Establishment of Primary Adult Skin Fibroblast Cell Lines from African Savanna Elephants (Loxodonta africana).

Following population declines of the African savanna elephant (Loxodonta africana) across the African continent, the establishment of primary cell lines of endangered wildlife species is paramount for the preservation of their genetic resources. In addition, it allows molecular and functional studies on the cancer suppression mechanisms of elephants, which have previously been linked to a redundancy of tumor suppressor gene TP53. This methodology describes the establishment of primary elephant dermal fibroblast (EDF) cell lines from skin punch biopsy samples (diameter: ±4 mm) of African savanna elephants (n = 4, 14-35 years). The applied tissue collection technique is minimally invasive and paves the way for future remote biopsy darting. On average, the first explant outgrowth was observed after 15.75 ± 6.30 days. The average doubling time (Td) was 93.02 ± 16.94 h and 52.39 ± 0.46 h at passage 1 and 4, respectively. Metaphase spreads confirmed the diploid number of 56 chromosomes. The successful establishment of EDF cell lines allows for future elephant cell characterization studies and for research on the cancer resistance mechanisms of elephants, which can be harnessed for human cancer prevention and treatment and contributes to the conservation of their genetic material.

Telomerase Inhibition by MST-312 Sensitizes Breast Cancer Cells to the Anti-cancer Properties of Plumbagin.

Breast cancer is the most common cause of malignancy and the second most common cause of death due to cancer in women. This heterogeneous disease is currently broadly classified as estrogen receptor (ER), progesterone receptor (PR) positive luminal tumors, human epidermal growth factor receptor 2 (HER2) amplified tumors and triple-negative breast cancers (TNBC). Phytochemicals are proven to be promising anti-cancer chemotherapeutics agents with minimal cytotoxic effects on normal cells. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) is a phytochemical derived from the roots of Plumbago zeylanica and it is known to possess anti-cancer properties similar to other compounds of naphthoquinones. In about 90% of cancer cells, the telomerase enzyme activity is revived to add telomeric repeats to evade apoptosis. In this study, a combinatorial approach of combining the anti-cancer compound plumbagin to induce genotoxicity and a potent telomerase inhibitor, MST-312 (synthetic derivative of tea catechins), was used to determine the combinational treatment-induced lethality in breast cancer cells such as MDA-MB-231 (TNBC) and MCF-7 (lumina) cells. MDA-MB-231 cells were responsive to combination treatment in both short-term (48 h) and long-term treatment (14 days) in a synergistic manner, whereas in MCF-7, the combination treatment was more effective in the long-term regimen. Furthermore, the cytotoxic effects of the plumbagin and MST-312 combination treatment were not recoverable after the short-term treatment. In conclusion, a combination treatment of MST-312 and plumbagin is proven to be more effective than a single plumbagin compound treatment in inducing DNA damage and telomere dysfunction leading to greater genome instability, cell cycle arrest and eventually cell death in cancer cells.

Dietary acid load and esophageal cancer risk: A case-control study.

BACKGROUND: A high dietary acid load (DAL) can produce metabolic acidosis, which is linked to cancer development through mechanisms of inflammation and cell transformation. There is limited epidemiological evidence linking DAL and cancer risk; however, none of the published studies focused on DAL and esophageal cancer (EC) risk in particular. Therefore, we sought to explore this association in the present study. METHODS: A case-control study was performed in 1295 male patients (185 squamous cell EC cases and 1110 age-frequency and urban/rural residence matched controls) through a multitopic inquiry, including a food frequency questionnaire. Food-derived nutrients were calculated from available databases. The DAL was calculated based on two validated measures: Potential renal acid load (PRAL) score and net endogenous acid production (NEAP) score. Odds ratios (OR) and their 95% confidence intervals (95% CI) were estimated by unconditional logistic regression, adjusting for confounders. RESULTS: We found direct, significant associations between dietary acid load and EC risk: (OR = 2.28, 95% CI: 1.44-3.61, ptrend <0.0001) and (OR = 2.17, 95% CI: 1.38-3.41, ptrend <0.0001) for highest PRAL and NEAP tertiles, respectively. Our data raise the possibility that a high DAL may contribute to EC development. Both acid load scores were directly associated with animal-based foods (mainly meat) and inversely associated with the intake of plant-based foods. CONCLUSION: To the best of our knowledge, this is the first epidemiological case-control study analyzing associations of DAL and squamous cell EC risk. Further research is warranted to confirm our findings.

Role of Xeroderma pigmentosum D (XPD) protein in genome maintenance in human cells under oxidative stress.

Xeroderma pigmentosum D (XPD) protein plays a pivotal role in the nucleotide excision repair pathway. XPD unwinds the local area of the damaged DNA by virtue of constituting transcription factor II H (TFIIH) and is important not only for repair but also for basal transcription. Although cells deficient in XPD have shown to be defective in oxidative base-lesion repair, the effects of the oxidative assault on primary fibroblasts from patients suffering from Xeroderma Pigmentosum D have not been fully explored. Therefore, we sought to investigate the role of XPD in oxidative DNA damage-repair by treating primary fibroblasts derived from a patient suffering from Xeroderma Pigmentosum D, with hydrogen peroxide. Our results show dose-dependent increase in genotoxicity with minimal effect on cytotoxicity with H2O2 in XPD deficient cells compared to control cells. XPD deficient cells displayed increased susceptibility and reduced repair capacity when subjected to DNA damage induced by oxidative stress. XPD deficient fibroblasts exhibited increased telomeric loss after H2O2 treatment. In addition, we demonstrated that chronic oxidative stress induced accelerated premature senescence characteristics. Gene expression profiling revealed alterations in genes involved in transcription and nucleotide metabolisms, as well as in cellular and cell cycle processes in a more significant way than in other pathways. This study highlights the role of XPD in the repair of oxidative stress and telomere maintenance. Lack of functional XPD seems to increase the susceptibility of oxidative stress-induced genotoxicity while retaining cell viability posing as a potential cancer risk factor of Xeroderma Pigmentosum D patients.

Oxidative Damage Induced Telomere Mediated Genomic Instability in Cells from Ataxia Telangiectasia Patients.

Our cellular genome is susceptible to cytotoxic lesions which include single strand breaks and double strand breaks among other lesions. Ataxia telangiectasia mutated (ATM) protein was one of the first DNA damage sensor proteins to be discovered as being involved in DNA repair and as well as in telomere maintenance. Telomeres help maintain the stability of our chromosomes by protecting the ends from degradation. Cells from ataxia telangiectasia (AT) patients lack ATM and accumulate chromosomal alterations. AT patients display heightened susceptibility to cancer. In this study, cells from AT patients (called as AT -/- and AT +/- cells) were characterized for genome stability status and it was observed that AT -/- cells show considerable telomere attrition. Furthermore, DNA damage and genomic instability were compared between normal (AT +/+ cells) and AT -/- cells exhibiting increased frequencies of spontaneous DNA damage and genomic instability markers. Both AT -/- and AT +/- cells were sensitive to sodium arsenite (1.5 and 3.0 µg/ml) and ionizing radiation-induced (2 Gy, gamma rays) oxidative stress. Interestingly, telomeric fragments were detected in the comet tails as revealed by comet-fluorescence in situ hybridization analysis, suggestive of telomeric instability in AT -/- cells upon exposure to sodium arsenite or radiation. Besides, there was an increase in the number of chromosome alterations in AT -/- cells following arsenite treatment or irradiation. In addition, complex chromosome aberrations were detected by multicolor fluorescence in situ hybridization in AT -/- cells in comparison to AT +/- and normal cells. Telomere attrition and chromosome alterations were detected even at lower doses of sodium arsenite. Peptide nucleic acid - FISH analysis revealed defective chromosome segregation in cells lacking ATM proteins. The data obtained in this study substantiates the role of ATM in telomere stability under oxidative stress.

Dietary acid load and lung cancer risk: A case-control study in men.

BACKGROUND: Dysregulation of the endogenous acid-base balance can contribute to inflammation and cancer development if metabolic acidosis is sustained. The epidemiologic evidence on the association between diet-dependent acid load and cancer risk is scarce and inconsistent. We aim to explore the possible role of dietary acid load in lung cancer (LC) risk. METHODS: A case-control study was performed on 843 LC cases and 1466 controls by using a multi-topic questionnaire, including a food frequency questionnaire. Controls were matched to cases by age-frequency, urban/rural residence, and region. Food-derived nutrients were calculated from available databases. The dietary acid load was calculated using validated measures as potential renal acid load (PRAL) score and net endogenous acid production (NEAP) score. Odds ratios (ORs) were estimated by logistic regression. RESULTS: We found direct associations between dietary acid load and LC risk. The highest quartile of the NEAP score was significantly associated (OR=2.22, ptrend<0.001). The PRAL score displayed similar associations in simpler regression models, but there was no association when a more complex one was used (OR=0.99, ptrend =0.94). The NEAP score was associated with a significant risk increase in all cell types, except for small cell cancers, but the PRAL score did not show any association. CONCLUSIONS: The NEAP scores, directly associated with meat intake and inversely associated with plant-based foods intake, suggest that a high acid load dietary style may increase LC risk. Studies focused on food groups, and nutritional patterns are in line with our findings. Although the data shown here represent the first one to be published on this issue, further studies are needed to confirm these findings.

Selective anticancer effects of Serjania marginata Casar. extract in gastric cells are mediated by antioxidant response.

Gastric cancer is the fifth most common malignancy worldwide. Serjania marginata Casar. (SM) displays anti-inflammatory properties and has been used to treat gastrointestinal disorders. In the current study, we examined whether the hydroethanolic extract of SM leaves exerted cytotoxic, mutagenic, and protective effects in non-tumor gastric epithelium cells (MNP01) and gastric adenocarcinoma cells (ACP02) in vitro and analyzed whether its action was selective. Initially, cell viability (MTT assay), cell cycle kinetics (flow cytometry), and cell proliferation (total protein content) were analyzed. In addition, genomic instability (cytokinesis-block micronucleus cytome assay), anti/pro-oxidant status (CM-H2 DCFDA probe), and transcriptional expression (RT-qPCR) of genes related to cell cycle, cell death, and antioxidant defense were also evaluated. The SM extract was cytotoxic toward MNP01 and ACP02 cells at concentrations greater than 300 and 100 µg·ml-1 , respectively, and decreased protein content only toward ACP02 cells at 200 µg ml-1 . In ACP02 cells, the SM extract at 100 µg·ml-1 associated with doxorubicin (DXR; 0.2 µg ml-1 ) clearly promoted cell cycle arrest at the G2/M phase. The extract alone was not mutagenic to either cell type and reversed DXR-induced DNA damage and H2 O2 -induced oxidative stress in MNP01 cells. The gene expression experiments showed that SM hydroethanolic extract exerts an antioxidant response via NFE2L2 activation in non-tumor gastric cells, and cell cycle arrest (G2/M) in ACP02 gastric cancer cells via the TP53 pathway. The selective action of SM indicates that it is a promising therapeutic agent to treat gastric diseases and merits further studies.

Investigations on the new mechanism of action for acetaldehyde-induced clastogenic effects in human lung fibroblasts.

Acetaldehyde (AA) has been classified as a probable human carcinogen by the International Agency for Research on Cancer (IARC, WHO) and by the US Environmental Protection Agency due to its ability to cause tumours following inhalation or alcohol consumption in animals. Humans are constantly exposed to AA through inhalation from the environment through cigarette smoke, vehicle fumes and industrial emissions as well as by persistent alcohol ingestion. Individuals with deficiencies in the enzymes that are involved in the metabolism of AA are more susceptible to its toxicity and constitute a vulnerable human population. Studies have shown that AA induces DNA damage and cytogenetic abnormalities. A study was undertaken to elucidate the clastogenic effects induced by AA and any preceding DNA damage that occurs in normal human lung fibroblasts as this will further validate the detrimental effects of inhalation exposure to AA. AA exposure induced DNA damage, involving DNA double strand breaks, which could possibly occur at the telomeric regions as well, resulting in a clastogenic effect and subsequent genomic instability, which contributed to the cell cycle arrest. The clastogenic effect induced by AA in human lung fibroblasts was evidenced by micronuclei induction and chromosomal aberrations, including those at the telomeric regions. Co-localisation between the DNA double strand breaks and telomeric regions was observed, suggesting possible induction of DNA double strand breaks due to AA exposure at the telomeric regions as a new mechanism beyond the clastogenic effect of AA. From the cell cycle profile following AA exposure, a G2/M phase arrest and a decrease in cell viability were also detected. Therefore, these effects due to AA exposure via inhalation may have implications in the development of carcinogenesis in humans.

An Automated Microscopic Scoring Method for the γ-H2AX Foci Assay in Human Peripheral Blood Lymphocytes.

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, γ-H2AX. The use of an automated microscopy platform to score the number of γ-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the γ-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive γ-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

Phytochemical Profile, and Antiproliferative and Proapoptotic Effects of Pouteria ramiflora (Mart.) Radlk. Leaf Extract, and Its Synergism with Cisplatin in HepG2 Cells.

Different species of the genus Pouteria have been used in folk medicine for the treatment of inflammation, fever, ulcers, diabetes, and diarrhea. We analyzed the phytochemical profile of the hydroethanolic extract from Pouteria ramiflora leaves by electrospray ionization ion trap tandem mass spectrometry and high-performance liquid chromatography-diode array detection, and examined whether it alone and in combination with cisplatin interfered with cell proliferation and death processes in HepG2 (human hepatocellular carcinoma) and FGH (human gingival fibroblasts) cells. Five compounds were identified in the extract: gallic acid, myricetin-3-O-α-l-arabinopyranoside, quercetin-3-O-ß-d-galactopyranoside, myricetin-3-O-α-l-rhamnopyranoside, and myricetin-3-O-ß-d-galactopyranoside. The extract was cytotoxic to both cell lines by inducing apoptotic cell death and acted in synergy with cisplatin; such effect was stronger in HepG2 cells than in FGH cells, demonstrating some selectivity to tumor cells. In HepG2 cells, the extract exerted antiproliferative effect mediated by induction of cell cycle arrest at the S and G2/M phases. Association of the extract with cisplatin enhanced the latter's antiproliferative effect, arrested the cell cycle at the S phase by CDK2 modulation, and reduced the number of anti-cyclin D1-stained HepG2 cells. Simultaneous treatment with the extract and cisplatin increased the latter's cytotoxicity, apoptotic cell death, and BAX expression in HepG2 cells. Altogether, the results reported herein indicate that P. ramiflora extract is a possible adjuvant to cancer therapy, which can circumvent the cisplatin-mediated resistance mechanisms in cancer cells.

Production and antiproliferative effect of violacein, a purple pigment produced by an Antarctic bacterial isolate.

We studied the production and the potential use of a purple-pigment produced by an Antarctic bacterial isolate. This pigment was identified as violacein, a metabolite produced by many bacterial strains and reported that it has antiproliferative activity in many cell lines. We analyzed the effect of temperature and the composition of the growth medium on pigment production, achieving the highest yield at 20 °C in Tryptic Soy Broth medium supplemented with 3.6 g/L glucose. We doubled the yield of the pigment production when the process was scaled up in a 5 L bioreactor (77 mg/L of crude pigment). The pigment was purified and identified by mass spectrometry (DI-EI-MS) and Nuclear Magnetic Resonance (NMR) spectroscopy as violacein. We performed survival assays that showed that the pure pigment has antiproliferative activity and sensitize HeLa cells (cervix cell carcinoma) to cisplatin. Besides, the pigment did not show genotoxic activity in HeLa cells as found performing micronucleus assays. These results suggest that this pigment may be used as anticancer or sensitizer to cisplatin drug in cervix cancer.

Chemosensitizer effect of cisplatin-treated bladder cancer cells by phenazine-5,10-dioxides.

We determined the chemosensitizer effect of phenazine dioxide derivatives to cisplatin and the possible mechanism of action on bladder cancer cells. Anti-proliferative activity of nine phenazine dioxide derivatives in presence or absence of cisplatin was evaluated in two bladder tumor human cells T24 and 253 J and one non tumor cell line V79-4. The sensitizer effect of the combined treatment was determined by chromosomal aberrations and micronucleus test. A possible mechanism of action of the sensitizer compounds as HDACi was also investigated.The phenazine dioxide 2c combined with cisplatin induced a cell cycle arrest on bladder cancer cells and resensitize the invasive and cisplatin resistant 253 J cell line. The HDAC inhibitory activity appears as one of the mechanism of action of the compound. The low toxicity levels against normal cells point out the phenazine dioxide derivative 2c as a very good scaffold for further design of HDACi sensitizer agents.

Furoxans and tocopherol analogs-furoxan hybrids as anticancer agents.

We determined the antiproliferative and nitric oxide (NO)-releasing activity of furoxans and tocopherol analogs-furoxan hybrids by tandem Griess/resazurin/sulforhodamin B assays in HeLa, 253J, T24, and HepG2 cancer cells. In addition, to investigate the NO implications in the inhibition of cell growth, cells were pretreated with the NO scavenger hemoglobin and the genotoxic damage was determined. The compounds 1 and 3 emerged as good anticancer agents for bladder cancer treatment. The NO-releasing activity of these compounds appears to be necessary to obtain the antiproliferative effect. Although compound 1 exerted a DNA damage mechanism of action, compound 3 seemed to act in a different way. The low toxicity levels against normal cell line HaCaT point them out as a very promising scaffold for the further design of new anticancer agents.

A highly efficient and cost-effective recombinant production of a bacterial photolyase from the Antarctic isolate Hymenobacter sp. UV11.

Photolyases are DNA-repairing flavoproteins that are represented in most phylogenetic taxa with the exception of placental mammals. These enzymes reduce the ultraviolet-induced DNA damage; thus, they have features that make them very attractive for dermatological or other medical uses, such as the prevention of human skin cancer and actinic keratosis. In this work, we identified a 50.8 kDa photolyase from the UVC-resistant Antarctic bacterium Hymenobacter sp. UV11. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and its activity was analyzed using different approaches: detection of cyclobutane pyrimidine dimers (CPDs) by immunochemistry, high-performance liquid chromatography and comet assays using Chinese Hamster Ovary (CHO) and immortalized nontumorigenic human epidermal (HaCat) cells. The information supports that the recombinant protein has the ability to repair the formation of CPDs, on both double- and single-stranded DNA. This CPD-photolyase was fully active on CHO and HaCat cell lines, suggesting that this enzyme could be used for medical or cosmetic purposes. Results also suggest that the UV11 CPD-photolyase uses MTHF as chromophore in the antenna domain. The potential use of this recombinant enzyme in the development of new inventions with pharmaceutical and cosmetic applications is discussed during this work.

Pouteria ramiflora (Mart.) Radlk. extract: Flavonoids quantification and chemopreventive effect on HepG2 cells.

Pouteria ramiflora (Mart.) Radlk., popularly known as curriola, is commonly used in Brazil as medicinal plant to treat worm infections, dysentery, pain, inflammation, hyperlipidemia, and obesity. At present the safety of this extract when used therapeutically in human remains to be determined. Thus, the aim of this study was to examine cytotoxicity, antiproliferative, and antimutagenic actions of this extract. The hydroalcoholic extract from P. ramiflora leaves consisted of flavonoids identified and quantified as myricetin-3-O-ß-D-galactopyranoside (13.55 mg/g) and myricetin-3-O-α-L-rhamnopyranoside (9.61 mg/g). The extract exhibited cytotoxicity at concentrations higher than 1.5 µg/ml in human hepatocarcinoma (HepG2)and 2.5 µg/ml in non-tumoral primary gastric (GAS) cells using the MTT assay, and at concentrations higher than 3 µg/ml in HepG2 and 3.5 µg/ml in GAS cells by the neutral red assay. The extract did not show antiproliferative effect as evidenced by the nuclear division index (NDI). However, in the presence of benzo[a]pyrene (BaP) (positive control), an enhanced cytostatic effect in the NDI and flow cytometry was noted. It is of interest that when the extract was co-incubated with BaP a significant decrease in DNA damage was observed indicating an antimutagenic action. This protective effect might be attributed to myricetin and gallic acid found in P. ramiflora extract. The low cytotoxicity action and protective effect observed in the present study encourage further studies regarding other biological effects of P. ramiflora, as well as its potential use as a chemopreventive agent.

Occupational Exposure to Pesticides in Tobacco Fields: The Integrated Evaluation of Nutritional Intake and Susceptibility on Genomic and Epigenetic Instability.

Pesticides used at tobacco fields are associated with genomic instability, which is proposed to be sensitive to nutritional intake and may also induce epigenetic changes. We evaluated the effect of dietary intake and genetic susceptibility polymorphisms in MTHFR (rs1801133) and TERT (rs2736100) genes on genomic and epigenetic instability in tobacco farmers. Farmers, when compared to a nonexposed group, showed increased levels of different parameters of DNA damage (micronuclei, nucleoplasmic bridges, and nuclear buds), evaluated by cytokinesis-block micronucleus cytome assay. Telomere length (TL) measured by quantitative PCR was shorter in exposed individuals. Global DNA methylation was significantly decreased in tobacco farmers. The exposed group had lower dietary intake of fiber, but an increase in cholesterol; vitamins such as B6, B12, and C; ß-carotene; and α-retinol. Several trace and ultratrace elements were found higher in farmers than in nonfarmers. The MTHFR CT/TT genotype influenced nucleoplasmic bridges, nuclear buds, and TL in the exposed group, whereas TERT GT/TT only affected micronucleus frequency. We observed a positive correlation of TL and lipids and an inverse correlation of TL and fibers. The present data suggest an important role of dietary intake and subjects' genetic susceptibility to xenobiotics-induced damages and epigenetic alterations in tobacco farmers occupationally exposed to mixtures of pesticides.

Evaluation of chromosomal aberrations induced by 188Re-dendrimer nanosystem on B16f1 melanoma cells.

PURPOSE: To study the rhenium-188 labeling of polyamidoamine (PAMAM) generation 4 (G4) dendrimer and its evaluation on biodistribution and chromosomal aberrations in melanoma cells induced by ionizing radiation as potential treatment agent. MATERIALS AND METHODS: Dendrimers were first conjugated with Suc-HYNIC (succinimidyl 6-hydrazinopyridine-3-carboxylic acid hydrochloride). Dendrimer-HYNIC was then incubated with 188ReO4-. Biodistribution was performed administrating 188Re-dendrimer to normal (NM) or melanoma-bearing mice (MBM). Chromosome aberration test was conducted in order to measure treatment capacity of 188Re-dendrimer in melanoma cells. RESULTS: Radiolabeling yield of dendrimer was approx. 70%. Biodistribution studies in NM showed blood clearance with hepatic and renal depuration. MBM showed a similar pattern of biodistribution with tumor uptake of 6% of injected dose. Aberrant metaphases quantified in control cells were 7%, increasing to 29.5% in cells treated with 15µCi (0.555 MBq) of 188Re-dendrimer for 24 h. CONCLUSIONS: 188Re-dendrimer can produce double-stranded breaks in DNA induced by ionizing radiation in melanoma cells in vitro.

Chronic occupational exposure endured by tobacco farmers from Brazil and association with DNA damage.

Tobacco farming is an important economic income in Brazil, although it has been challenged as regard the occupational exposure to both pesticides and nicotine endured by farmers. Chronic occupational exposure to complex mixtures can lead to health hazardous. We examined genomic instability and epigenetic changes in tobacco farmers occupationally exposed to pesticide mixtures and nicotine at tobacco fields. DNA damage was assessed by alkaline comet assay in blood cells. Genomic DNA was isolated, and telomere length was measured using quantitative polymerase chain reaction assay. We measured 5-methyl-2'-deoxycytidine, a marker of global DNA methylation, and p16 promoter methylation. The oxidative profile was evaluated by trolox equivalent antioxidant capacity and lipid peroxidation (thiobarbituric acid reactive substances) in serum. Exposure parameters, plasma cotinine and inorganic element levels, were also measured. DNA damage was significantly elevated for farmers in relation to unexposed group (P < 0.001; Mann-Whitney test) and positively associated with years of exposure. Inverse relationship between DNA damage and total equivalent antioxidant activity was demonstrated for exposed and unexposed groups. Exposed group showed significantly shorter telomeres (P < 0.001; unpaired t-test) and DNA hypomethylation (P < 0.001; unpaired t-test), as well as p16 hypermethylation (P = 0.003; Mann-Whitney test). Lipid peroxidation was increased for exposed group in relation to unexposed one (P = 0.02; Mann-Whitney test) and presented a positive correlation with global DNA methylation (P = 0.0264). Farmers have increased plasma cotinine levels (P < 0.001) and inorganic elements (phosphorus, sulphur and chlorine) in relation to unexposed group. Elevated oxidative stress levels due to chronic occupational pesticide mixtures and nicotine exposure in tobacco farmers were associated with higher DNA damage, shorter telomeres and altered DNA methylation. Telomere-accelerated attrition due to exposure may be potential intermediate step before a disease state.

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.