RESUMO
This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L-buthionine-(S,R)-sulfoximine (BSO) or 1-chloro-2,4-dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O2 , 3% and 20%, were employed during a 1 h exposure of the cells to 25 J cm(-2) of UVA radiation (338-400 nm wavelength, peak at 365 nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O2 compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA-induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA-induced cell damage.
Assuntos
Células Epiteliais/efeitos da radiação , Glutationa/metabolismo , Cristalino/efeitos da radiação , Tiorredoxina Redutase 1/metabolismo , Catalase/metabolismo , Contagem de Células , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dinitroclorobenzeno/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Glutationa/antagonistas & inibidores , Humanos , Cristalino/citologia , Cristalino/efeitos dos fármacos , Cristalino/metabolismo , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacologia , Estresse Oxidativo , Oxigênio/farmacologia , Tiorredoxinas/metabolismo , Raios UltravioletaRESUMO
PURPOSE: To compare levels of S-glutathiolation and S-cysteinylation occurring at more than 60 cysteine residues of 12 different guinea pig lens water-soluble nuclear crystallins following treatment of the animals with hyperbaric oxygen (HBO). METHODS: Guinea pigs (initially 18 months old) were treated 30X (3X per week for 10 weeks) with HBO (2.5 atm 100% O(2) for 2.5 h) as a model to study the formation of nuclear cataract. This treatment produces a moderate increase in lens nuclear light scatter (compared to denser scatter occurring after 80 HBO treatments), with five- to sixfold increases in levels of protein-bound glutathione (PSSG) and protein-bound cysteine (PSSC). Trypsin digests of lens nuclear water-soluble proteins were analyzed with two-dimensional liquid chromatography and mass spectrometry to identify specific cysteine residues binding either glutathione or cysteine. Lens nuclei of age-matched untreated animals were used as controls. RESULTS: All major crystallins, except αB, were modified to some extent by either S-glutathiolation or S-cysteinylation. Overall, 72% of the cysteine residues of guinea pig lens nuclear crystallins were shown to be capable of binding glutathione, cysteine, or both molecules. The crystallin with the highest level of modification was ßA1/A3 (six of eight -SH groups), and that with the lowest (two of five -SH groups) was ßA2. O(2)-induced increases in PSSG levels were 2.8, 2.4, and 4.1 times control for γA-, γB-, and γC-crystallins, respectively. Comparable increases in PSSC levels for the three γ-crystallins were 2.3, 2.7, and 2.4 times control, respectively. ßB2-crystallin showed the highest amount of O(2)-induced PSSG formation of any of the crystallins, as well as a substantial level of control PSSG, and nearly all of this was due to a single residue, C67, a site also present in human ßB2-crystallin. Overall, 32 of the 44 modified cysteine residues were homologous with the human. CONCLUSIONS: This large-scale study successfully identified lens crystallin cysteine residues that bound glutathione and/or cysteine under normal or oxidative stress conditions. The high percentage of protein -SH groups that are modified by S-thiolation in the guinea pig lens nucleus demonstrates the substantial protein sulfhydryl redox buffer capability present in the center of the lens. The results suggest that PSSG and PSSC formation may act to delay O(2)-induced insolubilization of γA-, γB-, and γC-crystallins, and ß-crystallins, but with a greater effect on the γ-crystallins at an early stage of oxidative stress. The study has shown that technological approaches are now available to investigate in considerable detail the role of specific lens -SH groups in nuclear cataractogenesis.
Assuntos
Cristalinas/química , Cristalinas/metabolismo , Núcleo do Cristalino/metabolismo , Proteômica/métodos , Animais , Sítios de Ligação , Catarata/etiologia , Catarata/metabolismo , Cisteína/química , Modelos Animais de Doenças , Glutationa/química , Cobaias , Hiperóxia/metabolismo , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Solubilidade , Compostos de Sulfidrila/química , Espectrometria de Massas em TandemRESUMO
It is known that fluorescence, much of it caused by UVA light excitation, increases in the aging human lens, resulting in loss of sharp vision. This study used an in vivo animal model to investigate UVA-excited fluorescence in the rabbit lens, which contains a high level of the UVA chromophore NADH, existing both free and bound to λ-crystallin. Also, the ability of a Class I (senofilcon A) soft contact lens to protect against UVA-induced effects on the rabbit lens was tested. Rabbit eyes were irradiated with UVA light in vivo (100 mW/cm(2) on the cornea) for 1 h using monochromatic 365 nm light. Irradiation was conducted in the presence of either a senofilcon A contact lens, a minimally UV-absorbing lotrafilcon A contact lens, or no contact lens at all. Eyes irradiated without a contact lens showed blue 365 nm-excited fluorescence initially, but this changed to intense yellow fluorescence after 1 h. Isolated, previously irradiated lenses exhibited yellow fluorescence originating from the lens nucleus when viewed under 365 nm light, but showed normal blue fluorescence arising from the cortex. Previously irradiated lenses also exhibited a faint yellow color when observed under visible light. The senofilcon A contact lens protected completely against the UVA-induced effects on fluorescence and lens yellowing, whereas the lotrafilcon A lens showed no protection. The UVA-exposure also produced a 53% loss of total NADH (free plus bound) in the lens nucleus, with only a 13% drop in the anterior cortex. NADH loss in the nucleus was completely prevented with use of a senofilcon A contact lens, but no significant protection was observed with a lotrafilcon A lens. Overall, the senofilcon A lens provided an average of 67% protection against UVA-induced loss of four pyridine nucleotides in four different regions of the lens. HPLC analysis with fluorescence detection indicated a nearly six-fold increase in 365 nm-excited yellow fluorescence arising from lens nuclear λ-crystallin after the in vivo UVA exposure. It is concluded that UVA-induced loss of free NADH (which fluoresces blue) may have allowed the natural yellow fluorescence of λ-crystallin and other proteins in the lens nucleus to become visible. Increased fluorescence exhibited by UVA-exposed λ-crystallin may have been the result of a UVA-induced change in the conformation of the protein occurring during the initial UVA-exposure in vivo. The results demonstrate the greater susceptibility of the lens nucleus to UVA-induced stress, and may relate to the formation of human nuclear cataract. The senofilcon A contact lens was shown to be beneficial in protecting the rabbit lens against effects of UVA light, including changes in fluorescence, increased yellowing and loss of pyridine nucleotides.
Assuntos
Lentes de Contato Hidrofílicas , Fluorescência , Hidrogéis , Núcleo do Cristalino/efeitos da radiação , NAD/metabolismo , Lesões Experimentais por Radiação/prevenção & controle , Silicones , Raios Ultravioleta/efeitos adversos , Animais , Catarata/enzimologia , Catarata/prevenção & controle , Cromatografia Líquida de Alta Pressão , Cristalinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Olho/efeitos da radiação , Núcleo do Cristalino/patologia , Malato Desidrogenase/metabolismo , Coelhos , Lesões Experimentais por Radiação/enzimologia , Lesões Experimentais por Radiação/patologia , Proteção Radiológica/instrumentaçãoRESUMO
PURPOSE: UVB radiation from sunlight is known to be a risk factor for human cataract. The purpose in this study was to investigate the ability of a class I UV-blocking soft contact lens to protect against UVB-induced effects on the ocular tissues of the rabbit in vivo. METHODS: Eyes of rabbits were exposed to UVB light for 30 minutes (270-360 nm, peak at 310 nm, 1.7 mW/cm(2) on the cornea). Eyes were irradiated in the presence of either a UV-blocking senofilcon A contact lens, a minimally UV-blocking lotrafilcon A contact lens, or no contact lens at all. Effects on the cornea and lens were evaluated at various times after exposure. RESULTS: Eyes irradiated with no contact lens protection showed corneal epithelial cell loss plus lens epithelial cell swelling, vacuole formation, and DNA single-strand breaks, as well as lens anterior subcapsular opacification. The senofilcon A lens protected nearly completely against the UVB-induced effects, whereas the lotrafilcon A lens showed no protection. CONCLUSIONS: The results indicate that use of a senofilcon A contact lens is beneficial in protecting ocular tissues of the rabbit against the harmful effects of UVB light, including photokeratitis and cataract.
Assuntos
Catarata/prevenção & controle , Lentes de Contato Hidrofílicas , Olho/efeitos da radiação , Hidrogéis , Lesões Experimentais por Radiação/prevenção & controle , Proteção Radiológica/instrumentação , Silicones , Raios Ultravioleta/efeitos adversos , Animais , Catarata/etiologia , Catarata/patologia , Morte Celular , Dano ao DNA/efeitos da radiação , Células Epiteliais/efeitos da radiação , Células Epiteliais/ultraestrutura , Epitélio Corneano/patologia , Epitélio Corneano/efeitos da radiação , Ceratite/etiologia , Ceratite/patologia , Ceratite/prevenção & controle , Cristalino/efeitos da radiação , Cristalino/ultraestrutura , Coelhos , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/patologiaAssuntos
Inibidores da Angiogênese/efeitos adversos , Endoftalmite/etiologia , Infecções Oculares/etiologia , Injeções Intravítreas/efeitos adversos , Infecções Estafilocócicas/etiologia , Antibacterianos/uso terapêutico , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados , Aptâmeros de Nucleotídeos/efeitos adversos , Bevacizumab , Ceftazidima/uso terapêutico , Quimioterapia Combinada , Endoftalmite/tratamento farmacológico , Infecções Oculares/tratamento farmacológico , Humanos , Ranibizumab , Fatores de Risco , Infecções Estafilocócicas/tratamento farmacológico , Vancomicina/uso terapêutico , Corpo Vítreo/microbiologiaRESUMO
Zeta-crystallin is an NADPH-binding protein consisting of four identical 35kD subunits. The protein possesses quinone oxidoreductase activity, and is present in large amounts in the lenses of camelids, certain hystricomorphic rodents, and the Japanese tree frog, and in lower catalytic amounts in certain tissues of various species. In this study, recombinant methods were used to produce substantial quantities of his-tagged recombinant mouse zeta-crystallin, which was then purified to homogeneity. The yield of pure recombinant mouse zeta-crystallin was five times that obtained previously for purification of recombinant guinea pig zeta-crystallin. The quinone oxidoreductase activity of purified his-tagged recombinant mouse zeta-crystallin was comparable to that of purified native guinea pig lens zeta-crystallin, and to that previously reported for recombinant guinea pig zeta-crystallin. The method permits production of substantial amounts of recombinant zeta-crystallin for conducting studies on the biological role of this interesting protein, which exists in such high concentration in the lenses of certain species.
Assuntos
Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , zeta-Cristalinas/isolamento & purificação , zeta-Cristalinas/metabolismo , Animais , Cobaias , Cristalino/química , Cristalino/metabolismo , Camundongos , NADP/metabolismo , Quinona Redutases/metabolismo , Proteínas Recombinantes/genética , zeta-Cristalinas/genéticaRESUMO
PURPOSE: To determine if enzymatic induction of a posterior vitreous detachment (PVD) and/or vitreous liquefaction affects O2 concentration in the vitreous cavity in animals with vascularized and avascular retinal circulations. METHODS: Either microplasmin or hyaluronidase was injected intravitreally into guinea pigs (avascular retinal circulation), brown Norway rats (vascularized retinal circulation without fovea), or cats (vascularized retinal circulation with fovea) with the contralateral eye used as a control. One to 2 weeks post injection, vitreal oxygen concentration was measured using a highly sensitive, platinum-based fluorophore O2 sensor. In addition, control and microplasmin-injected rats, guinea pigs, and cats were exposed to 100% oxygen and vitreal O2 levels were measured over time. Scanning electron microscopy (SEM) was used to evaluate the vitreoretinal interface for the presence of a PVD. RESULTS: In animals with a vascularized retinal circulation (brown Norway rats and cats), intravitreal injection of microplasmin with induction of a PVD significantly increased baseline O2 concentration in the vitreous cavity compared to hyaluronidase injected eyes and controls in rats (35, 25, and 23 mm Hg, P < 0.001 and P < 0.001, respectively) and cats (26, 18, and 16 mm Hg, P < 0.01 and P < 0.001, respectively). Interestingly, intravitreal injection of hyaluronidase (vitreous liquefaction without induction of a PVD) did not significantly increase vitreal O2 levels in any of the animal species (P > 0.1). Upon exposure to 100% oxygen by facemask, microplasmin injected animals showed a rapid increase in vitreal oxygen levels compared to hyaluronidase injected animals and controls, indicating that the presence of a PVD allows rapid O2 exchange within the vitreous cavity. Similarly, once O2 was discontinued, the O2 concentration decreased in a similarly rapid rate. SEM showed smooth retinal surfaces in microplasmin-injected cat eyes, indicating the presence of a PVD which was not present in hyaluronidase injected or control eyes. CONCLUSION: The results suggest that enzymatic-assisted PVD with microplasmin increases vitreal O2 levels and increases the rate of O2 exchange within the vitreous cavity.
Assuntos
Fibrinolisina/farmacologia , Fibrinolíticos/farmacologia , Oxigênio/metabolismo , Fragmentos de Peptídeos/farmacologia , Corpo Vítreo/efeitos dos fármacos , Descolamento do Vítreo/metabolismo , Animais , Gatos , Cobaias , Hialuronoglucosaminidase/farmacologia , Injeções , Microscopia Eletrônica de Varredura , Neovascularização Fisiológica , Ratos , Ratos Endogâmicos BN , Retina/ultraestrutura , Corpo Vítreo/ultraestrutura , Descolamento do Vítreo/induzido quimicamenteRESUMO
Our laboratory treats guinea pigs with hyperbaric oxygen (HBO) as a model for investigating the formation of nuclear cataract. Previous analyses of lens supernatants using this model have shown an increase in disulfide (-SS-) and loss of sulfhydryl (-SH) in the lens nucleus of O(2)-treated animals. In this paper, we have used the non-invasive technique of Raman spectroscopy to confirm these findings in intact, freshly-excised lenses. Guinea pigs were treated 3 times per week with HBO for a total of 50 (4 months of treatment) or 85 (7 months of treatment) times to induce an increased level of lens nuclear light scattering. Intact lenses were analyzed by Raman spectroscopy using a 514.5 nm laser and collecting the scattered light in a 90 degrees geometry. The laser beam was focused either in the lens nucleus or equatorial cortex. Changes in the levels of -SS- (503 cm(-1)) and -SH (2577 cm(-1)) vibrations were measured. Raman spectra were analyzed by fitting Lorentzian profiles to the observed data in the -SS- and -SH regions. -SS- levels in the O(2)-treated nucleus were found to have increased by a factor of 2.1 (p=0.0001) and 2.5 (p=0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. Based on previous biochemical analyses, the -SS- increase was due mainly to the formation of protein disulfide (PSSP) with contribution also from protein/thiol mixed disulfides, but not from oxidized glutathione. -SH levels in the O(2)-treated nucleus decreased by 13% (p=0.007) and 35% (p=0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. No significant increase in -SS- or loss of -SH was observed in the lens cortex of the O(2)-treated guinea pigs. The Raman spectroscopy results rule out the possibility that artifactual production of -SS- and loss of -SH occurred during homogenization of lenses in previous studies. The data provide additional evidence to support a link between O(2), disulfide-crosslinking of lens crystallins in the nucleus, and nuclear cataract.
Assuntos
Catarata/metabolismo , Dissulfetos/análise , Núcleo do Cristalino/química , Análise Espectral Raman , Envelhecimento , Animais , Catarata/patologia , Modelos Animais de Doenças , Cobaias , Oxigenoterapia Hiperbárica , Núcleo do Cristalino/patologia , Masculino , Compostos de Sulfidrila/análiseRESUMO
PURPOSE: Heavy metals and other forms of oxidative stress have been implicated as key factors in the formation of age-related cataract in humans. Metallothioneins are a group of proteins known to play important roles in defending cells against the cytotoxic effects of heavy metals. However, little is known about their involvement in defending against other forms of oxidative stress. Here, we examined the ability of metallothionein IIa (MTIIa) to protect human lens epithelial cells against cadmium and tertiary butyl hydroperoxide (TBHP)-induced oxidative stress. METHODS: MTIIa over-expressing human lens epithelial cells (SRA01/04) were created by retroviral mediated gene transfer. Normal and MTIIa over-expressing cells were exposed to various concentrations of cadmium and TBHP and subsequently monitored for cell death, changes in cell phenotype and differences in growth rate. In addition, expression levels of three other important antioxidant genes, heme oxygenase-1, thioredoxin reductase-1, and manganese superoxide dismutase were monitored by real-time RT-PCR following exposure to TBHP. RESULTS: Analysis of the over expressing cell lines revealed an approximate 3-4 fold increase in MTIIa expression relative to control cells, resulting in as much as 20% protection against cadmium-induced oxidative stress (p<0.001). The MTIIa over expressing cells were also significantly more resistant to TBHP treatment while control cells exhibited significant shrinking and rounding-up following 3-6 h TBHP treatment, no changes were observed in TBHP-treated over expressing cells. When control cells were treated for 3 h or overnight with TBHP, 40-45% cell death occurred by day three. However, no cell death was observed at this time for the treated MTIIa over-expressing cell line. In addition, TBHP induced the expression of MTIIa, heme oxygenase-1, thioredoxin reductase-1, and MnSOD in both normal and MTIIa over-expressed cell lines. Interestingly the latter three genes were induced at 2-3 fold higher levels in TBHP-treated MTIIa over-expressing cells, compared to treated control cells (p=0.001, p=0.02, and p=0.01, respectively). CONCLUSIONS: These data indicate that over-expression of MTIIa in human lens epithelial cells results in protection against cadmium and TBHP-induced oxidative stress. In addition, the results suggest that MTIIa, and/or its ability to chelate metals, may play a role in regulating expression of other important antioxidant genes in response to oxidative stress.
Assuntos
Cádmio/farmacologia , Cristalino/efeitos dos fármacos , Cristalino/metabolismo , Metalotioneína/farmacologia , Estresse Oxidativo/efeitos dos fármacos , terc-Butil Hidroperóxido/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Resistência a Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Heme Oxigenase-1/metabolismo , Humanos , Cristalino/citologia , Cristalino/fisiologia , Metalotioneína/genética , Metalotioneína/metabolismo , Superóxido Dismutase/metabolismo , Tiorredoxina Redutase 1 , Tiorredoxina Dissulfeto Redutase/metabolismo , TransfecçãoRESUMO
PURPOSE: The role of oxygen in the formation of lens high-molecular-weight (HMW) protein aggregates during the development of human nuclear cataract is not well understood. The purpose of this study was to investigate lens crystallin aggregate formation in hyperbaric oxygen (HBO)-treated guinea pigs by using in vivo and in vitro METHODS: methods. Guinea pigs were treated three times weekly for 7 months with HBO, and lens crystallin aggregation was investigated in vivo with the use of dynamic light-scattering (DLS) and in vitro by HPLC analysis of water-insoluble (WI) proteins. DLS measurements were made every 0.1 mm across the 4.5- to 5.0-mm optical axis of the guinea pig lens. RESULTS: The average apparent diameter of proteins in the nucleus (the central region) of lenses of HBO-treated animals was nearly twice that of the control animals (P < 0.001). Size distribution analysis conducted at one selected point in the nucleus and cortex (the outer periphery of the lens) after dividing the proteins into small-diameter and large-diameter groups, showed in the O2-treated nucleus a threefold increase in intensity (P < 0.001) and a doubling in apparent size (P = 0.03) of large-diameter aggregate proteins, compared with the same control group. No significant changes in apparent protein diameter were detected in the O2-treated cortex, compared with the control. The average diameter of protein aggregates at the single selected location in the O2-treated nucleus was estimated to be 150 nm, a size capable of scattering light and similar to the size of aggregates found in human nuclear cataracts. HPLC analysis indicated that one half of the experimental nuclear WI protein fraction (that had been dissolved in guanidine) consisted of disulfide cross-linked 150- to 1000-kDa aggregates, not present in the control. HPLC-isolated aggregates contained alphaA-, beta-, gamma-, and zeta-crystallins, but not alphaB-crystallin, which is devoid of -SH groups and thus does not participate in disulfide cross-linking. All zeta-crystallin present in the nuclear WI fraction appeared to be there as a result of disulfide cross-linking. CONCLUSIONS: The results indicate that molecular oxygen in vivo can induce the cross-linking of guinea pig lens nuclear crystallins into large disulfide-bonded aggregates capable of scattering light. A similar process may be involved in the formation of human nuclear cataract.
Assuntos
Catarata/metabolismo , Cristalinas/metabolismo , Modelos Animais de Doenças , Oxigenoterapia Hiperbárica , Núcleo do Cristalino/metabolismo , Oxigênio/fisiologia , Animais , Western Blotting , Catarata/patologia , Cromatografia Líquida de Alta Pressão , Cristalinas/química , Cobaias , Núcleo do Cristalino/química , Luz , Masculino , Ligação Proteica , Desnaturação Proteica , Espalhamento de Radiação , Compostos de Sulfidrila/químicaRESUMO
The main objective of this study was to investigate the effect of in vivo hyperbaric oxygen (HBO) treatment of albino guinea pigs on ocular refractive state and optical properties of the lens in vitro, as well as on the integrity of the mitochondria of the lens. The animals were treated 30-35 times (2.5-3 months) or 70 times (6 months) with HBO. An increased level of lens nuclear light scattering was evident by slit-lamp at 30 treatments, and this increased at 70 treatments. After 30-35 HBO treatments a myopic shift in refractive state of the eye was seen in two separate studies with two different refractionists. Also, the average back vertex distance of the lens was significantly shorter after 35 HBO treatments while spherical aberration (focal variability) increased after 70 treatments. No difference in refractive state was noted after 70 HBO treatments (a reversal of the initial myopic effect). The mitochondrial distribution and morphology of the lens epithelium and the superficial cortical fibre cells were normal after both 35 and 70 HBO treatments, highlighting that HBO treatment does not affect the superficial cortex of the lens. The results of the in vitro lens optical analysis carried out in this study correlate with the myopia observed after 30-35 HBO in vivo treatments. A similar reversible myopia and increase in lens nuclear light scattering is known to occur in humans treated with HBO for extended periods and the results suggest that the myopia was caused by a change in the refractive index of the lens. The significant loss of sharp focus after 70 HBO treatments can be correlated with previous reports of biochemical and morphological changes associated with HBO-induced loss of lens nuclear transparency in mature guinea pigs. The guinea pig HBO model may be a useful approach for the study of lens development and refractive error.
Assuntos
Oxigenoterapia Hiperbárica/efeitos adversos , Cristalino/fisiopatologia , Erros de Refração/etiologia , Animais , Modelos Animais de Doenças , Cobaias , Cristalino/ultraestrutura , Masculino , Microscopia Confocal , Mitocôndrias/ultraestrutura , Óptica e Fotônica , Refração Ocular , Erros de Refração/patologia , Erros de Refração/fisiopatologia , Espalhamento de RadiaçãoRESUMO
We have shown previously with in vivo and in vitro animal models that the lens epithelium, in contrast to the nucleus, is remarkably resistant to hyperoxia. The main purpose of this study was to investigate the mRNA response of cultured human lens epithelial cells (LECs) to challenge by a high level of hyperbaric oxygen. Cells were treated for 3 hr with 50 atm of 99% O2, and then cultured normally for various times up to 11 days. Although the cells appeared normal immediately after the O2-treatment, they failed to grow and suffered 50% cell loss, as well as significant mitochondrial damage, during normal post-culture. Growth of the cells resumed after 3 days and by day 11, the number of O2-treated cells was the same as the controls. Remarkably, the 3 hr O2-treatment produced no immediate effects on either the cellular level of GSH, or on the activities of a number of antioxidant enzymes including glyceraldehyde-3-phosphate dehydrogenase, which is generally regarded as being highly sensitive to oxidation. In contrast, the activity of thioredoxin reductase (TrxR) was severely affected by the O2, decreasing by 51% after the 3 hr exposure. O2-induced death of the cells appeared to be caused by loss of ATP since a 31% decrease in ATP level occurred immediately after the O2-treatment, in spite of a 46% increase in lactate production. Analysis with real-time PCR showed a maximum 3-6-fold increase in mRNA levels 9 hr after the 3 hr O2-exposure for the enzymes heme oxygenase-1 (HO-1), MnSOD and TrxR1 (the cytoplasmic form of TrxR). These results were confirmed with the use of one-step RT-PCR and Northern blotting. Initial upregulation of message for HO-1 occurred a few hours before any upregulation of MnSOD could be detected, suggesting that release of free iron from the degradation of heme by HO-1 may have played a role in the upregulation of the dismutase. No significant changes in mRNA levels were observed for the antioxidant enzymes catalase, CuZnSOD, glutathione reductase and glutathione peroxidase, or for the antioxidant protein thioredoxin. Recovery of TrxR activity over a 4-day period appeared to parallel the return of the cells to a normal rate of growth. The results indicate that damaging effects of hyperoxia on cultured LECs occur primarily in the mitochondria, rather than in the cytoplasm. Cells avoid O2-induced cell death, and return to a normal rate of proliferation by upregulating mRNA levels for HO-1, MnSOD and TrxR1. It appears that full activity of TrxR1, an enzyme required for the production of deoxyribonucletides for DNA synthesis, is essential for the normal growth of O2-challenged LECs.
Assuntos
Células Epiteliais/enzimologia , Cápsula do Cristalino/enzimologia , Oxigênio/toxicidade , Tiorredoxina Dissulfeto Redutase/fisiologia , Antioxidantes/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Cápsula do Cristalino/efeitos dos fármacos , Cápsula do Cristalino/ultraestrutura , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Estresse Oxidativo/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/genéticaRESUMO
n-Propyl gallate (nPG) is a food preservative that is generally regarded as safe by the US FDA. It suppresses oxidation in biological systems. The mechanism by which nPG acts in biological systems is uncertain. We investigated whether nPG protected cultured lens epithelial cells from H(2)O(2)-induced damage. Cells were treated with H(2)O(2) or with nPG and then H(2)O(2). H(2)O(2) inhibited growth, caused membrane blebbing, decreased lactate production, increased the level of GSSG, decreased the levels of GSH, ATP and NAD(+), and G3PDH activity, stimulated the hexose monophosphate shunt and induced single-strand breaks in DNA. nPG prevented the H(2)O(2)-induced growth inhibition, membrane blebbing, drop in NAD(+) and single-strand breaks in DNA. The mechanism by which nPG acts at the chemical level was investigated using electron paramagnetic resonance (EPR), direct spectrophotometric kinetic measurements, and cyclic voltammetry. When nPG at low concentrations (nM to microM) was mixed with a large excess of O(2)(-)*, the superoxide signal was destroyed as indicated by UV visible spectroscopy and EPR. Kinetic analysis indicated that nPG dismutated O(2)(-)* in repetitive additions of superoxide with little loss of activity. The rate constant for the overall reaction of nPG with O(2)(-)* was ca. 10(6)M(-1)s(-1). nPG had a very low specific binding constant for Fe(2+) as determined by cyclic voltammetry. The evidence indicates that nPG dismutates the superoxide ion in a catalytic manner.
Assuntos
Antioxidantes/farmacologia , Células Epiteliais/efeitos dos fármacos , Peróxido de Hidrogênio/antagonistas & inibidores , Cristalino/efeitos dos fármacos , Galato de Propila/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Peróxido de Hidrogênio/farmacologia , Cristalino/citologia , Cristalino/metabolismo , Coelhos , Superóxido Dismutase/fisiologia , Superóxidos/metabolismoRESUMO
The possible role of ultraviolet light in the formation of cataract is not well understood. In this study, guinea pigs were exposed to a chronic, low level of UVA light (0.5 mWcm(-2), 340-410 nm wavelength, peak at 365 nm) for 4-5 months. It is known that the lens of the guinea pig possesses unusually high levels of the UVA chromophore NADPH. In a preliminary analysis, it was found that isolated guinea pig corneas transmitted 70-90% of 340-400 nm light, and that UVA radiation was able to penetrate deep into the nucleus of the guinea pig lens, where it was absorbed. Exposure of guinea pigs to UVA in vivo produced a 60% inactivation of lens epithelial catalase; however, analysis by transmission electron microscopy (TEM) showed no apparent morphological effects on either the lens epithelium or the cortex. A number of UVA-induced effects were found in the nucleus of the guinea pig lens, but were observed either not at all or to a lesser extent in the cortex. The effects included an increase in light scattering (two-fold; slit-lamp examination), distention of intercellular spaces (TEM), an increase in lipid peroxidation (30-35%; infrared spectroscopy), a decrease in GSH level (30%), an increase in protein-thiol mixed disulfide levels (80%), loss of water-soluble protein (20%), an increase in the amount of protein disulfide (two-fold; two-dimensional diagonal electrophoresis), degradation of MIP26 (15%) and loss of cytoskeletal proteins including actin, alpha- and beta- tubulin, vimentin and alpha-actinin (60-100%). The results indicate that a 4-5 month exposure of guinea pigs to a biologically relevant level of UVA light produces deleterious effects on the central region of the lenses of the animals. UVA radiation, coupled presumably with the photoreactive UVA chromophore NADPH and trace amounts of O(2) present in the lens nucleus, produced significant levels of oxidized products in the nuclear region over a five month period. The data demonstrate the potentially harmful nature of UVA light with respect to the lens, and highlight the importance of investigating a possible role for this type of radiation in the formation of human cataract.