No description available.

It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

Para otimizar um modelo experimental para o estudo do desbalanço redox em porfirias relacionadas ao acúmulo de ácido 5-aminolevulínico-(ALA), via inibição da ALA desidratase-(ALA-D), ratos foram tratados com o éster metílico de succinilacetona-(SAME), um catabólito da tirosina que inibe fortemente a ALA-O, mimetízando o estado metabólico observado nos portadores de portirias e tirosinemias. Estabeleceram-se modelos de tratamento agudo por 36 e 18 h. No primeiro, os animais receberam 3 injeções de SAME (10, 40 ou 80 mg/kg, grupos Ali-IV). No segundo, os animais receberam 3 injeções de 40 mg/kg de SAME, ALA ou éster metílico de ALA (grupos BII-IV), ALA:SAME (30: 10 mg/kg, grupo BV), ou 10 mg/kg SAME (grupo BVI). Paralelamente, avaliou-se se os sintomas neurológicos característicos das portirias decorriam de danos oxidativos mitocondriais. Para isso, aplicou-se uma tecnologia óptica para medidas da difusão da depressão cortical que determinou a oxigenação e o estado redox do cit c em mitocôndrias do córtex cerebral de ratos submetidos ao tratamento crônico com ALA (40 mg/kg), SAME (10 e 40 mg/kg) e ALA:SAME (30: 1O mg/kg), a cada 48 h, durante 30 dias. Tratamento agudo/36 h: Os níveis de ALA no plasma, fígado, cérebro e urina e o clearance renal do ALA aumentaram nos grupos tratados. A atividade de ALA-D e a coproporfirina urinária reduziram. A marcação para proteínas carboniladas, ferro e ferritina aumentou no fígado e cérebro dos grupos tratados, especialmente no All. Os níveis de malondialdeído hepático aumentaram no grupo AIV. A razão GSH/GSH+GSSG e a atividade de GPx cerebrais aumentaram nos grupos AIV e AIII, respectivamente. Consistentemente com estes dados indicando um desbalanço oxidativo induzido pelo SAME, alterações mitocondriais e citosólicas ultraestruturais foram reveladas, especialmente no fígado. Tratamento agudo/18 h: Os níveis de ALA plasmáticos aumentaram nos grupos tratados, exceto em BIV. O grupo BII mostrou aumento dos níveis hepáticos de ALA. Interessantemente, a inibição da atividade de ALA-D não foi evidenciada. O conteúdo de ferro plasmático aumentou no grupo BII. Para os grupos tratados com 10 e 40 mg SAME/kg, a atividade de SOD hepática reduziu ~50% com a extensão do tratamento de 18 para 36 h, sugerindo que este último é mais efetivo em promover danos oxidativos induzidos pelo ALA. Tratamento crônico/30 dias: Embora nenhuma alteração tenha sido evidenciada no estado redox dos animais tratados, o tratamento com ALA reduziu o fluxo sanguíneo cerebral (CBF) e o consumo de oxigênio-(CMRO2), sugerindo uma vasoconstrição mediada pelo ALA, efeito este confirmado por ensaios de reatividade vascular conduzidos em anéis de aorta de ratos incubados com ALA. O tratamento com ALA:SAME restaurou os níveis de CBF e CMRO2. Interessantemente, a disponibilidade do radical superóxido-(O2•-) estava reduzida nos anéis de aorta incubados com ALA. Juntos, estes dados: a)validam o modelo de tratamento agudo/36 h para o estudo bioquímico e dos possíveis efeitos fisiológicos induzidos pelo ALA, e b)sugerem que as alterações mediadas pelo ALA exógeno levam à vasoconstrição. / To optimize an experimental model for studying redox imbalance in porphyrias related to 5-aminolevulinic acid (ALA) accumulation through the inhibition of ALA dehydratase (ALA-D), rats were treated with methyl ester of succinylacetone (SAME), a tyrosine catabolite that strongly inhibits ALA-D, what mimics the metabolic state observed in patients suffering from porphyrías and tyrosinemias. Models of acute treatment were established during 36 and 18 h. In the first model, animals received 3 injections of SAME (10, 40 or 80 mg/kg, groups Ali-IV). In the second model, animals received 3 injections of 40 mg/kg SAME, ALA or methyl ester of ALA (groups BII-IV), ALA:SAME (30:10 mg/kg, group BV), or 10 mg/kg SAME (group BVI). Concomitantly, we evaluated if the neurologic symptoms characteristics of porphyrias were a consequence of the oxidative mitochondrial impairment. For this, an optical technology for the measurement of cortical spreading depression was applied. This techonology determined the cerebral oxygenation and the redox state of cit c in mitochondria of the cerebral cortex of rats submitted to a chronic treatment with ALA (40 mg/kg), SAME (10 and 40 mg/kg) and ALASAME (30:10 mg/kg), alternate days, during 30 days. Acute treatment/36 h: ALA levels in plasma, liver and urine and clearance of renal ALA increased in treated groups. ALA-D activities and urinary coproporphyrin were found to be decreased. Liver and brain proteins carbonyl, iron and ferritin were higher in the liver of treated groups, especially in All. Liver malondialdehyde levels were higher in group AIV. Cerebral GSH/GSH+GSSG ratio and GPx activities increased in groups AIV and AIII, respectively. Consistently with these data indicating SAME-induced oxidative imbalance, mitochondrial and cytosolic ultrastructural changes were revealed, especially in the liver. Acute treatment/18 h: Plasma ALA levels increased in all treated groups but BIV. Group BII showed increased hepatic ALA levels. Interestingly, inhibition in ALA-D activities was not evidenced. Plasma iron content increased in group BII. For the groups treated with 10 and 40 mg SAME/kg, liver SOD activities reduced ~50% by extending the treatment from 18 to 36 h, suggesting that the latter is more effective in ALA-induced oxidative damage. Chronic treatment /30 days: Despite no changes in the redox state of treated animals were observed, the treatment with ALA reduced the cerebral blood flow (CBF) and the consumption of oxygen (CMRO2), suggesting a vasoconstriction mediated by ALA. This effetc was confirmed by vascular reactivity assay performed in aortic rings of rats incubated with ALA. The treatment with ALA:SAME recovered the CBF and CMRO2 levels. Interestingly, the availability of superoxide radical (O2•-) was reduced in the aortic rings incubated with ALA. Altogether, these data a) validate the model of acute treatment/36 h for studying biochemical and possibly physiological effects induced by ALA, and b)suggest that the changes mediated by exogenous ALA lead to vasoconstriction.

Para otimizar um modelo experimental para o estudo do desbalanço redox em porfirias relacionadas ao acúmulo de ácido 5-aminolevulínico-(ALA), via inibição da ALA desidratase-(ALA-D), ratos foram tratados com o éster metílico de succinilacetona-(SAME), um catabólito da tirosina que inibe fortemente a ALA-O, mimetízando o estado metabólico observado nos portadores de portirias e tirosinemias. Estabeleceram-se modelos de tratamento agudo por 36 e 18 h. No primeiro, os animais receberam 3 injeções de SAME (10, 40 ou 80 mg/kg, grupos Ali-IV). No segundo, os animais receberam 3 injeções de 40 mg/kg de SAME, ALA ou éster metílico de ALA (grupos BII-IV), ALA:SAME (30: 10 mg/kg, grupo BV), ou 10 mg/kg SAME (grupo BVI). Paralelamente, avaliou-se se os sintomas neurológicos característicos das portirias decorriam de danos oxidativos mitocondriais. Para isso, aplicou-se uma tecnologia óptica para medidas da difusão da depressão cortical que determinou a oxigenação e o estado redox do cit c em mitocôndrias do córtex cerebral de ratos submetidos ao tratamento crônico com ALA (40 mg/kg), SAME (10 e 40 mg/kg) e ALA:SAME (30: 1O mg/kg), a cada 48 h, durante 30 dias. Tratamento agudo/36 h: Os níveis de ALA no plasma, fígado, cérebro e urina e o clearance renal do ALA aumentaram nos grupos tratados. A atividade de ALA-D e a coproporfirina urinária reduziram. A marcação para proteínas carboniladas, ferro e ferritina aumentou no fígado e cérebro dos grupos tratados, especialmente no All. Os níveis de malondialdeído hepático aumentaram no grupo AIV. A razão GSH/GSH+GSSG e a atividade de GPx cerebrais aumentaram nos grupos AIV e AIII, respectivamente. Consistentemente com estes dados indicando um desbalanço oxidativo induzido pelo SAME, alterações mitocondriais e citosólicas ultraestruturais foram reveladas, especialmente no fígado. Tratamento agudo/18 h: Os níveis de ALA plasmáticos aumentaram nos grupos tratados, exceto em BIV. O grupo BII mostrou aumento dos níveis hepáticos de ALA. Interessantemente, a inibição da atividade de ALA-D não foi evidenciada. O conteúdo de ferro plasmático aumentou no grupo BII. Para os grupos tratados com 10 e 40 mg SAME/kg, a atividade de SOD hepática reduziu ~50% com a extensão do tratamento de 18 para 36 h, sugerindo que este último é mais efetivo em promover danos oxidativos induzidos pelo ALA. Tratamento crônico/30 dias: Embora nenhuma alteração tenha sido evidenciada no estado redox dos animais tratados, o tratamento com ALA reduziu o fluxo sanguíneo cerebral (CBF) e o consumo de oxigênio-(CMRO2), sugerindo uma vasoconstrição mediada pelo ALA, efeito este confirmado por ensaios de reatividade vascular conduzidos em anéis de aorta de ratos incubados com ALA. O tratamento com ALA:SAME restaurou os níveis de CBF e CMRO2. Interessantemente, a disponibilidade do radical superóxido-(O2•-) estava reduzida nos anéis de aorta incubados com ALA. Juntos, estes dados: a)validam o modelo de tratamento agudo/36 h para o estudo bioquímico e dos possíveis efeitos fisiológicos induzidos pelo ALA, e b)sugerem que as alterações mediadas pelo ALA exógeno levam à vasoconstrição. / To optimize an experimental model for studying redox imbalance in porphyrias related to 5-aminolevulinic acid (ALA) accumulation through the inhibition of ALA dehydratase (ALA-D), rats were treated with methyl ester of succinylacetone (SAME), a tyrosine catabolite that strongly inhibits ALA-D, what mimics the metabolic state observed in patients suffering from porphyrías and tyrosinemias. Models of acute treatment were established during 36 and 18 h. In the first model, animals received 3 injections of SAME (10, 40 or 80 mg/kg, groups Ali-IV). In the second model, animals received 3 injections of 40 mg/kg SAME, ALA or methyl ester of ALA (groups BII-IV), ALA:SAME (30:10 mg/kg, group BV), or 10 mg/kg SAME (group BVI). Concomitantly, we evaluated if the neurologic symptoms characteristics of porphyrias were a consequence of the oxidative mitochondrial impairment. For this, an optical technology for the measurement of cortical spreading depression was applied. This techonology determined the cerebral oxygenation and the redox state of cit c in mitochondria of the cerebral cortex of rats submitted to a chronic treatment with ALA (40 mg/kg), SAME (10 and 40 mg/kg) and ALASAME (30:10 mg/kg), alternate days, during 30 days. Acute treatment/36 h: ALA levels in plasma, liver and urine and clearance of renal ALA increased in treated groups. ALA-D activities and urinary coproporphyrin were found to be decreased. Liver and brain proteins carbonyl, iron and ferritin were higher in the liver of treated groups, especially in All. Liver malondialdehyde levels were higher in group AIV. Cerebral GSH/GSH+GSSG ratio and GPx activities increased in groups AIV and AIII, respectively. Consistently with these data indicating SAME-induced oxidative imbalance, mitochondrial and cytosolic ultrastructural changes were revealed, especially in the liver. Acute treatment/18 h: Plasma ALA levels increased in all treated groups but BIV. Group BII showed increased hepatic ALA levels. Interestingly, inhibition in ALA-D activities was not evidenced. Plasma iron content increased in group BII. For the groups treated with 10 and 40 mg SAME/kg, liver SOD activities reduced ~50% by extending the treatment from 18 to 36 h, suggesting that the latter is more effective in ALA-induced oxidative damage. Chronic treatment /30 days: Despite no changes in the redox state of treated animals were observed, the treatment with ALA reduced the cerebral blood flow (CBF) and the consumption of oxygen (CMRO2), suggesting a vasoconstriction mediated by ALA. This effetc was confirmed by vascular reactivity assay performed in aortic rings of rats incubated with ALA. The treatment with ALA:SAME recovered the CBF and CMRO2 levels. Interestingly, the availability of superoxide radical (O2•-) was reduced in the aortic rings incubated with ALA. Altogether, these data a) validate the model of acute treatment/36 h for studying biochemical and possibly physiological effects induced by ALA, and b)suggest that the changes mediated by exogenous ALA lead to vasoconstriction.