In turn, extract concentrations of 0.62, 1.25 and 2.5 mg/mL, exhibited a protective effect in pretreatment, compared to hydrogen peroxide alone. However, in simultaneous and post-treatment protocols, only the lowest concentration reduced the frequency of DNA damage induced by hydrogen peroxide. In addition, in the simultaneous treatment protocol, the highest artichoke extract concentration increased hydrogen peroxide genotoxicity. It can be concluded that artichoke is genotoxic, in vitro, to HepG2 cells, but can also modulate hydrogen peroxide DNA damage.

Surface Water Impacted by Rural Activities Induces Genetic Toxicity Related to Recombinagenic Events in Vivo

This investigation assessed the interaction of surface water samples with DNA to quantitatively and qualitatively characterize their mutagenic and/or recombinagenic activity. Samples were obtained at three different sites along the Tocantins River (Tocantins State, Brazil). The area has withstood the impact mainly of rural activities, which release different chemical compounds in the environment. The Drosophila melanogaster Somatic Mutation and Recombination Test (SMART) was performed in standard (ST) and high bioactivation (HB) crosses. SMART is useful for the detection of mutational and recombinational events induced by genotoxins of direct and indirect action. Results demonstrated that samples collected in both seasons were able to induce increments on the mutant spot frequencies in the larvae of the HB cross. Genotoxicity was related to a massive recombinagenic activity. The positive responses ascribed to only the HB cross means that it is linked to pro-genotoxins requiring metabolic activation. The SMART wing test in Drosophila melanogaster was shown to be highly sensitive to detect genotoxic agents present in the aquatic environment impacted by agriculture.

Evaluation of Toxicological Effects of an Aqueous Extract of Shells from the Pecan Nut Carya illinoinensis (Wangenh.) K. Koch and the Possible Association with Its Inorganic Constituents and Major Phenolic Compounds

Background. Industrial processing of the pecan nut Carya illinoinensis K. Koch generated a large amount of shells, which have been used to prepare nutritional supplements and medicinal products; however, the safe use of shells requires assessment. This study evaluated the toxic, genotoxic, and mutagenic effects of pecan shell aqueous extract (PSAE) and the possible contribution of phenolic compounds, ellagic and gallic acids, and inorganic elements present in PSAE to induce toxicity. Results. Levels of inorganic elements like K, P, Cl, and Rb quantified using the Particle-Induced X-Ray Emission method were higher in PSAE than in pecan shells, while Mg and Mn levels were higher in shells. Mice showed neurobehavioral toxicity when given high PSAE doses (200–2,000 mg kg−1). The LD50 was 1,166.3 mg kg−1. However, PSAE (50–200 mg·kg−1) and the phenolic compounds (10–100 mg·kg−1) did not induce DNA damage or mutagenicity evaluated using the comet assay and micronucleus test. Treatment with ellagic acid (10–100 mg·kg−1) decreased triglyceride and glucose levels, while treatments with PSAE and gallic acid had no effect. Conclusion. Pecan shell toxicity might be associated with high concentrations of inorganic elements such as Mn, Al, Cu, and Fe acting on the central nervous system, besides phytochemical components, suggesting that the definition of the safe dose should take into account the consumption of micronutrients.