RESUMO
Ultraviolet C (UVC) light has long been used as a sterilizing agent, primarily through devices that emit at 254 nm. Depending on the dose and duration of exposure, UV 254 nm can cause erythema and photokeratitis and potentially cause skin cancer since it directly modifies nitrogenated nucleic acid bases. Filtered KrCl excimer lamps (emitting mainly at 222 nm) have emerged as safer germicidal tools and have even been proposed as devices to sterilize surgical wounds. All the studies that showed the safety of 222 nm analyzed cell number and viability, erythema generation, epidermal thickening, the formation of genetic lesions such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts (6-4PPs) and cancer-inducing potential. Although nucleic acids can absorb and be modified by both UV 254 nm and UV 222 nm equally, compared to UV 254 nm, UV 222 nm is more intensely absorbed by proteins (especially aromatic side chains), causing photooxidation and cross-linking. Here, in addition to analyzing DNA lesion formation, for the first time, we evaluated changes in the proteome and cellular pathways, reactive oxygen species formation, and metalloproteinase (MMP) levels and activity in full-thickness in vitro reconstructed human skin (RHS) exposed to UV 222 nm. We also performed the longest (40 days) in vivo study of UV 222 nm exposure in the HRS/J mouse model at the occupational threshold limit value (TLV) for indirect exposure (25 mJ/cm2) and evaluated overall skin morphology, cellular pathological alterations, CPD and 6-4PP formation and MMP-9 activity. Our study showed that processes related to reactive oxygen species and inflammatory responses were more altered by UV 254 nm than by UV 222 nm. Our chronic in vivo exposure assay using the TLV confirmed that UV 222 nm causes minor damage to the skin. However, alterations in pathways related to skin regeneration raise concerns about direct exposure to UV 222 nm.
Assuntos
Dano ao DNA , Ácidos Nucleicos , Camundongos , Animais , Humanos , Espécies Reativas de Oxigênio/metabolismo , Dímeros de Pirimidina/metabolismo , Pele/efeitos da radiação , Raios Ultravioleta , Ácidos Nucleicos/metabolismo , EritemaRESUMO
The photolyase family consists of flavoproteins with enzyme activity able to repair ultraviolet light radiation damage by photoreactivation. DNA damage by the formation of a cyclobutane pyrimidine dimer (CPD) and a pyrimidine-pyrimidone (6-4) photoproduct can lead to multiple affections such as cellular apoptosis and mutagenesis that can evolve into skin cancer. The development of integrated applications to prevent the negative effects of prolonged sunlight exposure, usually during outdoor activities, is imperative. This study presents the functions, characteristics, and types of photolyases, their therapeutic and cosmetic applications, and additionally explores some photolyase-producing microorganisms and drug delivery systems.
Assuntos
Desoxirribodipirimidina Fotoliase , Reparo do DNA , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Flavoproteínas , Dímeros de Pirimidina , Pirimidinas , Pirimidinonas , Raios Ultravioleta/efeitos adversosRESUMO
Photolyases are proteins that enzymatically repair the UV-induced DNA damage by a protein-DNA electron transfer mechanism. They repair either cyclobutane pyrimidine dimers or pyrimidine (6-4) pyrimidone photoproducts or just (6-4)-photoproducts. In this work, we report the production and partial characterization of a recombinant (6-4)-photolyase (SphPhrB97) from a bacterial psychrotolerant Antarctic isolate identified as Sphingomonas sp. strain UV9. The spectrum analysis and the in silico study of SphPhrB97 suggest that this enzyme has similar features as compared to the (6-4)-photolyase from Agrobacterium tumefaciens (4DJA; PhrB), including the presence of three cofactors: FAD, DMRL (6,7-dimethyl-8-(1'-D-ribityl) lumazine), and an Fe-S cluster. The homology model of SphPhrB97 predicts that the DNA-binding pocket (area and volume) is larger as compared to (6-4)-photolyases from mesophilic microbes. Based on sequence comparison and on the homology model, we propose an electron transfer pathway towards the FAD cofactor involving the residues Trp342, Trp390, Tyr40, Tyr391, and Tyr399. The phylogenetic tree performed using curated and well-characterized prokaryotic (6-4)-photolyases suggests that SphPhrB97 may have an ancient evolutionary origin. The results suggest that SphPhrB97 is a cold-adapted enzyme, ready to cope with the UV irradiation stress found in a hostile environment, such as Antarctica.
Assuntos
Proteínas de Bactérias/química , Desoxirribodipirimidina Fotoliase , Sphingomonas/enzimologia , Regiões Antárticas , Proteínas de Bactérias/genética , Reparo do DNA , Desoxirribodipirimidina Fotoliase/química , Desoxirribodipirimidina Fotoliase/genética , Filogenia , Dímeros de Pirimidina , Proteínas Recombinantes , Sphingomonas/genética , Raios UltravioletaRESUMO
The DUSP3 phosphatase regulates cell cycle, proliferation, apoptosis and senescence of different cell types, lately shown as a mediator of DNA repair processes. This work evaluated the impact of DUSP3 loss of function (lof) on DNA repair-proficient fibroblasts (MRC-5), NER-deficient cell lines (XPA and XPC) and translesion DNA synthesis (TLS)-deficient cells (XPV), after UV-radiation stress. The levels of DNA strand breaks, CPDs and 6-4-PPs have accumulated over time in all cells under DUSP3 lof, with a significant increase in NER-deficient lines. The inefficient repair of these lesions increased sub-G1 population of XPA and XPC cells 24 hours after UV treatment, notably marked by DUSP3 lof, which is associated with a reduced cell population in G1, S and G2/M phases. It was also detected an increase in S and G2/M populations of XPV and MRC-5 cells after UV-radiation exposure, which was slightly attenuated by DUSP3 lof due to a discrete increase in sub-G1 cells. The cell cycle progression was accompanied by changes in the levels of the main Cyclins (A1, B1, D1 or E1), CDKs (1, 2, 4 or 6), and the p21 Cip1 inhibitor, in a DUSP3-dependent manner. DUSP3 lof affected the proliferation of MRC-5 and XPA cells, with marked worsening of the XP phenotype after UV radiation. This work highlights the roles of DUSP3 in DNA repair fitness and in the fine control of regulatory proteins of cell cycle, essential mechanisms to maintenance of genomic stability.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/genética , Fosfatase 3 de Especificidade Dupla/metabolismo , Instabilidade Genômica , Ciclo Celular/efeitos da radiação , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos da radiação , Inativação Gênica/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Humanos , Dímeros de Pirimidina/metabolismo , Estresse Fisiológico/efeitos da radiação , Raios UltravioletaRESUMO
UVA-induced mutagenesis was investigated in human pol eta-deficient (XP-V) cells through whole-exome sequencing. In UVA-irradiated cells, the increase in the mutation frequency in deficient cells included a remarkable contribution of C>T transitions, mainly at potential pyrimidine dimer sites. A strong contribution of C>A transversions, potentially due to oxidized bases, was also observed in non-irradiated XP-V cells, indicating that basal mutagenesis caused by oxidative stress may be related to internal tumours in XP-V patients. The low levels of mutations involving T induced by UVA indicate that pol eta is not responsible for correctly replicating T-containing pyrimidine dimers, a phenomenon known as the 'A-rule'. Moreover, the mutation signature profile of UVA-irradiated XP-V cells is highly similar to the human skin cancer profile, revealing how studies involving cells deficient in DNA damage processing may be useful to understand the mechanisms of environmentally induced carcinogenesis.
Assuntos
Mutagênese/genética , Estresse Oxidativo/genética , Dímeros de Pirimidina/genética , Xeroderma Pigmentoso/genética , Linhagem Celular , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , Humanos , Mutagênese/efeitos da radiação , Mutação/genética , Mutação/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Dímeros de Pirimidina/efeitos da radiação , Raios Ultravioleta , Sequenciamento do Exoma , Xeroderma Pigmentoso/etiologiaRESUMO
Ultraviolet radiation from sunlight causes DNA damage in skin cells by formation of photoproducts, mainly cyclobutane pyrimidine dimers (CPD), which are reverted by exogenous CPD-photolyase, preventing photoaging and skin cancer. High performance liquid chromatography tandem mass spectrometry method for quantification of CPD-photolyase activity was developed to search new enzymes sources for dermatology or clinical studies. The method was based in the enzymatic conversion of a 15mer oligonucleotide, containing a center cyclobutane thymidine dimer, to the restored 15mer oligonucleotide. Three ion pair reagent were evaluated by response surface methodology to increase mass intensities. Additionally, chromatographic separation of oligonucleotides was performed. The selected mobile phase was 15â¯mM diisopropylethylamine/20â¯mM hexafluoroisopropanol in methanol. The method allowed total separation between the oligonucleotides studied (resolution of 2.3) by using the core shell technology, which reduce the diffusion time of the analyte into the column, increasing the efficiency and minimizing the analysis time at 7â¯min. The mass spectrometry detection allowed a high selectivity and sensitivity. This is the first time where MRM modality has been employed with this specific purpose. Oligonucleotides recovery from reaction mixture was â¼ 94% and the limit of quantification was 13.4â¯nM for 15mer. The method was evaluated with a recombinant CPD-photolyase from Synechococcus leopoliensis using purified and crude protein extract. CPD-photolyase could be measured in terms of activity for enzymatic kinetics studies, for evaluation of UV-R effects in (micro)organisms and to identify new enzymes.
Assuntos
Proteínas de Bactérias/química , Cromatografia Líquida/métodos , Desoxirribodipirimidina Fotoliase/química , Ensaios Enzimáticos/métodos , Oligonucleotídeos/análise , Synechococcus/enzimologia , Espectrometria de Massas em Tandem/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Cinética , Dímeros de Pirimidina/química , Synechococcus/química , Synechococcus/genéticaRESUMO
Ultraviolet light crossing the ozone layer in the atmospheric barrier affects all forms of living beings on earth. In eukaryotic cells, the nucleotide excision repair (NER) pathway protects the DNA by removing cyclobutane pyrimidine dimers (CPDs) and 6-4-photoproduct (6-4-PP) lesions caused by ultraviolet (UV) light, allowing cells to proliferate. On the other hand, adhesion and invasion processes, primarily regulated by the typical Rho GTPases Rho, Rac, and Cdc42, are also affected by UV radiation effects. Studies focused on determining whether or not these GTPases might affect the NER pathway in different cell models are enlightening and should start with classical experimental methodologies. In this chapter we describe two methods (host cell reactivation assay, or HCR, and slot-blots for CPDs and 6-4-PPs) to assess the direct or indirect involvement of these three GTPases on the NER pathway.
Assuntos
Proliferação de Células/efeitos da radiação , Reparo do DNA , Dímeros de Pirimidina/metabolismo , Raios Ultravioleta/efeitos adversos , Proteínas rho de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Dímeros de Pirimidina/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
One approach to protect the human skin against harmful effects of solar ultraviolet (UV) radiation was to use natural products as photoprotectors. In this work, the extract from specie Phyllanthus orbicularis K was evaluated as a protective agent against the photodamage by UVB, UVA artificial lamps, and environmental sunlight exposure. The plasmid DNA solutions were exposed to radiations using the DNA dosimeter system in the presence of plant extract. The DNA repair enzymes, Escherichia coli Formamidopyrimidine-DNA glycosylase (Fpg) and T4 bacteriophage endonuclease V (T4-endo V), were employed to discriminate oxidized DNA damage and cyclobutane pyrimidine dimers (CPD), respectively. The supercoiled and relaxed forms of DNA were separated through electrophoretic migration in agarose gels. These DNA forms were quantified to determine strand break, representing the types of lesion levels. The results showed that, in the presence of P. orbicularis extract, the CPD and oxidative damage were reduced in irradiated DNA samples. The photoprotective effect of extract was more evident for UVB and sunlight radiation than for UVA. This work documented the UV absorbing properties of P. orbicularis aqueous extract and opened up new vistas in its characterization as protective agent against DNA damage induced by environmental sunlight radiation.
Assuntos
Antimutagênicos/farmacologia , Phyllanthus/química , Extratos Vegetais/farmacologia , Protetores contra Radiação/farmacologia , Luz Solar/efeitos adversos , Raios Ultravioleta/efeitos adversos , DNA/efeitos da radiação , Dano ao DNA , DNA-Formamidopirimidina Glicosilase/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Eletroforese em Gel de Ágar , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Plasmídeos , Dímeros de Pirimidina/metabolismo , Proteínas Virais/metabolismoRESUMO
UVA light is hardly absorbed by the DNA molecule, but recent works point to a direct mechanism of DNA lesion by these wavelengths. UVA light also excite endogenous chromophores, which causes DNA damage through ROS. In this study, DNA samples were irradiated with UVA light in different conditions to investigate possible mechanisms involved in the induction of DNA damage. The different types of DNA lesions formed after irradiation were determined through the use of endonucleases, which recognize and cleave sites containing oxidized bases and cyclobutane pyrimidine dimers (CPDs), as well as through antibody recognition. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxodG) was also studied in more detail using electrochemical detection. The results show that high NaCl concentration and concentrated DNA are capable of reducing the induction of CPDs. Moreover, concerning damage caused by oxidative stress, the presence of sodium azide and metal chelators reduce their induction, while deuterated water increases the amounts of oxidized bases, confirming the involvement of singlet oxygen in the generation of these lesions. Curiously, however, high concentrations of DNA also enhanced the formation of oxidized bases, in a reaction that paralleled the increase in the formation of singlet oxygen in the solution. This was interpreted as being due to an intrinsic photosensitization mechanism, depending directly on the DNA molecule to absorb UVA and generate singlet oxygen. Therefore, the DNA molecule itself may act as a chromophore for UVA light, locally producing a damaging agent, which may lead to even greater concerns about the deleterious impact of sunlight.