Genetic instability in farmers using pesticides: A study in Brazil with analysis combining alkaline comet and micronucleus assays.

The use of pesticides to prevent and control pests also increases food production. Pesticides are widely used by contemporary farmers, especially in Brazil, where the economy is based on agriculture. The objective of this study was to evaluate the genotoxic potential of pesticide use in rural workers in Maringá, Paraná, Brazil. DNA damage in whole blood cells was measured by the comet assay, while the frequency of cell types, abnormalities, and nuclear damage was estimated using the buccal micronucleus cytome assay. Samples of buccal mucosa were collected from 50 male volunteers (27 not exposed to pesticides and 23 occupationally exposed to pesticides). Among them, 44 volunteered for blood sampling (24 unexposed and 20 exposed). In the comet assay, the exposed farmers had a higher damage index than non-exposed ones. There were also statistically significant differences between the groups in the buccal micronucleus cytome assay. Farmers exhibited an increase in basal cell numbers, and cytogenetic alterations, represented by condensed chromatin and karyolitic cells. Comparisons between cell morphologies and epidemiological factors indicated an increased number of condensed chromatin and karyolitic cells in individuals who were responsible for preparation and transportation of pesticides to agricultural machines. Thus, the participants in this study who were exposed to pesticides were more sensitive to genetic damage, and thereby, more susceptible to diseases resulting from such damage. These results demonstrated that health policies should be developed for pesticide-exposed farmers to better mitigate risks and damage to their health.

Toxicogenic effects of the mushroom Ganoderma lucidum on human liver and kidney tumor cells and peripheral blood lymphocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum (Curtis) P. Karst., a bioactive mushroom with medicinal properties, is known to exert immunomodulatory, anti-inflammatory, hypocholesterolemic, hypoglycemic, and hepatoprotective effects. AIM OF THE STUDY: In this study, the effects of the G. lucidum fruiting body dry extract (GLE) on human liver (HepG2/C3A) and kidney (786-O) tumor cells and peripheral blood lymphocytes were evaluated. MATERIALS AND METHODS: MTT-based cytotoxicity, trypan blue-based cell viability, comet, and cytokinesis-block micronucleus cytome assays were performed, and the production of reactive oxygen species was evaluated in vitro. RESULTS: GLE was toxic to the tumor cells, decreasing their viability by increasing their production of reactive oxygen species and inducing damage to their DNA. By contrast, only high concentrations of GLE were toxic to lymphocytes and decreased their viability, whereas low concentrations increased lymphocyte viability. Moreover, primary DNA damage was induced by GLE only at the highest concentration tested. CONCLUSIONS: G. lucidum shows potential antitumor effects against cancerous kidney and liver cells, exhibiting cytotoxic and genotoxic activity at low concentrations, whereas the same effects in lymphocytes are mediated only at high concentrations. This mushroom has the potential to be biotechnologically developed into a therapeutic agent for diseases, such as cancer.

Buccal micronucleus cytome assay: Inter-laboratory scoring exercise and micronucleus and nuclear abnormalities frequencies in different populations from Brazil.

The Buccal Micronucleus Cytome Assay (BMCyt) has become an important biomonitoring tool for assessing cytogenetic damage in many studied populations. Each laboratory applies protocols that vary according to the method of collecting and preparing samples. Besides, Brazil is a country of great territorial extensions that received immigrants from various parts of the world with different genetic backgrounds. Therefore, the present study aimed to evaluate the inter-laboratory variation in scoring the same set of slides using the more comprehensive scoring criteria, to standardize the BMCyt protocol, to observe the basal alterations in populations of different Brazilian regions and to compare it with other places around the world. Our results showed that a valuable number of laboratories participated, ten laboratories from different regions of the country, for the validation of the BMCyt in human biomonitoring studies, resulting in the 804 healthy individuals. This was possible because we observed: a range of measures needs to be considered, such as the baseline frequency of DNA damage and cell death in non-exposed individuals; age when grouped showed an influence on DNA damage, although when evaluated by group we did not see an influence; association between smoking habit and all endpoints of the BMCyt (except karyolytic cells) was evident; the basal MN frequency, in the majority of groups, follows those around the world; and the BMCyt was confirmed as a good health status biomarker. We emphasize the need for constant discussions on the parameters of cell death due to greater difficulty among the analyzers.

Radioprotective efficacy of plastic polymer against the toxicogenomic effects of radiopharmaceutical 18F-FDG on human lymphocytes.

BACKGROUND: Healthcare workers occupationally exposed to 18F-FDG cannot wear protective equipment, such as lead aprons, since the interaction between high energy radiation (511 keV) and metal increases the dose of radiation absorption. The objective of this study was to evaluate the shielding efficacy of a plastic polymer against the toxicogenomic effects of ionizing radiation in human lymphocytes, using cytokinesis-block micronucleus assays. METHODS: Human peripheral blood lymphocytes were isolated from three subjects and cultured under standard conditions. The cultures were exposed to 300 mCi of 18F-FDG at a distance of 10 cm for 10 min, in the absence of shielding or with lead, polymer, and lead + polymer shields. RESULTS: Lead shielding was found to increase the number of counts detected by Geiger-Müller radiation monitors as a consequence of the photoelectron effect. Conversely, the lead + polymer shield reduced the number of counts. The lead, polymer, and lead + polymer shields significantly reduced the frequency of micronuclei, nucleoplasmic bridges, and nuclear buds induced by ionizing radiation. Regarding cytotoxicity, only the lead + polymer shield re-established the cell cycle at the level observed for the negative control. CONCLUSIONS: Lead aprons that are internally coated with polymer increased the radiological protection of individuals occupationally exposed to 18F-FDG PET/CT, especially during examinations.

Evaluation of hospital laundry effluents treated by advanced oxidation processes and their cytotoxic effects on Allium cepa L.

Hospital laundries are responsible for a significant part of the amount of wastewater that is generated in hospitals. Hospital laundry wastewater represents a complex mixture of chemicals that arouse concerns about possible environmental risks. The objective of the present study was to evaluate the cytotoxicity of different laundry effluents from the Regional University Hospital of Maringá, Paraná, Brazil, on Allium cepa L. meristematic root cells. The effluents were characterised as rinsing, wetting, prewashing, washing, softening, wastewater (the effluent generated at the end of the washing process), the wastewater that was treated by physicochemical (PC) processes and the wastewater that was treated by advanced oxidation processes (PC + UV, PC + H2O2 and PC + UV/H2O2). The mitotic indexes were calculated by scoring 5000 cells per group and the statistical analyses were performed by one-way ANOVA, followed by Tukey's post-test (α = 0.05). Results showed that the rinsing, wetting, prewashing and wastewater laundry effluents were cytotoxic at 24 h of exposure, significantly reducing the mitotic index. Despite the slight cytotoxicity of the PC + UV/H2O2 treatment, physicochemical and advanced oxidation processes efficiently reduced the critical parameters of wastewater, such as the biochemical and chemical oxygen demands, to tolerable levels of effluent discharge. It is essential to perform constant monitoring of these effluents in order to reduce the possible occurrence of environmental impacts.

Evaluation of textile laundry effluents and their cytotoxic effects on Allium cepa.

Industrial laundries have water as one of their main inputs and they release effluents in large amounts, with a high polluting load, which are usually discarded into the environment, or they are insufficiently treated for release into the neighboring water bodies. The aim of this study was to evaluate the efficiencies of the biological treatments in an industrial textile laundry and their environmental impact on the surface waters of the stream where the dump is usually made, by using cytotoxicity tests on the meristematic root cells of Allium cepa L. The results have shown, for the most part, that the treated effluents over a period of 24 h showed reductions in their mitotic index. The treatments on the raw effluents showed cytotoxic effects when compared to control, with cell division recoveries after 24 h in the waters. Cytotoxic effects were additionally observed in the stream waters, at a point before the dump, indicating that they received a pollutant load, before the effluent disposal site of the evaluated industrial laundry. Notably, the treatments that were being applied by the industrial laundry were effective throughout the processing, reducing the concentrations of the toxic substances. When considering the data presented, it is now understood that there is a constant need for the evaluation of industrial effluents, as well as for the waters of the streams and the rivers that receive these disposals, in order to preserve and maintain the quality of the waters, the organisms, and consequently, the ecosystems.

Cytotoxicity, mutagenicity, and antimutagenicity of the gentisic acid on HTC cells.

Gentisic acid (GA) exhibits antioxidant, anti-inflammatory, and antibiotic activities. This substance can be found in citrus fruits, grapes, olive oil, and peas. Considering that there are few studies in the literature on the toxicity of GA, the present work aimed to investigate its cytotoxic, mutagenic, and antimutagenic activities on HTC cells. GA was diluted in culture medium at the final concentration of 0.08, 0.16, 0.8, 1.6, and 8 µg/mL. The cytotoxicity was determined by the MTT assay and Trypan Blue exclusion method, with methyl methanesulfonate and doxorubicin as positive controls, respectively. The cytokinesis-block micronucleus assay determined the mutagenic/antimutagenic activity with benzo[a]pyrene as positive control. Negative control received culture medium only. GA (0.08-8 µg/mL) was not cytotoxic to HTC cells by the MTT assay nor the Trypan Blue exclusion method as no statistical difference was observed when compared to the control. Concentration of 0.08 and 0.8 µg/mL showed no mutagenic or clastogenic effects, as no significant micronuclei inductions were observed, different from 8 µg/mL, that was mutagenic. Furthermore, none of the concentrations presented an antiproliferative activity. The antimutagenic activity of GA (0.08 µg/mL) was observed at the simultaneous treatment, as it reduced the frequency of micronuclei by 76% (24 h) and 79% (48 h). Although pre- and post-treatments were not statistically different from the mutagen, they reduced the induced-damage by 11% and 21%, respectively. The present study indicated the absence of cytotoxicity and antiproliferative activities of GA, in addition to their antimutagenic/protective effects that may contribute to human health.

Genotoxicity and Antigenotoxicity Assessments of the Flavonoid Vitexin by the Drosophila melanogaster Somatic Mutation and Recombination Test.

Vitexin is a C-glycosylated flavone found in various medicinal plants with several proven biological properties such as anti-inflammatory, antispasmodic, antimicrobial, cytotoxic, and antioxidant activities. Considering that the human population consumes many foods that contain this flavonoid, the objective of this study was to investigate the genotoxic and the antigenotoxic potentials of vitexin by the SMART (Somatic Mutation and Recombination Test) in Drosophila melanogaster wings, in vivo. To evaluate the genotoxic activity, larvae obtained from standard (ST) and high bioactivation (HB) crosses were chronically treated with different concentrations of vitexin (0.15, 0.3, and 0.6 mM). For the evaluation of antigenotoxic activity, the same vitexin concentrations were associated with two damage inducing agents: doxorubicin (0.2 mM) and benzo[a]pyrene (1.0 mM). The results obtained for genotoxic activity showed that vitexin did not induce a statistically significant increment in the frequency of mutant spots, when compared to control. The results for the evaluation of antigenotoxicity indicated that the flavonoid statistically reduced the frequency of mutant spots, when compared to those treated with only the damage inducing agents. Thus, this article presents results that have demonstrated the antigenotoxic activity of vitexin, which could be applied in new studies for the development of drugs with chemoprotective effects.

ß-(1→3,1→6)-d-glucans produced by Diaporthe sp. endophytes: Purification, chemical characterization and antiproliferative activity against MCF-7 and HepG2-C3A cells.

This study reports the characterization and antiproliferative activity of exopolysaccharides (EPS) produced by submerged cultures of the endophytes Diaporthe sp. JF766998 and Diaporthe sp. JF767007 isolated from the medicinal plant Piper hispidum Sw. Both strains secreted a crude EPS that, upon size exclusion chromatography, showed to contain a heteropolysaccharide (galactose, glucose and mannose) and a high-molecular weight glucan. Data from methylation analysis, FTIR and NMR spectroscopy (1H, COSY, TOCSY and HSQC-DEPT) indicated that the purified glucan consisted of a main chain of glucopyranosyl ß-(1→3) linkages substituted at O-6 by glucosyl residues. According to MTT assay, some treatments of both ß-glucans have antiproliferative activity against human breast carcinoma (MCF-7) and hepatocellular carcinoma (HepG2-C3A) cells after 24 and 48h of treatment, exhibiting a degree of inhibition ratio that reached the highest values at 400µg/mL: 58.0% (24h) and 74.6% (48h) for MCF-7 cells, and 61.0% (24h) and 83.3% (48h) for HepG2-C3A cells. These results represent the first reports on the characterization and antiproliferative effect of ß-glucans from Diaporthe species and also expand the knowledge about bioactive polysaccharides from endophytic sources.

Citotoxicidade das águas do Rio do Peixe (São Paulo-Brasil), em células meristemáticas de raiz de Allium cepa L / Cytotoxicity of waters of the River Peixe (São Paulo-Brazil), in meristematic root cells of Allium cepa L

Nas últimas décadas, a qualidade da água do Rio do Peixe, localizado no interior do Estado de São Paulo, está diminuindo, principalmente porque o rio vem sofrendo com o desmatamento feito às suas margens, o que provocou o assoreamento e permitiu o despejo de todos os tipos de resíduos, em especial das indústrias que ficam ao seu redor. Assim, o objetivo deste trabalho foi avaliar o potencial citotóxico das águas do Rio do Peixe, coletadas em quatro propriedades rurais localizadas em Garça­SP (nascente), Tupã-SP, Flórida Paulista-SP e Ouro Verde-SP (jusante), usando como sistema teste vegetal as células meristemáticas de raiz de Allium cepa L. Os resultados obtidos mostraram que todas as amostras das águas coletadas não apresentaram efeito citotóxico após 24 horas de tratamento. Entretanto, as amostras obtidas em Tupã, Flórida Paulista e Ouro Verde (jusante) foram mutagênicas, pois causaram o aparecimento de alterações nas células, principalmente, dos tipos metáfases-colchicínicas, anáfases desorganizadas, multipolares e com ponte cromatídica, além de células micronucleadas. Portanto, os resultados obtidos alertam para o perigo eminente que os efluentes lançados indiscriminadamente no ambiente, de rios e lagos, podem representar não só aos organismos que ali habitam, mas a todos os outros que dele dependem, seja para lazer, limpeza, higiene ou alimentação.

(Cytotoxicity of waters of the River Peixe (São Paulo-Brazil), in meristematic root cells of Allium cepa L.) In recent decades, the water quality of the River Peixe, located within the State of São Paulo, has been decreasing, mainly because the river has suffered from deforestation made to its banks, causing silting and allowed the dumping of all types of waste, especially in industries that are around you. The objective of this study was to evaluate the cytotoxic potential of the waters of the River Peixe, collected at four farms located in Garça-SP (source), Tupã-SP, Flórida Paulista-SP and Ouro Verde-SP (downstream), using as a system test plant root meristematic cells of Allium cepa L. The results showed that all water samples collected was no cytotoxic effect after 24 hours of treatment. However, samples taken in Tupã, Flórida Paulista and Ouro Verde (downstream) were mutagenic, because it caused the appearance of changes in cells, mainly types of colchicine-metaphase, disorganized anaphase, multipolar and with chromatid bridge, addition of micronucleated cells. Therefore, the results warn of imminent danger that the effluents discharged indiscriminately into the environment may represent not only the organisms that live there, but for all others who depend on it, whether for leisure, cleaning, hygiene or food.

Effect of processing, post-harvest irradiation, and production system on the cytotoxicity and mutagenicity of Vitis labrusca L. juices in HTC cells.

The juices of grapes (Vitis labrusca L.) are similar to the fruit itself because the main constituents of the fruit are present in the juice. However, their quality characteristics may be modified by the harsh technological processes used for the production of integral food, such as production systems of raw materials and post-harvest treatment of grapes with ultraviolet (UV) irradiation. Therefore, the present study analyzed juices produced naturally (by liquefying the fruit) or by the technological process of extraction by steam distillation (90°C) of grapes from organic and conventional production systems that were untreated or treated with UV type C (65.6 J/m² for 10 minutes). Using cultures of Rattus norvegicus hepatoma cells (HTC) in vitro, cytotoxic effects were assayed by the MTT test and by calculating the cytokinesis blocked proliferation index (CBPI), and mutagenic effects were measured by the cytokinesis block micronucleus assay. The results of the MTT assay and the CBPIs indicated that none of the juices were cytotoxic, including those that induced cell proliferation. The results of the micronucleus assay showed that none of the juices were mutagenic. However, the average number of micronuclei was lower in the juices produced from organic grapes, and cell proliferation, soluble acids and phenolic compounds were significantly higher. Compared with the natural juices, the integral juices of conventional grapes showed a higher average number of micronuclei as well as lower stimulation of cell proliferation and lower levels of bioactive compounds. The results demonstrate a beneficial effect of UV-C irradiation of post-harvest grapes in stimulating the synthesis of nutraceutical compounds without generating cytotoxic or mutagenic substances. Taken together, our findings support the consumption of grape juice and the application of food production techniques that enhance its nutritional value and promote its production, marketing and consumption.

Evaluation of cytotoxicity and mutagenicity of the benzodiazepine flunitrazepam in vitro and in vivo

Flunitrazepam (FNZ) is a sedative benzodiazepine prescribed for the short-term treatment of insomnia. However, there are concerns regarding possible carcinogenic or genotoxic effects of this medicine. Thus, the aim of this study was to evaluate the cytotoxic, clastogenic and aneugenic effects of FNZ in hepatoma cells from Rattus norvegicus (HTC) in vitro and in bone marrow cells of Wistar rats in vivo. These effects were examined in vitro following treatment with 0.2, 1.0, 5.0 or 10 μg/mL FNZ using a micronucleus test with a cytokinesis block or in vivo using a chromosomal aberration test following treatment with 7, 15 or 30 μg/mL/kg body weight. The results showed that the benzodiazepine concentrations tested were not cytotoxic, aneugenic or clastogenic. However, considering the adverse effects of using this benzodiazepine, more studies are required.

Flunitrazepam (FNZ) é um sedativo benzodiazepínico prescrito para o tratamento da insônia em curto prazo. Entretanto, existe a preocupação com relação aos possíveis efeitos carcinogênicos ou genotóxicos causados por este fármaco. Então, o objetivo deste estudo foi avaliar os efeitos citotóxicos, clastogênicos e aneugênicos do FNZ em células de hepatoma de Rattus norvegicus (HTC) in vitro e em células de medula óssea de ratos Wistar in vivo. Foram testadas as concentrações de 0,2, 1,0 e 10 μg/mL de FNZ pelo teste do micronúcleo com bloqueio de citocinese in vitro e 7, 15 e 30 μg/mL/kg de peso corpóreo para o teste de aberração cromossômica in vivo. Os resultados mostraram que as concentrações do benzodiazepínico testadas não foram citotóxicas, aneugênicas ou clastogênicas. Entretanto, considerando os efeitos adversos do uso deste benzodiazepínico, mais estudos são necessários.

Evaluation of the cytotoxicity, mutagenicity and antimutagenicity of a natural antidepressant, Hypericum perforatum L. (St. John's wort), on vegetal and animal test systems.

BACKGROUND: St. John's wort (Hypericum perforatum L.) is an herbaceous plant that is native to Europe, West Asia and North Africa and that is recognized and used worldwide for the treatment of mild and moderate depression. It also has been shown to be therapeutic for the treatment of burns, bruises and swelling and can be used for its wound healing, antiviral, antimicrobial, antioxidant, analgesic, hepato-protective and anxiolytic properties. The aim of this study was to evaluate the potential cytotoxic, mutagenic and antimutagenic action of H. Perforatum. METHODS: Meristematic cells were used as the test system for Allium cepa L., and bone marrow cells from Rattus norvegicus, ex vivo, were used to calculate the mitotic index and the percentage of chromosomal aberration. Statistical analysis was performed using the chi-square test. RESULTS: This medicinal plant had no cytotoxic potential in the vegetal test system evaluated. In the animal test system, none of the acute treatments, including intraperitoneal gavage and subchronic gavage, were cytotoxic or mutagenic. Moreover, this plant presented antimutagenic activity against the clastogenic action of cyclophosphamide, as confirmed in pre-treatment (76% reduction in damage), simultaneous treatment (95%) and post-treatment (97%). CONCLUSIONS: Thus, the results of this study suggest that the administration of H. perforatum, especially by gavage similar to oral consumption used by humans, is safe and with beneficial antimutagenic potential.

Antimutagenic Effect of Medicinal Plants Achillea millefolium and Bauhinia forficata In Vivo.

The investigation of traditionally used medicinal plants is valuable both as a source of potential chemotherapeutic drugs and as a measure of safety for the continued use of these medicinal plants. Achillea millefolium L. (AM) is an ancient remedial herb native to Europe that is used to treat wounds, gastrointestinal and hepatobiliary disorders, inflammation, headaches, and pain. Bauhinia forficata Link (BF), an Asiatic plant, is one of the most commonly used plants in folk medicine against diabetes. The aim of this study was to evaluate the cytotoxic and antimutagenic potential of aqueous extracts of AM and BF on bone marrow cells of Wistar rats treated in vivo. These plant extracts possess considerable antioxidant activity due to the presence of flavonoids and phenolic compounds. These compounds were determinants to noncytotoxic and antimutagenic/protective action of these plants, that reduced statistically the percentage of chromosomal alterations induced by the chemotherapeutic agent cyclophosphamide in simultaneous (AM, 68%; BF, 91%), pre- (AM, 68%; BF, 71%), and post-treatment (AM, 67%; BF, 95%). Therefore, the results of this study indicate that extracts of A. millefolium and B. forficata have antimutagenic potential and that their consumption can benefit the health of those using them as an alternative therapy.