RESUMO
Aquatic environments are subject to ultraviolet B (UVB) radiation incidence, and its effects on organisms are dose-dependent. Besides DNA, mitochondria are an important target of this radiation that causes structural damage and impairs its functional dynamics. Here, we hypothesize that mitophagy acts as an organelle quality control mechanism to mitigate UVB impacts in embryonic cells. Then, freshwater prawn Macrobrachium olfersii embryos was used as a model to investigate the effects of UVB on genes (Tomm20, Opa1, Pink, Prkn, Sqstm1, and Map1lc3) and proteins (TOM20, PINK1, p62 and LC3B) involved in mitophagy modulation. The choice of genes and proteins was based on the identification of mitochondrial membrane (Tomm20, Opa1 and TOM20), mediation of mitophagy (Pink1, Prkn and PINK1), and recognition of mitochondria by the autophagosome membrane (Sqstm1, Map1lc3, p62 and LC3B). First, the phylogeny of all genes presented bootstrap values >80 and conserved domains among crustacean species. Gene expression was inherently modulated during development, with transcripts (Tomm20, Opa1, Pink, Prkn, Sqstm1, and Map1lc3) overexpressed in the initial and final stages of development. Moreover, UVB radiation induced upregulation of Tomm20, Opa1, Pink, Prkn, Sqstm1, and Map1lc3 genes at 6 h after exposure. Interestingly, after 12 h, the protein content of PINK1, p62, and LC3B increased, while TOM20 was not responsive. Despite UVB radiation's harmful effects on embryonic cells, the chronology of gene expression and protein content indicates rapid activation of mitophagy, serving as an organelle quality control mechanism, given the analyzed cells' integrity.
Assuntos
Mitofagia , Palaemonidae , Raios Ultravioleta , Animais , Raios Ultravioleta/efeitos adversos , Mitofagia/efeitos da radiação , Palaemonidae/efeitos da radiação , Palaemonidae/embriologia , Palaemonidae/genética , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Embrião não Mamífero/efeitos da radiação , Embrião não Mamífero/metabolismo , Proteínas de Artrópodes/metabolismo , Proteínas de Artrópodes/genética , Filogenia , Organelas/metabolismo , Organelas/efeitos da radiaçãoRESUMO
The extracellular matrix (ECM) is a non-cellular and three-dimensional structure, constituted by a macromolecular dynamic network that involves the cells in all animal tissues, including embryonic ones. Several studies with vertebrates and cell cultures have reported deleterious effects of ultraviolet-B (UVB) radiation on the components associated with the ECM. However, studies focusing on the UVB radiation effects on ECM components of crustaceans during embryonic development are very scarce. Thus, the aim of this study was to identify the coding sequences of components associated with the ECM and to evaluate the effect of UVB radiation on embryos of the ecologically-important decapod Macrobrachium olfersii. To evaluate the modulation of these ECM components during embryonic development, the transcript levels of Col4α1, Itgß, Lamα, Mmp1 and Timp in M. olfersii embryos were analyzed at early developmental stages (E1, E3 and E4), intermediate developmental stage (E7) and late developmental stages (E10 and E14). In addition, embryos at E7, which correspond to a landmark of crustacean development, were analyzed after 12 h of UVB exposure to verify UVB effects on the ECM components. The ECM component sequences were similar to other decapods, suggesting conservation of these genes among crustaceans. The results showed modulations of the ECM components of M. olfersii embryos that reflect the need for each component in the cellular mechanisms, necessary for normal embryonic development. After UVB exposure, embryos showed opacity of embryonic tissues and it was found the overexpression of Col4α1, Itgß, Mmp1 and Timp transcript levels (1.82-, 1.52-, 2.34- and 6.27-fold, respectively). These impairments can compromise important events for normal embryonic development, such as growth of optic lobes, caudal papilla, ramification of appendages and differentiation of organic systems. The results presented here, together with the effects on morphology, cell proliferation, differentiation, and apoptosis demonstrated previously, strengthen the knowledge of the complex impacts of UVB radiation on freshwater embryos. Nevertheless, our results encourage further investigations focusing on the assessment of UVB effects on different organisms in order to better understand the myriad of UVB effects on ECM components.
Assuntos
Embrião não Mamífero/efeitos da radiação , Desenvolvimento Embrionário/efeitos da radiação , Matriz Extracelular/efeitos da radiação , Palaemonidae/efeitos da radiação , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta , Animais , Apoptose/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Desenvolvimento Embrionário/genética , Matriz Extracelular/genética , Água Doce/química , Palaemonidae/genética , Palaemonidae/crescimento & desenvolvimentoRESUMO
BACKGROUND: Ultraviolet (UV) radiation is the main exogenous inductor of skin damage and so photoprotection is important to control skin disorders. The Antarctic moss Sanionia uncinata is an important source of antioxidants and the photoprotective activity of its organic extracts has been investigated. This study aimed to evaluate the potential photoprotection, cytotoxicity and embryotoxicity of residual aqueous fraction (AF) from the moss S. uncinata. METHODS: UV-visible spectrum and SPF (sun protection factor) were determined by spectrophotometry. Embryotoxicity potential was evaluated by Fish embryo-larval toxicity test using zebrafish (Danio rerio) as organism model. Cell death assays by water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) were investigated using HaCaT keratinocyte cell line cultured in monolayers and three dimensions (3D). Phototoxicity and association with UV-filters were performed by 3T3 neutral red uptake test. RESULTS: The AF showed sharp absorption bands in the UV region and less pronounced in the visible region. The SPF was low (2.5 ± 0.3), but the SPF values of benzophenone-3 and octyl-methoxycinnamate increased ~ 3 and 4 times more, respectively, in association with AF. The AF did not induce significant lethal and sublethal effects on zebrafish early-life stages. In monolayers, the HaCaT cell viability, evaluated by WST-1, was above 70% by ≤0.4 mg AF/mL after 48 and 72-h exposure, whereas ≤1 mg AF/mL after 24-h exposure. The LDH assay showed that the cell viability was above 70% by ≤0.4 mg AF/mL even after 72-h exposure, but ≤1 mg/mL after 24 and 48-h exposure. In 3D cell culture, an increased cell resistance to toxicity was observed, because cell viability of HaCaT cell by WST-1 and LDH was above ~ 90% when using ≤1 and 4 mg AF/mL, respectively. The AF demonstrated values of photo irritation factor < 2 and of photo effect < 0.1, even though in association with UV-filters. CONCLUSIONS: The residual AF absorbs UV-vis spectrum, increased SPF values of BP-3 and OMC and does not induce embryotoxicity to zebrafish early life-stage. The cell death assays allowed establishing non-toxic doses of AF and phototoxicity was not detected. AF of S. uncinata presents a good potential for skin photoprotection against UV-radiation.
Assuntos
Bryopsida/química , Embrião não Mamífero/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Protetores Solares/farmacologia , Raios Ultravioleta , Animais , Regiões Antárticas , Bryopsida/crescimento & desenvolvimento , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta a Droga , Embrião não Mamífero/efeitos da radiação , Humanos , Queratinócitos/efeitos da radiação , Extratos Vegetais/toxicidade , Fator de Proteção Solar , Protetores Solares/toxicidade , Peixe-ZebraRESUMO
PURPOSE: Some phytochemicals have shown the potential of being radiomodifiers, especially phenolic compounds, such as lichenic secondary metabolites. To evaluate the phytochemical usnic acid as a radiomodifier, embryonic cells of molluscs have been used due to their ease of collection, high sensitivity to physical and chemical agents, well-known embryology and low cost for analysis. MATERIALS AND METHODS: This study aimed to assess the radiosensitizing action of usnic acid on Biomphalaria glabrata embryos. Samples were irradiated with 4 Gy of gamma rays from a 60Co source (dose rate 2.906 Gy/h). An acute toxicity test was performed using B. glabrata embryos in the blastula stage, in order to determine the toxicity of usnic acid and to establish the lethal Concentration for 50% (LC50). Subsequently, the radiomodifing capacity of usnic acid was estimated using assays with B. glabrata embryos. RESULTS: Irradiation increased the number of non-viable embryos compared to unirradiated controls. Additionally, it was observed that embryos exposed to a non-toxic concentration of usnic acid (0.6 µg/mL) before irradiation showed a further enhancement in non-viable embryos when compared with exposure to ionizing radiation alone. CONCLUSION: The results presented here indicate that usnic acid makes cells more sensitive to the damaging effects of radiation.
Assuntos
Benzofuranos/farmacologia , Biomphalaria/embriologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/efeitos da radiação , Radiossensibilizantes/farmacologia , Animais , Benzofuranos/toxicidade , Radioisótopos de Cobalto , Raios gama , Dose Letal Mediana , Radiossensibilizantes/toxicidadeRESUMO
Our previous studies showed that embryos of the freshwater prawn Macrobrachium olfersii exposed to ultraviolet B (UVB) radiation exhibited DNA damage, excessive ROS production, mitochondrial dysfunction and increased hsp70 expression, which are able, independently or together, to induce apoptosis. Thus, we attempted to elucidate some key apoptosis-related genes (ARG) and apoptosis-related proteins (ARP) and their expression during different stages of embryonic development, as well as to characterize the chronology of ARG expression and ARP contents after UVB radiation insult. We demonstrate that p53, Bax and Caspase3 genes are active in the embryonic cells at early embryonic developmental stages, and that the Bcl2 gene is active from the mid-embryonic stage. After UVB radiation exposure, we found an increase in ARP such as p53 and Bak after 3h of exposure. Moreover, an increase in ARG transcript levels for p53, Bax, Bcl2 and Caspase3 was observed at 6h after UVB exposure. Then, after 12h of UVB radiation exposure, an increase in Caspase3 gene expression and protein was observed, concomitantly with an increased number of apoptotic cells. Our data reveal that ARG and ARP are developmentally regulated in embryonic cells of M. olfersii and that UVB radiation causes apoptosis after 12h of exposure. Overall, we demonstrate that embryonic cells of M. olfersii are able to active the cell machinery against environmental changes, such as increased incidence of UVB radiation in aquatic ecosystems.
Assuntos
Apoptose/efeitos da radiação , Dano ao DNA , Embrião não Mamífero/efeitos da radiação , Expressão Gênica/efeitos da radiação , Palaemonidae/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Ecossistema , Embrião não Mamífero/patologia , Água Doce/química , Palaemonidae/embriologia , Exposição à Radiação/efeitos adversosRESUMO
The type of embryonic development (mixed and direct) and its influence on the accumulation and translocation of photoprotective compounds from the mother to the encapsulated embryo was studied in the intertidal gastropods Crepipatella peruviana and Crepipatella dilatata during their reproductive peak. HPLC/MS was used to determine type and levels of sunscreen compounds (total carotenoids; TC/and mycosporine-like amino acid; MAA) in brooding females, capsule walls and early and pre-hatching embryos of both species. Photoprotective compounds were only quantified in nurse eggs of C. dilatata. Our results indicate that females of both species can accumulate TC and MAA at different levels, and they are able to transfer them selectively to capsule walls, embryos and nurse eggs. Palythine-serine (MW=244Da; λmax=320nm) and MAA-330 (MW=234Da; λmax=330nm) constitute total MAA pool in brooding females, whereas brooded embryos incorporate palythine (MW=244Da; λmax=320nm) to the MAA pool. Although TC was transferred from the mother to the embryo through the yolk in both species, MAA trespass showed differences. Females of C. peruviana transfer MAA to their embryos through the embryonic yolk; C. dilatata can transfer MAA only through their nurse eggs, which are consumed by embryos during the terminal stages of intracapsular development. Differences between mixed and direct embryonic development, as well as environmental UV-R levels, which the recently hatched larvae and juveniles of C. peruviana and C. dilatata are exposed to, would determine levels of sunscreen compounds in each species. Higher TC and MAA levels in pre-hatching larvae of C. peruviana compared to C. dilatata, indicate a necessity of C. peruviana for protection against UV-R radiation during approximately 15days when their veliger larvae remain in the water column before metamorphosis is complete. Conversely, low photoprotective levels in pre-hatching juveniles of C. dilatata could be related to low UV-R exposure levels due to the direct incorporation to the benthos and the presence of a protective shell.
Assuntos
Gastrópodes/embriologia , Gastrópodes/metabolismo , Raios Ultravioleta/efeitos adversos , Aminoácidos/metabolismo , Animais , Carotenoides/metabolismo , Relação Dose-Resposta à Radiação , Embrião não Mamífero/metabolismo , Embrião não Mamífero/efeitos da radiação , Feminino , Gastrópodes/efeitos da radiaçãoRESUMO
In South America, increased UVB radiation has become an important environmental issue that is potentially threatening aquatic ecosystems. Considering that species exhibit different degrees of sensitivity to UVB radiation and that embryos are more sensitive than organisms at later life stages, the aim of this study was to characterize the effects of UVB radiation on subcellular compartments of embryos of the freshwater prawn Macrobrachium olfersi. This species lives and reproduces in clear and shallow waters, where UV radiation can fully penetrates. Embryos were irradiated with a UVB 6W lamp for 30min and examined after 1h, 12h, 24h and 48h of exposure. The irradiance of the UVB used simulates the UV radiation that embryos receive in the natural environment. The subcellular compartment most affected by the UVB radiation was the mitochondria, which exhibited a circular shape, a decrease in mitochondrial cristae, rupture of membranes and a morphology compatible with fission. These impairments were observed simultaneously with increased ROS production, just after 1h of UVB exposure. Thus, we investigated proteins related to mitochondrial fission (Drp-1) and fusion (Mfn-1), which are essential to cell maintenance. We found a significant increase in Drp-1 expression at all analyzed time-points and a significant decrease in Mfn-1 expression only after 24h of UVB exposure. Additionally, a decrease in embryonic cell viability was verified via the mitochondrial integrity assay. To conclude, we observed important mitochondrial dysfunctions against the environmental stress caused by UVB radiation. Moreover, the cellular responses found are critical and should not be disregarded, because they impact embryos that can potentially compromise the aquatic ecosystems.
Assuntos
Ecossistema , Monitoramento Ambiental/métodos , Água Doce , Mitocôndrias/efeitos da radiação , Palaemonidae/efeitos da radiação , Raios Ultravioleta , Animais , Sobrevivência Celular/efeitos da radiação , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos da radiação , Dinâmica Mitocondrial/efeitos da radiação , Modelos Teóricos , Palaemonidae/embriologia , Palaemonidae/crescimento & desenvolvimento , América do SulRESUMO
Several studies have reported about the effects of magnetic fields (MFs) on vascular tissue. Extremely low frequency magnetic fields (ELF-MFs) can promote either inhibition or stimulation of vasculogenesis and angiogenesis, depending upon the intensity and time of exposure to the MF. To investigate the possible effects of ELF-MF on vascular processes, it is necessary to employ methods that allow parameterization of the vascular network. Vascular network is a structure with fractal geometry; therefore, fractal methods have been used to evaluate its morphometric complexity. Here, we used the lacunarity parameter (complementary method of fractal analysis) and multifractal analyses to investigate angiogenesis and vasculogenesis in the embryonic yolk sac membrane (YSM) of Japanese quails (Coturnix japonica) with and without exposure to an external MF of 1 mT and 60 Hz. Lacunarity results showed that the vascular density was lower for the group exposed to the magnetic field for 9 h/day. In addition, multifractal analysis showed reduced vascularization in the experimental groups (6 h/day and 9 h/day of exposure to MF). Furthermore, multifractal analysis showed difference between the groups exposed for 12 and 24 h/day. Using multifractal methods (generalized dimensions and singularity spectrum), it was possible to characterize the vascular network of the quail embryo YSM as a multifractal object, therefore proving this method to be a more appropriate application than the traditional monofractal methods.
Assuntos
Vasos Sanguíneos/embriologia , Coturnix/embriologia , Campos Magnéticos , Saco Vitelino/irrigação sanguínea , Animais , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/efeitos da radiação , Fractais , Modelos Estatísticos , Neovascularização Patológica , Probabilidade , Radiação não Ionizante , Fatores de Tempo , Saco Vitelino/efeitos da radiaçãoRESUMO
Magnetic fields (MF) can alter the dynamic behavior of vascular tissue and may have a stimulatory or inhibitory effect on blood vessel growth. Fractal geometry has been used in several studies as a tool to describe the development of blood vascular networks. Due to its self-similarity, irregularity, fractional dimension, and dependence on the scale of vessel dimensions, vascular networks can be taken as fractal objects. In this work, we calculated the fractal dimension by the methods of box counting (D(bc)) and information dimension (D(inf)) to evaluate the development of blood vessels of the yolk sac membrane (YSM) from quail embryos exposed to MF with a magnetic flux density of 1 mT and a frequency of 60 Hz. The obtained results showed that when the MF was applied to embryos aged between 48 and 72 h, in sessions of 2 h (6 h/day) and 3 h (9 h/day) with exposure intervals between 6 and 5 h, respectively, blood vascular formation was inhibited. Exposure sessions shorter than 2 h or longer than 3 h had no observable change on the vascular process. In contrast, the magnetic field had no observable change on the YSM vascular network for embryos aged between 72 and 96 h, irrespective of the exposure time. In conclusion, these results show a "window effect" regarding exposure time.
Assuntos
Vasos Sanguíneos/embriologia , Coturnix/embriologia , Animais , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/efeitos da radiação , Fractais , Campos Magnéticos , Radiação não Ionizante , Saco Vitelino/efeitos da radiaçãoRESUMO
The recent decrease of the stratospheric ozone has resulted in an increase of ultraviolet-B (UV-B) radiation reaching the Earth's surface. In freshwater ecosystems with transparent water, UV-B rays easily penetrate and potentially cause harmful effects to organisms. In this study, embryos of the prawn Macrobrachium olfersi were used to evaluate the impact of UV-B rays in freshwater environments. We observed three groups of embryos: the first was to assess whether UV-B radiation produced morphological defects and/or biochemical impairments in the laboratory. The second was to check whether embryos with the same impairments as those observed in the laboratory were found in their environment, under natural solar radiation. The third group was the non-irradiated control. The embryos irradiated with 310 mW cm(-2) UV-B for 30 min showed morphological alterations similar to those observed in embryos from the environmental control group. The most important effects of the UV-B radiation observed in M. olfersi embryos were morphological (1.2% of the total number of embryos from the environment and 2.8% of the total number of irradiated embryos), pigmentation changes in the eyes (78.0% of the total number of embryos from the environment and 98.9% of the total number of irradiated embryos), and disruption of the chromatophores (46.9% of the total number of embryos from the environment and 95.5% of the total number of irradiated embryos). We also observed an increase in egg volume, which was accompanied by a significant increase in water content in UV-B irradiated groups when compared with aquaria control embryos. In addition, a significant decrease in the mitotic index in eggs exposed to UV-B radiation was detected (0.17 for the embryos from the aquaria control, 0.10 for the embryos of the environmental control, and 0.04 for the irradiated groups). The low levels of NPSH and high levels of TBARS indicated that UV-B rays directly compromised the antioxidant function of the embryonic cells, leading to oxidative stress. Our combined morphological and biochemical analyses revealed important effects induced by UV-B on M. olfersi embryos, and the results suggest that the recent changes in global conditions may have injurious effects, at least on the embryos of freshwater prawns.