Limited contribution of photoenzymatic DNA repair in mitigating carry-over effects from larval UVB exposure: Implications for frog recruitment.

Solar ultraviolet-B (UVB) radiation has increased due to stratospheric ozone depletion, climate and ecosystem changes and is a driver of amphibian population declines. Photoenzymatic repair (PER) is a critical mechanism for limiting UVB lethality in amphibian larvae. However, the link between PER and the UVB-induced effects remains understudied through long-term investigations in vivo. Here, we assessed how larval PER determines the lethal and sublethal effects induced by environmentally relevant acute UVB exposure until the juvenile phase in the Neotropical frog Odontophrynus americanus. We conducted laboratory-based controlled experiments in which tadpoles were or were not exposed to UVB and subsequently were exposed to light (for PER activation) or dark treatments. Results showed that the rates of mortality and apoptosis observed in post-UVB dark treatment are effectively limited in post-UVB light treatment, indicating PER (and not dark repair, i.e. nucleotide excision repair) is critical to limit the immediate genotoxic impact of UVB-induced pyrimidine dimers. Nonetheless, even tadpoles that survived UVB exposure using PER showed sublethal complications that extended to the juvenile phase. Tadpole responses included alterations in morphology, chromosomal instability, increased skin susceptibility to fungal proliferation, as well as increased generation of reactive oxygen species. The short-term effects were carried over to later stages of life because metamorphosis time increased and juveniles were smaller. No body abnormalities were visualized in tadpoles, metamorphs, and juveniles, suggesting that O. americanus is UVB-resistant concerning these responses. This study reveals that even frog species equipped with an effective PER are not immune to carry-over effects from early UVB exposure, which are of great ecological relevance as late metamorphosis and smaller juveniles may impact individual performance and adult recruitment to breeding. Future ecological risk assessments and conservation and management efforts for amphibian species should exercise caution when linking PER effectiveness to UVB resistance.

Phytochemical and biological characterization of aqueous extract of Vassobia breviflora on proliferation and viability of melanoma cells: involvement of purinergic pathway.

Vassobia breviflora belongs to the Solanaceae family, possessing biological activity against tumor cells and is a promising alternative for therapy. The aim of this investigation was to determine the phytochemical properties V. breviflora using ESI-ToF-MS. The cytotoxic effects of this extract were examined in B16-F10 melanoma cells and the relationship if any to purinergic signaling was involved. The antioxidant activity of total phenols, (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was analyzed, as well as production of reactive oxygen species (ROS) and nitric oxide (NO) was determined. Genotoxicity was assessed by DNA damage assay. Subsequently, the structural bioactive compounds were docked against purinoceptors P2X7 and P2Y1 receptors. The bioactive compounds found in V. breviflora were N-methyl-(2S,4 R)-trans-4-hydroxy-L-proline, calystegine B, 12-O-benzoyl- tenacigenin A and bungoside B. In vitro cytotoxicity was demonstrated at concentration ranges of 0.1-10 mg/ml, and plasmid DNA breaks only at the concentration of 10 mg/ml. V. breviflora extracts affected hydrolysis by ectoenzymes, such as ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) and ectoadenosine deaminase (E-ADA) which control levels of degradation and formation of nucleosides and nucleotides. In the presence of substrates ATP, ADP, AMP and adenosine, the activities of E-NTPDase, 5´-NT or E-ADA were significantly modulated by V. breviflora. N-methyl-(2S,4 R)-trans-4-hydroxy-L-proline presented higher binding affinity (according to receptor-ligand complex estimated binding affinity as evidenced by ∆G values) to bind to both P2X7 and P2Y1purinergic receptors.Our results suggest a putative interaction of V. breviflora bioactive compounds with growth inhibitory potential in B16-F10 melanoma and suggest that may be considered as promising compounds in melanoma and cancer treatment.

Genotoxicity of surface waters in Brazil.

Brazil has abundant surface water resources, huge aquatic biodiversity and is home to 213 million people. Genotoxicity assays are sensitive tools to detect the effects of contaminants in surface waters and wastewaters, as well as to determine potential risks of contaminated waters to aquatic organisms and human health. This work aimed to survey the articles published in 2000-2021 that evaluated the genotoxicity of surface waters within Brazilian territory to unveil the profile and trends of this topic over time. In our searches, we considered articles focused on assessing aquatic biota, articles that conducted experiments with caged organisms or standardized tests in the aquatic sites, as well as articles that transported water or sediment samples from aquatic sites to the laboratory, where exposures were performed with organisms or standardized tests. We retrieved geographical information on the aquatic sites evaluated, the genotoxicity assays used, the percentage of genotoxicity detected, and, when possible, the causative agent of aquatic pollution. A total of 248 articles were identified. There was a trend of increase in the number of publications and annual diversity of hydrographic regions evaluated over time. Most articles focused on rivers from large metropolises. A very low number of articles were conducted on coastal and marine ecosystems. Water genotoxicity was detected in most articles, regardless of methodological approach, even in little-studied hydrographic regions. The micronucleus test and the alkaline comet assay were widely applied with blood samples, mainly derived from fish. Allium and Salmonella tests were the most frequently used standard protocols. Despite most articles did not confirm polluting sources and genotoxic agents, the detection of genotoxicity provides useful information for the management of water pollution. We discuss key points to be assessed to reach a more complete picture of the genotoxicity of surface waters in Brazil.

Genotoxic risk assessment of solar UV radiation in tadpoles from Brazilian wetlands.

Solar ultraviolet (UV) radiation is an environmental genotoxic factor linked to amphibian decline. Here we assessed the genotoxic risk of UVB and UVA exposure for tadpoles from open ponds in southern Brazil, a mid-latitude region influenced by stratospheric ozone depletion. Daily UV doses were measured on the surface of a pond in Taim Ecological Station (TAIM; 32°49'24''S; 52°38'31''W) on a cloudless summer day to predict the worst-case scenario for UV-induced DNA damage. Pond descriptors were related to the use of microhabitats by Boana pulchella tadpoles in two ponds over the climate seasons of 2013 and 2014. Our results indicate that shaded microhabitats were more frequent than unshaded ones in autumn, winter, and spring but not in summer. Hence, the penetration of UV radiation into the water of unshaded microhabitats was evaluated through laboratory experiments with artificial UV sources and pond water samples. Physical and biological sensors were applied in the experiments to measure the incident UV radiation and its genotoxic action. By integrating field and laboratory data, we demonstrate that low doses of biologically effective UV radiation reached the tadpoles in autumn, winter, spring, and early summer due to a high proportion of shaded microhabitats and a high concentration of solids in unshaded microhabitats. However, the relative reduction of shaded microhabitats jointly with a declining water level in late summer may have exposed tadpoles to high UV doses. Our experiments also indicate that solar UVB radiation, but not UVA, is primarily responsible for the induction of DNA pyrimidine dimers in organisms living under the surface of aquatic ecosystems. The present work highlights the determinant role of wetland descriptors for minimizing the genotoxic potential of UV radiation and its consequences for amphibians.

Effects of isolated and combined exposures of Boana curupi (Anura: Hylidae) tadpoles to environmental doses of trichlorfon and ultraviolet radiation.

The biodiversity collapse strongly affects the amphibian group and many factors have been pointed out as catalytic agents. It is estimated that several events in the amphibian population decline worldwide may have been caused by the interaction of multiple drivers. Thus, this study aimed to evaluate the stressful effects of the exposure to environmental doses of trichlorfon (TCF) pesticide (0.5 µg/L; and an additional 100-fold concentration of 50 µg/L) and ultraviolet radiation (UV) (184.0 kJ/m² of UVA and 3.4 kJ/m² of UVB, which correspond to 5% of the daily dose) in tadpoles of the Boana curupi species (Anura: Hylidae). The isolated and combined exposures to TCF happened within 24 h of acute treatments under laboratory-controlled conditions. In the combined treatments, we adopted three different moments (M) of tadpole irradiation from the beginning of the exposures to TCF (0 h - M1; 12 h - M2; and 24 h - M3). Then, we evaluated tadpole survival, change in morphological characters, induction of apoptotic cells, lipid peroxidation (LPO), protein carbonyl content (PCC), glutathione S-transferase (GST), non-protein thiols (NPSH), and acetylcholinesterase (AChE), as well as the induction of genomic DNA (gDNA) damage. UVB treatment alone resulted in high mortality, along with a high level of apoptosis induction. Both UVA, UVB, and TCF increased LPO, PC, and AChE, while decreased GST activity. Regarding co-exposures, the most striking effect was observed in the interaction between UVB and TCF, which surprisingly decreased UVB-induced tadpole mortality, apoptosis, and gDNA damage. These results reinforce the B. curupi sensitivity to solar UVB radiation and indicate a complex response in face of UVB interaction with TCF, which may be related to activation of DNA repair pathways and/or inhibition of apoptosis, decreasing UVB-induced tadpole mortality.

Development of a rapid electrophoretic assay for genomic DNA damage quantification.

Accuracy, sensitivity, simplicity, reproducibility, and low-cost are desirable requirements for genotoxicity assessment techniques. Here we describe a simple electrophoretic assay for genomic DNA lesions quantification (EAsy-GeL) based on subjecting DNA samples to rapid unwinding/renaturation treatments and neutral agarose gel electrophoresis. The experiments performed in this work involved different biological samples exposed to increasing environmental-simulated doses of ultraviolet-B (UVB) radiation, such as Escherichia coli, human leukocytes, and isolated human genomic DNA. DNA extraction was carried out using a universal and low-cost protocol, which takes about 4 h. Before electrophoresis migration, DNA samples were kept into a neutral buffer to detect double-strand breaks (DSBs) or subjected to a 5-min step of alkaline unwinding and neutral renaturation to detect single-strand breaks (SSBs) or incubated with the DNA repair enzyme T4-endonuclease V for the detection of cyclobutane pyrimidine dimers (CPDs) before the 5-min step of DNA unwinding/renaturation. Then, all DNA samples were separated by neutral agarose gel electrophoresis, the DNA average length of each lane was calculated through the use of free software, and the frequency of DNA breaks per kbp was determined by a simple rule of three. Dose-response experiments allowed the quantification of different levels of DNA damage per electrophoretic run, varying from a constant and low amount of DSBs/SSBs to high and dose-dependent levels of CPDs. Compared with other assays based on alkaline unwinding and gel electrophoresis, EAsy-GeL has important advantages for both environmental monitoring and laboratory testing purposes. The simplicity and applicability of this protocol to other types of DNA lesions, biological models, and agents make it ideal for genotoxicity, DNA repair studies, as well as for assessing exposure risks to ecosystems and human health.

Impact of solar UV radiation on amphibians: focus on genotoxic stress.

Solar UV radiation is one of the most important environmental genotoxic factors. Its incidence increased due to stratospheric ozone depletion, climate changes, and deforestation, and plays a crucial role in the worldwide decline of the populations of amphibians. Even sublethal effects of UV-induced genotoxicity may cause drastic consequences in the performance and fitness of amphibians. We reviewed the existing literature searching for research papers focused on DNA damage (and responses) in various species by environmental relevant UVB and UVA doses. We found twenty one papers relative to this topic, but only four of them concerned direct measurements of DNA lesions in vivo. Finally, we identify knowledge gaps and provide recommendations for future investigations concerning the impact of the genotoxicity induced by sunlight on amphibians.

Sunlight-induced genotoxicity and damage in keratin structures decrease tadpole performance.

The increased incidence of solar ultraviolet (UV) radiation, an environmental genotoxic agent, due to ozone depletion or deforestation may help to explain the enigmatic decline of amphibian populations in specific localities. In this work, we evaluated the importance of DNA repair performed by photolyases to maintain the performance of treefrog tadpoles after acute and chronic treatments with environmental-simulated doses of solar UVB and UVA radiation. Immediately after UV treatments, tadpoles were exposed to a visible light source to activate photolyases or kept in dark containers. The biological effects of UV treatments were evaluated through morphological, histological, locomotor and survival analyzes of Boana pulchella tadpoles (Anura: Hylidae). The results indicate that tadpole body weight suffered influence after both UVB and UVA treatments, although the body length was bit affected. The locomotor performance of UVB-exposed tadpoles was significantly reduced. In addition, UVB radiation induced a severe impact on tadpole skin, as well as on keratinized structures of mouth (tooth rows and jaw), indicating that these should be important effects of solar UV radiation in the reduction of tadpole performance. Furthermore, photolyases activation was fundamental for the maintenance of tadpole performance after chronic UVB exposures, but it was relatively inefficient after acute exposures to UVB, but not to UVA radiation. Therefore, this work demonstrates how the UV-induced genotoxicity and structural alterations in the skin and oral apparatus affect tadpole performance and survival.

Impacts of UVB radiation on food consumption of forest specialist tadpoles.

Solar ultraviolet radiation B (UVB) is an important environmental stressor for amphibian populations due to its genotoxicity, especially in early developmental stages. Nonetheless, there is an absence of works focused on the UVB effects on tadpoles' food consumption efficiency. In this work, we investigated the effects of the exposure to a low environmental-simulated dose of UVB radiation on food consumption of tadpoles of the forest specialist Hypsiboas curupi [Hylidae, Anura] species. After UVB treatment tadpoles were divided and exposed to a visible light source or kept in the dark, in order to indirectly evaluate the efficiency of DNA repair performed by photolyases and nucleotide excision repair (NER), respectively. The body mass and the amount of food in tadpoles' guts were verified in both conditions and these data were complemented by the micronuclei frequency in blood cells. Furthermore, the keratinized labial tooth rows were analyzed in order to check for possible UVB-induced damage in this structure. Our results clearly show that the body weight decrease induced by UVB radiation occurs due to the reduction of tadpoles' food consumption. This behavior is directly correlated with the genotoxic impact of UVB light, since the micronuclei frequency significantly increased after treatments. Surprisingly, the results indicate that photoreactivation treatment was ineffective to restore the food consumption activity and body weight values, suggesting a low efficiency of photolyases enzymes in this species. In addition, UVB treatments induced a higher number of breaks in the keratinized labial tooth rows, which could be also associated with the decrease of food consumption. This work contributes to better understand the process of weight loss observed in tadpoles exposed to UVB radiation and emphasizes the susceptibility of forest specialist amphibian species to sunlight-induced genotoxicity.