Dry tobacco leaves: an in vivo and in silico approach to the consequences of occupational exposure.

Exposure of tobacco workers handling dried tobacco leaves has been linked to an increased risk of toxicity and respiratory illness due to the presence of nicotine and other chemicals. This study aimed to evaluate the DNA damage caused by the exposure of tobacco growers during the dry leaf classification process and the relation to cellular mechanisms. A total of 86 individuals participated in the study, divided into a group exposed to dry tobacco (n = 44) and a control group (n = 42). Genotoxicity was evaluated using the alkaline comet assay and lymphocyte micronucleus (MN) assay (CBMN-Cyt), and measurement of telomere length. The levels of oxidative and nitrosative stress were evaluated through the formation of thiobarbituric acid reactive species, and nitric oxide levels, respectively. The inorganic elements were measured in the samples using particle-induced X-ray emission method. The combination of variables was demonstrated through principal component analysis and the interactions were expanded through systems biology. Comet assay, MN, death cells, thiobarbituric acid reactive species, and nitrosative stress showed a significant increase for all exposed groups in relation to the control. Telomere length showed a significant decrease for exposed women and total exposed group in relation to men and control groups, respectively. Bromine (Br) and rubidium (Rb) in the exposed group presented higher levels than control groups. Correlations between nitrate and apoptosis; Br and MN and necrosis; and Rb and telomeres; besides age and DNA damage and death cells were observed. The systems biology analysis demonstrated that tobacco elements can increase the nuclear translocation of NFKB dimers inducing HDAC2 expression, which, associated with BRCA1 protein, can potentially repress transcription of genes that promote DNA repair. Dry tobacco workers exposed to dry leaves and their different agents showed DNA damage by different mechanisms, including redox imbalance.

Exposure in the tobacco fields: Genetic damage and oxidative stress in tobacco farmers occupationally exposed during harvest and grading seasons.

Agricultural workers engaged in tobacco cultivation are constantly exposed to large amounts of harmful agents, such as pesticides and nicotine. Furthermore, most of the flue-cured tobacco leaves are manually graded exposing workers to agents such as tobacco-specific nitrosamines. This study aimed to evaluate genetic damage and oxidative stress in tobacco farmers occupationally exposed during the harvest and grading seasons. We obtained data on DNA damage detected in Comet assay in blood cells and micronucleus experiment with buccal cells from 241 individuals. The serum cotinine levels and nitrates were also evaluated. The Comet Assay results showed a showed an increased visual score for males and females during harvest time and tobacco grading. An increase of micronucleated and binucleated cells was observed in the grading group compared to the control and harvest groups. The oxidative stress measurements showed a clear increase of thiobarbituric acid reactive substances (TBARS) in tobacco farmers during harvest time, and trolox equivalent antioxidant capacity (TEAC) in individuals during harvest and grading time compared to the controls. Significant increases of the cotinine levels were observed during the harvest and grading period (harvest>grading), and nitrates for the grading period compared to the control. In this study, tobacco farmers presented compromised DNA integrity associated with enhanced oxidative stress levels.

Genotoxic effect induced by dried nicotiana tabacum leaves from tobacco barns (kiln-houses) in chinese hamster lung fibroblast cells (V79).

Nicotiana tabacum is the most cultivated tobacco species in the state of Rio Grande do Sul, Brazil. Workers who handle the plant are exposed to the leaf components during the harvesting process and when separating and classifying the dried leaves. In addition to nicotine, after the drying process, other components may be found including tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, as well as pesticides residues. The objective of this study was to examine the genotoxicity attributed to the aqueous extract of dried tobacco leaves obtained from tobacco barns using Chinese hamster lung fibroblast cells (V79) as a model system by employing alkaline comet assay, micronucleus (MN) and Ames test. MTT assay was used to assess cytotoxicity and establish concentrations for this study. Data demonstrated cell viability > 85% for concentrations of 0.625-5 mg/ml while the comet assay indicated a significant increase in DNA damage at all concentrations tested. A significant elevation of MN and nuclear buds (NBUD) was found for 5 mg/ml compared to control and other dry tobacco leaves concentrations (0.625-2.5 mg/ml). Mutagenicity was not found using the Salmonella/Microsome test (TA98, TA100, and TA102 strains) with and without metabolic activation. The concentration of inorganic elements was determined employing the PIXE technique, and 13 inorganic elements were detected. Using CG/MS nicotine amounts present were 1.56 mg/g dry tobacco leaf powder. Due to the observed genotoxicity in V79 cells, more investigations are needed to protect the health of tobacco workers exposed daily to this complex mixture of toxic substances present in dry tobacco leaves.

Evaluation of soils under the influence of coal mining and a thermoelectric plant in the city of Candiota and vicinity, Brazil.

Coal burning generates gases, particles, and condensation by-products that are harmful to soil, water, and to the atmosphere. The aim of this study was to characterize and identify the cytotoxic and mutagenic potential of soil samples from the cities of Aceguá, Bagé, Candiota and Pinheiro Machado, near a large coal-fired power plant. Our study describes soil characteristics and contributes to the evaluation of the genotoxic activity of coal mining and burning, using the Comet Assay and Micronucleus test in V79 cells, as well as mutagenicity assays with Salmonella typhimurium strains. Comet Assay results show that the winter soil samples of Candiota and Pinheiro Machado induced a significant increase of the Damage Index for cells, as well as for the Aceguá summer sample. The micronucleus test did not detect differences between cities and seasons. A component analysis indicates associations between results obtained in Comet Assay and Ti and phenanthene concentrations for Pinheiro Machado during the winter, and Al for Aceguá during the summer and Zn during the winter. Results of Salmonella/microsome assays were negative, only Candiota and Pinheiro Machado samples showed a statistical increase of his + colonies in TA102. Our work describes biological data on these cells exposed to coal-contaminated soil, confirming the sensitivity of the Comet Assay in V79 cells and Salmonella/microsome assay for the evaluation of the effects of complex mixtures. These findings help to understand the spatial distribution of contaminants in the local soil related to a power plant, which is important for planning public safety actions.

Environmental exposure to mineral coal and by-products: Influence on human health and genomic instability.

Environmental exposure to pollution generated by mining and burning coal is inevitable for people living nearby. Therefore, the aim of this study was to evaluate the influence of coal dust on health conditions and genomic instability of individuals who live near coal mines and thermoelectric power plants, and to relate the results to inorganic elements and inflammatory responses. Thus, we evaluated 284 individuals from four cities in the south of Brazil around a region with coal mines and a thermoelectric power plant (one city was considered a negative control). The results of the Comet assay and Micronucleus (MN) test did not show a genotoxic or mutagenic effect related to environmental exposure to coal, but the inflammatory cytokine tumor necrosis factor-α (TNF-α) was increased in all cities around the power plant when compared to the control conditions. Higher levels of MN were associated with body mass index and cardiovascular risk, and higher levels of Damage Index (DI), TNF-α and interleukin1ß (IL-1ß) with number of cigarettes/day. Principal component analysis (PCA) was used to integrate DNA damage and inflammatory results with inorganic elements. This study also demonstrated the relationship between zinc and MN, copper, and interleukin10 (IL-10), and among silicon and sulfur with DI and nucleoplasmic bridge. A relationship was also observed between the reduction of inorganic elements and both aging and quality of health. The use of different methodologies and the relationship between the results obtained in these studies, including different lifestyles, can increase the understanding of the interaction between this mineral and the health status of residents of regions affected by coal pollution.

Evaluation of the cytotoxic and genotoxic effects of Sida planicaulis Cav extract using human neuroblastoma cell line SH-SY5Y.

Sida planicaulis is a weed thought to have originated in Brazil, where it is present in abundant quantities, but also this plant is also found in south-central Florida, Indian Ocean Islands, and the Pacific Islands. Sida planicaulis produces neurotoxicity that adversely affects livestock breeding with heavy animal losses and consequent negative impact on Brazil's economy. The aim of this study was to determine the chemical profile, cytotoxic and genotoxic effects of ethanolic extracts of S. planicaulis collected in winter (leaf extract) and summer (leaf extract and leaf + flower extract) using an in vitro model of human neuroblastoma cell line SH-SY5Y. Phytochemical screening demonstrated the presence of alkaloids, flavonoids, and apolar compounds. Rutin, quercetin, and swainsonine were detected by HPLC and GC/MS, respectively. Phosphorus, potassium, iron, and zinc were the inorganic elements found. Extracts produced cytotoxicity at all concentrations tested (7-4,000 µg/ml) as evidenced by the colorimetric assay [3-(4,5-dimethyl-thiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT)]. Based upon the alkaline comet assay extracts were found to induce genotoxicity at concentrations ranging from 0.437 to 7 µg/ml. DNA damage produced by extracts was affirmed using a modified comet assay with the enzymes Endo III and FPG in a concentration dependent manner. Further, enzyme-modified comet assay showed both oxidized purines and pyrimidines, and consequently oxidative stress was related to genomic instability and cell death. Data suggest that low concentrations of ethanolic extracts of S. planicaulis (different seasons) induced increased DNA damage related to oxidative stress and chemical composition.

Cytotoxic and genotoxic evaluation of cotinine using human neuroblastoma cells (SH-SY5Y).

Cotinine is the main metabolite of nicotine, which is metabolized in the liver through a cytochrome P450 enzyme. Different studies point to genetic instability caused by nicotine, such as single and double DNA strand breaks and micronuclei formation, but little is known about the effect of cotinine. Therefore, the present in vitro study assessed the effects of cotinine on cell viability and DNA damage in SH-SY5Y neuroblastoma cells, as well as genotoxicity related to oxidative stress mechanisms. Comparisons with nicotine were also performed. An alkaline comet assay modified by repair endonucleases (FPG, OGG1, and Endo III) was used to detect oxidized nucleobases. SH-SY5Y neuronal cells were cultured under standard conditions and exposed for 3 h to different concentrations of cotinine and nicotine. Cytotoxicity was observed at higher doses of cotinine and nicotine in the MTT assay. In the trypan blue assay, cells showed viability above 80% for both compounds. Alkaline comet assay results demonstrated a significant increase in damage index and frequency for cells treated with cotinine and nicotine, presenting genotoxicity. The results of the enzyme-modified comet assay suggest a DNA oxidative damage induced by nicotine. Unlike other studies, our results demonstrated genotoxicity induced by both cotinine and nicotine. The similar effects observed for these two pyridine alkaloids may be due to the similarity of their structures.

Low DNA damage in peripheral lymphocytes of healthy elderly individuals with inverted CD4:CD8 ratio.

The aim of this study was to evaluate the DNA damage in peripheral lymphocytes and the frequencies of CD8+ T cells expressing CD25, CD28 and CD45ro in aged individuals with inverted CD4:CD8 ratio. Blood samples of elderly individuals (aged >65) with normal CD4:CD8 ratio (n = 8) and inverted CD4:CD8 ratio (n = 8) were collected to identify the expression of CD25+, CD28+ and CD45ro+ in CD8+ T cells. DNA damage index was evaluated by the alkaline comet assay which was performed in lymphocytes treated with different concentrations of methyl methanesulfonate (MMS) (control non-treatment, 2 × 10-5 M, 4 × 10-5 M) for 1, 2 or 24 h. Elderly individuals with inverted CD4:CD8 ratio presented low frequency of CD8+ CD28+. Moreover, low DNA damage was observed in lymphocytes of elderly with inverted CD4:CD8 ratio in different doses of MMS. Aged individuals with inverted CD4:CD8 ratio presented lower DNA damage events in peripheral lymphocytes, suggesting a resistance for cell death in T cells of individuals with immune risk profile.

Amido Black 10B a widely used azo dye causes DNA damage in pro- and eukaryotic indicator cells.

Acid Black 10B (AB10B) is widely used for the production of textiles, leather and prints. It is a representative of azo dyes and it is well documented that some of these compounds are mutagenic per se, and that cleavage products (in particular aromatic amines) may cause damage of the genetic material and cancer. Since no toxicological data on AB10B have been published, we evaluated its mutagenic activity in Salmonella/microsome assays and studied its acute toxic and genotoxic properties in a human derived liver cell line (HepG2) which retained the activities of drug metabolizing enzymes. The compound did not cause cytotoxicity (MTT assay), but clear genotoxic effects were detected in pro- and eukaryotic indicator cells. Dose dependent induction of his+ revertants was seen in strain TA98 which detects frameshift mutations without metabolic activation; a more pronounced effect was seen in its derivative YG1024 which overexpresses N-acetyltransferase. Induction of single/double strand breaks by Comet assay was detected with concentrations > 0.125 mg/mL in liver derived cells; as well as increased rates for micronucleus (reflecting structural and numeric chromosomal aberrations) and nuclear buds which are a consequence of gene amplifications were seen with a higher dose (2.0 mg/mL) (p < 0.05; Tukey's test). The mutational pattern which was observed in the bacterial tests indicates that the cleavage product p-nitroaniline may cause the genotoxic effects of the dye. Our findings indicate that exposure of humans and the release of the compound into the environment may lead to adverse effects due to its DNA damaging activity.