Prior Glutathione Depletion Research Articles

Metabolism and disposition of [14C]dibromoacetonitrile in rats and mice following oral and intravenous administration

Tissue distribution, metabolism, and disposition of oral (0.2–20 mg/kg) and intravenous (0.2 mg/kg) doses of [2-14C]dibromoacetonitrile (DBAN) were investigated in male rats and mice.[14C]DBAN reacts rapidly with rat blood in vitro and binds covalently. Prior depletion of glutathione (GSH) markedly diminished loss of DBAN. Chemical reaction with GSH readily yielded glutathionylacetonitrile.About 90% of the radioactivity from orally administered doses of [14C]DBAN was absorbed. After intravenous administration, 10% and 20% of the radioactivity was recovered in mouse and rat tissues, respectively, at 72 h. After oral dosing, three to four times less radioactivity was recovered, but radioactivity in stomach was mostly covalently bound.Excretion of radioactivity into urine exceeded that in feces; 9–15% was exhaled as labeled carbon dioxide and 1–3% as volatiles in 72 h.The major urinary metabolites were identified by liquid chromatography-mass spectrometry, and included acetonitrile mercaptoacetate (mouse), acetonitrile mercapturate, and cysteinylacetonitrile.The primary mode of DBAN metabolism is via reaction with GSH, and covalent binding may be due to reaction with tissue sulphydryls.

Super-induction of HO-1 in macrophages stimulated with lipopolysaccharide by prior depletion of glutathione decreases iNOS expression and NO production

In the LPS-stimulated macrophages undergoing oxidative burst, intracellular storage of glutathione (GSH) is depleted, expression of iNOS is enhanced, and NO is overproduced. In response to the depletion of GSH, expression of HO-1 is induced and HO activity is elevated. Thus, in macrophages treated with LPS, productions of NO and CO, catalyzed, respectively, by accumulated iNOS and HO-1, are increased in sequence [Biochem. Pharmacol. 68 (2004) 1709]. In support of this, HO-1 is induced in macrophages treated only with buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis depleting the GSH level. Alternatively, when the macrophages were exposed to spermine NONOate, an exogenous NO-donor, HO-1, was induced also. When the GSH-depleted or BSO-pretreated macrophages were exposed to NO, delivered either exogenously from spermine NONOate or endogenously from LPS-derived elevation of iNOS, super-induction of HO-1 was observed. Moreover, both the BSO and LPS treatments increased the accumulation of HO-1 inducing redox-sensitive transcription factor Nrf2 in the nuclear protein fraction. Thus, when the depletion of GSH is combined with NO delivery, expression of HO-1 is enhanced to a greater extent than that enhanced either by GSH depletion or by NO delivery. In these macrophages with super-induced HO-1 and elevated HO activity, LPS-derived increase in iNOS expression was down-regulated and NO production was suppressed. This indicated that induction of HO-1 caused by the NO overproduced from up-regulated iNOS, in turn, produces a causative inhibition on iNOS expression and NO production. Thus, it appears that there is a reciprocal cross-talk between inductions of HO-1 and iNOS in macrophages stimulated with LPS leading to their survival.

Endogenous production and exogenous exposure to nitric oxide augment doxorubicin cytotoxicity for breast cancer cells but not cardiac myoblasts

We studied the effect of nitric oxide (NO) on the anticancer activity of doxorubicin. When MCF-7 human breast cancer cells were exposed to an aqueous solution of NO delivered as a bolus 30 min prior to doxorubicin, the cytotoxic effect as measured in a clonogenic assay was increased (doxorubicin alone, 40% survival, doxorubicin plus NO, 5% survival). The NO donor diethylamine nitric oxide, but not inactivated donor, also yielded an increase in doxorubicin cytotoxicity. The sequence was important since the simultaneous application of NO with doxorubicin yielded only a small augmentation of effect, and the exposure of the cells to doxorubicin prior to the NO obliterated the augmentation. Prior depletion of glutathione by incubation of the cells for 24 h with d,l-buthionine-S,R-sulfoximine (BSO) further increased the cytotoxicity so that BSO plus NO plus doxorubicin killed all of the clones. MCF-7 cells transduced with inducible nitric oxide synthase gene (iNOS) through an adenoviral vector overexpressed iNOS and produced increased amounts of nitrite, an indicator of increased NO production. These iNOS transduced cells were more susceptible to doxorubicin than vector control or wild-type cells. Cell cycle progression of iNOS transduced cells was not different from controls. Likewise, iNOS transduction resulted in no change in cellular glutathione levels. For comparison, we examined the effect of iNOS transduction on the sensitivity of MCF-7 to edelfosine, a membrane-localizing anticancer drug without direct DNA interaction. Insertion of the iNOS had no effect on killing of the MCF-7 cells by this ether lipid class drug. We also tested the effect of iNOS transduction on doxorubicin sensitivity of H9c2 rat heart-derived myoblasts. We found no augmentation of cytotoxicity by NO, and this observation offers potential therapeutic tumor selectivity by using NO with doxorubicin. Therefore, we conclude that NO produced intracellularly by iNOS overexpression or delivered as a bolus sensitizes human breast cancer cells in culture to doxorubicin, but not to a cardiac cell line or to edelfosine. This augmentation is not due to a modulation of cell cycle distribution or measurable cellular glutathione resulting from the transduction.

Endogenous production and exogenous exposure to nitric oxide augment doxorubicin cytotoxicity for breast cancer cells but not cardiac myoblasts

We studied the effect of nitric oxide ( NO) on the anticancer activity of doxorubicin. When MCF-7 human breast cancer cells were exposed to an aqueous solution of NO delivered as a bolus 30 min prior to doxorubicin, the cytotoxic effect as measured in a clonogenic assay was increased (doxorubicin alone, 40% survival, doxorubicin plus NO, 5% survival). The NO donor diethylamine nitric oxide, but not inactivated donor, also yielded an increase in doxorubicin cytotoxicity. The sequence was important since the simultaneous application of NO with doxorubicin yielded only a small augmentation of effect, and the exposure of the cells to doxorubicin prior to the NO obliterated the augmentation. Prior depletion of glutathione by incubation of the cells for 24 h with d, l-buthionine- S, R-sulfoximine (BSO) further increased the cytotoxicity so that BSO plus NO plus doxorubicin killed all of the clones. MCF-7 cells transduced with inducible nitric oxide synthase gene (iNOS) through an adenoviral vector overexpressed iNOS and produced increased amounts of nitrite, an indicator of increased NO production. These iNOS transduced cells were more susceptible to doxorubicin than vector control or wild-type cells. Cell cycle progression of iNOS transduced cells was not different from controls. Likewise, iNOS transduction resulted in no change in cellular glutathione levels. For comparison, we examined the effect of iNOS transduction on the sensitivity of MCF-7 to edelfosine, a membrane-localizing anticancer drug without direct DNA interaction. Insertion of the iNOS had no effect on killing of the MCF-7 cells by this ether lipid class drug. We also tested the effect of iNOS transduction on doxorubicin sensitivity of H9c2 rat heart-derived myoblasts. We found no augmentation of cytotoxicity by NO, and this observation offers potential therapeutic tumor selectivity by using NO with doxorubicin. Therefore, we conclude that NO produced intracellularly by iNOS overexpression or delivered as a bolus sensitizes human breast cancer cells in culture to doxorubicin, but not to a cardiac cell line or to edelfosine. This augmentation is not due to a modulation of cell cycle distribution or measurable cellular glutathione resulting from the transduction.

Modulation of hepatocyte thiol content by medium composition: implications for toxicity studies

Toxicity of compounds requiring glutathione for detoxification, thiol content and synthesis were determined in 24-h rat hepatocytes cultured in medium containing different concentrations of the sulphur amino acids. Glutathione synthesis was determined following prior depletion of glutathione with diethylmaleate. L-15 medium, which has high levels of cysteine and methionine (1 m m of each), provided some protection against dichloroacetone, dibromopropanol and dichloropropanol toxicity, and had a small effect on increasing glutathione content and synthesis, relative to Williams' medium E (WE) which has low levels (less than 0.5 m m) of both amino acids. However, WE containing N-acetylcysteine (NAC) (1 m m final cysteine concentration), with or without methionine (final concentration 1 m m), was a better cytoprotectant medium than L-15, markedly reducing toxicity of all three compounds, and rapidly (within 1.5 h) increasing cellular glutathione content. WE supplemented with methionine alone stimulated glutathione synthesis after an initial lag phase, and protected cultures against dichloropropanol, but not dibromopropanol or dichloroacetone, both of which are highly reactive in these cultures. There was a clear association between glutathione content at early time points in culture and toxicity observed at later time points, and overall these results indicate that differences in culture medium composition can alter intracellular glutathione content and xenobiotic toxicity.

Clofibrate-induced in vitro hepatoprotection against acetaminophen is not due to altered glutathione homeostasis.

Prior induction of peroxisome proliferation protects mice against the in vivo hepatotoxicity of acetaminophen and various other bioactivation-dependent toxicants. The mechanisms underlying such chemoresistance are poorly understood, although they have been suggested to involve alterations in glutathione homeostasis. To clarify the role of glutathione in this phenomenon, we isolated hepatocytes from mice in which hepatic peroxisome proliferation had been induced with clofibrate. The cells were incubated with a range of acetaminophen concentrations and the extent of cell killing after up to 8 h was assessed by measuring lactate dehydrogenase leakage from the cells. Hepatocytes from clofibrate-pretreated mice were much less susceptible to acetaminophen than cells from vehicle-treated controls. However, the extent of glutathione depletion during exposure to acetaminophen was similar in both cell types, as were rates of excretion of the product of glutathione-mediated detoxication of acetaminophen's quinoneimine metabolite, 3-glutathionyl-acetaminophen. The glutathione-replenishing ability of clofibrate-pretreated cells after a brief exposure to diethyl maleate also resembled that of control cells. More importantly, prior depletion of glutathione by diethyl maleate did not abolish the resistance of clofibrate-pretreated cells to acetaminophen. Taken together, these findings indicate that although glutathione-dependent pathways may contribute to hepatoprotection during peroxisome proliferation, the resistance phenomenon is not due exclusively to this mechanism.

A Role for Bioactivation and Covalent Binding within Epidermal Keratinocytes in Sulfonamide-Induced Cutaneous Drug Reactions

Cutaneous reactions are the most common manifestation of delayed-type hypersensitivity caused by sulfamethoxazole and dapsone. In light of the recognized metabolic and immunologic activity of the skin, we investigated the potential role of normal human epidermal keratinocytes in the development of these reactions. Adult and neonatal normal human epidermal keratinocytes metabolized sulfamethoxazole and dapsone to N-4-hydroxylamine and N-acetyl derivatives in a time-dependent manner. The latter was catalyzed by N-acetyltransferase 1 alone as normal human epidermal keratinocytes did not express mRNA for N-acetyltransferase 2. Investigation of metabolism-dependent toxicity of sulfamethoxazole and dapsone, and subsequent incubation of normal human epidermal keratinocytes with the respective hydroxylamine metabolites, demonstrated that these cells were resistant to the cytotoxic effects of sulfamethoxazole hydroxylamine but not dapsone hydroxylamine. With prior depletion of glutathione, however, normal human epidermal keratinocytes became susceptible to the toxicity of sulfamethoxazole hydroxylamine. Covalent adduct formation by sulfamethoxazole hydroxylamine was detected in normal human epidermal keratinocytes, even in the absence of cell death, and was increased with glutathione depletion. Major protein targets of sulfamethoxazole hydroxylamine were observed in the region of 160, 125, 95, and 57 kDa. Dapsone hydroxylamine also caused covalent adduct formation in normal human epidermal keratinocytes. Together, these observations provide a basis for our hypothesis that normal human epidermal keratinocytes are involved in the initiation and propagation of a cutaneous hypersensitivity response to these drugs.

Role of the cellular redox state in modulating acute ethanol toxicity in isolated hepatocytes

Objectives: To propose a mechanism for ethanol induced hepatocytotoxicity. Design and methods: Hepatocytotoxicity was determined at various concentrations of oxygen and agents involved in NADH metabolism. Results: At 1% O 2, hepatocytes were nearly 8-fold more susceptible to ethanol than at 95% O 2 (carbogen). Cytotoxicity at 1% O 2 was enhanced in the presence of glycolytic substrates that generate NADH (e.g., sorbitol or xylitol), and prevented by glycolytic substrates that reoxidise NADH ( e.g., fructose or dihydroxyacetone). Susceptibility to ethanol correlated with the cytosolic redox state (lactate; pyruvate ratio). Cytotoxicity also correlated with reactive oxygen species (ROS) formation. Cytotoxicity was averted by ROS scavengers or the ferric chelator desferoxamine but was increased by hydroxylamine, a catalase inhibitor, or by prior glutathione depletion. Ethanol induced cytotoxicity was also decreased by inhibitors of alcohol/aldehyde dehydrogenases or CYP2E1, an alcohol inducible cytochrome P450. Conclusions: A cytotoxic mechanism was proposed where the sustained increase in NADH levels, resulting from ethanol metabolism, maintains CYP2E1 in a more reduced state that increases ROS formation.

Detoxification of 1-Chloro-2,4-dinitrobenzene in MCF7 Breast Cancer Cells Expressing Glutathione S-Transferase P1-1 and/or Multidrug Resistance Protein 1

We examined the roles of glutathione S-transferase (GST) P1-1 and the glutathione S-conjugate (GS-X) transporter, multidrug resistance protein 1 (MRP1), singly or in combination, in the detoxification of 1-chloro-2,4-dinitrobenzene (CDNB). Derivatives of MCF7 breast carcinoma cells expressing GST P1-1 and MRP1 alone or in combination were developed. Detoxification was measured in cells as formation of the glutathione conjugate of CDNB, S-(2,4-dinitrophenyl)-glutathione (DNP-SG), efflux of DNP-SG, and ultimately protection from CDNB cytotoxicity. MRP1 expression in the absence of GST P1-1 confers a three- to fourfold resistance to CDNB, which is associated with a >10-fold increase in the maximum rate of DNP-SG efflux. DNP-SG efflux in MRP1-expressing MCF7 cells was ATP-dependent and exhibited an apparent Km for DNP-SG of 95 μM. MRP1 expression alone, however, had no effect on DNP-SG formation. Combined expression of GST P1-1 and MRP1 increased the rates of DNP-SG formation when cells were exposed to 10 μM CDNB. Moreover, combined expression of GSTP1-1 with MRP1 moderately augmented MRP1-mediated resistance to CDNB but only during short term (10 min) exposures to CDNB where IC50 values were in the 8–10 μM range. In contrast, expression of GST P1-1 in the absence of MRP1 slightly sensitized cells to the toxicity of CDNB (10 min exposures), despite increasing rates of DNP-SG formation. The sensitization to CDNB in cells expressing GST P1-1 alone was associated with increased intracellular accumulation of DNP-SG, indicating that DNP-SG may contribute to CDNB toxicity. The potential toxicity of DNP-SG is also suggested by the finding that inhibition of DNP-SG formation by prior glutathione depletion confers resistance to CDNB cytotoxicity in MRP1-poor MCF7 cells. Altogether, our results demonstrate that glutathione conjugation and MRP1-mediated conjugate efflux can operate together to confer resistance to CDNB. The data indicate that MRP1-mediated conjugate efflux is required for cytoprotection from CDNB because its conjugate (DNP-SG), when present at high intracellular levels, may also be toxic to cells.

The photoprotective effect of 1,25-dihydroxyvitamin D 3 on ultraviolet light B-induced damage in keratinocyte and its mechanism of action

We investigated the photoprotective effect of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) both in vivo and in vitro, revealing its relationship with glutathione, a well-known antioxidant. We also probed into the possible mechanism of photoprotection of 1,25(OH) 2D 3 through immunohistochemical study for metallothionein (MT). At the same time, endogenous antioxidant effect of 1,25(OH) 2D 3 was examined. Survival of cultured human keratinocytes was decreased when the cells were irradiated with ultraviolet light-B (UVB) at doses above 30 mJ/cm 2. But in the presence of 1,25(OH) 2D 3 (12 nM), the decrease of survival of keratinocytes by UVB was diminished. The formation of sunburn cells by UVB irradiation in the skin of ICR mice was inhibited by topical application of 1,25(OH) 2D 3, regardless of prior glutathione depletion. Immunohistochemical staining revealed that 1,25(OH) 2D 3 induced the expression of MT, a potent radical scavenger, mainly in the basal layer of ICR mice skin. 1,25(OH) 2D 3 neither inhibited peroxidation of plasma lipids nor interacted with superoxide, nor removed hydrogen peroxide as an antioxidant. These findings suggest that 1,25(OH) 2D 3 has photoprotective effect not related with glutathione or its endogenous antioxidant property. Rather, it could be attributed to 1,25(OH) 2D 3-induced MT and its capacity to prevent radical-related damage in UVB irradiation.

Peroxynitrite modulates tyrosine phosphorylation and phosphoinositide signalling in human neuroblastoma SH-SY5Y cells: attenuated effects in human 1321N1 astrocytoma cells.

Peroxynitrite may contribute to oxidative stress involving neurodegeneration in several disorders, including Alzheimer's disease. As with other reactive oxygen species, peroxynitrite might affect neuronal signalling systems, actions that could contribute to adaptive or deleterious cellular outcomes, but such effects have not previously been studied. To address this issue directly, peroxynitrite (50-500 microM) was administered to human neuroblastoma SH-SY5Y cells to assess its effects on protein tyrosine nitration, phosphoinositide signalling and protein tyrosine phosphorylation. Peroxynitrite rapidly increased the nitrotyrosine immunoreactivity of numerous proteins, primarily in the cytosol. Peroxynitrite inhibited, in a concentration-dependent manner, phosphoinositide hydrolysis stimulated by activation of muscarinic receptors with carbachol and the inhibition was greater after the depletion of cellular glutathione. In comparison, muscarinic receptor-stimulated phosphoinositide hydrolysis in human astrocytoma 1321N1 cells was less vulnerable to inhibition by peroxynitrite either without or with prior depletion of glutathione. There was a large, rapid and reversible increase in the tyrosine phosphorylation of the p120 Src substrate in peroxynitrite-treated SH-SY5Y cells, a response that was potentiated by glutathione depletion; in contrast, peroxynitrite decreased the tyrosine phosphorylation of focal adhesion kinase and paxillin. Tyrosine phosphorylation of p120 in 1321N1 astrocytoma cells was less sensitive to modulation by peroxynitrite. Thus alterations in phosphoinositide signalling and protein tyrosine phosphorylation were greater in neuroblastoma than astrocytoma cells, and modulation of these signalling processes probably contributes to neuronal mechanisms of the response to peroxynitrite.

Biotransformation and Hepatotoxicity of HCFC-123 in the Guinea Pig: Potentiation of Hepatic Injury by Prior Glutathione Depletion

The chlorofluorocarbon substitute 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) is a structural analog of halothane. Both are oxidatively metabolized by CYP2EI, producing a reactive trifluoroacyl acid chloride intermediate and have been shown to cause acute liver necrosis in the guinea pig. With halothane, liver injury has been associated with the degree of reactive intermediate binding to hepatic protein. This injury can be potentiated by prior glutathione (GSH) depletion. Thus, the combination of GSH depletion and HCFC-123 exposure was evaluated for its hepatotoxic potential in this species. Male outbred Hartley guinea pigs were injected with either 0.8 g/kg l-buthionine-(S,R)-sulfoximine (BSO) to deplete hepatic glutathione or vehicle control solution 24 hr before a 4-hr inhalation exposure to 1.0% (v/v) HCFC-123 with 40% O2. HCFC-123 caused minimal liver injury with only 1 of 8 exposed animals displaying confluent zone 3 necrosis. GSH depletion potentiated injury producing submassive to massive liver necrosis in some animals. This potentiation was associated with a 36% increase in covalent binding of reactive HCFC-123 intermediates to hepatic protein. These results were not due to alterations in the biotransformation of HCFC-123 as indicated by plasma concentrations of the metabolites trifluoroacetic acid and fluoride ion which were not affected by BSO pretreatment. HCFC-123 was also found to cause a decrease in liver GSH concentrations following exposure. These findings demonstrate a role for hepatic GSH in helping to prevent covalent binding by the trifluoroacyl acid chloride intermediate. Inhalation of HCFC-123 can cause acute hepatic injury in the guinea pig that is worsened by low hepatic GSH concentrations.

Buthionine Sulfoximine Reduces the Protective Capacity of Myocytes to Withstand Peroxide-Derived Free Radical Attack

Mammalian heart myocytes have a limited capacity to withstand the deleterious effects of free radical generating compounds. To assess the role of the glutathione redox cycle relative to this capacity, rat heart cell cultures were subjected for 90 min to 80 μmol/1 cumene hydroperoxide (CHPO) without and with prior glutathione depletion by buthionine sulfoximine. Preincubation of cultures with 125 μmol/1 buthionine sulfoximine for 2 h and 17 h caused a reduction of glutathione by 33% and 82%, respectively, without concomitant increase of glutathione disulfide. Subsequent incubation with CHPO for 90 min caused slowing of NADPH consumption (in the first 20 min 27 pmol vs 68 pmol without pretreatment with buthionine sulfoximine for 17 h), which indicates that glutathione depletion reduced the turnover rate of the glutathione redox cycle. Pretreatment with buthionine sulfoximine for 17 h exaggerated the negative chronotropic effects of CHPO: the time elapsed to 50% of baseline contraction frequency fell from 5.7 ± 1.4 min without buthionine sulfoximine to 3.7 ± 0.4 min after pretreatment with buthionine sulfoximine (P < 0.02). The severity of CHPO-induced lipid peroxidation as assessed by malondialdehyde formation (2.23 ± 0.51 vs 0.99 ± 0.05 nmol in the first 20 min; P < 0.05) was increased by buthionine sulfoximine pretreatment, as was the extent of cell necrosis as assessed by release of α-hydroxybutyrate dehydrogenase (39.5 ± 5.1 vs 29.0 ± 12.9% in the first 45 min). A "sublethal" dose of 10 μM CHPO for 60 min caused no substantial HBDH release, no formation of malondialdehyde, and no exhaustion of cellular GSH (35 nmol/U HBDHi = 0). However, following pretreatment with buthionine sulfoximine, 10 μM CHPO for 60 min produced 12% HBDH release and extensive lipid peroxidation (1.95 nmol malondialdehyde/U HBDHi = 0). As the deleterious effects of CHPO were aggravated by glutathione depletion, we conclude that the glutathione redox cycle plays a major role in the protection of myocytes against peroxide-induced free redical attack.

Effect of Glutathione Depletion on the In Vivo Inhibition of Drug Metabolism by Agents Forming an Inactive Cytochrome P-450 Fe(ll):Metabolite Complex. Studies with Amiodarone and Troleandomycin

The relative contribution of competitive inhibition versus formation of a P-450:metabolite complex to the in vivo inhibition of drug metabolism for several agents is unclear. The present investigation examined the contribution of these two mechanisms to the in vivo inhibition of drug metabolism by amiodarone through manipulation of glutathione turnover. In vivo P-450-dependent metabolism in rats was assessed by determining antipyrine clearance. Pretreatment with amiodarone (50 mg/kg, iv) decreased antipyrine clearance with or without prior glutathione depletion. Depletion of glutathione by buthionine sul- foximine (1.6 g/kg, ip) did not enhance the magnitude of inhibition of antipyrine clearance by amiodarone. Moreover, administration of a normally subinhibitory dose of amiodarone after buthionine sulfoximine pretreatment did not influence antipyrine clearance. Similarly, depletion of glutathione via buthionine sulfoximine or diethylmaleate (1 mUkg, po) did not influence the magnitude of inhibition caused by a single po dose of troleandomycin (500 or 350 mg/kg, respectively). These data indicate that glutathione content may not be a critical determinant for the in vivo inhibition of drug metabolism by agents which form a P-450:metabolite complex.