Occupational exposure to pesticides: DNA damage in horticulturist from Nativitas, Tlaxcala in Mexico.

Mexico is a country where agricultural activity is of great importance, but biomonitoring data are still scarce. With more intensive pesticides use per unit area/surface in horticultural productivity, there is a higher impact on environmental contamination and workers' health. Considering that exposure to various pesticide and pesticide mixtures represents an additional genotoxic risk, the appropriate characterization of exposure, confounding factors and the risk itself are very much needed. We compared genetic damage in 42 horticulturists and 46 unexposed controls (Nativitas, Tlaxcala) using alkaline comet (whole blood) and micronucleus (MN) test with nuclear abnormalities (NA) (buccal epithelial cells). Workers demonstrated significantly higher levels of damage (TI%=14.02 ± 2.49 vs. 5.37 ± 0.46; MN=10.14 ± 5.15 vs. 2.40 ± 0.20), with more than 90% of them not using protective clothing nor gloves during application. Combined DNA damage techniques and periodic monitoring together with educational programs for safe pesticide application is the best strategy to assess and prevent workers' health risks.

The wild plant Gnaphalium lavandulifolium as a sentinel for biomonitoring the effects of environmental heavy metals in the metropolitan area of México Valley.

Biomonitoring is a valuable tool for assessing the presence and effects of air pollutants such as heavy metals (HM); due to their toxicity and stability, these compounds can affect human health and the balance of ecosystems. To assess its potential as a sentinel organism of HM pollution, the wild plant Gnaphalium lavandulifolium was exposed to four sites in the metropolitan area of México Valley (MAMV): Altzomoni (ALT) Coyoacán (COY), Ecatepec (ECA), and Tlalnepantla (TLA) during 2, 4, and 8 weeks, between October and November 2019. Control plants remained under controlled conditions. The chemical analysis determined twelve HM (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, V, and Zn) in the leaves. Macroscopic damage to the leaves, later determined in semi-thin sections under light microscopy, lead to a finer analysis. Transmission electron microscope (TEM) showed major structural changes: chromatin condensation, protoplast shrinkage, cytoplasm vacuolization, cell wall thinning, decreased number and size of starch grains, and plastoglobules in chloroplasts. All these characteristics of stress-induced programed cell death (sPCD) were related to the significant increase of toxic HM in the leaves of the exposed plants compared to the control (p < 0.05). Immunohistochemistry revealed a significant amount of proteases with caspase 3-like activity in ECA and TLA samples during long exposure times. Ultrastructural changes and sPCD features detected confirmed the usefulness of G. lavandulifolium as a good biomonitor of HM contamination. They supported the possibility of considering subcellular changes as markers of abiotic stress conditions in plants.

Robinsonecio gerberifolius as a sentinel organism for atmospheric pollution by heavy metals in several sites of Mexico city and its metropolitan area.

Evaluate the effect of heavy metals (HM) on sentinel organisms such as vascular plants represent a model to estimate toxic hazard due to environmental pollution. In the present study, the plant Robinsonecio gerberifolius was used to evaluate the toxic effects of the HM contained in the leaves of plants that were exposed to 4 different sites in Mexico city and its metropolitan area, during the rainy and dry seasons in the period 2017-2019. The comet assay to evaluate genotoxicity revealed an increase with respect to control (p < 0.05), in 2nd and 8th week of exposure, in all 4 study sites and in both seasons, more significant in the rainy period. An increase in the induction of oxidative stress was also observed in the exposed leaves from the 4 study sites when compared with the control; in some cases, the increases were significant (p < 0.05). In general, α- and ß-carotenoids were increased at 8th week of exposure, in all plants exposed in both seasons, while miR398 increased in plants exposed in 2 study sites (p < 0.05). Finally, toxic HM like aluminum, vanadium, and cadmium, increased significantly in the rainy season, while lead increased in the dry season. We conclude that R. gerberifolius can be considered a sentinel plant for evaluating the presence and general toxic effects caused by the presence of toxic HM that have been documented in the atmosphere of Mexico City and its metropolitan area.

Moss (Hypnum amabile) as biomonitor of genotoxic damage and as bioaccumulator of atmospheric pollutants at five different sites of Mexico City and metropolitan area.

Mexico City has been classified as one megacity, its altitude, thermal inversions, and high seasonal radiation are factors that prevent dispersion of pollutants, which effects are detrimental to health. Therefore, it is important to have an organism that allows evaluate the damage caused by such exposure, as is the case of mosses that obtain nutrients from the atmosphere; this property makes them excellent biomonitors to evaluate genotoxic damage caused by exposure to pollutants, in addition to its large accumulation capacity. For these reasons and to relate the effects of atmospheric pollution with a biological response, we propose to use the moss Hypnum amabile as a bioaccumulator of atmospheric pollutants and biomonitor of the genotoxic effect that the air pollution can induce it through the comet assay. Mosses were placed in five localities of Mexico City and the metropolitan area on the first days of each month of the dry (cold and warm) and rainy seasons, with a 30-day exposure, after which they were changed for a new sample (for 8 months). Each month, the moss exposed was collected and nuclei were isolated to perform comet assay. To demonstrate heavy metal bioaccumulation capacity, samples were observed in a transmission electron microscope and qualitative microanalysis by scanning electron microscopy was carried out parallel. The chemical analysis detected 14 heavy metals by mass spectrometry method with inductively coupled plasma source. Additionally, 22 polycyclic aromatic hydrocarbons were also determined by gas chromatography-mass spectrometry. Analysis of variance and Kruskal-Wallis test were performed to compare DNA damage of each station against control, which was maintained in the laboratory in a chamber with filtered air. This is the first study on the genotoxicity of mosses exposed to the atmosphere of Mexico City and metropolitan area that in addition to proving their accumulation capacity shows their ability to respond to atmospheric pollutants.

In vitro cytotoxicity and genotoxicity of Furia®180 SC (zeta-cypermethrin) and Bulldock 125®SC (ß-cyfluthrin) pyrethroid insecticides in human peripheral blood lymphocytes.

In the present study, human peripheral blood lymphocytes were exposed in vitro to 0, 6, 12, 18, 24, and 30 µg/mL Furia®180 SC (zeta-cypermethrin) and 0, 6.3, 12.5, 18.8, 25, and 31.3 µg/mL Bulldock®125 SC (ß-cyfluthrin). Exposure to 32 µg/mL bleomycin for 24 h served as a positive control. The cytotoxic and genotoxic effects of each insecticide were analyzed using alkaline comet and trypan blue dye exclusion assays. DNA damage was evaluated through three genotoxicity parameters: tail length (TL), tail moment (TM) and tail intensity (TI). Furia®180 SC and Bulldock®125 SC pyrethroid insecticides and bleomycin significantly increased DNA damage in a concentration-dependent manner. Bulldock®125 SC induced more DNA damage than Furia. Lymphocyte viability did not change after exposure to different concentrations of the two pyrethroid insecticides and bleomycin. Moreover, genotoxic results demonstrated that Furia®180 SC and Bulldock®125 SC insecticides caused in vitro DNA damage in human peripheral lymphocytes.

Indicators of environmental contamination by heavy metals in leaves of Taraxacum officinale in two zones of the metropolitan area of Mexico City.

The present study was designed to detect the effect of heavy metals in two zones of the Metropolitan Area of Mexico City (MAMC), the Centro de Ciencias de la Atmósfera (CCA), and the Altzomoni station in the Iztaccíhuatl-Popocatépetl National Park. Taraxacum officinale was selected as the indicator organism of responses to atmospheric contamination by heavy metals. Determinations of heavy metals were performed, and total mRNA was extracted to quantify the expression of microRNA398 (miR398), superoxide dismutase 2 (CSD2), and the amounts of free radicals using the bromide of 3-(4,5-dimethylthiazole-2-ilo)-2,5-diphenyltetrazole (MTT) salts reduction assay. Results from the Altzomoni station showed high concentrations of five heavy metals, especially Aluminum, while three heavy metals were identified in the CCA-UNAM zone, most importantly, Vanadium, both in the dry season; miR398 expression presented subtle changes but was greater in the leaves from the stations with higher concentrations of heavy metals. Observations included a significant expression of CSD2, mainly in the dry season in both study zones, where levels were significant with respect to controls (p < 0.05). Reduced MTT was also higher in the dry season than in the rainy season (p < 0.05). In conclusion, the increase in heavy metals on the leaves of Taraxacum officinale induces increased expression of the CSD2 gene and reduced MTT; thus, they can be used as indicators for biomonitoring heavy metal concentrations.

Cytogenotoxicity of selected organophosphate insecticides on HaCaT keratinocytes and NL-20 human bronchial cells.

Organophosphate insecticides (OI) are widely used. To humans the main routes of exposure are skin and inhalation. For this, keratinocytes (HaCaT) and bronchial cells (NL-20) were used as cell culture models to evaluate the effects of OI. The aim of this study was to evaluate the effect of four OI on HaCaT and NL-20 cells: azinphos-methyl, (AM); parathion-methyl (PM); omethoate (OM); and methamidophos (MET). Cells were exposed to 0.1, 1 and 10 µg/µL of each. Results showed a decrease in cell viability in both cell lines. Viability of the NL-20 cell line decreased with the three concentrations of OM. All differences were significant (p < 0.05). Genotoxic damage, evaluated through the comet assay, was observed in both cell lines with AM. NL-20 cell line was more sensitive than HaCaT. Higher concentrations of the insecticides except MET, induced cell death. MET caused DNA damage in HaCaT cells at all concentrations. Differences were significant (p < 0.05). Both cell lines revealed the presence of single membrane vacuoles of different sizes when exposed to 1 µg/µL of each insecticide. Quantitative real time-polymerase chain reaction (RT-qPCR) showed an increase of BN1 gene in HaCaT by effect of AM and MET at 1 µg/µL. In conclusion, all the insecticides induced different levels of cyto and genotoxic effects in both cell lines.

Aflatoxin (B1 , B2 , G1 , and G2 ) contamination in rice of Mexico and Spain, from local sources or imported.

Rice is an important cereal but it is often contaminated with aflatoxins (AFs). The purpose of this study was to identify and quantify AF (B1 , B2 , G1 , and G2 ) in 67 rice samples cultivated in Mexico and Spain, and from imported crops collected in 2008 and 2009. The methodology was validated, the rice samples were concentrated and purified with immunoaffinity columns and were quantified by high-pressure liquid chromatography (HPLC). The average total AF (AFt) in the Spanish rice was 37.3 µg/kg, the range was from 1.6 to 1383 µg/kg, the most contaminated samples being from San Juan de Aznalfarache, Sevilla (AFt = 138.6 µg/kg), from Tortosa, Tarragona (AFt = 104.6 µg/kg), and Calasparra, Murcia (AFt = 103.9 µg/kg). The rice imported from France to Spain had AFt of 26.6 µg/kg and from Pakistan AFt of 18.4 µg/kg, showing less AF contamination than the local one. The rice which originated from Mexico contained (AFt = 16.9 µg/kg), and those imported from the United States (AFt = 14.4 µg/kg) and Uruguay (AFt = 15.6 µg/kg). The imported rice had better quality in terms of the presence of AFs.

The role of plant metabolism in the mutagenic and cytotoxic effects of four organophosphorus insecticides in Salmonella typhimurium and in human cell lines.

This study used a cell/microbe co-incubation assay to evaluate the effect of four organophosphorus insecticides (parathion-methyl, azinphos-methyl, omethoate, and methamidophos) metabolized by coriander (Coriandrum sativum). The reverse mutation of Salmonella typhimurium strains TA98 and TA100 was used as an indicator of genetic damage. Treatments with these insecticides inhibited peroxidase activity in plant cells by between 17% (omethoate) and 98% (azinphos-methyl) and decreased plant protein content by between 36% (omethoate) and 99.6% (azinphos-methyl). Azinphos-methyl was the most toxic when applied directly. In the Ames test, treatments applied directly to strain TA100 killed the bacteria; however, the presence of plant metabolism detoxified the system and permitted the growth of bacteria. In strain TA98, plant metabolites of insecticides were mutagenic. This result suggests that the tested pesticides produce mutations through frameshifting. The same pesticides were applied to human skin (HaCaT) and lung (NL-20) cell lines to evaluate their effects on cell viability. Pesticides applied directly were more cytotoxic than the combination of pesticide plus coriander metabolic fraction. Omethoate and methamidophos did not affect the viability of HaCaT cells, but azinphos-methyl and parathion-methyl at 100 and 1000µgmL(-1) significantly decreased viability (p<0.05). The NL-20 cell line was remarkably sensitive to the direct application of insecticides. All of the treatment conditions caused decreases in NL-20 cell viability (e.g., viability decreased to 12.0% after parathion-methyl treatment, to 14.7% after azinphos-methyl treatment, and to 6.9% after omethoate treatment). Similar to the Ames test, all of the insecticides showed decreased toxicity in human cells when they were cultured in the presence of plant metabolism. In conclusion, when the studied organophosphorus insecticides were plant-metabolized, they induced mutations in the bacterial strain TA98. In human cell lines, plant metabolism reduced the cytotoxic properties of the insecticides, and human keratinocytes were more resistant to mortality than bronchial cells.

Evaluation of genotoxic and cytotoxic effects in human peripheral blood lymphocytes exposed in vitro to neonicotinoid insecticides news.

Calypso (thiacloprid), Poncho (clothianidin), Gaucho (imidacloprid), and Jade (imidacloprid) are commercial neonicotinoid insecticides, a new class of agrochemicals in México. However, genotoxic and cytotoxic studies have not been performed. In the present study, human peripheral blood lymphocytes (PBL) were exposed in vitro to different concentrations of the four insecticides. The genotoxic and cytotoxic effects were evaluated using the alkaline comet and trypan blue dye exclusion assays. DNA damage was evaluated using two genotoxicity parameters: tail length and comet frequency. Exposure to 9.5 × 10(-6) to 5.7 × 10(-5) M Jade; 2.8 × 10(-4) to 1.7 × 10(-3) M Gaucho; 0.6 × 10(-1) to 1.4 × 10(-1) M Calypso; 1.2 × 10(-1) to 9.5 × 10(-1) M Poncho for 2 h induced a significant increase DNA damage with a concentration-dependent relationship. Jade was the most genotoxic of the four insecticides studied. Cytotoxicity was observed in cells exposed to 18 × 10(-3) M Jade, 2.0 × 10(-3) M Gaucho, 2.0 × 10(-1) M Calypso, 1.07 M Poncho, and cell death occurred at 30 × 10(-3) M Jade, 3.3 × 10(-3) M Gaucho, 2.8 × 10(-1) M Calypso, and 1.42 M Poncho. This study provides the first report of genotoxic and cytotoxic effects in PBL following in vitro exposure to commercial neonicotinoid insecticides.

Metabolic activation of herbicide products by Vicia faba detected in human peripheral lymphocytes using alkaline single cell gel electrophoresis.

Ametryn and metribuzin S-triazines derivatives and EPTC thiocarbamate are herbicides used extensively in Mexican agriculture, for example in crops such as corn, sugar cane, tomato, wheat, and beans. The present study evaluated the DNA damage and cytotoxic effects of three herbicides after metabolism by Vicia faba roots in human peripheral lymphocytes using akaline single cell gel electrophoresis. Three parameters were scored as indicators of DNA damage: tail length, percentage of cells with DNA damage (with comet), and level DNA damage. The lymphocytes were treated for 2 h with 0.5-5.0 microg/ml ametryn or metribuzin and 1.5-10 microg/ml EPTC. Lymphocytes also were coincubated for 2 h with 20 microl V. faba roots extracts that had been treated for 4 h with 50-500 mg/l of the two triazines or with the thiocarbamate herbicide or with ethanol (3600 mg/l), as positive control. The lymphocytes treated with three pesticides without in vivo metabolic activation by V. faba root did not show significant differences in the mean values between genotoxic parameters compared with negative control. But when human cells were exposed to three herbicides after they had been metabolized the frequency of cell comet, tail length and level DNA damage all increased. At highest concentrations of the three herbicides produced severe DNA damage compared with S10 fraction and negative control. The linear regression analysis of the tail length values of three herbicides indicated that there was genotoxic effect concentration-response relationship with ametryn and ametribuzin but no EPTC. The ethanol induced major increase DNA damage compared with S10 fraction and the three pesticides. There were not effects in cell viability with treatment EPTC and metribuzin whether or not it had been metabolized. High concentrations of ametryn alone and after it had been metabolized decreased cell viability compared with the negative control. The results demonstrated that the three herbicides needed to be activated by the V. faba root metabolism to produce DNA damage in human peripheral lymphocyte. The alkaline comet technique is a rapid and sensitive assay, to quickly evaluate DNA damage the metabolic activation of herbicide products by V. faba root in human cells in vitro.

Antimutagenicity of coriander (Coriandrum sativum) juice on the mutagenesis produced by plant metabolites of aromatic amines.

Aromatic amines are metabolically activated into mutagenic compounds by both animal and plant systems. The 4-nitro-o-phenylenediamine (NOP) is a well-known direct-acting mutagen whose mutagenic potential can be enhanced by plant metabolism; m-phenylenediamine (m-PDA) is converted to mutagenic products detected by the Salmonella typhimurium TA98 strain, and 2-aminofluorene (2-AF) is the plant-activated promutagen most extensively studied. Plant cells activate both 2-AF and m-PDA into potent mutagens producing DNA frameshift mutations. Coriander (Coriandrum sativum) is a common plant included in the Mexican diet, usually consumed uncooked. The antimutagenic activity of coriander juice against the mutagenic activity of 4-nitro-o-phenylenediamine, m-phenylenediamine and 2-aminofluorene was investigated using the Ames reversion mutagenicity assay (his- to his+) with the S. typhimurium TA98 strain as indicator organism. The plant cell/microbe coincubation assay was used as the activating system for aromatic transformation and plant extract interaction. Aqueous crude coriander juice significantly decreased the mutagenicity of metabolized aromatic amines (AA) in the following order: 2-AF (92.43%) > m-PDA (87.14%) > NOP (83.21%). The chlorophyll content in vegetable juice was monitored and its concentration showed a positive correlation with the detected antimutagenic effect. Protein content and peroxidase activity were also determined. The concentration of coriander juice (50-1000 microl/coincubation flask) was neither toxic nor mutagenic. The similar shape of the antimutagenic response curves obtained with coriander juice and chlorophyllin (used as a subrogate molecule of chlorophyll) indicated that comparable mechanisms of mutagenic inhibition could be involved. The negative correlation between chlorophyll content and mutagenic response of the promutagenic and direct-acting used amines allows us to deduce that a chemical interaction takes place between the two molecules, leading to the inactivation of mutagenic moiety.