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We examined the reaction of hydroxyl radicals (HOâ¢) and sulfate radical anions (SO4â¢-), which is generated by ionizing radiation in aqueous solutions under anoxic conditions, with an alternating GC doubled-stranded oligodeoxynucleotide (ds-ODN), i.e., the palindromic 5'-d(GCGCGC)-3'. In particular, the optical spectra of the intermediate species and associated kinetic data in the range of ns to ms were obtained via pulse radiolysis. Computational studies by means of density functional theory (DFT) for structural and time-dependent DFT for spectroscopic features were performed on 5'-d(GCGC)-3'. Comprehensively, our results suggest the addition of HO⢠to the G:C pair moiety, affording the [8-HO-G:C]⢠detectable adduct. The previous reported spectra of one-electron oxidation of a variety of ds-ODN were assigned to [G(-H+):C]⢠after deprotonation. Regarding 5'-d(GCGCGC)-3' ds-ODN, the spectrum at 800 ns has a completely different spectral shape and kinetic behavior. By means of calculations, we assigned the species to [G:C/C:G]â¢+, in which the electron hole is predicted to be delocalized on the two stacked base pairs. This transient species was further hydrated to afford the [8-HO-G:C]⢠detectable adduct. These remarkable findings suggest that the double-stranded alternating GC sequences allow for a new type of electron hole stabilization via delocalization over the whole sequence or part of it.
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Radical Hidroxila , Oligonucleotídeos , Oligonucleotídeos/química , Elétrons , Radicais Livres/química , Oxirredução , OligodesoxirribonucleotídeosRESUMO
Chlamydomonas reinhardtii (C. reinhardtii) is one of the most well-studied microalgae organisms that revealed important information for the photosynthetic and metabolic processes of plants and eukaryotes. Numerous extensive studies have also underpinned its great potential as a biochemical factory, capable of producing various highly desired molecules with a direct impact on human health and longevity. Polysaccharides, lipids, functional proteins, pigments, hormones, vaccines, and antibodies are among the valuable biomolecules that are produced spontaneously or under well-defined conditions by C. reinhardtii and can be directly linked to human nutrition and diet. The aim of this review is to highlight the recent advances in the field focusing on the most relevant applications related to the production of important biomolecules for human health that are also linked with human nutrition and diet. The limitations and challenges are critically discussed along with the potential future applications of C. reinhardtii biomass and processed products in the field of nutraceuticals and food supplements. The increasing need for high-value and low-cost biomolecules produced in an environmentally and economy sustainable manner also underline the important role of C. reinhardtii.
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Chlamydomonas reinhardtii , Humanos , Chlamydomonas reinhardtii/metabolismo , Fotossíntese , Suplementos Nutricionais , PlantasRESUMO
In recent years, nucleic acids have emerged as powerful biomaterials, revolutionizing the field of biomedicine. This review explores the multifaceted applications of nucleic acids, focusing on their pivotal role in various biomedical applications. Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), possess unique properties such as molecular recognition ability, programmability, and ease of synthesis, making them versatile tools in biosensing and for gene regulation, drug delivery, and targeted therapy. Their compatibility with chemical modifications enhances their binding affinity and resistance to degradation, elevating their effectiveness in targeted applications. Additionally, nucleic acids have found utility as self-assembling building blocks, leading to the creation of nanostructures whose high order underpins their enhanced biological stability and affects the cellular uptake efficiency. Furthermore, this review delves into the significant role of oligonucleotides (ODNs) as indispensable tools for biological studies and biomarker discovery. ODNs, short sequences of nucleic acids, have been instrumental in unraveling complex biological mechanisms. They serve as probes for studying gene expression, protein interactions, and cellular pathways, providing invaluable insights into fundamental biological processes. By examining the synergistic interplay between nucleic acids as powerful biomaterials and ODNs as indispensable tools for biological studies and biomarkers, this review highlights the transformative impact of these molecules on biomedical research. Their versatile applications not only deepen our understanding of biological systems but also are the driving force for innovation in diagnostics and therapeutics, ultimately advancing the field of biomedicine.
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Ácidos Nucleicos , Ácidos Nucleicos/uso terapêutico , Oligonucleotídeos/uso terapêutico , RNA , Materiais Biocompatíveis/uso terapêutico , Transporte BiológicoRESUMO
Mitochondrial (mt) DNA and nuclear (n) DNA have known structures and roles in cells; however, they are rarely compared under specific conditions such as oxidative or degenerative environments that can create damage to the DNA base moieties. Six purine lesions were ascertained in the mtDNA of wild type (wt) CSA (CS3BE-wtCSA) and wtCSB (CS1AN-wtCSB) cells and defective counterparts CS3BE and CS1AN in comparison with the corresponding total (t) DNA (t = n + mt). In particular, the four 5',8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. The 8-oxo-Pu levels were found to be in the range of 25-50 lesions/107 nucleotides in both the mtDNA and tDNA. The four cPu were undetectable in the mtDNA both in defective cells and in the wt counterparts (CSA and CSB), contrary to their detection in tDNA, indicating a nonappearance of hydroxyl radical (HOâ¢) reactivity within the mtDNA. In order to assess the HO⢠reactivity towards purine nucleobases in the two genetic materials, we performed γ-radiolysis experiments coupled with the 8-oxo-Pu and cPu quantifications on isolated mtDNA and tDNA from wtCSB cells. In the latter experiments, all six purine lesions were detected in both of the DNA, showing a higher resistance to HO⢠attack in the case of mtDNA compared with tDNA, likely due to their different DNA helical topology influencing the relative abundance of the lesions.
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Síndrome de Cockayne , Humanos , Dano ao DNA , DNA Mitocondrial/genética , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , PurinasRESUMO
Oxygen is important for lipid metabolism, being involved in both enzymatic transformations and oxidative reactivity, and is particularly influent when genetic diseases impair the repair machinery of the cells, such as described for Cockayne syndrome (CS). We used two cellular models of transformed fibroblasts defective for CSA and CSB genes and their normal counterparts, grown for 24 h under various oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%) to examine the fatty acid-based membrane remodeling by GC analysis of fatty acid methyl esters derived from membrane phospholipids. Overall, we first distinguished differences due to oxygen tensions: (a) hyperoxia induced a general boost of desaturase enzymatic activity in both normal and defective CSA and CSB cell lines, increasing monounsaturated fatty acids (MUFA), whereas polyunsaturated fatty acids (PUFA) did not undergo oxidative consumption; (b) hypoxia slowed down desaturase activities, mostly in CSA cell lines and defective CSB, causing saturated fatty acids (SFA) to increase, whereas PUFA levels diminished, suggesting their involvement in hypoxia-related signaling. CSB-deprived cells are the most sensitive to oxidation and CSA-deprived cells are the most sensitive to the radical-based formation of trans fatty acids (TFA). The results point to the need to finely differentiate biological targets connected to genetic impairments and, consequently, suggest the better definition of cell protection and treatments through accurate molecular profiling that includes membrane lipidomes.
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Síndrome de Cockayne , Síndrome de Cockayne/genética , Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Insaturados/farmacologia , Humanos , Hipóxia , Lipidômica , OxigênioRESUMO
Despite the widespread use of silver nanoparticles (AgNPs) in different fields and the amount of investigations available, to date, there are many contradictory results on their potential toxicity. In the present study, extensively characterized 20-nm AgNPs were investigated using optimized protocols and standardized methods to test several toxicological endpoints in different cell lines. The agglomeration/aggregation state of AgNPs in culture media was measured by dynamic light scattering (DLS). DNA and chromosomal damage on BEAS-2B and RAW 264.7 cells were evaluated by comet and micronucleus assays, while oxidative DNA damage by modified comet assay and 8-oxodG/8-oxodA detection. We also investigated immunotoxicity and immunomodulation by cytokine release and NO production in RAW 264.7 and MH-S cells, with or without lipopolysaccharide (LPS) stimulus. Transmission electron microscope (TEM) analysis was used to analyze cellular uptake of AgNPs. Our results indicate different values of AgNPs hydrodynamic diameter depending on the medium, some genotoxic effect just on BEAS-2B and no or slight effects on function of RAW 264.7 and MH-S in absence or presence of LPS stimulus. This study highlights the relevance of using optimized protocols and multiple endpoints to analyze the potential toxicity of AgNPs and to obtain reliable and comparable results.
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Técnicas In Vitro/métodos , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Testes de Toxicidade/métodos , Linhagem Celular , Ensaio Cometa , Testes para MicronúcleosRESUMO
Hydroxyl radical (HOâ¢) is the most reactive toward DNA among the reactive oxygen species (ROS) generated in aerobic organisms by cellular metabolisms. HO⢠is generated also by exogenous sources such as ionizing radiations. In this review we focus on the purine DNA damage by HO⢠radicals. In particular, emphasis is given on mechanistic aspects for the various lesion formation and their interconnections. Although the majority of the purine DNA lesions like 8-oxo-purine (8-oxo-Pu) are generated by various ROS (including HOâ¢), the formation of 5',8-cyclopurine (cPu) lesions in vitro and in vivo relies exclusively on the HO⢠attack. Methodologies generally utilized for the purine lesions quantification in biological samples are reported and critically discussed. Recent results on cPu and 8-oxo-Pu lesions quantification in various types of biological specimens associated with the cellular repair efficiency as well as with distinct pathologies are presented, providing some insights on their biological significance.
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Radical Hidroxila/metabolismo , Purinas/metabolismo , Dano ao DNA , Radical Hidroxila/química , Purinas/química , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5',8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3-6 lesions/106 nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage.
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Síndrome de Cockayne/patologia , Dano ao DNA , Oxigênio/metabolismo , Purinas/metabolismo , Linhagem Celular , Síndrome de Cockayne/genética , DNA/isolamento & purificação , Humanos , Mutação/genética , Purinas/química , Estereoisomerismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Chronic inflammation is estimated to be a causative factor in a variety of diseases. Under inflammatory conditions reactive oxygen species (ROS) and nitrogen species (RNS) are released leading to DNA damage accumulation and genomic instability. Purine 5',8-cyclo-2'-deoxynucleosides (cPu) are oxidative DNA lesions, exclusively derived from the attack of HO⢠radicals, which are known to have cytotoxic and mutagenic properties. Herein, we have analyzed the presence of cPu in genomic DNA isolated from fresh colon and visceral adipose tissue biopsies collected from inflammatory bowel diseases (IBD)-affected patients and severely obese subjects, respectively, versus what observed in the control specimens. In colon biopsies, characterized by a higher gene expression level of inducible nitric oxide synthase (iNOS), a significant increase of 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) lesions and an accumulation of both diastereomeric cPu have been detected. In contrast, the 8-oxo-dA and 8-oxo-dG levels were extremely lower compared to the colon tissues values and no accumulation of cPu, in the inflamed visceral adipose tissue biopsies isolated from bariatric patients versus the lean counterpart was reported. In addition, in adipose tissue undetectable levels of iNOS have been found. These data suggest a potential involvement of cPu in the colon cancer susceptibility observed in IBD patients.
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DNA , Doenças Inflamatórias Intestinais , 8-Hidroxi-2'-Desoxiguanosina , Dano ao DNA , Desoxiguanosina , Humanos , Doenças Inflamatórias Intestinais/genética , Espécies Reativas de OxigênioRESUMO
The reaction of HO⢠radical with DNA is intensively studied both mechanistically and analytically for lesions formation. Several aspects related to the reaction paths of purine moieties with the formation of 5',8-cyclopurines (cPu), 8-oxopurines (8-oxo-Pu), and their relationship are not well understood. In this study, we investigated the reaction of HO⢠radical with a 21-mer double-stranded oligodeoxynucleotide (ds-ODNs) in γ-irradiated aqueous solutions under various oxygen concentrations and accurately quantified the six purine lesions (i.e., four cPu and two 8-oxo-Pu) by LC-MS/MS analysis using isotopomeric internal standards. In the absence of oxygen, 8-oxo-Pu lesions are only â¼4 times more than cPu lesions. By increasing oxygen concentration, the 8-oxo-Pu and the cPu gradually increase and decrease, respectively, reaching a gap of â¼130 times at 2.01 × 10-4 M of O2. Kinetic treatment of the data allows to estimate the C5' radical competition between cyclization and oxygen trapping in ds-ODNs, and lastly the rate constants of the four cyclization steps. Tailored computational studies by means of dispersion-corrected DFT calculations were performed on the CGC and TAT in their double-strand models for each cPu diastereoisomer along with the complete reaction pathways of the cyclization steps. Our findings reveal unheralded reaction mechanisms that resolve the long-standing issues with C5' radical cyclization in purine moieties of DNA sequences.
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The originally published article [...].
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The reaction of hydroxyl radical (HOâ¢) with DNA produces many primary reactive species and many lesions as final products. In this study, we have examined the optical spectra of intermediate species derived from the reaction of HO⢠with a variety of single- and double-stranded oligodeoxynucleotides and ct-DNA in the range of 1 µs to 1 ms by pulse radiolysis using an Intensified Charged Coupled Device (ICCD) camera. Moreover, we applied our published analytical protocol based on an LC-MS/MS system with isotopomeric internal standards to enable accurate and precise measurements of purine lesion formation. In particular, the simultaneous measurement of the four purine 5',8-cyclo-2'-deoxynucleosides (cPu) and two 8-oxo-7,8-dihydro-2'-deoxypurine (8-oxo-Pu) was obtained upon reaction of genetic material with HO⢠radicals generated either by γ-radiolysis or Fenton-type reactions. Our results contributed to the debate in the literature regarding absolute level of lesions, method of HO⢠radical generation, 5'R/5'S diastereomeric ratio in cPu, and relative abundance between cPu and 8-oxo-Pu.
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DNA/química , Radical Hidroxila/química , Oligodesoxirribonucleotídeos/química , Animais , Dano ao DNA/genética , Humanos , Peróxido de Hidrogênio/química , Ferro/química , Purinas/químicaRESUMO
Xeroderma Pigmentosum (XP) is a DNA repair disease characterized by nucleotide excision repair (NER) malfunction, leading to photosensitivity and increased incidence of skin malignancies. The role of XP-A in NER pathways has been well studied while discrepancies associated with ROS levels and the role of radical species between normal and deficient XPA cell lines have been observed. Using liquid chromatography tandem mass spectrometry we have determined the four 5',8-cyclopurines (cPu) lesions (i.e., 5'R-cdG, 5'S-cdG, 5'R-cdA and 5'S-cdA), 8-oxo-dA and 8-oxo-dG in wt (EUE-pBD650) and XPA-deficient (EUE-siXPA) human embryonic epithelial cell lines, under different oxygen tension (hyperoxic 21%, physioxic 5% and hypoxic 1%). The levels of Fe and Cu were also measured. The main findings of our study were: (i) the total amount of cPu (1.82-2.52 lesions/106 nucleotides) is the same order of magnitude as 8-oxo-Pu (3.10-4.11 lesions/106 nucleotides) in both cell types, (ii) the four cPu levels are similar in hyperoxic and physioxic conditions for both wt and deficient cell lines, whereas 8-oxo-Pu increases in all cases, (iii) both wt and deficient cell lines accumulated high levels of cPu under hypoxic compared to physioxic conditions, whereas the 8-oxo-Pu levels show an opposite trend, (iv) the diastereoisomeric ratios 5'R/5'S are independent of oxygen concentration being 0.29 for cdG and 2.69 for cdA for EUE-pBD650 (wt) and 0.32 for cdG and 2.94 for cdA for EUE-siXPA (deficient), (v) in deficient cell lines Fe levels were significantly higher. The data show for the first time the connection of oxygen concentration in cells with different DNA repair ability and the levels of different DNA lesions highlighting the significance of cPu. Membrane lipidomic data at 21% O2 indicated differences in the fatty acid contents between wild type and deficient cells, envisaging functional effects on membranes associated with the different repair capabilities, to be further investigated.
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Reparo do DNA/fisiologia , Oxigênio/metabolismo , Xeroderma Pigmentoso/metabolismo , Linhagem Celular , Cromatografia Líquida/métodos , Cobre/metabolismo , DNA/metabolismo , Dano ao DNA , Humanos , Hipóxia/metabolismo , Ferro/metabolismo , Purinas/metabolismo , Espectrometria de Massas em Tandem/métodos , Xeroderma Pigmentoso/patologiaRESUMO
Purine 5',8-cyclo-2'-deoxynucleosides (cPu) are tandem-type lesions observed among the DNA purine modifications and identified in mammalian cellular DNA in vivo. These lesions can be present in two diasteroisomeric forms, 5'R and 5'S, for each 2'-deoxyadenosine and 2'-deoxyguanosine moiety. They are generated exclusively by hydroxyl radical attack to 2'-deoxyribose units generating C5' radicals, followed by cyclization with the C8 position of the purine base. This review describes the main recent achievements in the preparation of the cPu molecular library for analytical and DNA synthesis applications for the studies of the enzymatic recognition and repair mechanisms, their impact on transcription and genetic instability, quantitative determination of the levels of lesions in various types of cells and animal model systems, and relationships between the levels of lesions and human health, disease, and aging, as well as the defining of the detection limits and quantification protocols.
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Dano ao DNA , Purinas/química , Purinas/metabolismo , Animais , Reparo do DNA , Humanos , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Espécies Reativas de Oxigênio/metabolismo , Bibliotecas de Moléculas PequenasRESUMO
The anticancer activity of acridone derivatives has attracted increasing interest, therefore, a variety of substituted analogs belonging to this family have been developed and evaluated for their anti-cancer properties. A series of N-alkyl-acridones 1-6 and N,N'-dialkyl-9,9'-biacridylidenes 7-12 with variable alkyl chains were examined for their topoisomerase I activity at neutral and acidic conditions as well as for their binding capacity to calf thymus and possible radical trapping antioxidant activity. It was found that at a neutral pH, topoisomerase I activity of both classes of compounds was similar, while under acidic conditions, enhanced intercalation was observed. N-alkyl-acridone derivatives 1-6 exhibited stronger, dose-dependent, cytotoxic activity against MCF-7 human breast epithelial cancer cells than N,N'-dialkyl-9,9'-biacridylidenes 7-12, revealing that conjugation of the heteroaromatic system plays a significant role on the effective distribution of the compound in the intracellular environment. Cellular investigation of long alkyl derivatives against cell migration exhibited 40-50% wound healing effects and cytoplasm diffusion, while compounds with shorter alkyl chains were accumulated both in the nucleus and cytoplasm. All N,N'-dialkyl-9,9'-biacridylidenes showed unexpected high scavenging activity towards DPPH or ABTS radicals which may be explained by higher stabilization of radical cations by the extended conjugation of heteroaromatic ring system.
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Acridinas/farmacologia , Acridonas/farmacologia , Neoplasias da Mama/patologia , DNA Topoisomerases Tipo I/metabolismo , Sequestradores de Radicais Livres/farmacologia , Acridinas/química , Acridonas/química , Benzotiazóis/química , Compostos de Bifenilo/química , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Feminino , Humanos , Células MCF-7 , Picratos/química , Ácidos Sulfônicos/química , Cicatrização/efeitos dos fármacosRESUMO
5',8-Cyclo-2'-deoxyadenosine (cdA), in the 5'R and 5'Sdiastereomeric forms, are typical non strand-break oxidative DNA lesions, induced by hydroxyl radicals, with emerging importance as a molecular marker. These lesions are exclusively repaired by the nucleotide excision repair (NER) mechanism with a low efficiency, thus readily accumulating in the genome. Poly(ADP-ribose) polymerase1 (PARP1) acts as an early responder to DNA damage and plays a key role as a nick sensor in the maintenance of the integrity of the genome by recognizing nicked DNA. So far, it was unknown whether the two diastereomeric cdA lesions could induce specific PARP1 binding. Here, we provide the first evidence of PARP1 to selectively recognize the diastereomeric lesions of 5'S-cdA and 5'R-cdA in vitro as compared to deoxyadenosine in model DNA substrates (23-mers) by using circular dichroism, fluorescence spectroscopy, immunoblotting analysis, and gel mobility shift assay. Several features of the recognition of the damaged and undamaged oligonucleotides by PARP1 were characterized. Remarkably, PARP1 exhibits different affinities in binding to a double strand (ds) oligonucleotide, which incorporates cdA lesions in R and S diastereomeric form. In particular, PARP1 proved to bind oligonucleotides, including a 5'S-cdA, with a higher affinity constant for the 5'S lesion in a model of ds DNA than 5'R-cdA, showing different recognition patterns, also compared with undamaged dA. This new finding highlights the ability of PARP1 to recognize and differentiate the distorted DNA backbone in a biomimetic system caused by different diastereomeric forms of a cdA lesion.
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Biomimética , Desoxiadenosinas/química , Modelos Biológicos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Dano ao DNA , Fluorescência , Humanos , Peso Molecular , Oligonucleotídeos/metabolismo , Ligação Proteica , Estereoisomerismo , Especificidade por Substrato , TemperaturaRESUMO
The nonbulky 5',8-cyclopurine DNA lesions (cP) and the bulky, benzo[ a]pyrene diol epoxide-derived stereoisomeric cis- and trans- N2-guanine adducts (BPDE-dG) are good substrates of the human nucleotide excision repair (NER) mechanism. These DNA lesions were embedded at the In or Out rotational settings near the dyad axis in nucleosome core particles reconstituted either with native histones extracted from HeLa cells (HeLa-NCP) or with recombinant histones (Rec-NCP). The cP lesions are completely resistant to NER in human HeLa cell extracts. The BPDE-dG adducts are also NER-resistant in Rec-NCPs but are good substrates of NER in HeLa-NCPs. The four BPDE-dG adduct samples are excised with different efficiencies in free DNA, but in HeLa-NCPs, the efficiencies are reduced by a common factor of 2.2 ± 0.2 relative to the NER efficiencies in free DNA. The NER response of the BPDE-dG adducts in HeLa-NCPs is not directly correlated with the observed differences in the thermodynamic destabilization of HeLa-NCPs, the Förster resonance energy transfer values, or hydroxyl radical footprint patterns and is weakly dependent on the rotational settings. These and other observations suggest that NER is initiated by the binding of the DNA damage-sensing NER factor XPC-RAD23B to a transiently opened BPDE-modified DNA sequence that corresponds to the known footprint of XPC-DNA-RAD23B complexes (≥30 bp). These observations are consistent with the hypothesis that post-translational modifications and the dimensions and properties of the DNA lesions are the major factors that have an impact on the dynamics and initiation of NER in nucleosomes.
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Adutos de DNA/química , Dano ao DNA , Reparo do DNA , DNA/química , Nucleossomos/química , Purinas/química , Adutos de DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Nucleossomos/genéticaRESUMO
The use of copper complexes for redox and oxidative-based mechanisms in therapeutic strategies is an important field of multidisciplinary research. Here, a novel Cu(II) complex [Cu(TPMA)(Phen)](ClO4)2 (Cu-TPMA-Phen, where TPMA = tris-(2-pyridylmethyl)amine and Phen = 1,10-phenanthroline) was studied using both the free and encapsulated forms. A hollow pH-sensitive drug-delivery system was synthesized, characterized, and used to encapsulate and release the copper complex, thus allowing for the comparison with the free drug. The human neuroblastoma-derived cell line NB100 was treated with 5 µM Cu-PMA-Phen for 24 h, pointing to the consequences on mono- and polyunsaturated fatty acids (MUFA and PUFA) present in the membrane lipidome, coupled with cell viability and death pathways (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium viability assay, flow cytometry, microscopy, caspase activation). In parallel, the Cu-TPMA-Phen reactivity with the fatty acid moieties of phospholipids was studied using the liposome model to work in a biomimetic environment. The main results concerned: (i) the membrane lipidome in treated cells, involving remodeling with a specific increase of saturated fatty acids (SFAs) and a decrease of MUFA, but not PUFA; (ii) cytotoxic events and lipidome changes did not occur for the encapsulated Cu-TPMA-Phen, showing the influence of such nanocarriers on drug activity; and (iii) the liposome behavior confirmed that MUFA and PUFA fatty acid moieties in membranes are not affected by oxidative and isomerization reactions, proving the different reactivities of thiyl radicals generated from amphiphilic and hydrophilic thiols and Cu-TPMA-Phen. This study gives preliminary but important elements of copper(II) complex reactivity in cellular and biomimetic models, pointing mainly to the effects on membrane reactivity and remodeling based on the balance between SFA and MUFA in cell membranes that are subjects of strong interest for chemotherapeutic activities as well as connected to nutritional strategies.
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Fatty acids, as structural components of membranes and inflammation/anti-inflammatory mediators, have well-known protective and regulatory effects. They are studied as biomarkers of pathological conditions, as well as saturated and unsaturated hydrophobic moieties in membrane phospholipids that contribute to homeostasis and physiological functions. Lifestyle, nutrition, metabolism and stress-with an excess of radical and oxidative processes-cause fatty acid changes that are examined in the human body using blood lipids. Fatty acid-based membrane lipidomics represents a powerful diagnostic tool for assessing the quantity and quality of fatty acid constituents and also for the follow-up of the membrane fatty acid remodeling that is associated with different physiological and pathological conditions. This review focuses on fatty acid biomarkers with two examples of recent lipidomic research and health applications: (i) monounsaturated fatty acids and the analytical challenge offered by hexadecenoic fatty acids (C16:1); and (ii) the cohort of 10 fatty acids in phospholipids of red blood cell membranes and its connections to metabolic and nutritional status in healthy and diseased subjects.
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5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase ß (pol ß) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol ß wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol ß knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol ß plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol ß inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol ß, indicating a mutagenic effect induced by pol ß lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol ß generated an intermediate that failed to be extended by pol ß, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol ß plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol ß bypassing of a cdA lesion.