RESUMEN
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.
Asunto(s)
Antimutagênicos/farmacología , Phyllanthus/química , Extractos Vegetales/farmacología , Protectores contra Radiación/farmacología , Luz Solar/efectos adversos , Rayos Ultravioleta/efectos adversos , ADN/efectos de la radiación , Daño del ADN , ADN-Formamidopirimidina Glicosilasa/metabolismo , Desoxirribonucleasa (Dímero de Pirimidina)/metabolismo , Electroforesis en Gel de Agar , Escherichia coli/enzimología , Proteínas de Escherichia coli/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Plásmidos , Dímeros de Pirimidina/metabolismo , Proteínas Virales/metabolismoRESUMEN
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.
Asunto(s)
Daño del ADN , ADN/química , Desoxiguanosina/análogos & derivados , Oxígeno Singlete/química , Timo/fisiología , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Anticuerpos Antinucleares/metabolismo , Bovinos , Sistema Libre de Células , ADN/inmunología , ADN/efectos de la radiación , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Estrés Oxidativo , Trastornos por Fotosensibilidad , Dímeros de Pirimidina/química , Cloruro de Sodio/metabolismo , Luz Solar , Rayos Ultravioleta/efectos adversosRESUMEN
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.
Asunto(s)
Bioensayo/métodos , Protectores Solares/análisis , Rayos Ultravioleta , Apoptosis/efectos de los fármacos , Emparejamiento Base , Línea Celular Tumoral , ADN/metabolismo , Exposición a Riesgos Ambientales/análisis , Genoma Humano , Humanos , Mutágenos/toxicidad , Dímeros de Pirimidina/metabolismoRESUMEN
Substantially high rate of glycolysis, known as the Warburg effect, is a well-known feature of cancers, and emerging evidence suggests that it supports cancerous proliferation/tumor growth. Phosphoglycerate mutase (PGAM), a glycolytic enzyme, is commonly up-regulated in several cancers, and recent reports show its involvement in the Warburg effect. Here, a comprehensive analysis shows that PGAM is acetylated at lysines 100/106/113/138 in its central region, and a member of the Sirtuin family (class III deacetylase), SIRT2, is responsible for its deacetylation. Over-expression of SIRT2 or mutations at the acetylatable lysines of PGAM attenuates cancer cell proliferation with a concomitant decrease in PGAM activity. We also report that the acetyltransferase PCAF (p300/CBP-associated factor) interacts with PGAM and acetylates its C-terminus, but not the central region. As prior evidence suggests that SIRT2 functions as a tumor suppressor, our results would provide support for the mechanistic basis of this activity.
Asunto(s)
Fosfoglicerato Mutasa/metabolismo , Sirtuina 2/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Animales , Arginina/metabolismo , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Humanos , Lisina/metabolismo , Ratones , Mutación , Estructura Terciaria de Proteína , Sirtuina 2/genética , Sirtuinas/metabolismoRESUMEN
BACKGROUND: Due to the increase of solar ultraviolet radiation (UV) incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter. METHODOLOGY/PRINCIPAL FINDINGS: The Sun Protection Factor for DNA (DNA-SPF) is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD) and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF). Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations. CONCLUSIONS/SIGNIFICANCE: The application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage.
Asunto(s)
Daño del ADN , ADN/metabolismo , Mutágenos/toxicidad , Factor de Protección Solar , Protectores Solares/farmacología , Análisis de Varianza , Humanos , Radiometría , Luz SolarRESUMEN
Despite growing knowledge on the biological effects of ultraviolet (UV) radiation on human health and ecosystems, it is still difficult to predict the negative impacts of the increasing incidence of solar UV radiation in a scenario of global warming and climate changes. Hence, the development and application of DNA-based biological sensors to monitor the solar UV radiation under different environmental conditions is of increasing importance. With a mind to rendering a molecular view-point of the genotoxic impact of sunlight, field experiments were undertaken with a DNA-dosimeter system in parallel with physical photometry of solar UVB/UVA radiation, at various latitudes in South America. On applying biochemical and immunological approaches based on specific DNA-repair enzymes and antibodies, for evaluating sunlight-induced DNA damage profiles, it became clear that the genotoxic potential of sunlight does indeed vary according to latitude. Notwithstanding, while induction of oxidized DNA bases is directly dependent on an increase in latitude, the generation of 6-4PPs is inversely so, whereby the latter can be regarded as a biomolecular marker of UVB incidence. This molecular DNA lesion-pattern largely reflects the relative incidence of UVA and UVB energy at any specific latitude. Hereby is demonstrated the applicability of this DNA-based biosensor for additional, continuous field experiments, as a means of registering variations in the genotoxic impact of solar UV radiation.
Asunto(s)
Daño del ADN , ADN/efectos de la radiación , Luz Solar/efectos adversos , Rayos Ultravioleta/efectos adversos , Humanos , Oxidación-ReducciónRESUMEN
Solar ultraviolet (UV) radiation is widely known as a genotoxic environmental agent that affects Earth ecosystems and the human population. As a primary consequence of the stratospheric ozone layer depletion observed over the last decades, the increasing UV incidence levels have heightened the concern regarding deleterious consequences affecting both the biosphere and humans, thereby leading to an increase in scientific efforts to understand the role of sunlight in the induction of DNA damage, mutagenesis, and cell death. In fact, the various UV-wavelengths evoke characteristic biological impacts that greatly depend on light absorption of biomolecules, especially DNA, in living organisms, thereby justifying the increasing importance of developing biological sensors for monitoring the harmful impact of solar UV radiation under various environmental conditions. In this review, several types of biosensors proposed for laboratory and field application, that measure the biological effects of the UV component of sunlight, are described. Basically, the applicability of sensors based on DNA, bacteria or even mammalian cells are presented and compared. Data are also presented showing that on using DNA-based sensors, the various types of damage produced differ when this molecule is exposed in either an aqueous buffer or a dry solution. Apart from the data thus generated, the development of novel biosensors could help in evaluating the biological effects of sunlight on the environment. They also emerge as alternative tools for using live animals in the search for protective sunscreen products.