Glutathione regulates transforming growth factor-beta-stimulated collagen production in fibroblasts

Transforming growth factor-beta (TGF-beta) is a potent fibrogenic cytokine. The molecular mechanism underlying TGF-beta fibrogenesis, however, has not been completely elucidated. In this study, we showed that TGF beta decreased the intracellular GSH content in murine embryo fibroblasts (NIH 3T3), which was followed by an increase in collagen I mRNA content and collagen protein production. Prevention of GSH depletion with N-acetylcysteine (NAC), GSH, or GSH ester abrogated TGF-beta-stimulated collagen production, whereas a decrease in intracellular GSH content with L-buthionine-S,R-sulfoximine, an inhibitor of de novo GSH synthesis, enhanced TGF-beta-stimulated collagen production. These results suggest that GSH depletion induced by TGF-beta may mediate TGF-beta-stimulated collagen production. In addition, we showed that TGF-beta stimulated superoxide production and increased release of H2O2 from the cells, whereas GSH ester decreased basal and TGF-beta + glucose oxidase-stimulated H2O2 release. H2O2, exogenously added or continuously generated by glucose oxidase, enhanced TGF-beta-stimulated collagen production, whereas suppression of superoxide production by diphenyliodonium, an NAD(P)H oxidase inhibitor, blocked TGF-beta-stimulated collagen production. These data further suggest that reactive oxygen species are involved in TGF-beta-stimulated collagen production and that the effect of GSH depletion on TGF-beta-stimulated collagen production may be mediated by facilitating reactive oxygen species signaling.     

DLPC decreases TGF-beta1-induced collagen mRNA by inhibiting p38 MAPK in hepatic stellate cells

Dilinoleoylphosphatidylcholine (DLPC), the active component of polyenylphosphatidylcholine extracted from soybeans, decreases collagen accumulation induced by TGF-beta1 in cultured hepatic stellate cells (HSCs). Because DLPC exerts antioxidant effects and TGF-beta1 generates oxidative stress, we evaluated whether the antifibrogenic effect of DLPC is linked to its antioxidant action. In passage 1 culture of rat HSCs, TGF-beta1 induced a concentration-dependent increase in procollagen-alpha(1)(I) mRNA levels and enhanced intracellular H(2)O(2) and superoxide anion formation and lipid peroxidation but decreased GSH levels. These changes were prevented by DLPC. Upregulation of collagen mRNA by TGF-beta1 was likewise inhibited by catalase and p38 MAPK inhibitor SB-203580, suggesting involvement of H(2)O(2) and p38 MAPK signaling in this process. TGF-beta1 or addition of H(2)O(2) to HSCs activated p38 MAPK with a rise in procollagen mRNA level; these changes were blocked by catalase and SB-203580 and likewise by DLPC. alpha-Smooth muscle actin abundance in HSCs was not altered by TGF-beta1 treatment (with or without DLPC), indicating that downregulation of procollagen mRNA by DLPC was not due to alteration in HSC activation. These results demonstrate that DLPC prevents TGF-beta1-induced increase in collagen mRNA by inhibiting generation of oxidative stress and associated H(2)O(2)-dependent p38 MAPK activation, which explains its antifibrogenic effect. DLPC, an innocuous phospholipid, may be considered for prevention and treatment of liver fibrosis.     

Apoptosis in arsenic trioxide-treated Calu-6 lung cells is correlated with the depletion of GSH levels rather than the changes of ROS levels

Arsenic trioxide (ATO) can regulate many biological functions such as apoptosis and differentiation in various cells. We investigated an involvement of ROS such as H(2)O(2) and O(2)(*-), and GSH in ATO-treated Calu-6 cell death. The levels of intracellular H(2)O(2) were decreased in ATO-treated Calu-6 cells at 72 h. However, the levels of O(2)(*-) were significantly increased. ATO reduced the intracellular GSH content. Many of the cells having depleted GSH contents were dead, as evidenced by the propidium iodine staining. The activity of CuZn-SOD was strongly down-regulated by ATO at 72 h while the activity of Mn-SOD was weakly up-regulated. The activity of catalase was decreased by ATO. ROS scavengers, Tiron and Trimetazidine did not reduce levels of apoptosis and intracellular O(2)(*-) in ATO-treated Calu-6 cells. Tempol showing a decrease in intracellular O(2)(*-) levels reduced the loss of mitochondrial transmembrane potential (DeltaPsi(m)). Treatment with NAC showing the recovery of GSH depletion and the decreased effect on O(2)(*-) levels in ATO-treated cells significantly inhibited apoptosis. In addition, BSO significantly increased the depletion of GSH content and apoptosis in ATO-treated cells. Treatment with SOD and catalase significantly reduced the levels of O(2)(*-) levels in ATO-treated cells, but did not inhibit apoptosis along with non-effect on the recovery of GSH depletion. Taken together, our results suggest that ATO induces apoptosis in Calu-6 cells via the depletion of the intracellular GSH contents rather than the changes of ROS levels.     

Role of glutathione in the adaptive tolerance to H2O2

Endogenous antioxidant defense systems are enhanced by various physiological stimuli including sublethal oxidative challenges, which induce tolerance to subsequent lethal oxidative injuries. We sought to evaluate the contributions of catalase and the glutathione system to the adaptive tolerance to H2O2. For this purpose, H9c2 cells were stimulated with 100 microM H2O2, which was the maximal dose at which no significant acute cell damage was observed. Twenty-four hours after stimulation, control and pretreated cells were challenged with a lethal concentration of H2O2 (300 microM). Compared with the control cells, pretreated cells were significantly tolerant of H2O2, with reduced cell lysis and improved survival rate. In pretreated cells, glutathione content increased to 48.20 +/- 6.38 nmol/mg protein versus 27.59 +/- 2.55 nmol/mg protein in control cells, and catalase activity also increased to 30.82 +/- 2.64 versus 15.46 +/- 1.29 units/mg protein in control cells, whereas glutathione peroxidase activity was not affected. Increased glutathione content was attributed to increased gamma-glutamylcysteine synthetase activity, which is known as the rate-limiting enzyme of glutathione synthesis. To elucidate the relative contribution of the glutathione system and catalase to tolerance of H2O2, control and pretreated cells were incubated with specific inhibitors of gamma-glutamyl cysteine synthetase (L-buthionine sulfoximine) or catalase (3-amino-1,2,4-triazole), and challenged with H2O2. Cytoprotection by the low-dose H2O2 pretreatment was almost completely abolished by L-buthionine sulfoximine, while it was preserved after 3-amino-1,2,4-triazole treatment. From these results, it is concluded that both the glutathione system and catalase can be enhanced by H2O2 stimulation, but increased glutathione content rather than catalase activity was operative in the tolerance of lethal oxidative stress.     

Indomethacin attenuation of hepatic perfusion and plasma 6-ketoPGF1 alpha elevations following glutathione depletion in rabbits

Glutathione (GSH) is important in detoxification and regulating cyclooxygenase activity. Since the liver has high levels of GSH, xenobiotic-induced changes in hepatic GSH could affect hepatic tissue blood perfusion (HP) via alterations in prostaglandin synthesis. In anesthetized male New Zealand rabbits, elevating GSH with GSH monoethyl ester had no affect on HP. Treatment of rabbits with diethyl maleate to deplete GSH also had no affect on HP in animals previously given GSH monoethyl ester. However, HP increased within 20 min in rabbits treated with diethyl maleate prior to GSH monoethyl ester. In another experiment, a similar rise in HP following GSH depletion was accompanied by arterial plasma 6-ketoPGF1 alpha (the stable metabolite of prostacyclin) levels that were 4-times higher than in the controls. Plasma TxB2 (the stable metabolite of thromboxane) also increased following diethyl maleate, but only to levels that were 25-times lower than for 6-ketoPGF1 alpha. Since indomethacin blocked the rise in HP, as well as the increases in 6-ketoPGF1 alpha and TxB2, these results indicate changes in HP may occur following GSH depletion as a result of increased synthesis of one or more arachidonic acid metabolites and implicate prostacyclin as a possible mediator of this phenomenon.     

Roles of RhoA and phospholipase A2 in the elevation of intracellular H2O2 by transforming growth factor-beta in Swiss 3T3 fibroblasts.

We have investigated the mechanisms by which transforming growth factor-beta (TGF-beta) increased intracellular H2O2 in Swiss 3T3 fibroblasts. Increase of intracellular H2O2 by TGF-beta was maximal at 30 min and blocked by catalase from Aspergillus niger. Scrape-loading of C3 transferase, which down-regulated RhoA, inhibited the production of H2O2 in response to TGF-beta. TGF-beta stimulated release of arachidonic acid, which was completely inhibited by mepacrine, a phospholipase A2 inhibitor. Mepacrine also blocked the increase of H2O2 by TGF-beta. In addition, arachidonic acid increased intracellular H2O2. Furthermore, TGF-beta stimulated stress fibre formation, which was blocked by catalase, without membrane ruffling. Catalase also inhibited stimulation of thymidine incorporation by TGF-beta. These results suggested that TGF-beta increased intracellular H2O2 through RhoA and phospholipase A2, and also suggested that intracellular H2O2 was required for the stimulation of stress fibre formation and DNA synthesis in response to TGF-beta.     

Hyperoxia increases H2O2 production by brain in vivo

Hyperoxia and hyperbaric hyperoxia increased the rate of cerebral hydrogen peroxide (H2O2) production in unanesthetized rats in vivo, as measured by the H2O2-mediated inactivation of endogenous catalase activity following injection of 3-amino-1,2,4-triazole. Brain catalase activity in rats breathing air (0.2 ATA O2) decreased to 75, 61, and 40% of controls due to endogenous H2O2 production at 30, 60, and 120 min, respectively, after intraperitoneal injection of 3-amino-1,2,4-triazole. The rate of catalase inactivation increased linearly in rats exposed to 0.6 ATA O2 (3 ATA air), 1.0 ATA O2 (normobaric 100% O2) and 3.0 ATA O2 (3 ATA 100% O2) compared with 0.2 ATA O2 (room air). Catalase inactivation was prevented by pretreatment of rats with ethanol (4 g/kg), a competitive substrate for the reactive catalase-H2O2 intermediate, compound I. This confirmed that catalase inactivation by 3-amino-1,2,4-triazole was due to formation of the catalase-H2O2 intermediate, compound I. The linear rate of catalase inactivation allows estimates of the average steady-state H2O2 concentration within brain peroxisomes to be calculated from the formula: [H2O2] = 6.6 pM + 5.6 ATA-1 X pM X [O2], where [O2] is the concentration of oxygen in ATA that the rats are breathing. Thus the H2O2 concentration in brains of rats exposed to room air is calculated to be about 7.7 pM, rises 60% when O2 tension is increased to 100% O2, and increases 300% at 3 ATA 100% O2, where symptoms of central nervous system toxicity first become apparent. These studies support the concept that H2O2 is an important mediator of O2-induced injury to the central nervous system.     

Toxicity by pyruvate in HepG2 cells depleted of glutathione: role of mitochondria.

Several studies have shown that pyruvate can scavenge H(2)O(2) and protect from H(2)O(2)-mediated cell injury. Mitochondria are critical participants in the control of apoptotic and necrotic cell death. Mitochondrial GSH plays an important role in the maintenance of cell functions and viability by metabolism of oxygen free radicals generated by the respiratory chain. Since loss of GSH, especially mitochondrial GSH, is associated with increased production of reactive oxygen species and cell toxicity, the ability of pyruvate to protect against these actions was evaluated. Adding pyruvate to HepG2 cells depleted of GSH by treatment with l-buthionine sulfoximine (BSO) surprisingly caused loss of viability after 24 and 48 h of incubation. Anoxia, treatment with antioxidants, and infection with cytosolic catalase, and interestingly, catalase expressed in the mitochondrial compartment were able to rescue the HepG2 cells from this pyruvate plus BSO injury, suggesting a key role for H(2)O(2), and lipid peroxides as mediators in the cytotoxicity. This toxicity and cell death observed was linked to damage to the mitochondria as evidenced by the increased lipid peroxidation in total homogenate and mitochondrial fraction, loss of mitochondrial membrane potential, and a decrease in protein-sulfhydryl groups. The type of cell death observed under these conditions was a mixture of apoptosis and necrosis. These results suggest that the protective ability of pyruvate against oxidant damage requires a functional GSH pool, especially in the mitochondrial compartment, and that in the absence of GSH, pyruvate increases cell injury by damaging the mitochondria, presumably as a consequence of enhanced electron flow and reactive oxygen production by the respiratory chain.     

Release of H2O2 and phosphorylation of 30 kilodalton proteins as early responses of cell cycle-dependent inhibition of DNA synthesis by transforming growth factor beta 1.

Transforming growth factor beta 1 (TGF-beta 1) and H2O2 both inhibited DNA synthesis of mouse osteoblastic (MC3T3) cells in the late G1 phase of the cell cycle. TGF-beta 1 stimulated cells to release H2O2 in the late G1 phase, but not in the G0 phase, even though TGF-beta 1 receptors were present in both phases. The inhibition of DNA synthesis caused by TGF-beta 1 was partly decreased by the addition of catalase. TGF-beta 1 and H2O2 increased the phosphorylation of the same proteins with a molecular weight of 30,000 in cells in the late G1 phase, and the increase by TGF-beta 1 was abolished at least partly by catalase. The results suggest that H2O2 is one of the mediators of inhibition of DNA synthesis by TGF-beta 1.     

Inhibition of the low-Km mitochondrial aldehyde dehydrogenase by diethyl maleate and phorone in vivo and in vitro. Implications for formaldehyde metabolism.

Formaldehyde can be oxidized primarily by two different enzymes, the low-Km mitochondrial aldehyde dehydrogenase and the cytosolic GSH-dependent formaldehyde dehydrogenase. Experiments were carried out to evaluate the effects of diethyl maleate or phorone, agents that deplete GSH from the liver, on the oxidation of formaldehyde. The addition of diethyl maleate or phorone to intact mitochondria or to disrupted mitochondrial fractions produced inhibition of formaldehyde oxidation. The kinetics of inhibition of the low-Km mitochondrial aldehyde dehydrogenase were mixed. Mitochondria isolated from rats treated in vivo with diethyl maleate or phorone had a decreased capacity to oxidize either formaldehyde or acetaldehyde. The activity of the low-Km, but not the high-Km, mitochondrial aldehyde dehydrogenase was also inhibited. The production of CO2 plus formate from 0.2 mM-[14C]formaldehyde by isolated hepatocytes was only slightly inhibited (15-30%) by incubation with diethyl maleate or addition of cyanamide, suggesting oxidation primarily via formaldehyde dehydrogenase. However, the production of CO2 plus formate was increased 2.5-fold when the concentration of [14C]formaldehyde was raised to 1 mM. This increase in product formation at higher formaldehyde concentrations was much more sensitive to inhibition by diethyl maleate or cyanamide, suggesting an important contribution by mitochondrial aldehyde dehydrogenase. Thus diethyl maleate and phorone, besides depleting GSH, can also serve as effective inhibitors in vivo or in vitro of the low-Km mitochondrial aldehyde dehydrogenase. Inhibition of formaldehyde oxidation by these agents could be due to impairment of both enzyme systems known to be capable of oxidizing formaldehyde. It would appear that a critical amount of GSH, e.g. 90%, must be depleted before the activity of formaldehyde dehydrogenase becomes impaired.     

Effects of menadione and hydrogen peroxide on glutathione status in growing Escherichia coli

Menadione (MD) and H2O2 caused distinct effects on glutathione status in growing Escherichia coli. Treatment of E. coli AB1157 with 1-25 mM H2O2 did not result in an appreciable decrease in intracellular total glutathione (reduced glutathione [GSH] + oxidized glutathione [GSSG]). Only when cells were treated with 25 mM H2O2 an increase in GSSG and a decrease in the GSH:GSSG ratio were observed. In cells deficient in catalase HPI, such effect was observed even at 10 mM H2O2. The exposure of E. coli AB1157 to MD caused a dose-dependent decrease in intracellular total glutathione, an increase in GSSG, and a decrease in the ratio of GSH:GSSG. In E. coli deficient in cytosolic superoxide dismutase activity, a decrease in total glutathione after incubation with 0.2 mM MD was not accompanied by an increase in GSSGin, and the ratio of GSHin:GSSGin was three times higher than in the wild-type cells. The changes in the redox status of extracellular glutathione under the action of both oxidants were similar. Although the catalase activity increased several times after exposure to both oxidants, there were little or no changes in the activity of enzymes related to glutathione metabolism. A possible role of changes in redox status of glutathione under oxidative stress is discussed.     

Intracellular GSH level is a factor in As4.1 juxtaglomerular cell death by arsenic trioxide

Arsenic trioxide has been known to regulate many biological functions such as cell proliferation, apoptosis, differentiation, and angiogenesis in various cell lines. We investigated the involvement of GSH and ROS such as H(2)O(2) and O(2)(*-) in the death of As4.1 cells by arsenic trioxide. The intracellular ROS levels were changed depending on the concentration and length of incubation with arsenic trioxide. The intracellular O(2)(*-) level was significantly increased at all the concentrations tested. Arsenic trioxide reduced the intracellular GSH content. Treatment of Tiron, ROS scavenger decreased the levels of ROS in 10 microM arsenic trioxide-treated cells. Another ROS scavenger, Tempol did not decrease ROS levels in arsenic trioxide-treated cells, but slightly recovered the depleted GSH content and reduced the level of apoptosis in these cells. Exogenous SOD and catalase did not reduce the level of ROS, but did decrease the level of O(2)(*-). Both of them inhibited GSH depletion and apoptosis in arsenic trioxide-treated cells. In addition, ROS scavengers, SOD and catalase did not alter the accumulation of cells in the S phase induced by arsenic trioxide. Furthermore, JNK inhibitor rescued some cells from arsenic trioxide-induced apoptosis, and this inhibitor decreased the levels of O(2)(*-) and reduced the GSH depletion in these cells. In summary, we have demonstrated that arsenic trioxide potently generates ROS, especially O(2)(*-), in As4.1 juxtaglomerular cells, and Tempol, SOD, catalase, and JNK inhibitor partially rescued cells from arsenic trioxide-induced apoptosis through the up-regulation of intracellular GSH levels.     

Glutathione oxidation and PTPase inhibition by hydrogen peroxide in Caco-2 cell monolayer

The role of H(2)O(2) and protein thiol oxidation in oxidative stress-induced epithelial paracellular permeability was investigated in Caco-2 cell monolayers. Treatment with a H(2)O(2) generating system (xanthine oxidase + xanthine) or H(2)O(2) (20 microM) increased the paracellular permeability. Xanthine oxidase-induced permeability was potentiated by superoxide dismutase and prevented by catalase. H(2)O(2)-induced permeability was prevented by ferrous sulfate and potentiated by deferoxamine and 1,10-phenanthroline. GSH, N-acetyl-L-cysteine, dithiothreitol, mercaptosuccinate, and diethylmaleate inhibited H(2)O(2)-induced permeability, but it was potentiated by 1,3-bis(2-chloroethyl)-1-nitrosourea. H(2)O(2) reduced cellular GSH and protein thiols and increased GSSG. H(2)O(2)-mediated reduction of GSH-to-GSSG ratio was prevented by ferrous sulfate, GSH, N-acetyl-L-cysteine, diethylmaleate, and mercaptosuccinate and potentiated by 1,10-phenanthroline and 1, 3-bis(2-chloroethyl)-1-nitrosourea. Incubation of soluble fraction of cells with GSSG reduced protein tyrosine phosphatase (PTPase) activity, which was prevented by coincubation with GSH. PTPase activity was also lower in H(2)O(2)-treated cells. This study indicates that H(2)O(2), but not O(2)(-). or.OH, increases paracellular permeability of Caco-2 cell monolayer by a mechanism that involves oxidation of GSH and inhibition of PTPases.     

A superoxide anion generator, pyrogallol induces apoptosis in As4.1 cells through the depletion of intracellular GSH content

We investigated the involvement of ROS such as H2O2 and O2*-, and GSH in As4.1 cell death induced by pyrogallol. The intracellular H2O2 levels were decreased or increased depending on the concentration and incubation time of pyrogallol. The levels of O2*- were significantly increased. Pyrogallol reduced the intracellular GSH content. And ROS scavengers, Tempol, Tiron, Trimetazidine and NAC could not significantly down-regulate the production of H2O2 and O2*-. However, these ROS scavengers slightly inhibited apoptosis. Interestingly, Tempol showing the recovery of GSH depletion induced by pyrogallol significantly decreased apoptosis without the significant reduction of intracellular O2*- levels. SOD and catalase did not change the level of H2O2 but decreased the level of O2*-. The inhibition of GSH depletion by these was accompanied with the decrease of apoptosis, as evidenced by sub-G1 DNA content, annexin V staining, mitochondria membrane potential (DeltaPsi(m)) and Western data. In addition, ROS scavengers and SOD did not alter a G2 phase accumulation of the cell cycle induced by pyrogallol. However, catalase changed the cell cycle distributions of pyrogallol-treated cells to those of pyrogallol-untreated cells. In summary, we have demonstrated that pyrogallol potently generates ROS, especially O2*-, in As4.1 JG cells, and Tempol, SOD and catalase could rescue to a lesser or greater extent cells from pyrogallol-induced apoptosis through the up-regulation of intracellular GSH content.     

Cytoprotective effect of reduced glutathione in hydrogen peroxide-induced endothelial cell injury

The kinetic effects of hydrogen peroxide (H2O2) on cultured endothelial cells isolated from bovine carotid artery were studied. The cytoprotective effects of glutathione (GSH) on H2O2-induced cell injury were also investigated. H2O2-induced a dose- and time-dependent cell injury in cultured endothelial cells. H2O2-induced cell injury was blocked by simultaneous treatment by catalase, but not by superoxide dismutase. H2O2 also induced endogenous PGI2 biosynthesis, and the maximum PGI2 production was reached after 1 h treatment. Stimulation of PGI2 production was parallel with arachidonate release from H2O2-treated cells. However the prostaglandin biosynthesis enzyme activity in cells was inhibited by H2O2 treatment. When the cells were treated with GSH, the intracellular GSH reached a plateau after 3 h treatment. Both H2O2-induced cell injury and PGI2 production were significantly inhibited by the 3 h pretreatment with GSH. The cytoprotective effect of GSH was completely inhibited by buthionine sulfoximine which is a specific inhibitor of gamma-glutamylcysteine synthetase. The results indicate that the cytoprotective effect of GSH on H2O2-induced cell injury in cultured bovine carotid artery endothelial cells depends on the increase in intracellular GSH content.     

Tumoricidal activity of endothelial cells. Inhibition of endothelial nitric oxide production abrogates tumor cytotoxicity induced by hepatic sinusoidal endothelium in response to B16 melanoma adhesion in vitro

The mechanism of NO- and H(2)O(2)-induced tumor cytotoxicity was examined during B16 melanoma (B16M) adhesion to the hepatic sinusoidal endothelium (HSE) in vitro. We used endothelial nitric-oxide synthetase gene disruption and N(G)-nitro-l-arginine methyl ester-induced inhibition of nitric-oxide synthetase activity to study the effect of HSE-derived NO on B16M cell viability. Extracellular H(2)O(2) was removed by exogenous catalase. H(2)O(2) was not cytotoxic in the absence of NO. However, NO-induced tumor cytotoxicity was increased by H(2)O(2) due to the formation of potent oxidants, likely ( small middle dot)OH and (-)OONO radicals, via a trace metal-dependent process. B16M cells cultured to low density (LD cells), with high GSH content, were more resistant to NO and H(2)O(2) than B16M cells cultured to high density (HD cells; with approximately 25% of the GSH content found in LD cells). Resistance of LD cells decreased using buthionine sulfoximine, a specific GSH synthesis inhibitor, whereas resistance increased in HD cells using GSH ester, which delivers free intracellular GSH. Because NO and H(2)O(2) were particularly cytotoxic in HD cells, we investigated the enzyme activities that degrade H(2)O(2). NO and H(2)O(2) caused an approximately 75% (LD cells) and a 60% (HD cells) decrease in catalase activity without affecting the GSH peroxidase/GSH reductase system. Therefore, B16M resistance to the HSE-induced cytotoxicity appears highly dependent on GSH and GSH peroxidase, which are both required to eliminate H(2)O(2). In agreement with this fact, ebselen, a GSH peroxidase mimic, abrogated the increase in NO toxicity induced by H(2)O(2).     

Suppression of arsenic trioxide-induced apoptosis in HeLa cells by N-acetylcysteine

Arsenic trioxide (ATO) can affect many biological functions such as apoptosis and differentiation in various cells. We investigated the involvement of ROS and GSH in ATO-induced HeLa cell death using ROS scavengers, especially N-acetylcysteine (NAC). ATO increased intracellular O(2)(*-) levels and reduced intracellular GSH content. The ROS scavengers, Tempol, Tiron and Trimetazidine, did not significantly reduce levels of ROS or GSH depletion in ATO-treated HeLa cells. Nor did they reduce the apoptosis induced by ATO. In contrast, treatment with NAC reduced ROS levels and GSH depletion in the ATO-treated HeLa cells and prevented ATO-induced apoptosis. Treatment with exogenous SOD and catalase reduced the depletion of GSH content in ATO-treated cells. Catalase strongly protected the cells from ATO-induced apoptosis. In addition, treatment with SOD, catalase and NAC slightly inhibited the G1 phase accumulation induced by ATO. In conclusion, NAC protects HeLa cells from apoptosis induced by ATO by up-regulating intracellular GSH content and partially reducing the production of O(2)(*-).     

Susceptibility to hydrogen peroxide and catalase activity of root nodule bacteria

The root nodule bacteria (free-living cells) tested had higher susceptibility to hydrogen peroxide (H2O2) than the other genera of aerobic or facultative anaerobic bacteria tested. The catalase activities tended to have a positive correlation with H2O2 resistance among all bacteria tested. Addition of a catalase inhibitor such as 3-amino-1, 2, 4-triazole increased the susceptibility to H2O2. These results suggest that the lower catalase activity brings about the higher susceptibility of root nodule bacteria to H2O2. Root nodule bacteria seemed to have two or three catalase isozymes during growth and their catalase activities were higher in log phase than in stationary phase, contrary to other genera of bacteria tested.     

Oxidative protein cross-linking reactions involving L-tyrosine in transforming growth factor-beta1-stimulated fibroblasts

The mechanisms by which ligand-stimulated generation of reactive oxygen species in nonphagocytic cells mediate biologic effects are largely unknown. The profibrotic cytokine, transforming growth factor-beta1 (TGF-beta1), generates extracellular hydrogen peroxide (H2O2) in contrast to intracellular reactive oxygen species production by certain mitogenic growth factors in human lung fibroblasts. To determine whether tyrosine residues in fibroblast-derived extracellular matrix (ECM) proteins may be targets of H2O2-mediated dityrosine-dependent cross-linking reactions in response to TGF-beta1, we utilized fluorophore-labeled tyramide, a structurally related phenolic compound that forms dimers with tyrosine, as a probe to detect such reactions under dynamic cell culture conditions. With this approach, a distinct pattern of fluorescent labeling that seems to target ECM proteins preferentially was observed in TGF-beta1-treated cells but not in control cells. This reaction required the presence of a heme peroxidase and was inhibited by catalase or diphenyliodonium (a flavoenzyme inhibitor), similar to the effect on TGF-beta1-induced dityrosine formation. Exogenous addition of H2O2 to control cells that do not release extracellular H2O2 produced a similar fluorescent labeling reaction. These results support the concept that, in the presence of heme peroxidases in vivo, TGF-beta1-induced H2O2 production by fibroblasts may mediate oxidative dityrosine-dependent cross-linking of ECM protein(s). This effect may be important in the pathogenesis of human fibrotic diseases characterized by overexpression/activation of TGF-beta1.