Endogenous defenses against the cytotoxicity of hydrogen peroxide in cultured rat hepatocytes.
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The catalase activity of cultured rat hepatocytes was inhibited by 90% pretreatment with 20 mM aminotriazole without effect on the activities of glutathione peroxidase or glutathione reductase, or on the viability of the cells over the subsequent 24 h. Glutathione reductase was inhibited by 85% by pretreatment with 300 microM 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) without effect on glutathione peroxidase, catalase, or on viability. Both pretreatments sensitized the hepatocytes to the cytotoxicity of H2O2 generated either by glucose oxidase (0.05-0.5 units/ml) or by the autoxidation of the one-electron-reduced state of menadione (50-250 microM). Aminotriazole pretreatment had no effect on the GSH content of the hepatocytes. BCNU reduced GSH levels by 50%. Depletion of GSH levels to less than 20% of control by treatment with diethyl maleate, however, did not sensitize the cells to either glucose oxidase or menadione, indicating that the effect of BCNU is related to inhibition of the GSH-GSSG redox cycle rather than to the depletion of GSH. With glucose oxidase, most of the cell killing in hepatocytes pretreated with either aminotriazole or BCNU occurred between 1 and 3 h. The antioxidant diphenylphenylenediamine (DPPD) had no effect on viability at 3 h. Catalase added to the culture medium 1 h after the addition of glucose oxidase prevented the cell killing measured at 3 h. The sulfhydryl reagents dithiothreitol (200 microM), N-acetyl-L-cysteine (4 mM), and alpha-mercaptopropionyl-L-glycine (2.5 mM) prevented the cell killing with exogenous H2O2 in hepatocytes sensitized by the inhibition of catalase or glutathione reductase. With menadione, there was no killing of nonpretreated hepatocytes at 1 h, and DPPD did not prevent the cell death after 3 h. Aminotriazole pretreatment enhanced the cell killing at 3 h but not at 1 h, and DPPD was not protective. Catalase added to the medium at 1 h inhibited the cell death measured at 3 h. In contrast, menadione killed hepatocytes pretreated with BCNU within 1 h. DPPD prevented cell death at 1 h, and there was evidence of lipid peroxidation in the accumulation of malondialdehyde in the culture medium. Catalase added with menadione did not prevent the cell killing at 1 h but did prevent it at 3 h. These data indicate that catalase and the GSH-GSSG cycle are active in the defense of hepatocytes against the toxicity of H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)Keywords:
Glutathione reductase
Buthionine sulfoximine
Menadione
Glucose oxidase
Dithiothreitol
Viability assay
Dithiothreitol
Acetaminophen
Thiol
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The intracellular superoxide and glutathione disulphide concentrations increased in Penicillium chrysogeum treated with 50,250 or 500 μM menadione (MQ). A significant increase in the intracellular peroxide concentration was also observed when mycelia were exposed to 250 or 500 μM MQ. The specific activity of Cu,Zn and Mn superoxide dismutases, glutathione reductase and glutathione S-transferase as well as the glutathione producing activity increased in the presence of MQ while glutathione peroxidase and γ-glutamyltranspeptidase were only induced by high intracellular peroxide levels. The glucose-6-phosphate dehydrogenase and catalase activities did not respond to the oxidative stress caused by MQ.
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Because cell-mediated reduction of menadione leads to the generation of reactive oxygen species (ROS), this quinone is widely used to investigate the effects of ROS on cellular functions. We report that A549 human lung epithelial cells exposed to menadione demonstrate a dose-dependent increase in both intracellular calcium ([Ca2+]i) and ROS formation. The concentrations of menadione required to initiate these two events are markedly different, with ROS detection requiring higher levels of menadione. Modulators of antioxidant defences (e.g. buthionine sulphoximine, 3-amino-1,2,4-triazole) have little effect on the [Ca2+]i response to menadione, suggesting that ROS formation does not account for menadione-dependent alterations in [Ca2+]i. Additional evidence suggests that menadione photochemistry may be responsible for the observed [Ca2+]i effects. Specifically: (a) EPR studies with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) show that light exposure (maximum effect at 340nm) stimulates menadione-dependent formation of the DMPO/•OH spin adduct that was not sensitive to antioxidant interventions; (b) DMPO inhibits menadione and light-dependent increases in [Ca2+]i; and (c) light (maximum effect at 340nm) augments the deleterious effects of menadione on cell viability as determined by 51Cr release. These photo effects do not appear to involve formation of singlet oxygen by menadione, but rather are the result of the oxidizing chemistry initiated by menadione in the triplet state. This work demonstrates that menadione species generated by photo-irradiation can exert biological effects on cellular functions and points to the potential importance of photochemistry in studies of menadione-mediated cell damage.
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Dithiothreitol
Buthionine sulfoximine
Thiol
Lymnaea stagnalis
Proteostasis
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Sodium bisulfite
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Abstract A spectrophotometric method is described for the determination of menadione and menadione sodium bisulfite in bulk and in solution for injection. The method measures the intensity of the violet color (λmax 540 nm) developed when menadione reacts with thiosemicarbazide in alkaline medium. Beer's law is obeyed in the concentration range 4-40 μg/m L (r = 0.9995). The method is simple, sensitive, and particularly suited for routine analysis of official menadione sodium bisulfite injection. Results are comparable with the USP method.
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Abstract Hepatocytes isolated from phenobarbital‐pretreated and naive male Sprague‐Dawley rats were preincubated with 80 μM N, N ‐bis (2‐chloroethyl)‐ N ‐nitrosourea and subsequently exposed to varying concentrations of menadione. We observed that the reduced glutathione levels of the hepatocytes isolated from the sodium phenobarbital(PB)‐pretreated, but not the naive rats, recovered to near‐control levels after exposure to 200 μM menadione. Since this recovery occurred in the presence of N, N ‐bis (2‐chloroethyl)‐ N ‐nitrosourea (an inhibitor of glutathione reductase), we hypothesized that this represented a PB‐mediated increase in de novo synthesis of glutathione. To test this hypothesis and to further assess the possible contribution of glutathione reductase in the recovery of the glutathione levels, we preincubated hepatocytes isolated from PB‐pretreated and naive rats with 2 mM buthionine sulfoximine, with or without N, N ‐bis (2‐chloroethyl)‐ N ‐nitrosourea. Following exposure to menadione, samples were periodically removed for glutathione assessment. Consistent with our hypothesis, the addition of buthionine sulfoximine abrogated the ability of the PB‐pretreated hepatocytes to restore glutathione levels following a menadione challenge. Buthionine sulfoximine in combination with N, N ‐bis (2‐chloroethyl)‐ N ‐nitrosourea completely abolished hepatocellular glutathione homeostasis for all of the concentrations of menadione employed. The findings from this investigation underscore the importance of phenobarbital‐mediated increases in glutathione synthesis, as well as the enhanced levels of glutathione reductase, in maintaining the pool of reduced glutathione and ultimately mitigating the consequences of oxidative stress. In addition, these findings suggest that PB pretreatment increases the reserve capacity of the hepatocyte for glutathione synthesis via a hitherto undescribed hormetic mechanism, a reserve expressed fully only on an oxidative stress of sufficient magnitude.
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Glutathione reductase
Phenobarbital
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Because cell-mediated reduction of menadione leads to the generation of reactive oxygen species (ROS), this quinone is widely used to investigate the effects of ROS on cellular functions. We report that A549 human lung epithelial cells exposed to menadione demonstrate a dose-dependent increase in both intracellular calcium ([Ca(2+)](i)) and ROS formation. The concentrations of menadione required to initiate these two events are markedly different, with ROS detection requiring higher levels of menadione. Modulators of antioxidant defences (e.g. buthionine sulphoximine, 3-amino-1,2,4-triazole) have little effect on the [Ca(2+)](i) response to menadione, suggesting that ROS formation does not account for menadione-dependent alterations in [Ca(2+)](i). Additional evidence suggests that menadione photochemistry may be responsible for the observed [Ca(2+)](i) effects. Specifically: (a) EPR studies with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) show that light exposure (maximum effect at 340 nm) stimulates menadione-dependent formation of the DMPO/(.)OH spin adduct that was not sensitive to antioxidant interventions; (b) DMPO inhibits menadione and light-dependent increases in [Ca(2+)](i); and (c) light (maximum effect at 340 nm) augments the deleterious effects of menadione on cell viability as determined by (51)Cr release. These photo effects do not appear to involve formation of singlet oxygen by menadione, but rather are the result of the oxidizing chemistry initiated by menadione in the triplet state. This work demonstrates that menadione species generated by photo-irradiation can exert biological effects on cellular functions and points to the potential importance of photochemistry in studies of menadione-mediated cell damage.
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A simple method is presented for determination of menadione in vitamin premixes and feedstuffs by normal-phase liquid chromatography (LC). Vitamin K3 is extracted and converted to free menadione, which can be determined directly by LC analysis. Peak area or height is measured at 251 nm, and menadione is quantitated by comparison with the working standard. Menadione can be estimated with a detection limit of 2.5 ppm. Recoveries for premixes ranged from 97.3 to 98.3% and for feedstuffs from 93.7 to 96.8%. The method allows the assay of all commercial K3 compounds in pure or stabilized form and is applicable to a wide variety of feeds and premixes.
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Liquid phase
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Dithiothreitol
Buthionine sulfoximine
Thiol
Primary culture
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