André Passaglia Schuch

The genotoxic effects of DNA lesions induced by artificial UV-radiation and sunlight.

Solar radiation sustains and affects all life forms on Earth. The increase in solar UV-radiation at environmental levels, due to depletion of the stratospheric ozone layer, highlights serious issues of social concern. This becomes still more dramatic in tropical and subtropical regions where radiation-intensity is still higher. Thus, there is the need to evaluate the harmful effects of solar UV-radiation on the DNA molecule as a basis for assessing the risks involved for human health, biological productivity and ecosystems. In order to evaluate the profile of DNA damage induced by this form of radiation and its genotoxic effects, plasmid DNA samples were exposed to artificial-UV lamps and directly to sunlight. The induction of cyclobutane pyrimidine dimer photoproducts (CPDs) and oxidative DNA damage in these molecules were evaluated by means of specific DNA repair enzymes. On the other hand, the biological effects of such lesions were determined through the analysis of the DNA inactivation rate and mutation frequency, after replication of the damaged pCMUT vector in an Escherichia coliMBL50 strain. The results indicated the induction of a significant number of CPDs after exposure to increasing doses of UVC, UVB, UVA radiation and sunlight. Interestingly, these photoproducts are those lesions that better correlate with plasmid inactivation as well as mutagenesis, and the oxidative DNA damages induced present very low correlation with these effects. The results indicated that DNA photoproducts play the main role in the induction of genotoxic effects by artificial UV-radiation sources and sunlight.

Biological Sensors for Solar Ultraviolet Radiation

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.

Sunlight damage to cellular DNA: Focus on oxidatively generated lesions

The routine and often unavoidable exposure to solar ultraviolet (UV) radiation makes it one of the most significant environmental DNA-damaging agents to which humans are exposed. Sunlight, specifically UVB and UVA, triggers various types of DNA damage. Although sunlight, mainly UVB, is necessary for the production of vitamin D, which is necessary for human health, DNA damage may have several deleterious consequences, such as cell death, mutagenesis, photoaging and cancer. UVA and UVB photons can be directly absorbed not only by DNA, which results in lesions, but also by the chromophores that are present in skin cells. This process leads to the formation of reactive oxygen species, which may indirectly cause DNA damage. Despite many decades of investigation, the discrimination among the consequences of these different types of lesions is not clear. However, human cells have complex systems to avoid the deleterious effects of the reactive species produced by sunlight. These systems include antioxidants, that protect DNA, and mechanisms of DNA damage repair and tolerance. Genetic defects in these mechanisms that have clear harmful effects in the exposed skin are found in several human syndromes. The best known of these is xeroderma pigmentosum (XP), whose patients are defective in the nucleotide excision repair (NER) and translesion synthesis (TLS) pathways. These patients are mainly affected due to UV-induced pyrimidine dimers, but there is growing evidence that XP cells are also defective in the protection against other types of lesions, including oxidized DNA bases. This raises a question regarding the relative roles of the various forms of sunlight-induced DNA damage on skin carcinogenesis and photoaging. Therefore, knowledge of what occurs in XP patients may still bring important contributions to the understanding of the biological impact of sunlight-induced deleterious effects on the skin cells.

Identification of influential events concerning the Antarctic ozone hole over southern Brazil and the biological effects induced by UVB and UVA radiation in an endemic treefrog species

The increased incidence of solar ultraviolet radiation (UV) due to ozone depletion has been affecting both terrestrial and aquatic ecosystems and it may help to explain the enigmatic decline of amphibian populations in specific localities. In this work, influential events concerning the Antarctic ozone hole were identified in a dataset containing 35 years of ozone measurements over southern Brazil. The effects of environmental doses of UVB and UVA radiation were addressed on the morphology and development of Hypsiboas pulchellus tadpole (Anura: Hylidae), as well as on the induction of malformation after the conclusion of metamorphosis. These analyzes were complemented by the detection of micronucleus formation in blood cells. 72 ozone depletion events were identified from 1979 to 2013. Surprisingly, their yearly frequency increased three-fold during the last 17 years. The results clearly show that H. pulchellus tadpole are much more sensitive to UVB than UVA light, which reduces their survival and developmental rates. Additionally, the rates of micronucleus formation by UVB were considerably higher compared to UVA even after the activation of photolyases enzymes by a further photoreactivation treatment. Consequently, a higher occurrence of malformation was observed in UVB-irradiated individuals. These results demonstrate the severe genotoxic impact of UVB radiation on this treefrog species and its importance for further studies aimed to assess the impact of the increased levels of solar UVB radiation on declining species of the Hylidae family.

DNA Dosimetry Assessment for Sunscreen Genotoxic Photoprotection

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.

DNA damage profiles induced by sunlight at different latitudes

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. Onapplying 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. Environ. Mol. Mutagen. 2012.

Highly Sensitive Biological Assay for Determining the Photoprotective Efficacy of Sunscreen

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.

Molecular and sensory mechanisms to mitigate sunlight-induced DNA damage in treefrog tadpoles

ABSTRACT The increased incidence of solar ultraviolet B (UVB) radiation has been proposed as an environmental stressor, which may help to explain the enigmatic decline of amphibian populations worldwide. Despite growing knowledge regarding the UV-induced biological effects in several amphibian models, little is known about the efficacy of DNA repair pathways. In addition, little attention has been given to the interplay between these molecular mechanisms with other physiological strategies that avoid the damage induced by sunlight. Here, DNA lesions induced by environmental doses of solar UVB and UVA radiation were detected in genomic DNA samples of treefrog tadpoles (Hypsiboas pulchellus) and their DNA repair activity was evaluated. These data were complemented by monitoring the induction of apoptosis in blood cells and tadpole survival. Furthermore, the tadpoles’ ability to perceive and escape from UV wavelengths was evaluated as an additional strategy of photoprotection. The results show that tadpoles are very sensitive to UVB light, which could be explained by the slow DNA repair rates for both cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6,4) pyrimidone photoproducts (6,4PPs). However, they were resistant to UVA, probably as a result of the activation of photolyases during UVA irradiation. Surprisingly, a sensory mechanism that triggers their escape from UVB and UVA light avoids the generation of DNA damage and helps to maintain the genomic integrity. This work demonstrates the genotoxic impact of both UVB and UVA radiation on tadpoles and emphasizes the importance of the interplay between molecular and sensory mechanisms to minimize the damage caused by sunlight. Highlighted Article: A UV-avoidance sensory mechanism complements the low DNA repair efficiency of treefrog tadpoles to mitigate the genotoxic effects of solar UV radiation.

Protective effect of a Phyllanthus orbicularis aqueous extract against UVB light in human cells

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/m2, 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.

An UV-sensitive anuran species as an indicator of environmental quality of the Southern Atlantic Rainforest

The Southern Atlantic rainforest is continuously suffering from wood extraction activity, which results in the increase of clearings within the forest. Although the direct impacts of deforestation on landscape are already well described, there is an absence of studies focused on the evaluation of its indirect effects, such as the increase of solar UV radiation levels inside forest environment and its consequences for forest specialist anuran species. The results presented in this work clearly show that the threatened tree frog species Hypsiboas curupi presents severe traits of sensitivity to UV wavelengths of sunlight, making it a vulnerable species to this environmental stressor, as well as a biological indicator of the quality of forest canopy coverage. In addition, the measurement of solar UVB and UVA radiation incidence upon H. curupi breeding site and the analyses of a 20-year dataset of satellite images regarding the management of canopy coverage indicate that the photoprotection provided by trees of the Southern Atlantic rainforest is critical for the conservation of this forest specialist anuran species. Therefore, this work demonstrates that the deforestation process enhances the exposure of H. curupi embryos to solar UVB and UVA radiation, negatively affecting their embryonic development, inducing mortality and population decline.