Publications by authors named "Wilner Martínez-López"

28 Publications

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

Cancer Treat Res Commun 2021 Apr 25;28:100382. Epub 2021 Apr 25.

Unit of Thoracic Oncology, National Cancer Institute, Joanicó 3265, Montevideo 11600, Uruguay.

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, p<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, p =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.
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http://dx.doi.org/10.1016/j.ctarc.2021.100382DOI Listing
April 2021

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

Environ Toxicol 2021 Apr 22. Epub 2021 Apr 22.

Department of General Biology, Center of Biological Sciences, State University of Londrina (UEL), Londrina, Brazil.

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-H 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 , respectively, and decreased protein content only toward ACP02 cells at 200 μg ml . In ACP02 cells, the SM extract at 100 μg·ml associated with doxorubicin (DXR; 0.2 μg ml ) 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 H O -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.
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http://dx.doi.org/10.1002/tox.23151DOI Listing
April 2021

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

Mutat Res 2021 Jan-Feb;861-862:503303. Epub 2020 Dec 30.

Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Vellore Institute of Technology, Vellore, India; Mangalore University, India; Tembusu College, National University of Singapore, Singapore. Electronic address:

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.
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http://dx.doi.org/10.1016/j.mrgentox.2020.503303DOI Listing
March 2021

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

J Med Food 2021 May 4;24(5):452-463. Epub 2020 Aug 4.

Department of General Biology, Biological Science Center, Londrina State University-UEL, Londrina, PR, Brazil.

Different species of the genus 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 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---l-arabinopyranoside, quercetin-3---d-galactopyranoside, myricetin-3---l-rhamnopyranoside, and myricetin-3---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 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 expression in HepG2 cells. Altogether, the results reported herein indicate that extract is a possible adjuvant to cancer therapy, which can circumvent the cisplatin-mediated resistance mechanisms in cancer cells.
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http://dx.doi.org/10.1089/jmf.2020.0045DOI Listing
May 2021

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

World J Microbiol Biotechnol 2020 Jul 18;36(8):120. Epub 2020 Jul 18.

Biochemistry and Molecular Biology, Faculty of Sciences, Universidad de la República, Igua 4225, Montevideo, Uruguay.

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.
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http://dx.doi.org/10.1007/s11274-020-02893-4DOI Listing
July 2020

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

Environ Toxicol Pharmacol 2019 Jul 19;69:9-15. Epub 2019 Mar 19.

Grupo de Química Medicinal, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay. Electronic address:

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.
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http://dx.doi.org/10.1016/j.etap.2019.03.015DOI Listing
July 2019

Furoxans and tocopherol analogs-furoxan hybrids as anticancer agents.

Anticancer Drugs 2019 04;30(4):330-338

Epigenetics and Genomic Instability Laboratory.

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.
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http://dx.doi.org/10.1097/CAD.0000000000000721DOI Listing
April 2019

Influence of chromatin remodeling in the removal of UVC-induced damage in TCR proficient and deficient Chinese hamster cells.

Mutat Res Genet Toxicol Environ Mutagen 2018 Dec 28;836(Pt A):124-131. Epub 2018 Aug 28.

Department of Ecological & Biological Sciences (DEB), University of Tuscia, Viterbo, Italy.

In mammalian cells, nucleotide excision repair system is constituted of two sub-pathways, global genomic repair (GGR) and transcription coupled repair (TCR). Deficiency of TCR pathway leads to Cockyane syndrome (CS) which is a rare human autosomal recessive disorder. Owing to the pivotal role of CSB gene in TCR, it's mutation causes severe repair and transcriptional defects in CSB patients. CSB protein belongs to the ATP chromatin remodeling complex, hence presumably an improper chromatin remodeling in CSB cells could be at the source of inefficient removal of pyrimidine dimers (CPDs) after UVC exposure in these patients. In this study, we evaluated the role of chromatin remodeling process on UVC induced CPDs and the ensuing effect on chromosomal aberrations in UV61 cells (TCR deficient) and its parental cell line, AA8 (TCR proficient). We observed that post 2 h UVC irradiation, both cell lines underwent pronounced chromatin relaxation but was lower in CSB deficient UV61 cells. Since the deficiency in chromatin remodeling in CSB-mutated cells was accompanied by a decrease in the histone acetylation level, the histone deacetylase inhibitor trichostatin A (TSA) was employed to improve the removal of UVC-induced lesions by increasing the histone acetylation level. Contrary to expectations, TSA increased the induction of chromosomal aberrations and apoptotic cells along with amounts of CPDs after UVC-irradiation, indicating that changes in histone acetylation levels might contribute to the failure in the removal of UVC-induced lesions. Also, it has been shown earlier that the expression of genes regulated by CSB is affected by the increase in the acetylation level produced by TSA. Taken all together, we hypothesize that failure in the removal of UVC induced lesions in CSB-deficient cells can be caused by an imbalance in histone acetylation levels leading to chromatin conformation changes and hence interaction defects among repair proteins and DNA lesions.
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http://dx.doi.org/10.1016/j.mrgentox.2018.08.003DOI Listing
December 2018

DNA Damage: Health and Longevity.

Oxid Med Cell Longev 2018;2018:9701647. Epub 2018 Sep 13.

Laboratory of Genetic Toxicology, PPGBioSaúde and PPGGTA, Lutheran University of Brazil (ULBRA), Canoas, RS, Brazil.

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http://dx.doi.org/10.1155/2018/9701647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158954PMC
December 2018

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

Extremophiles 2019 Jan 28;23(1):49-57. Epub 2018 Sep 28.

Biochemistry and Molecular Biology, Faculty of Sciences, Universidad de la República (UdelaR), Iguá 4225, 11400, Montevideo, Uruguay.

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.
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http://dx.doi.org/10.1007/s00792-018-1059-yDOI Listing
January 2019

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

J Toxicol Environ Health A 2018 12;81(16):792-804. Epub 2018 Jul 12.

a Departamento de Biologia Geral, Centro de Ciências Biológicas , Universidade Estadual de Londrina - UEL , Londrina , Paraná , Brazil.

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-β-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.
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http://dx.doi.org/10.1080/15287394.2018.1491911DOI Listing
August 2019

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

Oxid Med Cell Longev 2018 3;2018:7017423. Epub 2018 Jun 3.

Laboratory of Toxicological Genetics, Post-Graduate Program in Cellular and Molecular Biology Applied to Health (PPGBioSaúde), Lutheran University of Brazil (ULBRA), Av. Farroupilha, 8001 Canoas, RS, Brazil.

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 (rs1801133) and (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 B, B, and C; -carotene; and -retinol. Several trace and ultratrace elements were found higher in farmers than in nonfarmers. The genotype influenced nucleoplasmic bridges, nuclear buds, and TL in the exposed group, whereas 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.
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http://dx.doi.org/10.1155/2018/7017423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6009020PMC
October 2018

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

Int J Radiat Biol 2018 07 19;94(7):664-670. Epub 2018 Jun 19.

a Area de Radiofarmacia, Facultad de Ciencias , Centro de Investigaciones Nucleares, Universidad de la República , Montevideo , Uruguay.

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 ReO. Biodistribution was performed administrating Re-dendrimer to normal (NM) or melanoma-bearing mice (MBM). Chromosome aberration test was conducted in order to measure treatment capacity of Re-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 Re-dendrimer for 24 h.

Conclusions: Re-dendrimer can produce double-stranded breaks in DNA induced by ionizing radiation in melanoma cells in vitro.
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http://dx.doi.org/10.1080/09553002.2018.1478161DOI Listing
July 2018

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

Mutagenesis 2018 04;33(2):119-128

Laboratory of Toxicological Genetics, Post-Graduate Program in Cellular and Molecular Biology Applied to Health (PPGBioSaúde), Lutheran University of Brazil (ULBRA), Av. Farroupilha, São José, Canoas, Rio Grande do Sul, Brazil.

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.
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http://dx.doi.org/10.1093/mutage/gex045DOI Listing
April 2018

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

Mutagenesis 2018 02;33(1):87-95

Department of Genetic Toxicology and Chromosome Pathology, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay.

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.
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http://dx.doi.org/10.1093/mutage/gex035DOI Listing
February 2018

Phytochemical study and evaluation of cytotoxicity, mutagenicity, cell cycle kinetics and gene expression of Bauhinia holophylla (Bong.) Steud. in HepG2 cells in vitro.

Cytotechnology 2018 Apr 11;70(2):713-728. Epub 2017 Dec 11.

Department of General Biology, Biological Sciences Center, State University of Londrina - UEL, Rod Celso Garcia Cid PR 445, Km 380, University Campus, P.O. Box 6001, Londrina, PR, CEP 86057-970, Brazil.

Bauhinia holophylla (Bong.) Steud. (Fabaceae) is a plant used in Brazilian folk medicine to treat diabetes and inflammation. This study evaluated the phytochemical properties, cytotoxic, apoptotic, mutagenic/antimutagenic effects and alterations in gene expression (RNAm) in HepG2 cells treated with the B. holophylla extract. The phytochemical profile highlight the presence of flavonoids isorhamentin and quercetin derivates. The MTT assay was used to evaluate the cytotoxicity of different concentrations for different treatment times. Three concentrations (7.5, 15, 30 µg/mL) were chosen for assessment of apoptosis (AO/EB), mutagenicity (micronucleus), and cell cycle kinetics (flow cytometry). Thereafter, the concentration of 7.5 µg/mL was chosen to evaluate the protective effects against DNA damage induced by benzo[a]pyrene (B[a]P). At concentrations higher than 7.5 µg/mL (between 10 and 50 µg/mL), the extract was cytotoxic, induced apoptosis, and caused antiproliferative effects. However, it did not induce micronucleus and a reduction of apoptotic and micronucleated cells was observed in treatments that included the extract and B[a]P. The protective effect is attributable to the presence of flavonoids, described as antioxidants, inhibitors of DNA adduct and activators of detoxifying enzymes. The results of the present study such as absence of cytotoxic and mutagenic effects and protective effects against known carcinogens suggest that B. holophylla has potential for use soon as herbal medicine.
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http://dx.doi.org/10.1007/s10616-017-0173-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5851965PMC
April 2018

Chemical characterization and cytotoxic, genotoxic, and mutagenic properties of Baccharis trinervis (Lam, Persoon) from Colombia and Brazil.

J Ethnopharmacol 2018 Mar 1;213:210-220. Epub 2017 Nov 1.

Departamento de Biofísica/Centro de Biotecnologia-UFRGS, Porto Alegre- RS-Brasil.; Programa de Pós Graduação em Biologia Celular e Molecular (PPGBCM), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil; Centro de Ciências Exatas e de Tecnologia, Instituto de Biotecnologia, Universidade de Caxias do Sul - UCS Caxias do Sul - RS, Brazil. Electronic address:

Pharmacology Relevance: Baccharis trinervis (Lam, Persoon) leaves are used in the traditional medicine for the treatment of high fevers, edema, inflammation, sores and muscle cramps, snakebites and as antiseptic.

Aim Of The Study: To investigate the cytotoxic, genotoxic, and mutagenic effects of extracts and fractions of B. trinervis from Brazil and Colombia in Chinese Hamster Ovary (CHO) cells, and to examine the mutagenic activity in Salmonella typhimurium.

Material And Methods: Aqueous extracts (AE) of aerial parts of B. trinervis from Brazil (B) and Colombia (C) were fractioned in ethyl acetate fraction (EAF), butanol extract (BF), and aqueous residue fraction (ARF). Qualitative chemical screening and determination of total flavonoid content were made. Identification of chemical constituents was performed by High Performance Liquid Chromatography (HPLC) and High Resolution Mass Spectrometry (HRMS). For the in vitro tests, CHO cells were treated for 3h with extracts and fractions. The cytotoxic activity was evaluated by clonal survival and 3-(4.5-dimethylthiazole-2-yl)-2.5-biphenyl tetrazolium bromide reduction assay (MTT). Genotoxic and mutagenic effects were evaluated by the alkaline comet assay and Cytokinesis-blockage micronucleus test (CBMN), respectively. Additionally, Salmonella/microsome assay was carried out to determinate the mutagenic effects in EAF from Brazil and Colombia.

Results: Phytochemical analyses indicated the presence of saponins and flavonoids. AE and EAF were the samples with the highest quantity of total flavonoids. HPLC showed the presence of luteolin only in AEC, and caffeic acid, ellagic acid, rosmarinic acid, and rutin were identified in AEB and AEC (AEC>AEB). The HRMS in positive mode of EAFB and EAFC showed presence of two carboxylic acids, coumarin, and two terpenoids. In addition, were identified one terpenoid and two carboxylic acids in AE, BF and ARF of B. trinervis from both countries in negative mode. Dose-dependent cytotoxic effects were observed in CHO cells treated with B. trinervis extracts and fractions by using clonal survival and MTT at concentrations higher than 0.05mg/mL. All the extracts and fractions induced DNA strand breaks in CHO cells with dose-dependent response, mostly EAFB and EAFC. The EAF from Brazil and Colombia showed mutagenic effect at 0.5mg/mL, while the other fractions did not show a significant difference in relation to the control. No mutagenic effects were found in EAF from both countries by the Salmonella/microsome assay.

Conclusions: Cytotoxic and genotoxic effects were demonstrated in all extracts and fractions used, although only EAF showed mutagenic effects by CBMN, but not by Salmonella/microsome assay. Our results suggest that flavonoids, phenylpropanoids, coumarins, and diterpenes may be responsible for the cytotoxic, genotoxic and mutagenic effects observed.
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http://dx.doi.org/10.1016/j.jep.2017.10.027DOI Listing
March 2018

Effects of Valproic Acid on Radiation-Induced Chromosomal Aberrations in Human Lymphocytes.

Genome Integr 2017 23;8. Epub 2017 Jan 23.

Clemente Estable Biological Research Institute, Montevideo, Uruguay; Epigenetics and Genomic Instability Laboratory, Clemente Estable Biological Research Institute, Montevideo, Uruguay.

One of the most widely employed histone deacetylases inhibitors in the clinic is the valproic acid (VA), proving to have a good tolerance and low side effects on human health. VA induces changes in chromatin structure making DNA more susceptible to damage induction and influence DNA repair efficiency. VA is also proposed as a radiosensitizing agent. To know if VA is suitable to sensitize human lymphocytes γ-irradiation , different types of chromosomal aberrations in the lymphocytes, either in the absence or presence of VA, were analyzed. For this purpose, blood samples from four healthy donors were exposed to γ-rays at a dose of 1.5 Gy and then treated with two different doses of VA (0.35 or 0.70 mM). Unstable and stable chromosomal aberrations were analyzed by means of fluorescence hybridization. Human lymphocytes treated with VA alone did not show any increase in the frequency of chromosomal aberrations. However, a moderate degree of sensitization was observed, through the increase of chromosomal aberrations, when 0.35 mM VA was employed after γ-irradiation, whereas 0.70 mM VA did not modify chromosomal aberration frequencies. The lower number of chromosomal aberrations obtained when VA was employed at higher dose after γ-irradiation, could be related to the induction of a cell cycle arrest, a fact that should be taken into consideration when VA is employed in combination with physical or chemical agents.
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http://dx.doi.org/10.4103/2041-9414.198909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320781PMC
January 2017

Searching for novel photolyases in UVC-resistant Antarctic bacteria.

Extremophiles 2017 Mar 11;21(2):409-418. Epub 2017 Feb 11.

Biochemistry and Molecular Biology, Faculty of Sciences, Universidad de la República, Igua 4225, 11400, Montevideo, Uruguay.

Ultraviolet (UV) light irradiation has serious consequences for cell survival, including DNA damage by formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6,4) pyrimidone photoproducts. In general, the Nucleotide Excision Repair pathway repairs these lesions; however, all living forms, except placental mammals and some marsupials, produce a flavoprotein known as photolyase that directly reverses these lesions. The aim of this work was the isolation and identification of Antarctic UVC-resistant bacteria, and the search for novel photolyases. Two Antarctic water samples were UVC-irradiated (254 nm; 50-200 J m) and 12 UVC-resistant bacteria were isolated and identified by 16S rDNA amplification/analysis as members of the genera Pseudomonas, Janthinobacterium, Flavobacterium, Hymenobacter and Sphingomonas. The UVC 50% lethal dose and the photo-repair ability of isolates were analyzed. The occurrence of photolyase coding sequences in Pseudomonas, Hymenobacter and Sphingomonas isolates were searched by PCR or by searching in the draft DNA genome. Results suggest that Pseudomonas and Hymenobacter isolates produce CDP-photolyases, and Sphingomonas produces two CPD-photolyases and a 6,4-photolyase. Results suggest that the Antarctic environment is an important source of genetic material for the identification of novel photolyase genes with potential biotechnological applications.
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http://dx.doi.org/10.1007/s00792-016-0914-yDOI Listing
March 2017

RENEB intercomparisons applying the conventional Dicentric Chromosome Assay (DCA).

Int J Radiat Biol 2017 01 21;93(1):20-29. Epub 2016 Oct 21.

y Radiation and Nuclear Safety Authority , Helsinki , Finland.

Purpose: Two quality controlled inter-laboratory exercises were organized within the EU project 'Realizing the European Network of Biodosimetry (RENEB)' to further optimize the dicentric chromosome assay (DCA) and to identify needs for training and harmonization activities within the RENEB network.

Materials And Methods: The general study design included blood shipment, sample processing, analysis of chromosome aberrations and radiation dose assessment. After manual scoring of dicentric chromosomes in different cell numbers dose estimations and corresponding 95% confidence intervals were submitted by the participants.

Results: The shipment of blood samples to the partners in the European Community (EU) were performed successfully. Outside the EU unacceptable delays occurred. The results of the dose estimation demonstrate a very successful classification of the blood samples in medically relevant groups. In comparison to the 1st exercise the 2nd intercomparison showed an improvement in the accuracy of dose estimations especially for the high dose point.

Conclusions: In case of a large-scale radiological incident, the pooling of ressources by networks can enhance the rapid classification of individuals in medically relevant treatment groups based on the DCA. The performance of the RENEB network as a whole has clearly benefited from harmonization processes and specific training activities for the network partners.
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http://dx.doi.org/10.1080/09553002.2016.1233370DOI Listing
January 2017

RENEB accident simulation exercise.

Int J Radiat Biol 2017 01 25;93(1):75-80. Epub 2016 Aug 25.

t National Centre for Scientific Research Demokritos , Athens , Greece.

Purpose: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event.

Materials And Methods: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners.

Results: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes).

Conclusions: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested.
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http://dx.doi.org/10.1080/09553002.2016.1206230DOI Listing
January 2017

A New Cytogenetic Biodosimetry Image Repository for the Dicentric Assay.

Radiat Prot Dosimetry 2016 Dec 13;172(1-3):192-200. Epub 2016 Jul 13.

Public Health England, Chilton, UK.

The BioDoseNet was founded by the World Health Organization as a global network of biodosimetry laboratories for building biodosimetry laboratory capacities in countries. The newly established BioDoseNet image repository is a databank of ~25 000 electronically captured images of metaphases from the dicentric assay, which have been previously analysed by international experts. The detailed scoring results and dose estimations have, in most cases, already been published. The compilation of these images into one image repository provides a valuable tool for training and research purposes in biological dosimetry. No special software is needed to view and score the image galleries. For those new to the dicentric assay, the BioDoseNet Image Repository provides an introduction to and training for the dicentric assay. It is an excellent instrument for intra-laboratory training purposes or inter-comparisons between laboratories, as recommended by the International Organization for Standardisation standards. In the event of a radiation accident, the repository can also increase the surge capacity and reduce the turnaround time for dose estimations. Finally, it provides a mechanism for the discussion of scoring discrepancies in difficult cases.
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http://dx.doi.org/10.1093/rpd/ncw158DOI Listing
December 2016

LDH, proliferation curves and cell cycle analysis are the most suitable assays to identify and characterize new phytotherapeutic compounds.

Cytotechnology 2016 Dec 25;68(6):2729-2744. Epub 2016 Jun 25.

Department of General Biology, State University of Londrina, PR 445 Km 380, s/n - Campus Universitário, Londrina, PR, CEP 86057-970, Brazil.

Brazilian flora biodiversity has been widely investigated to identify effective and safe phytotherapeutic compounds. Among the investigated plant species, the Byrsonima genus exhibits promising biological activities. This study aimed at evaluating the cytotoxicity of B. correifolia, B. verbascifolia, B. fagifolia and B. intermedia extracts using different assays in two cell lines (primary gastric and HepG2 cells). The different extract concentrations effects on cell viability were assayed using the MTT, aquabluer, neutral red and LDH assays. Non-cytotoxic concentrations were selected to generate cell proliferation curves and to assess cell cycle kinetics by flow cytometry. Byrsonima extracts differentially affected cell viability depending on the metabolic cellular state and the biological parameter evaluated. B. fagifolia and B. intermedia extracts exhibited lower cytotoxic effects than B. correifolia and B. verbascifolia in all assays. The results obtained with LDH and flow cytometry assays were more reliable, suggesting that they can be useful in the screening for herbal medicine and to further characterize these extracts as phytotherapeutic compounds.
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http://dx.doi.org/10.1007/s10616-016-9998-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5101344PMC
December 2016

Antimutagenicity and induction of antioxidant defense by flavonoid rich extract of Myrcia bella Cambess. in normal and tumor gastric cells.

J Ethnopharmacol 2015 Dec 6;176:345-55. Epub 2015 Nov 6.

Department of Biological Sciences, Faculty of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara 14801-902, Brazil.

Ethnopharmacological Relevance: The Brazilian "Cerrado" is an important source of natural products, such as Myrcia bella Cambess (MB, also known as "mercurinho"). MB leaves are popularly used for the treatment of diabetes and gastrointestinal disorders; however, only its hypoglycemic activity has been experimentally described.

Aim Of The Study: Because MB is used to treat gastrointestinal disorders, the present study characterized biological activities of hydroalcoholic MB extract in human normal and tumor gastric cells.

Materials And Methods: Cytotoxic, antiproliferative, genotoxic and protective effects were evaluated, as well as the effects of the MB extract on gene expression.

Results: The MB extract induced cytotoxicity in tumor cells at lower concentrations compared with normal cells as assessed by the MTT assay. Moreover, the MB extract induced necrosis based on acridine orange/ethidium bromide staining. An antiproliferative effect was evidenced through an arrest in the G2/M phase detected by flow cytometry and a decrease in the nuclear division index using the cytokinesis-block micronucleus cytome assay. Cells treated with MB extract combined with doxorubicin (DXR) showed increased NUBDs, which may be related to the gene amplification of CCND1. Antimutagenic effects were also observed and may be associated with the antioxidant activities detected using the CM-H2DCFDA probe.

Conclusions: Our findings showed the following: (a) high concentrations of MB induced cytotoxicity and cell death by necrosis; (b) its antiproliferative effect was associated with G2/M arrest; and (c) its antioxidant activity could be responsible for the observed antimutagenic effects and for protective effects against gastrointestinal disorders previously described to MB. Although these effects are not specific to normal or tumor cells, they provide a panel of biological activities for further exploration.
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http://dx.doi.org/10.1016/j.jep.2015.11.003DOI Listing
December 2015

Chemical and biological characterisation of Machaerium hirtum (Vell.) Stellfeld: absence of cytotoxicity and mutagenicity and possible chemopreventive potential.

Mutagenesis 2016 Mar 27;31(2):147-60. Epub 2015 Aug 27.

Department of General Biology, Center of Biologic Sciences, State University of Londrina - UEL, Londrina, PR, Brazil,

Machaerium hirtum (Vell.) Stellfeld (M.hirtum) is a plant known as 'jacarandá-bico-de-pato' whose bark is commonly used against diarrhea, cough and cancer. The aim of this study was to phytochemically characterise the hydroethanolic extract of this plant, investigate its antimutagenic activities using the Ames test and evaluate its effects on cell viability, genomic instability, gene expression and cell protection in human hepatocellular carcinoma cells (HepG2). Antimutagenic activity was assessed by simultaneous pre- and post-treatment with direct and indirect mutagens, such as 4-nitro-o-phenylenediamine (NPD), mitomycin C (MMC), benzo[a]pyrene (B[a]P) and aflatoxin B1 (AFB1), using the Ames test, cytokinesis blocking micronucleus and apoptosis assays. Only 3 of the 10 concentrations evaluated in the MTT assay were cytotoxic in HepG2 cells. Micronucleated or apoptotic cells were not observed with any of the tested concentrations, and there were no mutagenic effects in the bacterial system. However, the Nuclear Division Index and flow cytometry data showed a decrease in cell proliferation. The extract showed an inhibitory effect against direct (NPD) and indirect mutagens (B[a]P and AFB1). Furthermore, pre- and post-treated cells showed significant reduction in the number of apoptotic and micronucleated cells. This effect is not likely to be associated with the modulation of antioxidant genes, as shown by the RT-qPCR results. Six known flavonoids were identified in the hydroethanolic extract of Machaerium hirtum leaves, and their structures were elucidated by spectroscopic and spectrophotometric methods. The presence of the antioxidants apigenin and luteolin may explain these protective effects, because these components can inhibit the formation of reactive species and prevent apoptosis and DNA damage. In conclusion, the M.hirtum extract showed chemopreventive potential and was not hazardous at the tested concentrations in the experiments presented here. Moreover, this extract should be investigated further as a chemopreventive agent.
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http://dx.doi.org/10.1093/mutage/gev066DOI Listing
March 2016

UV-triggered p21 degradation facilitates damaged-DNA replication and preserves genomic stability.

Nucleic Acids Res 2013 Aug 30;41(14):6942-51. Epub 2013 May 30.

Cell Cycle and Genomic Stability Laboratory, Fundación Instituto Leloir-CONICET, Buenos Aires C1405BWE, Argentina.

Although many genotoxic treatments upregulate the cyclin kinase inhibitor p21, agents such as UV irradiation trigger p21 degradation. This suggests that p21 blocks a process relevant for the cellular response to UV. Here, we show that forced p21 stabilization after UV strongly impairs damaged-DNA replication, which is associated with permanent deficiencies in the recruitment of DNA polymerases from the Y family involved in translesion DNA synthesis), with the accumulation of DNA damage markers and increased genomic instability. Remarkably, such noxious effects disappear when disrupting the proliferating cell nuclear antigen (PCNA) interacting motif of stable p21, thus suggesting that the release of PCNA from p21 interaction is sufficient to allow the recruitment to PCNA of partners (such as Y polymerases) relevant for the UV response. Expression of degradable p21 only transiently delays early replication events and Y polymerase recruitment after UV irradiation. These temporary defects disappear in a manner that correlates with p21 degradation with no detectable consequences on later replication events or genomic stability. Together, our findings suggest that the biological role of UV-triggered p21 degradation is to prevent replication defects by facilitating the tolerance of UV-induced DNA lesions.
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http://dx.doi.org/10.1093/nar/gkt475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3737556PMC
August 2013

Chromatin-remodelling mechanisms in cancer.

Mutat Res 2008 Mar-Apr;658(3):191-214. Epub 2008 Feb 17.

Genetic Toxicology Department, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay.

Chromatin-remodelling mechanisms include DNA methylation, histone-tail acetylation, poly-ADP-ribosylation, and ATP-dependent chromatin-remodelling processes. Some epigenetic modifications among others have been observed in cancer cells, namely (1) local DNA hypermethylation and global hypomethylation, (2) alteration in histone acetylation/deacetylation balance, (3) increased or decreased poly-ADP-ribosylation, and (4) failures in ATP-dependent chromatin-remodelling mechanisms. Moreover, these alterations can influence the response to classical anti-tumour treatments. Drugs targeting epigenetic alterations are under development. Currently, DNA methylation and histone deacetylase inhibitors are in use in cancer therapy, and poly-ADP-ribosylation inhibitors are undergoing clinical trials. Epigenetic therapy is gaining in importance in pharmacology as a new tool to improve anti-cancer therapies.
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http://dx.doi.org/10.1016/j.mrrev.2008.01.008DOI Listing
July 2008