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 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
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
The fields of telomere biology and DNA repair have enjoyed a great deal of cross-fertilization and convergence in recent years. Telomeres function at chromosome ends to prevent them from being falsely recognized as chromosome breaks by the DNA damage response and repair machineries. Conversely, both canonical and nonconical functions of numerous DNA repair proteins have been found to be critical for preserving telomere structure and function. In 2009, Elizabeth Blackburn, Carol Greider and Jack Szostak were awarded the Nobel prize in Physiology or Medicine for the discovery of telomeres and telomerase. Four years later, pioneers in the field of DNA repair, Aziz Sancar, Tomas Lindahl and Paul Modrich were recognized for their seminal contributions by being awarded the Nobel Prize in Chemistry. This review is part of a special issue meant to celebrate this amazing achievement, and will focus in particular on the convergence of nucleotide excision repair and telomere biology, and will discuss the profound implications for human health.
Assuntos
Reparo do DNA , Telômero , Aberrações Cromossômicas , Dano ao DNA , Desoxirribodipirimidina Fotoliase/metabolismo , Humanos , Prêmio Nobel , Dímeros de Pirimidina/metabolismo , Telomerase/metabolismo , Raios UltravioletaRESUMO
Ultraviolet-induced 6-4 photoproducts (6-4PP) and cyclobutane pyrimidine dimers (CPD) can be tolerated by translesion DNA polymerases (TLS Pols) at stalled replication forks or by gap-filling. Here, we investigated the involvement of Polη, Rev1 and Rev3L (Polζ catalytic subunit) in the specific bypass of 6-4PP and CPD in repair-deficient XP-C human cells. We combined DNA fiber assay and novel methodologies for detection and quantification of single-stranded DNA (ssDNA) gaps on ongoing replication forks and postreplication repair (PRR) tracts in the human genome. We demonstrated that Rev3L, but not Rev1, is required for postreplicative gap-filling, while Polη and Rev1 are responsible for TLS at stalled replication forks. Moreover, specific photolyases were employed to show that in XP-C cells, CPD arrest replication forks, while 6-4PP are responsible for the generation of ssDNA gaps and PRR tracts. On the other hand, in the absence of Polη or Rev1, both types of lesion block replication forks progression. Altogether, the data directly show that, in the human genome, Polη and Rev1 bypass CPD and 6-4PP at replication forks, while only 6-4PP are also tolerated by a Polζ-dependent gap-filling mechanism, independent of S phase.
Assuntos
Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Proteínas Nucleares/genética , Nucleotidiltransferases/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Linhagem Celular Transformada , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribodipirimidina Fotoliase , Fibroblastos/citologia , Fibroblastos/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Genoma Humano , Humanos , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/metabolismo , Dímeros de Pirimidina/metabolismo , Fase S/genética , Transdução Genética , Raios UltravioletaRESUMO
The protective effect of sunscreens has been extensively evaluated in vivo as a measure of erythema induced in human skin and is expressed as Sun Protection Factor (SPF). In vitro alternatives that use human cells might overcome the limitations of testing on human beings. Here is proposed a broad and accurate in vitro approach for evaluating the efficacy of commercial sunscreens even under environmental conditions. This Cell dosimeter allowed the determination of Sun Protection Factor for DNA (DNA-SPF), using specific DNA repair enzymes and antibodies, and Sun Protection Factor for Lethal Damage (LD-SPF), by measuring cell viability and apoptosis induced after the irradiation of human cells. The use of xeroderma pigmentosum (XP) cells, which are deficient in DNA repair, rendered this assay more sensitive. The results revealed significant protection against the effects elicited by UVB radiation; however, there was no efficient protection from DNA lesions and cell death induced by UVA radiation or natural sunlight. This work demonstrates the environmental application of this biodosimeter for measuring UV-induced biological damage to human cells and supports the need for better evaluation of the UVA protection efficacy conferred by commercial sunscreens, in terms of induction of DNA lesions and cell death.
Assuntos
Bioensaio/métodos , Protetores Solares/análise , Raios Ultravioleta , Apoptose/efeitos dos fármacos , Pareamento de Bases , Linhagem Celular Tumoral , DNA/metabolismo , Exposição Ambiental/análise , Genoma Humano , Humanos , Mutagênicos/toxicidade , Dímeros de Pirimidina/metabolismoRESUMO
UV-resistant Acinetobacter sp. Ver3 isolated from High-Altitude Andean Lakes (HAAL) in Argentinean Puna, one of the highest UV exposed ecosystems on Earth, showed efficient DNA photorepairing ability, coupled to highly efficient antioxidant enzyme activities in response to UV-B stress. We herein present the cloning, expression, and functional characterization of a cyclobutane pyrimidine dimer (CPD)-class I photolyase (Ver3Phr) from this extremophile to prove its involvement in the previously noted survival capability. Spectroscopy of the overexpressed and purified protein identified flavin adenine dinucleotide (FAD) and 5,10-methenyltetrahydrofolate (MTHF) as chromophore and antenna molecules, respectively. All functional analyses were performed in parallel with the ortholog E. coli photolyase. Whereas the E. coli enzyme showed the FAD chromophore as a mixture of oxidised and reduced states, the Ver3 chromophore always remained partly (including the semiquinone state) or fully reduced under all experimental conditions tested. Functional complementation of Ver3Phr in Phr(-)-RecA E. coli strains was assessed by traditional UFC counting and measurement of DNA bipyrimidine photoproducts by HPLC coupled with electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) detection. The results identified strong photoreactivation ability in vivo of Ver3Phr while its nonphotoreactivation function, probably related with the stimulation of nucleotide excision repair (NER), was not as manifest as for EcPhr. Whether this is a question of the approach using an exogenous photolyase incorporated in a non-genuine host or a fundamental different behaviour of a novel enzyme from an exotic environment will need further studies.
Assuntos
Acinetobacter/enzimologia , Acinetobacter/efeitos da radiação , Altitude , Desoxirribodipirimidina Fotoliase/metabolismo , Lagos/microbiologia , Dímeros de Pirimidina/metabolismo , Raios Ultravioleta , Acinetobacter/isolamento & purificação , Desoxirribodipirimidina Fotoliase/química , Desoxirribodipirimidina Fotoliase/classificação , Dados de Sequência Molecular , FilogeniaRESUMO
CONTEXT: One approach to protect human skin against the dangerous effects of solar ultraviolet (UV) irradiation is the use of natural products, such as photoprotectors. Phyllanthus orbicularis Kunth (Euphorbiaceae) is a Cuban endemic plant used in popular medicine. Its antigenotoxicity effect against some harmful agents has been investigated. However, the effect in ultraviolet B (UVB)-irradiated human cells has not been previously assessed. OBJECTIVE: The protective effect of a P. orbicularis extract against UVB light-induced damage in human cells was evaluated. MATERIALS AND METHODS: DNA repair proficient (MRC5-SV) and deficient (XP4PA, complementation group XPC) cell-lines were used. Damaging effects of UVB light were evaluated by clonogenic assay and apoptosis induction by flow cytometry techniques. The extent of DNA repair itself was determined by the removal of cyclobutane pyrimidine dimers (CPDs). The CPDs were detected and quantified by slot-blot assay. RESULTS: Treatment of UVB-irradiated MRC5-SV cells with P. orbicularis extract increased the percentage of colony-forming cells from 36.03 ± 3.59 and 4.42 ± 1.45 to 53.14 ± 8.8 and 14.52 ± 1.97, for 400 and 600 J/m(2), respectively. A decrease in apoptotic cell population was observed in cells maintained within the extract. The P. orbicularis extract enhanced the removal of CPD from genomic DNA. The CPDs remaining were found to be about 27.7 and 1.1%, while with plant extract, treatment these values decreased to 16.1 and 0.2%, for 3 and 24 h, respectively. DISCUSSION AND CONCLUSION: P. orbicularis aqueous extract protects human cells against UVB damage. This protective effect is through the modulation of DNA repair effectiveness.
Assuntos
Reparo do DNA/efeitos dos fármacos , Phyllanthus/química , Extratos Vegetais/farmacologia , Raios Ultravioleta/efeitos adversos , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Cuba , Reparo do DNA/efeitos da radiação , Citometria de Fluxo , Humanos , Dímeros de Pirimidina/metabolismo , Fatores de TempoRESUMO
Deleterious effects of UV-B radiation on DNA include the formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). These lesions must be repaired to maintain the integrity of DNA and provide genetic stability. Of the several repair systems involved in the recognition and removal of UV-B-induced lesions in DNA, the focus in the present study was on the mismatch repair system (MMR). The contribution of MutSα (MSH2-MSH6) to UV-induced DNA lesion repair and cell cycle regulation was investigated. MSH2 and MSH6 genes in Arabidopsis and maize are up-regulated by UV-B, indicating that MMR may have a role in UV-B-induced DNA damage responses. Analysis of promoter sequences identified MSH6 as a target of the E2F transcription factors. Using electrophoretic mobility shift assays, MSH6 was experimentally validated as an E2F target gene, suggesting an interaction between MMR genes and the cell cycle control. Mutations in MSH2 or MSH6 caused an increased accumulation of CPDs relative to wild-type plants. In addition, msh2 mutant plants showed a different expression pattern of cell cycle marker genes after the UV-B treatment when compared with wild-type plants. Taken together, these data provide evidence that plant MutSα is involved in a UV-B-induced DNA damage response pathway.