RESUMO
The effects of CDA-II (cell differentiation agent II; a urinary preparation) on both aflatoxin B(1) (AFB(1))-induced cell injury and DNA damage were investigated using cultured rat hepatocytes. CDA-II was able to suppress both the lipid peroxidation and lactate dehydrogenase leakage induced by AFB(1). Glutathione (GSH) depletion by AFB(1) was replenished by CDA-II treatment. Under these experimental conditions, CDA-II enhanced the activity of GSH peroxidase, but not GSH S-transferase. By evaluation of unscheduled DNA synthesis, CDA-II reduced AFB(1)-induced DNA damage in hepatocyte cultures. These findings suggest that CDA-II can inhibit cytotoxicity of AFB(1) through enhancing the activity of GSH peroxidase and preventing GSH depletion.
Assuntos
Dano ao DNA/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/farmacologia , Fenilacetatos/farmacologia , Substâncias Protetoras/farmacologia , Aflatoxina B1/toxicidade , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Sequestradores de Radicais Livres/administração & dosagem , Hepatócitos/efeitos dos fármacos , Masculino , Peptídeos/administração & dosagem , Fenilacetatos/administração & dosagem , Substâncias Protetoras/administração & dosagem , Ratos , Ratos Sprague-DawleyRESUMO
Oxidative stress causes modification of cellular macromolecules and leads to cell damage. The objective of this study was to identify protein modifications that relate to thiol groups in human red blood cells under oxidative stress. With t-butyl hydroperoxide (t-BH) treatment, results of isoelectric focusing (IEF) analysis showed that two dithiothreitol-reversible modifications are observed, one toward the cathode and the other to the anode. Protein change toward the cathode was demonstrated to be hemoglobin oxidation, which gains a net positive charge, based on the same focus on IEF gels as hemoglobin and methemoglobin and molecular weight analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Otherwise, the change toward the anode was the result of mixed disulfide formation between GSH and protein thiols. Based on the results of molecular weight analysis and its reversion from methemoglobin, protein formed mixed disulfides with GSH were also regarded as hemoglobin. As red blood samples were treated with diamide or GSSG, in addition to the mixed disulfides observed in t-BH-treated cells, additional hemoglobin-GSH mixed disulfide appeared. But the disappearance of this diamide-induced additional mixed disulfide by treating cells with t-BH after diamide treatment suggests that the increase of negative charges from GSH are offset by ferrohemoglobin oxidation to ferrihemoglobin. Additionally, other dithiothreitol-reversible modifications of one cell membrane protein, spectrin, were also observed from the formation of high molecular weight molecules as detected by SDS-PAGE. Results indicate that protein thiols in human red blood cells are susceptible to modification under oxidative stress. IEF analysis provides a useful tool to measure methemoglobin and hemoglobin GSH mixed disulfide formation.
Assuntos
Proteínas Sanguíneas/metabolismo , Eritrócitos/metabolismo , Estresse Oxidativo , Peróxidos/farmacologia , Adulto , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Glutationa/metabolismo , Humanos , Focalização Isoelétrica , Compostos de Sulfidrila/metabolismo , terc-Butil HidroperóxidoRESUMO
To investigate the role of disulfide bonds in the capsid structure, a recombinant JC virus-like particle (VLP) was used. The major capsid protein, VP1, of the JC virus was expressed in yeast cells. The yeast-expressed VP1 was self-assembled into a VLP. Disulfide bonds were found in the VLP which caused dimeric and trimeric VP1 linkages as demonstrated by non-reducing SDS-PAGE. The VLP remained intact when disulfide bonds were reduced by dithiothreitol. The VLP without disulfide bonds could be disassembled into capsomeres by EGTA alone, but those with disulfide bonds could not be disassembled by EGTA. Capsomeres were reassembled into VLPs in the presence of calcium ions. Capsomeres formed irregular aggregations instead of VLPs when treated with diamide to reconstitute the disulfide bonds. These results indicate that disulfide bonds play an important role in maintaining the integrity of the JC VLP by protecting calcium ions from chelation.
Assuntos
Cálcio/metabolismo , Proteínas do Capsídeo , Capsídeo/metabolismo , Quelantes/metabolismo , Dissulfetos/metabolismo , Vírus JC/metabolismo , Capsídeo/química , Quelantes/química , Diamida/química , Diamida/farmacologia , Dissulfetos/química , Ácido Egtázico/química , Ácido Egtázico/metabolismo , Eletroforese em Gel de Poliacrilamida , Vírus JC/química , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Substâncias Redutoras/química , Substâncias Redutoras/farmacologia , Saccharomyces cerevisiae , Transfecção , Vírion/química , Vírion/efeitos dos fármacos , Vírion/metabolismoRESUMO
The effect of dietary lipid on gamma-glutamyl transferase-positive (GGT-positive) foci was investigated. Female Sprague-Dawley rats were dosed with diethylnitrosamine (15 mg/kg) at 24 h of age. After weaning, they were fed nutritionally complete semipurified diets for 3 months. Rats fed 15% corn oil had significantly lower hepatic phospholipid eicosapentaenoate and docosahexaenoate than rats fed 7.5% corn oil plus 7.5% fish oil, 5% corn oil plus 10% fish oil (P < 0.05). However, rats fed 15% corn oil had significantly greater hepatic phospholipid arachidonate than rats fed the other two diets (P < 0.05), suggesting that n-3 polyunsaturated fatty acids were incorporated into hepatic phospholipid at the expense of n-6 polyunsaturated fatty acids. Hepatic PGF2alpha content was significantly greater in rats fed 15% corn oil than in rats fed the other two diets (P < 0.05). Rats fed fish oil had significantly lower hepatic vitamin E content than rats fed corn oil (P < 0.05). Hepatic lipid peroxidation (TBARS) tended to increase with increased dietary fish oil (P < 0.05). Dietary lipid did not influence GGT-positive foci area or number. In conclusion, dietary lipid affected hepatic PGF2alpha production, however, showed no effect on GGT-positive foci area and number. This may suggest that PGF2alpha is not the underlying mechanism for GGT-positive foci during hepatocarcinogenesis.
Assuntos
Gorduras Insaturadas na Dieta/farmacologia , Dinoprosta/análise , Neoplasias Hepáticas Experimentais/etiologia , Fígado/química , gama-Glutamiltransferase/metabolismo , Animais , Ácido Araquidônico/análise , Óleo de Milho/farmacologia , Dietilnitrosamina/administração & dosagem , Ácidos Docosa-Hexaenoicos/análise , Ácido Eicosapentaenoico/análise , Feminino , Óleos de Peixe/farmacologia , Glutationa Transferase/análise , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/enzimologia , Neoplasias Hepáticas Experimentais/química , Neoplasias Hepáticas Experimentais/enzimologia , Ácido Oleico/análise , Palmitatos/análise , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Estearatos/análise , Vitamina E/análiseRESUMO
Whether the alterations in the synthesis of thromboxane A2 (TXA2) is the direct mechanism underlying the blood pressure-lowering effect of fish oil was investigated in this study. Six groups of 11 male spontaneously hypertensive rats were fed semipurified diets containing corn or fish oils and graded levels (50, 5000 or 15,000 ppm) of dietary vitamin E for 8 weeks. Plasma TXA2, assayed by RIA, was significantly greater in the corn oil group than in the fish oil group (P < 0.05). Compared to 50 ppm dietary vitamin E, 5000 and 15 000 ppm dietary vitamin E, respectively, significantly decreased plasma TXA2 (P < 0.05). Systolic, mean or diastolic blood pressure, evaluated by the tail cuff method, were significantly higher in the corn oil group than in the fish oil group (P < 0.05). However, vitamin E had no effect on blood pressure. No relationship between TXA2 and blood pressure was found. Experimental results indicated that the alterations in the synthesis of TXA2 were not the direct antihypertensive effect of fish oil.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Óleos de Peixe/farmacologia , Hipertensão/sangue , Tromboxano A2/sangue , Animais , Óleo de Milho/farmacologia , Dieta , Modelos Animais de Doenças , Ácidos Graxos/sangue , Ácidos Graxos/química , Ácidos Graxos Insaturados/farmacologia , Hipertensão/fisiopatologia , Peroxidação de Lipídeos , Masculino , Peróxidos/farmacologia , Ratos , Ratos Endogâmicos , Vitamina E/farmacologia , terc-Butil HidroperóxidoRESUMO
Weanling male spontaneously hypertensive rats were fed semipurified diets containing either corn or fish oil for 8 weeks. Rats fed on fish oil diet had significantly lower plasma triglyceride, total cholesterol and HDL-cholesterol levels than rats fed on corn oil diet (P < 0.05). Moreover, rats fed on fish oil diet had significantly lower liver total lipid and triglyceride concentrations than rats fed on corn oil diet (P < 0.05). Dietary lipids were reflected in plasma fatty acid composition. Rats fed on fish oil diet had significantly greater plasma eicosapentaenoate (EPA) and docosahexaenoate (DHA) (n-3 PUFAs) with an accompanying decrease in plasma linoleate (LA) and arachidonate (AA) (n-6 PUFAs), in comparison with the rats fed corn oil (P < 0.05). Those results would suggest that the n-3 PUFAs were incorporated into plasma lipids at the expense of the n-6 PUFAs. Rats fed on corn oil diet had significantly greater liver DNA content than rats fed on fish oil diet (P < 0.05), thereby implying that the n-3 PUFAs in fish oil had an inhibitory effect on liver cell proliferation. Furthermore, rats fed on fish oil diet had significantly greater hepatic microsomal protein content than rats fed on corn oil diet (P < 0.05), indicating that fish oil exerted a stimulatory effect on hepatic microsomal enzymes.
Assuntos
Lipídeos/sangue , Lipídeos/farmacologia , Fígado/citologia , Microssomos Hepáticos/metabolismo , Proteínas/metabolismo , Animais , Colesterol/sangue , Óleo de Milho/farmacologia , DNA/análise , Ácidos Graxos/sangue , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6 , Ácidos Graxos Insaturados/farmacologia , Óleos de Peixe/farmacologia , Alimentos Fortificados , Metabolismo dos Lipídeos , Lipoproteínas HDL/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Proteínas/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Triglicerídeos/sangue , Aumento de Peso/efeitos dos fármacosRESUMO
A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.
Assuntos
Cromatografia de Afinidade/métodos , Glutationa Transferase/isolamento & purificação , Schistosoma japonicum/enzimologia , Animais , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/genética , Focalização Isoelétrica , Metais , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
OBJECTIVE: In this work, we have measured the plasma vitamins A and E and red blood cell fatty acid profile in newborns and their mothers and have determined whether there are any relationships between maternal blood and cord blood for the nutrients measured. SETTING: The study was performed at the Chung Shan Memorial Hospital, Taichung, Taiwan. SUBJECTS: Twenty-nine pairs of mothers and their term infants. INTERVENTIONS: Maternal venous blood was collected in the first trimester and at delivery, and cord blood was collected at delivery. Plasma vitamin A and E levels were determined by high performance liquid chromatography and red blood cell fatty acid profile was estimated by gas chromatography. RESULTS: Mothers had significantly greater plasma vitamin A and E levels and vitamin E/total lipid than their term neonates did (P < 0.05). Maternal plasma vitamin E and vitamin E/total lipid were significantly greater in the first trimester than at delivery (P < 0.05). Red blood cell phospholipid oleate and linoleate were significantly greater in maternal red blood cell than in cord blood (P < 0.05), however, stearate and arachidonate were significantly greater in the cord blood than in the maternal blood (P < 0.05). Maternal vitamin E, vitamin E/total lipid, palmitate, linoleate, arachidonate and docosahexaenoate were found positively correlated to those in their neonates (P < 0.05). CONCLUSIONS: The results suggest that there is a relationship between maternal blood and cord blood for some nutrients. Therefore, the nutritional status of mothers may affect the nutritional outcome of their neonates.
Assuntos
Eritrócitos/química , Ácidos Graxos/análise , Sangue Fetal/química , Vitamina A/sangue , Vitamina E/sangue , Adulto , Feminino , Sangue Fetal/citologia , Idade Gestacional , Humanos , Recém-Nascido , Masculino , Troca Materno-Fetal , Cooperação do Paciente , Gravidez , Primeiro Trimestre da Gravidez/sangue , Estudos ProspectivosRESUMO
Plasma alpha-tocopherol, ascorbate, retinol, uric acid, and lipid peroxides were investigated in 39 male smokers and 64 male non-smokers. The average level of plasma alpha-tocopherol of 35-45-year-old smokers (1.74+/-0.49 microg/mg total lipid) was significantly lower than that of age-matched non-smokers (2.55+/-0.88 microg/mg total lipid, P = 0.032). Similarly, the plasma alpha-tocopherol of smokers aged above 45 (1.66+/-0.29 microg/mg total lipid) was lower than that of the age-matched non-smokers (2.38+/-1.26 microg/mg total lipid, P = 0.014). However, no difference in plasma level of alpha-tocopherol was found between smokers and non-smokers below the age of 35. The average concentration of ascorbate in plasma was significantly decreased only in those smokers older than 45 (0.33+/-0.16 mg/dl vs non-smokers 0.53+/-0.19 mg/dl, P = 0.003). The average level of lipid peroxides (measured as malondialdehyde,MDA) in the plasma of smokers (2.77+/-0.51 nmol/ml) was higher than that of non-smokers (2.35+/-0.21 nmol/ml) aged above 45 (P = 0.034). No differences in the plasma levels of uric acid and retinol were noted between smokers and non-smokers in all age groups. Using partial correlation analysis under age control, we found that the plasma level of alpha-tocopherol was negatively correlated with the plasma level of MDA (r = -0.523, P = 0.038). In contrast, the plasma level of ascorbate was only weakly correlated with the plasma level of MDA (r = -0.341, P = 0.094). Moreover, we found a negative correlation between the plasma level of alpha-tocopherol and smoking index (r = -0.414, P = 0.006) under age control, but there was no correlation between plasma level of ascorbate and smoking index (r=0.221, P = 0.193). These results indicate that adequate levels of alpha-tocopherol and ascorbate may protect the plasma from oxidative damage elicited by smoking-mediated reactive oxygen species (ROS) and free radicals in young smokers. However, the antioxidant activities of alpha-tocopherol and ascorbate may be overwhelmed by the long-standing oxidative stress elicited by cigarette smoking in elderly subjects.
Assuntos
Antioxidantes/metabolismo , Fumar/sangue , Adulto , Animais , Ácido Ascórbico/sangue , Humanos , Peroxidação de Lipídeos , Masculino , Pessoa de Meia-Idade , Peso Molecular , Estresse Oxidativo/fisiologia , Ratos , Fumar/efeitos adversos , Ácido Úrico/sangue , Vitamina A/sangue , Vitamina E/sangueRESUMO
A total of 59 healthy male subjects (32 smokers and 27 nonsmokers) who had no reported systemic disease and did not take alcohol and vitamin supplementation were included. The levels of autoantibody to oxidized low-density lipoproteins (ox-LDL) in smokers and age-matched nonsmokers were compared. The plasma levels of antioxidants that can affect the formation of ox-LDL were also measured, and correlation analyses between anti ox-LDL IgG and plasma antioxidants, controlling for age and body mass index (BMI), were performed. Plasma alpha-tocopherol and uric acid concentrations of nonsmokers (2.78+/-1.09 microg/mg total lipid and 6.96+/-1.69 mg/dl, respectively) were significantly higher than those of smokers (1.68+/-0.48 microg/mg total lipid and 6.15+/-1.14 mg/dl, respectively) (P<0.05). Although plasma ascorbate and retinol levels were not significantly different between smokers and nonsmokers, smokers older than 45 years old had significantly lower plasma ascorbate levels (0.32+/-0.17 mg/dl) than age-matched nonsmokers (0. 53+/-0.14 mg/dl) (P=0.036). Higher level of plasma anti ox-LDL IgG was noted in the group of smokers compared with nonsmokers (515+/-409 mU/ml vs. 407+/-268 mU/ml, respectively) under the statistic method of Chi-Square test (P=0.049). A significant negative correlation was found between plasma anti ox-LDL IgG and alpha-tocopherol in the combined population as well as in the smoker group (r=-0.26, p=0.047; r=-0.48, p=0.006; respectively). However, there was no correlation between plasma anti ox-LDL IgG and the levels of other antioxidants. These results suggest that reduced concentrations of alpha-tocopherol are associated with cigarette smoking. The significantly negative correlation between plasma anti ox-LDL IgG and alpha-tocopherol in the entire study population as well as in the smoker group suggests that plasma alpha-tocopherol may be partially effective if not totally at protecting LDL from oxidative damage caused by cigarette smoking and dietary supplementation with alpha-tocopherol may provide a protective effect against LDL oxidation, especially in smokers.
Assuntos
Autoanticorpos/sangue , Lipoproteínas LDL/imunologia , Fumar/imunologia , Adulto , Antioxidantes/metabolismo , Ácido Ascórbico/sangue , Autoanticorpos/biossíntese , Índice de Massa Corporal , Dieta , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Masculino , Fumar/sangue , Ácido Úrico/sangue , Vitamina A/sangue , Vitamina E/sangueRESUMO
Protein S-glutathionation has been demonstrated to be one of the cellular responses under oxidative stress and may be involved in many cellular metabolisms. In this study, the effect of redox cycling bipyridylium compounds, paraquat and diquat, on this protein modification was investigated. Male Sprague-Dawley rats were administered i.p. either paraquat at 20 or 40 mg/kg body wt. or diquat at 85 or 170 mg/kg body wt., respectively. The liver was examined at different time points for taking the measurement of the S-glutathionation of carbonic anhydrase III (CA III), thiobarbituric acid-reactive substances (TBARS), vitamin E depletion, glutathione (GSH) and glutathione disulfide (GSSG) contents. The extent of S-glutathionation of CA III was chosen as a marker and was determined by a method combining isoelectric focusing analysis with immunoblotting. Those results indicated that paraquat and diquat significantly increased the generation of TBARS and showed a time-dependent response. The significant effect on vitamin E depletion was only obtained in rats treated with a high dose of diquat for 2 h. Hepatic cellular GSSG contents did not increase but tended to decrease all of the treatments. Although oxidative damage was actually generated in liver, based on the increase of TBARS generation and vitamin E depletion, no increase of CA III S-glutathionation was observed. We propose that the reason for this observation under this circumstance is probably due to the reversible characteristic of CA III S-glutathionation, which has been demonstrated in our previous study (Chai et al., 1991) Arch. Biochem. Biophys. 384, 270-278) and named as dethiolation.
Assuntos
Anidrases Carbônicas/metabolismo , Diquat/toxicidade , Glutationa/metabolismo , Fígado/efeitos dos fármacos , Paraquat/toxicidade , Animais , Fígado/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Vitamina E/metabolismoRESUMO
The objective of this study was to investigate the effect of the active principles in garlic-- diallyl sulfide (DAS) and diallyl disulfide (DADS)--on aflatoxin B(1) (AFB(1))-induced DNA damage in primary rat hepatocytes. Primary rat hepatocytes, induced with DNA damage using 10 microM AFB(1) were used as an experimental model. According to the results of LDH leakage, 0.5 and 2 mM of DAS or 0.5 and 1 mM of DADS significantly increased the viability of hepatocytes compared with the AFB(1) controls after 4, 8 and 24 h treatment (P<0.05). According to the results of unscheduled DNA synthesis (UDS) test, 0.5 and 2 mM of DAS or 0.5 and 1 mM of DADS could significantly decrease the DNA damage induced by AFB(1) (P<0.05). Furthermore, 0.5 and 2 mM DAS or 0.5 and 1 mM DADS could increase the glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities as compared with the AFB(1) controls after 24 h treatment (P<0.05). Results of immunoblot analysis of cytosolic GST isoenzyme indicate that the levels of GST isoform Ya, Yb2 and Yc were markedly increased after treatment with 0.5 and 2 mM DAS or 0.5 and 1 mM DADS compared with the AFB(1) control. These results indicate that 0.5 and 2 mM DAS or 0.5 and 1 mM DADS might protect hepatocytes from AFB(1)-induced DNA damage via increasing the activities of GST and GPx.
Assuntos
Compostos Alílicos/farmacologia , Dano ao DNA , Dissulfetos/farmacologia , Hepatócitos/efeitos dos fármacos , Sulfetos/farmacologia , Aflatoxina B1/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , DNA/efeitos dos fármacos , DNA/genética , DNA/metabolismo , Relação Dose-Resposta a Droga , Alho/química , Glutationa Transferase/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , L-Lactato Desidrogenase/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Plantas Medicinais , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Effects of vitamin E on cell morphology and cellular protein thiols under oxidative stress was investigated in cultured rat hepatocytes with different vitamin E status. Hepatocytes were incubated in the presence or absence of 100 microM alpha-tocopherol succinate for 24 h then treated with 1.5 mM t-butyl hydroperoxide (t-BH) for different time intervals. Lipid peroxidation, as determined by thiobarbituric acid-reactive substances, was completely inhibited over 60 min of treatment in cells incubated with alpha-tocopherol. The change of cell morphology, as determined by surface blebs formation, was correlated with cellular vitamin E status. Surface blebs were formed in 25.1 +/- 5.2 min in the presence of alpha-tocopherol in contrast to 11.1 +/- 2.9 min in its absence. In cells with alpha-tocopherol, surface blebs were induced even though lipid peroxidation was inhibited. Comparing the depletion of membrane protein thiols with t-BH treatment, twice as many (40%) thiols were lost over 60 min in the absence of alpha-tocopherol whereas 20% were lost in the presence of alpha-tocopherol. In addition, the extent of thiol modification of carbonic anhydrase III, as determined by combining isoelectric focusing analysis with immunoblotting, further demonstrated that alpha-tocopherol helps maintain protein thiols in the reduced state. Results indicate that vitamin E protects cell morphology and prevents the loss of protein thiols with t-BH treatment, and on cell morphology protection is associated with protein thiols rather than membrane lipids.
Assuntos
Membrana Celular/efeitos dos fármacos , Fígado/efeitos dos fármacos , Compostos de Sulfidrila , Vitamina E/farmacologia , Animais , Anidrases Carbônicas/química , Membrana Celular/patologia , Células Cultivadas , Immunoblotting/métodos , Focalização Isoelétrica/métodos , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/citologia , Masculino , Estresse Oxidativo , Ratos , Ratos Sprague-DawleyRESUMO
This study examined the effect of vitamin E on maintaining the protein reactive thiols under oxidative stress. Hepatocytes were prepared from male Sprague-Dawley rats fed diets containing three levels of vitamin E (0,100 and 15,000 mg/kg) for 12 wk. Cells were isolated by collagenase perfusion and treated with 0.5 Him tert-butyl hydroperoxide (t-BuOOH) after 24 hr in culture. Carbonic anhydrase III (CA III) having two reactive thiols that can react with GSH under oxidative stress was chosen as the study subject. CA III S-glutathionation was measured by isoelectric focusing/immunoblotting. Results indicated that thiol modification of CA III was induced by t-BuOOH and the pattern of modification was dependent on the vitamin E status. With t-BuOOH treatment, CA III S-glutathionation was quickly induced and the maximum modification was achieved at 3 min in cells isolated from rats fed high levels of vitamin E; however, modification was continuously increased and reached the maximum at 9 min of vitamin E-normal or -deficient cells. Following the maximum modification, a reversion occurred (dethiolation); the rate of reversion was also related to vitamin E status. As shown by image analysis, twofold more (40 v. 20%) CA III was modified in vitamin E-deficient hepatocytes than in cells from rats fed high vitamin E. Glutathione was also abruptly converted to the oxidized state at 3 min in all cells, then gradually reverted to the reduced state. As with the dethiolation of CA III, the rate of glutathione disulfide reduction was correlated to vitamin E status. The production of thiobarbituric acid-reactive substances corresponded to vitamin E status as well and was significantly inhibited in cells from rats fed high vitamin E. These results suggest that vitamin E not only inhibits lipid peroxidation but also plays a role in maintaining the protein thiols under oxidative stress.
RESUMO
Numerous root canals filling materials are available in the field of dentistry, based on various formulas that contain a variety of different and partly mutagenic components, such as epoxy resin sealers, Ca(OH)2-based materials, and zinc oxide-eugenol cements. AH Plus root canal sealer will not release formaldehyde according to the manufacturer, although AH26 does. The purpose of this study was to analyze the leakage of lactate dehydrogenase (LDH) from rat hepatocytes after treatment with AH26 and AH Plus root canal sealers in vitro. Hepatocytes from male Sprague-Dawley rats were used to test the cytotoxicity of AH26 and AH Plus. The root canal sealers were mixed and then dissolved in the dimethyl sulfoxide to final concentrations of 0.01%, 0.04%, and 0.1% (wt/vol), with a dimethyl sulfoxide concentration of < 0.05%. Dosage-dependent and time-dependent lactate dehydrogenase leakage values were measured and tested by one-way ANOVA. The results showed that both AH26 and AH Plus are toxic to rat hepatocytes. At a low concentration, AH26 had a higher toxicity than AH Plus to rat hepatocytes.
Assuntos
Bismuto/toxicidade , Resinas Epóxi/toxicidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Metenamina/toxicidade , Materiais Restauradores do Canal Radicular/toxicidade , Prata/toxicidade , Titânio/toxicidade , Análise de Variância , Animais , Morte Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Relação Dose-Resposta a Droga , Combinação de Medicamentos , L-Lactato Desidrogenase/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Testes de ToxicidadeRESUMO
The effects of fish oil and corn oil diets on diethylnitrosamine initiation/phenobarbital promotion of hepatic enzyme-altered foci in female Sprague-Dawley rats were investigated. Groups of 12 rats were initiated with diethylnitrosamine (15 mg/kg) at 24 h of age. After weaning, they received diets containing either 13.5% fish oil plus 1. 5% corn oil or 15% corn oil for 24 weeks. Rats fed fish oil had significantly greater liver weight, relative liver weight, spleen weight, and relative spleen weight than rats fed corn oil (p < 0.05). Hepatic phospholipid fatty-acid profile was significantly affected by the type of dietary lipid. The rats fed fish oil had significantly greater hepatic phospholipid 20:5 and 22:6 than rats fed corn oil; in contrast, the rats fed corn oil had significantly greater hepatic phospholipid 18:2 and 20:4 than rats fed fish oil (p < 0.05). Rats fed fish oil had significantly lower hepatic vitamin E and PGE(2) content but significantly greater hepatic lipid peroxidation than rats fed corn oil (p < 0.05). The hepatic levels of antioxidant enzymes (GSH reductase and GST) were significantly greater in rats fed fish oil than in rats fed corn oil (p < 0.05). Except for PGST-positive foci (foci area/tissue area), all the other foci parameters (GGT-positive foci area/tissue area, GGT-positive foci no./cm(2), GGT-positive foci no./cm(3), PGST-positive foci no. /cm(2), and PGST-positive foci no./cm(3)) measured in the fish oil group were 10-30% of those in the corn oil group (p < 0.05). Analyses of Pearson correlation coefficient revealed a positive correlation between hepatic GGT- or PGST-positive foci number (no. /cm(2)) and PGE(2) content (r = 0.66, P = 0.01; r = 0.56, P = 0.02, respectively) but a negative correlation between GGT- and PGST-positive foci (no./cm(2)) and lipid peroxidation (r = -0.8, P = 0.0006; r = -0.58, P = 0.01, respectively), GSH/(GSH + GSSG) ratio (r = -0.61, P = 0.05; r = -0.4, P = 0.14, respectively), GSH reductase (r = -0.75, P = 0.002; r = -0.53, P = 0.02, respectively), and GST activities (r = -0.65, P = 0.01; r = -0.44, P = 0.07, respectively). Similar correlation between foci number (no./cm(3)) and PGE(2), lipid peroxidation, GSH/(GSH + GSSG) ratio, GSH reductase, and GST activities were obtained. The results of this study show that dietary fish oil significantly inhibited hepatic enzyme-altered foci formation compared with corn oil in rats. These results suggest that the possible mechanisms involved in this process are the stimulation of hepatic detoxification system, changes in membrane composition, inhibition of PGE(2) synthesis, the enhancement of GSH-related antioxidant capacity, and the enhancement of lipid peroxidation by fish oil.
Assuntos
Óleo de Milho/farmacologia , Óleos de Peixe/farmacologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Fígado/efeitos dos fármacos , Animais , Feminino , Glutationa Transferase/metabolismo , Fígado/enzimologia , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/patologia , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/patologia , Gravidez , Ratos , Ratos Sprague-Dawley , gama-Glutamiltransferase/metabolismoRESUMO
Multiple bleb formation on cell membrane is common during cell death. The effects of alpha-tocopherol and glutathione (GSH) on tert-butyl hydroperoxide (TBH)-induced membrane changes in rat hepatocytes were studied. Over 60 min of exposure, TBH (0.5-2.0 mM) caused a dose-dependent membrane blebbing. Cells pretreated with buthionine sulfoximine, a GSH synthesis inhibitor, had significantly greater blebbing and lactate dehydrogenase (LDH) leakage under 0.5 mM TBH treatment as compared to cells without pretreatment. However, the protective effect of GSH disappeared when the TBH concentration was increased to 2.0 mM. In the presence of alpha-tocopheryl succinate (TS) pretreatment, it was noted that bleb formation, expressed as the percentage of cells bearing blebs, the average bleb size, or the onset of blebbing, was partially suppressed even when TBH concentration was 2.0 mM. TBH-induced thiobarbituric acid reactive substances and LDH leakage were completely abolished by TS pretreatment. Accompanying bleb formation, membrane-insoluble actin was noted to decrease by immunoblot assay. The decrease in actin was also suppressed by TS. These results indicated that intracellular GSH and alpha-tocopherol status are important to the TBH-induced cell membrane abnormality. Furthermore, TS plays a defensive role against blebbing when GSH is exhausted by TBH.
Assuntos
Glutationa/farmacologia , Hepatócitos/ultraestrutura , Vitamina E/farmacologia , terc-Butil Hidroperóxido/toxicidade , Actinas/análise , Animais , Butionina Sulfoximina/farmacologia , Membrana Celular/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Glutationa/análise , Dissulfeto de Glutationa/análise , Hepatócitos/efeitos dos fármacos , Processamento de Imagem Assistida por Computador , Immunoblotting , L-Lactato Desidrogenase/análise , L-Lactato Desidrogenase/metabolismo , Microscopia de Contraste de Fase , Ratos , Ratos Sprague-Dawley , Espectrometria de Fluorescência , Substâncias Reativas com Ácido Tiobarbitúrico/análiseRESUMO
In this genotoxic study, the Ames Salmonella microsome test showed that an aqueous extract of betel quid did not induce mutagenicity in Salmonella typhimurium strains TA98 and TA100. Mammalian cell studies (Chinese hamster ovary K1 cell; CHO-K1 cell) revealed that only higher concentrations (100 and 1000 microg/ml) of aqueous extract weekly increased the frequencies of sister-chromatid exchange (SCE) in the absence of S9. Animal (male Sprague-Dawley rat) studies showed that low-dose feeding (0.53 g dry aqueous extract/kg diet) significantly increased the activities of glutathione (GSH) peroxidase and cytoplasmic glutathione S-transferase (cGST) of liver, high-dose feeding (26.5 g dry aqueous extract/kg diet) lowered the contents of GSH and total glutathione. The effect of an aqueous extract of betel quid on the oxidation of 2'-deoxyguanosine (2'-dG) to 8-hydroxy-2'-deoxyguanosine (8-OH-dG) evaluated that this aqueous extract may act as a pro-oxidant at lower dosage and may be dependent on the iron ions in the model system. However, the aqueous extract of betel quid showed antioxidant activity at higher doses by the ability of the scavenging effect of the hydroxyl radicals.
Assuntos
Alcaloides/química , Alcaloides/toxicidade , Areca/química , Areca/toxicidade , Plantas Medicinais , Animais , Células CHO/efeitos dos fármacos , Cricetinae , Feminino , Glutationa Peroxidase/biossíntese , Glutationa Transferase/biossíntese , Masculino , Microssomos/efeitos dos fármacos , Testes de Mutagenicidade , Extratos Vegetais/química , Extratos Vegetais/toxicidade , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Salmonella typhimurium/genética , Troca de Cromátide Irmã , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The modulation of garlic oil (GO) and three allyl compounds, diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS), on the antioxidation system in rat livers and red blood cells was examined. Rats were orally administered GO (200 mg/kg body weight), DAS (20, 80 mg/kg body weight), DADS (80 mg/kg body weight) or DATS (70 mg/kg body weight) three times a week for 6 weeks. Control rats received corn oil (2 ml/kg body weight) alone. GO, DADS and DATS treatment significantly increased the glutathione (GSH) content (48-84%) in red blood cells (P < 0.05). DATS displayed a greater enhancement than GO and DADS (P < 0.05). Hemolysis induced by tert-butyl hydroperoxide was not suppressed by GO or allyl compound treatment although higher GSH content was evident. Hepatic GSH was not influenced by garlic components. In rat livers, DADS and DATS significantly increased the activity of GSH reductase (46 and 54%, respectively) and of GSH S-transferase (GST) (63 and 103%, respectively), but decreased the GSH peroxidase activity (27 and 28%, respectively). In contrast, GSH reductase and GST activities in the DAS group, either 20 or 80 mg/kg body weight, were similar to the control group. A decrease of GSH peroxidase activity was observed in rats dosed with 80 mg/kg body weight (P < 0.05). An increase in GST activity and a decrease in GSH peroxidase activities were also noted in GO-treated rats (P < 0.05). In red blood cells, three GSH-related antioxidant enzyme activities were not affected by garlic oil and its organosulfur components. Immunoblot assay showed that, accompanying the increase in hepatic GST activity, GO, DADS, DAS (80 mg/kg body weight) and DATS increased the expression of GST Ya, Yb1 and Yc proteins. Results indicate that GO and three allyl compounds play a differential role in modulation of the GSH-related antioxidant system in rat livers and red blood cells.
Assuntos
Compostos Alílicos/farmacologia , Eritrócitos/enzimologia , Fígado/enzimologia , Sulfetos/farmacologia , Compostos Alílicos/administração & dosagem , Animais , Anticarcinógenos/farmacologia , Antioxidantes , Western Blotting , Peso Corporal/efeitos dos fármacos , Dissulfetos/farmacologia , Eletroforese em Gel de Poliacrilamida , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Glutationa Redutase/metabolismo , Glutationa Transferase/metabolismo , Hemólise , Peroxidação de Lipídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Oxirredução , Ratos , Ratos Sprague-Dawley , Sulfetos/administração & dosagem , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The objectives of this study were to investigate the effects of various concentrations and incubation time intervals of diallyl sulfide (DAS), an active principle of garlic, on cell viability, and glutathione (GSH) concentration and its related enzymes activities in rat hepatocytes. According to the results of lactate dehydrogenase (LDH) leakage and microscopic examination, 0.5 or 1 mM DAS treatment did not have any adverse effects on the viability of hepatocytes. Intracellular GSH contents of cells treated with 0.5 and 1 mM DAS (58.6 and 66.4 nmol GSH/mg protein, respectively) were higher than in the controls (54.2 nmol GSH/mg protein), around 8-23%, at 24 hr of incubation; a significant difference (P < 0.05) was observed for 1 mM DAS treatment at 48 hr. This phenomenon is beneficial to the detoxification and antioxidation capabilities of hepatocytes. Further, when the hepatocytes were treated with 0.5 or 1 mM DAS, the activities of glutathione S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GRd) were almost the same as those of the controls. On the other hand, treatment with 5 mM DAS was associated with a significant decrease (P < 0.05) in cell viability, namely in increased LDH leakage (50% at 24-hr treatment), significant changes in the morphology of the hepatocytes, low intracellular GSH level (45% lower than in the controls at 24-hr treatment), and low activities of GST, GPx and Grd.