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1.
Chem Res Toxicol ; 33(10): 2668-2674, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-32894672

RESUMO

Inflammation is an immune response to protect against various types of infections. When unchecked, acute inflammation can be life-threatening, as seen with the current coronavirus pandemic. Strong oxidants, such as peroxynitrite produced by immune cells, are major mediators of the inflammation-associated pathogenesis. Cellular thiols play important roles in mitigating inflammation-associated macromolecular damage including DNA. Herein, we have demonstrated a role of glutathione (GSH) and other thiols in neutralizing the effect of peroxynitrite-mediated DNA damage through stable GSH-DNA adduct formation. Our observation supports the use of thiol supplements as a potential therapeutic strategy against severe COVID-19 cases and a Phase II (NCT04374461) open-label clinical trial launched in early May 2020 by the Memorial Sloan Kettering Cancer Center.


Assuntos
Adutos de DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Glutationa/farmacologia , Inflamação/fisiopatologia , Ácido Peroxinitroso/efeitos adversos , Doença Aguda , Animais , Betacoronavirus , Bovinos , Infecções por Coronavirus/tratamento farmacológico , DNA/química , Adutos de DNA/química , Dano ao DNA , Glutationa/química , Células HEK293 , Humanos , Mutagênicos/química , Mutagênicos/farmacologia , Pandemias , Ácido Peroxinitroso/química , Pneumonia Viral/tratamento farmacológico , Salmonella typhimurium/genética
2.
Nat Commun ; 11(1): 4714, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948754

RESUMO

The application of forces and torques on the single molecule level has transformed our understanding of the dynamic properties of biomolecules, but rare intermediates have remained difficult to characterize due to limited throughput. Here, we describe a method that provides a 100-fold improvement in the throughput of force spectroscopy measurements with topological control, which enables routine imaging of 50,000 single molecules and a 100 million reaction cycles in parallel. This improvement enables detection of rare events in the life cycle of the cell. As a demonstration, we characterize the supercoiling dynamics and drug-induced DNA break intermediates of topoisomerases. To rapidly quantify distinct classes of dynamic behaviors and rare events, we developed a software platform with an automated feature classification pipeline. The method and software can be readily adapted for studies of a broad range of complex, multistep enzymatic pathways in which rare intermediates have escaped classification due to limited throughput.


Assuntos
DNA/química , Fenômenos Magnéticos , Magnetismo/métodos , Nanotecnologia , Análise Espectral/métodos , Ciprofloxacino/farmacologia , DNA/efeitos dos fármacos , Quebras de DNA/efeitos dos fármacos , Conformação de Ácido Nucleico , Pinças Ópticas , Fenômenos Físicos , Software
3.
Nucleic Acids Res ; 48(18): 10383-10396, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32941607

RESUMO

In the constant evolutionary battle against mobile genetic elements (MGEs), bacteria have developed several defense mechanisms, some of which target the incoming, foreign nucleic acids e.g. restriction-modification (R-M) or CRISPR-Cas systems. Some of these MGEs, including bacteriophages, have in turn evolved different strategies to evade these hurdles. It was recently shown that the siphophage CAjan and 180 other viruses use 7-deazaguanine modifications in their DNA to evade bacterial R-M systems. Among others, phage CAjan genome contains a gene coding for a DNA-modifying homolog of a tRNA-deazapurine modification enzyme, together with four 7-cyano-7-deazaguanine synthesis genes. Using the CRISPR-Cas9 genome editing tool combined with the Nanopore Sequencing (ONT) we showed that the 7-deazaguanine modification in the CAjan genome is dependent on phage-encoded genes. The modification is also site-specific and is found mainly in two separate DNA sequence contexts: GA and GGC. Homology modeling of the modifying enzyme DpdA provides insight into its probable DNA binding surface and general mode of DNA recognition.


Assuntos
Bacteriófagos/genética , DNA/genética , Motivos de Nucleotídeos/genética , Pirimidinonas/farmacologia , Pirróis/farmacologia , Bacteriófagos/efeitos dos fármacos , Sequência de Bases/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , DNA/efeitos dos fármacos , Enzimas de Restrição-Modificação do DNA/efeitos dos fármacos , Escherichia coli/virologia , Edição de Genes , Guanina/análogos & derivados , Guanina/farmacologia , Humanos , Sequenciamento por Nanoporos , Motivos de Nucleotídeos/efeitos dos fármacos , Siphoviridae/genética
4.
PLoS One ; 15(8): e0236104, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32776939

RESUMO

There is an increasing emphasis on effects-based monitoring to document responses associated with exposure to complex mixtures of chemicals, climate change, pathogens, parasites and other environmental stressors in fish populations. For decades aquatic monitoring programs have included the collection of tissues preserved for microscopic pathology. Consequently, formalin-fixed, paraffin-embedded (FFPE) tissue can be an important reservoir of nucleic acids as technologies emerge that utilize molecular endpoints. Despite the cross-linking effects of formalin, its impact on nucleic acid quality and concentration, amplification, and sequencing are not well described. While fresh-frozen tissue is optimal for working with nucleic acids, FFPE samples have been shown to be conducive for molecular studies. Laser capture microdissection (LCM) is one technology which allows for collection of specific regions or cell populations from fresh or preserved specimens with pathological alterations, pathogens, or parasites. In this study, smallmouth bass (Micropterus dolomieu) liver was preserved in three different fixatives, including 10% neutral buffered formalin (NBF), Z-Fix® (ZF), and PAXgene® (PG) for four time periods (24 hr, 48 hr, seven days, and 14 days). Controls consisted of pieces of liver preserved in RNALater® or 95% ethanol. Smallmouth bass were chosen as they are an economically important sportfish and have been utilized as indicators of exposure to endocrine disruptors and other environmental stressors. Small liver sections were cut out with laser microdissection and DNA and RNA were purified and analyzed for nucleic acid concentration and quality. Sanger sequencing and the NanoString nCounter® technology were used to assess the suitability of these samples in downstream molecular techniques. The results revealed that of the formalin fixatives, NBF samples fixed for 24 and 48 hr were superior to ZF samples for both Sanger sequencing and the Nanostring nCounter®. The non-formalin PAXgene® samples were equally successful and they showed greater stability in nucleic acid quality and concentration over longer fixation times. This study demonstrated that small quantities of preserved tissue from smallmouth bass can be utilized in downstream molecular techniques; however, future studies will need to optimize the methods presented here for different tissue types, fish species, and pathological conditions.


Assuntos
Bass/genética , DNA/efeitos dos fármacos , Monitoramento Ambiental/métodos , Fixadores/efeitos adversos , RNA/efeitos dos fármacos , Animais , Clivagem do DNA/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Formaldeído/efeitos adversos , Perfilação da Expressão Gênica/métodos , Fígado/efeitos dos fármacos , Fígado/patologia , Microdissecção , Desnaturação de Ácido Nucleico/efeitos dos fármacos , RNA/isolamento & purificação , Estabilidade de RNA/efeitos dos fármacos , Análise de Sequência de DNA , Fatores de Tempo , Fixação de Tecidos/métodos , West Virginia
5.
PLoS One ; 15(8): e0236808, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750061

RESUMO

BACKGROUND: Ataxia with oculomotor apraxia type 1 (AOA1) is a rare autosomal recessive cerebellar ataxia, caused by mutations in the APTX gene. The disease is characterized by early-onset cerebellar ataxia, oculomotor apraxia and severe axonal polyneuropathy. The aim of this study was to detect the disease-causing variants in two unrelated consanguineous Jordanian families with cerebellar ataxia using whole exome sequencing (WES), and to correlate the identified mutation(s) with the clinical and cellular phenotypes. METHODS: WES was performed in three affected individuals and segregation analysis of p.W279* APTX candidate variant was performed. Expression levels of APTX were measured in patients' skin fibroblasts and peripheral blood mononuclear cells, followed by western blot analysis in skin fibroblasts. Genotoxicity assay was performed to detect the sensitivity of APTX mutated cells to H2O2, MMC, MMS and etoposide. RESULTS: A recurrent homozygous nonsense variant in APTX gene (c.837G>A, p.W279*) was revealed in all affected individuals. qRT-PCR showed normal APTX levels in peripheral blood and lower levels in fibroblast cells. However, western blot showed the absence of APTX protein in patients' skin fibroblasts. Significant hypersensitivity to H2O2, MMC and etoposide and lack of sensitivity to MMS were noted. CONCLUSIONS: This is the first study to report the identification of a nonsense variant in the APTX gene (c.837G>A; p.W279*) in AOA1 patients within the Jordanian population. This study confirmed the need of WES to assist in the diagnosis of cerebellar ataxia and it emphasizes the importance of studying the pathophysiology of the APTX gene.


Assuntos
Ataxia Cerebelar/genética , Códon sem Sentido , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Criança , Pré-Escolar , Consanguinidade , DNA/efeitos dos fármacos , Feminino , Humanos , Masculino , Mutagênicos/farmacologia , Sequenciamento Completo do Exoma
6.
Am J Pathol ; 190(10): 2155-2164, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32679231

RESUMO

Aberrant DNA methylation is an epigenetic hallmark of melanoma, but the expression of DNA methyltransferase (Dnmt)-1 in melanocytic tumors is unknown. Dnmt1 expression was analyzed in primary melanocytes, melanoma cell lines, and 83 melanocytic tumors, and its associations with proliferation, mutational status, and response to B-Raf and mitogen-activated protein kinase kinase (MEK) inhibition were explored. Dnmt1 expression was increased incrementally from nevi [mean fluorescence intensity (MFI), 48.1; interquartile range, 41.7 to 59.6] to primary melanomas (MFI, 68.8; interquartile range, 58.4 to 77.0) and metastatic melanomas (MFI, 87.5; interquartile range, 77.1 to 114.5) (P < 0.001). Dnmt1 expression was correlated with Ki-67 expression (Spearman correlation, 0.483; P < 0.001) and was independent of BRAF mutation status (P = 0.55). In BRAF-mutant melanoma, Dnmt1 was down-regulated during response to B-Raf and MEK inhibition and was again up-regulated on drug resistance in vitro and in vivo. Degradation of Dnmt1 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid was associated with decreased cell viability in B-Raf inhibitor-sensitive and -resistant cell lines. This study demonstrates that Dnmt1 expression is correlated with proliferation in melanocytic tumors, is increased with melanoma progression, and is associated with response to B-Raf and MEK inhibition. Given its strong expression in metastatic melanoma, Dnmt1 may be a promising target for combined epigenetic and immunotherapy.


Assuntos
Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Melanoma/metabolismo , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Cutâneas/metabolismo , Linhagem Celular Tumoral , DNA/efeitos dos fármacos , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanoma/genética , Melanoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Vorinostat/farmacologia
7.
PLoS One ; 15(7): e0235717, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658905

RESUMO

Vernonia amygdalina (VA) has been reported to have antioxidant potential; however, its DNA protection and anti-inflammatory properties remain unclear. We aimed to investigate whether aqueous (WEVAL) and alcoholic (EEVAL) VA extracts exert similar antioxidant, DNA protection and anti-inflammatory effects and attempted to explore the mechanism underlying the anti-inflammatory effects. These results demonstrated that WEVAL had greater polyphenolic and flavonoid contents, as well as a stronger reducing power, DPPH radical scavenging and DNA protective activity. Moreover, both extracts reduced lipopolysaccharide (LPS)-induced expression of COX-II, iNOS, pro-inflammatory factors, including NO, TNF-α, IL-1ß, and IL-10. Compared with WEVAL, EEVAL was a more potent inflammatory inhibitor. Both extracts similarly inhibited LPS-induced MAPK (p38) and NF-κB expression. Our findings indicate that WEVAL and EEVAL have diverse antioxidant and anti-inflammatory effects. WEVAL had a stronger antioxidant and DNA protection activity; contrastingly, EEVAL had a stronger anti-inflammatory ability. The anti-inflammatory activity involves reduced pro-inflammatory cytokines through NF-κB down-regulation and MAPK inhibition. These results demonstrated that production of WEVAL and EEVAL from VA leaves may provide a safe and efficacious source of pharmaceutical applications, with antioxidant, DNA protective and anti-inflammation activities.


Assuntos
Antioxidantes/farmacologia , DNA/efeitos dos fármacos , Inflamação/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Folhas de Planta/química , Vernonia/química , Proliferação de Células , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Flavonoides/farmacologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais
8.
Nucleic Acids Res ; 48(15): 8796-8807, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32652019

RESUMO

5-Formylcytosine (5fC) is a chemically edited, naturally occurring nucleobase which appears in the context of modified DNA strands. The understanding of the impact of 5fC on dsDNA physical properties is to date limited. In this work, we applied temperature-dependent 1H Chemical Exchange Saturation Transfer (CEST) NMR experiments to non-invasively and site-specifically measure the thermodynamic and kinetic influence of formylated cytosine nucleobase on the melting process involving dsDNA. Incorporation of 5fC within symmetrically positioned CpG sites destabilizes the whole dsDNA structure-as witnessed from the ∼2°C decrease in the melting temperature and 5-10 kJ mol-1 decrease in ΔG°-and affects the kinetic rates of association and dissociation. We observed an up to ∼5-fold enhancement of the dsDNA dissociation and an up to ∼3-fold reduction in ssDNA association rate constants, over multiple temperatures and for several proton reporters. Eyring and van't Hoff analysis proved that the destabilization is not localized, instead all base-pairs are affected and the transition states resembles the single-stranded conformation. These results advance our knowledge about the role of 5fC as a semi-permanent epigenetic modification and assist in the understanding of its interactions with reader proteins.


Assuntos
Citosina/análogos & derivados , DNA/efeitos dos fármacos , Conformação Molecular/efeitos dos fármacos , Termodinâmica , Pareamento de Bases/genética , Ilhas de CpG/genética , Citosina/química , Citosina/farmacologia , DNA/química , DNA/genética , DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/genética , Cinética , Espectroscopia de Ressonância Magnética , Temperatura de Transição
9.
Mutat Res ; 854-855: 503198, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32660822

RESUMO

TiO2 particles are broadly used in daily products, including cosmetics for their UV-absorbing property, food for their white colouring property, water and air purification systems, self-cleaning surfaces and photoconversion electrical devices for their photocatalytic properties. The toxicity of TiO2 nano- and microparticles has been studied for decades, and part of this investigation has been dedicated to the identification of their potential impact on DNA, i.e., their genotoxicity. This review summarizes data retrieved from their genotoxicity testing during the past 6 years, encompassing both in vitro and in vivo studies, mostly performed on lung and intestinal models. It shows that TiO2 particles, both nano- and micro-sized, produce genotoxic damage to a variety of cell types, even at low, realistic doses.


Assuntos
Mutagênicos/toxicidade , Titânio/toxicidade , Animais , DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Humanos , Testes de Mutagenicidade/métodos
10.
Nucleic Acids Res ; 48(12): 6715-6725, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32484547

RESUMO

DNA damage and epigenetic marks are well established to have profound influences on genome stability and cell phenotype, yet there are few technologies to obtain high-resolution genomic maps of the many types of chemical modifications of DNA. Here we present Nick-seq for quantitative, sensitive, and accurate mapping of DNA modifications at single-nucleotide resolution across genomes. Pre-existing breaks are first blocked and DNA modifications are then converted enzymatically or chemically to strand-breaks for both 3'-extension by nick-translation to produce nuclease-resistant oligonucleotides and 3'-terminal transferase tailing. Following library preparation and next generation sequencing, the complementary datasets are mined with a custom workflow to increase sensitivity, specificity and accuracy of the map. The utility of Nick-seq is demonstrated with genomic maps of site-specific endonuclease strand-breaks in purified DNA from Eschericia coli, phosphorothioate epigenetics in Salmonella enterica Cerro 87, and oxidation-induced abasic sites in DNA from E. coli treated with a sublethal dose of hydrogen peroxide. Nick-seq applicability is demonstrated with strategies for >25 types of DNA modification and damage.


Assuntos
Dano ao DNA/efeitos dos fármacos , Epigênese Genética/genética , Genoma Bacteriano/genética , Instabilidade Genômica/efeitos dos fármacos , Mapeamento Cromossômico , DNA/química , DNA/efeitos dos fármacos , Dano ao DNA/genética , Escherichia coli/genética , Genoma Bacteriano/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Peróxido de Hidrogênio/toxicidade , Nucleotídeos/química , Salmonella enterica/genética , Análise de Sequência de DNA
11.
Toxicol Lett ; 331: 57-64, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32442718

RESUMO

Electrophilic compounds present in humans, originating from endogenous processes or pollutant exposures, pose a risk to health though their reaction with nucleophilic sites in protein and DNA. Among this chemical class, aldehydes are mainly present in indoor air and they can also be produced by endogenous lipid peroxidation arising from oxidative stress. Known to be very reactive, aldehydes have the ability to form exocyclic adducts to DNA that, for the most if not repaired correctly, are mutagenic and by consequence potential agents involved in carcinogenesis. The aim of this work was to establish profiles of exocyclic DNA adducts induced by aldehyde mixtures, which could ultimately be considered as a genotoxic marker of endogenous and environmental aldehyde exposure. Adducts were quantified by an accurate, sensitive and validated ultra high performance liquid chromatography-electrospray ionization analytical method coupled to mass spectrometry in the tandem mode (UHPLC-ESI-MS/MS). We simultaneously measured nine exocyclic DNA adducts generated during the exposure in vitro of calf thymus DNA to different concentrations of each aldehyde along, as well as, to an equimolar mixture of these aldehydes. This approach has enabled us to establish dose-response relationships that allowed displaying the specific reactivity of aldehydes towards corresponding adducts formation. Profiles of these adducts determined in DNA of current smokers and non-smokers blood samples supported these findings. These first results are encouraging to explore genotoxicity induced by aldehyde mixtures and can furthermore be used as future reference for adductomic approaches.


Assuntos
Aldeídos/toxicidade , Adutos de DNA/sangue , DNA/efeitos dos fármacos , Mutagênicos/toxicidade , Fumar/sangue , Tabaco , DNA/genética , Relação Dose-Resposta a Droga , Humanos , Tabaco/química
12.
Nucleic Acids Res ; 48(11): 6108-6119, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32392345

RESUMO

Protamine proteins dramatically condense DNA in sperm to almost crystalline packing levels. Here, we measure the first step in the in vitro pathway, the folding of DNA into a single loop. Current models for DNA loop formation are one-step, all-or-nothing models with a looped state and an unlooped state. However, when we use a Tethered Particle Motion (TPM) assay to measure the dynamic, real-time looping of DNA by protamine, we observe the presence of multiple folded states that are long-lived (∼100 s) and reversible. In addition, we measure folding on DNA molecules that are too short to form loops. This suggests that protamine is using a multi-step process to loop the DNA rather than a one-step process. To visualize the DNA structures, we used an Atomic Force Microscopy (AFM) assay. We see that some folded DNA molecules are loops with a ∼10-nm radius and some of the folded molecules are partial loops-c-shapes or s-shapes-that have a radius of curvature of ∼10 nm. Further analysis of these structures suggest that protamine is bending the DNA to achieve this curvature rather than increasing the flexibility of the DNA. We therefore conclude that protamine loops DNA in multiple steps, bending it into a loop.


Assuntos
DNA/química , DNA/efeitos dos fármacos , Conformação de Ácido Nucleico/efeitos dos fármacos , Protaminas/química , Protaminas/farmacologia , DNA/ultraestrutura , Microscopia de Força Atômica , Maleabilidade
13.
Chemosphere ; 254: 126875, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32361544

RESUMO

In the present scenario, insecticides/pesticides are used intensively to control the various insect pests. Indiscriminate use of these insecticides/pesticides affects the structure and function of the ecosystem. In this context, a thorough toxicological study of each insecticide/pesticide is a must to understand the hazardous effect of these chemicals on the target and non-target organisms. The present study was aimed to understand the hazardous effect of thiamethoxam against the Spodoptera litura. Different concentrations (20-80 µg/mL) of thiamethoxam were prepared, and fourth instar larvae of S. litura were allowed to feed for 12-72 h. We first examined the interaction of thiamethoxam with DNA. Next, treated and non-treated larvae were assessed for different biological parameters such as mortality, emergence, fecundity, fertility, longevities, and biochemical parameters. Our result showed that thiamethoxam directly interacts with the DNA and significantly influenced the different biological and biochemical parameters of exposed the organisms. We observed a significant change in stress enzymes such as SOD, CAT, and GST. A similar observation was also made with the oxidative marker for lipid damage, MDA and DNA damage, 8-OHdG, respectively. In conclusion, our results suggest that improper use of synthetic chemical insecticides influenced both biological and biochemical parameters through oxidative stress and probably damage the genetic material.


Assuntos
DNA/efeitos dos fármacos , Inseticidas/toxicidade , Tiametoxam/toxicidade , Animais , Dano ao DNA , Ecossistema , Insetos , Larva/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Praguicidas/farmacologia , Spodoptera/efeitos dos fármacos
14.
Environ Mol Mutagen ; 61(7): 716-729, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32329115

RESUMO

Many environmental carcinogens cause DNA damage, which can result in mutations and other alterations in genomic DNA if not repaired promptly. Because of the bulkiness of the lesions, DNA-protein crosslinks (DPCs) are one of the types of toxic DNA damage with potentially deleterious consequences. Despite the importance of DPCs, how cells remove these complex DNA adducts has been incompletely understood. However, major progress in the DPC repair field over the past 5 years now supports the view that cells are equipped with multiple mechanisms to cope with DPCs. Here, we first provide an overview of environmental substances that induce DPCs, describing the sources of exposure and mechanisms of DPC formation. We then review current models of DPC repair and discuss their significance for environmental carcinogens.


Assuntos
Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , DNA/efeitos dos fármacos , DNA/genética , Exposição Ambiental/efeitos adversos , Animais , Humanos
15.
Anticancer Res ; 40(4): 2025-2032, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32234893

RESUMO

BACKGROUND/AIM: The winemaking procedure results in the generation of stems, a by-product that is harmful to the environment. Concomitantly, stems are rich in polyphenols and, hence, they are putatively beneficial for human health. MATERIALS AND METHODS: In this study, the grape stem extracts derived from three native Greek vine varieties, namely Mavrodaphne, Muscat and Rhoditis were examined for their chemical composition and antioxidant and antimutagenic properties using a battery of in vitro biomarkers. RESULTS: All extracts are rich in polyphenols. Moreover, they exhibit potent antioxidant and antimutagenic properties with the extract of Mavrodaphne being the strongest in reducing the DPPH• and O2 -• radicals and the Fe3+ and in protecting plasmid DNA from peroxyl radical-induced oxidative modification. CONCLUSION: Therefore, although they are serious pollutants, grape stems contain phytochemicals with important biological properties and can be used as (ingredients of) bio-functional foods to improve certain aspects of human health.


Assuntos
Antioxidantes/farmacologia , DNA/efeitos dos fármacos , Polifenóis/farmacologia , Vitis/química , DNA/metabolismo , Dano ao DNA/efeitos dos fármacos , Grécia , Humanos , Oxirredução , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Caules de Planta/química , Plasmídeos/efeitos dos fármacos , Plasmídeos/metabolismo , Polifenóis/química , Espécies Reativas de Oxigênio/metabolismo
16.
Mutat Res ; 850-851: 503136, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247553

RESUMO

Tumorigenesis induced by oxidative stress is thought to be initiated by mutagenesis, but via an indirect mechanism. The dose-response curves for agents that act by this route usually show a threshold, for unknown reasons. To gain insight into these phenomena, we have analyzed the dose response for mutagenesis induced by the oral administration of potassium bromate, a typical oxidative-stress-generating agent, to gpt delta mice. The agent was given orally for 90 d to either Nrf2+ or Nrf2-knockout (KO) mice and mutants induced in the small intestine were analyzed. In Nrf2+mice, the mutant frequency was significantly greater than in the vehicle controls at a dose of 0.6 g/L but not at 0.2 g/L, indicating that a practical threshold for mutagenesis lies between these doses. At 0.6 g/L, the frequencies of G-to-T transversions (landmark mutations for oxidative stress) and G-to-A transitions were significantly elevated. In Nrf2-KO mice, too, the total mutant frequency was increased only at 0.6 g/L. G-to-T transversions are likely to have driven tumorigenesis in the small intestine. A site-specific G-to-T transversion at guanine (nucleotide 406) in a 5'-TGAA-3' sequence in gpt, and our primer extension reaction showed that formation of the oxidative DNA base modification 8-oxo-deoxyguanosine (8-oxo-dG) at nucleotide 406 was significantly increased at doses of 0.6 and 2 g/L in the gpt delta mice. In the Apc oncogene, guanine residues in the same or similar sequences (TGAA or AGAA) are highly substituted by thymine (G-to-T transversions) in potassium bromate-induced tumors. We propose that formation of 8-oxo-dG in the T(A)GAA sequence is an initiating event in tumor formation in the small intestine in response to oxidative stress.


Assuntos
Bromatos/toxicidade , Mutagênese/genética , Estresse Oxidativo/genética , Pentosiltransferases/genética , 8-Hidroxi-2'-Desoxiguanosina/genética , Administração Oral , Animais , Bromatos/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , DNA/efeitos dos fármacos , DNA/genética , Relação Dose-Resposta a Droga , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Camundongos , Camundongos Knockout , Mutagênese/efeitos dos fármacos , Mutação , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos
17.
Eur J Med Chem ; 192: 112185, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32145644

RESUMO

Over the past decade, we described a novel tumour targeted approach that sought to design "combi-molecules" to hit two distinct targets in tumour cells. Here, to generate small combi-molecules with strong DNA damaging potential while retaining EGFR inhibitory potency, we developed the first synthetic strategy to access the 6-N, N-disubstituted quinazoline scaffold and designed JS61 to possess a nitrogen mustard function directly attached to the 6-position of the quinazoline ring. We compared its biological activity with that of structures containing either a hemi mustard or a non-alkylating substituent. Surprisingly, the results showed that JS61, while capable of inducing strong DNA damage, exhibited moderate EGFR inhibitory potency. In contrast, "combi-molecules" with no bulky substituent at the N-6 position (e.g. ZR2002 and JS84) showed stronger EGFR and growth inhibitory potency than JS61 in a panel of lung cancer cells. To rationalize these results, X-ray crystallography and molecular modeling studies were undertaken, and the data obtained indicated that bulkiness of the 6-N,N-disubstituted moieties hinder its binding to the ATP site and affects binding reversibility.


Assuntos
Antineoplásicos/farmacologia , DNA/efeitos dos fármacos , Quinazolinas/farmacologia , Células A549 , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Bovinos , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Quinazolinas/síntese química , Quinazolinas/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas
18.
Sensors (Basel) ; 20(7)2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32218227

RESUMO

Cancer is a multifactorial family of diseases that is still a leading cause of death worldwide. More than 100 different types of cancer affecting over 60 human organs are known. Chemotherapy plays a central role for treating cancer. The development of new anticancer drugs or new uses for existing drugs is an exciting and increasing research area. This is particularly important since drug resistance and side effects can limit the efficacy of the chemotherapy. Thus, there is a need for multiplexed, cost-effective, rapid, and novel screening methods that can help to elucidate the mechanism of the action of anticancer drugs and the identification of novel drug candidates. This review focuses on different label-free bioelectrochemical approaches, in particular, impedance-based methods, the solid supported membranes technique, and the DNA-based electrochemical sensor, that can be used to evaluate the effects of anticancer drugs on nucleic acids, membrane transporters, and living cells. Some relevant examples of anticancer drug interactions are presented which demonstrate the usefulness of such methods for the characterization of the mechanism of action of anticancer drugs that are targeted against various biomolecules.


Assuntos
Antineoplásicos/efeitos adversos , Técnicas Biossensoriais , DNA/isolamento & purificação , Espectroscopia Dielétrica , Antineoplásicos/uso terapêutico , DNA/efeitos dos fármacos , DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Tratamento Farmacológico , Humanos , Neoplasias/tratamento farmacológico
19.
ACS Appl Mater Interfaces ; 12(12): 14563-14568, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32129065

RESUMO

Nanopores have become an important tool for the detection and analysis of molecules at the single-molecule level. Surface modification of solid-state nanopores can improve their durability and efficiency. Peptides are ideal for surface modifications as they allow tailoring of multiple properties by a rational design of their sequence. Here, silicon nitride nanopores were coated by a dipeptide layer where a l-3,4-dihydroxyphenylalanine (DOPA) residue is the anchoring element and the other amino acid moiety is the functional element. DOPA binds tightly to many types of surfaces and allows a one-step functionalization of surfaces by simple immersion. As a result, the lifetime of coated nanopores increased from hours to months and the current-stability has significantly improved with respect to uncoated pores. This improvement is achieved by controlling the surface wettability and charge. Peptide-coated nanopores can be utilized as sensitive sensors that can be adjusted based on the choice of the functional moiety of the coated peptide. In addition, the coating slows down dsDNA translocation because of the DNA interaction with the pore coating.


Assuntos
Di-Hidroxifenilalanina/química , Dipeptídeos/química , Nanoporos/ultraestrutura , Nanotecnologia , DNA/efeitos dos fármacos , Dipeptídeos/genética , Compostos de Silício/química , Propriedades de Superfície/efeitos dos fármacos
20.
J Med Chem ; 63(6): 3090-3103, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32142285

RESUMO

Targeting G-quadruplex structures is currently viewed as a promising anticancer strategy. Searching for potent and selective G-quadruplex binders, here we describe a small series of new monohydrazone derivatives designed as analogues of a lead which was proved to stabilize G-quadruplex structures and increase R loop levels in human cancer cells. To investigate the G-quadruplex binding properties of the new molecules, in vitro biophysical studies were performed employing both telomeric and oncogene promoter G-quadruplex-forming sequences. The obtained results allowed the identification of a highly selective G-quadruplex ligand that, when studied in human cancer cells, proved to be able to stabilize both G-quadruplexes and R loops and showed a potent cell killing activity associated with the formation of micronuclei, a clear sign of genome instability.


Assuntos
Antineoplásicos/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Hidrazonas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Genoma/efeitos dos fármacos , Humanos , Hidrazonas/síntese química , Hidrazonas/metabolismo , Ligantes , Estruturas R-Loop/efeitos dos fármacos
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