Glutathione Synthesis Inhibitor Buthionine Sulfoximine Research Articles

Characterization of Aminobenzylphenols as Protein Disulfide Isomerase Inhibitors in Glioblastoma Cell Lines.

Disulfide bond formation is a critical post-translational modification of newly synthesized polypeptides in the oxidizing environment of the endoplasmic reticulum and is mediated by protein disulfide isomerase (PDIA1). In this study, we report a series of α-aminobenzylphenol analogues as potent PDI inhibitors. The lead compound, AS15, is a covalent nanomolar inhibitor of PDI, and the combination of AS15 analogues with glutathione synthesis inhibitor buthionine sulfoximine (BSO) leads to synergistic cell growth inhibition. Using nascent RNA sequencing, we show that an AS15 analogue triggers the unfolded protein response in glioblastoma cells. A BODIPY-labeled analogue binds proteins including PDIA1, suggesting that the compounds are cell-permeable and reach the intended target. Taken together, these findings demonstrate an extensive biochemical characterization of a novel series of highly potent reactive small molecules that covalently bind to PDI.

Development of an assay for cellular efflux of pharmaceutically active agents and its relevance to understanding drug interactions.

Drug interactions may dictate the failure or success of a treatment. Patients undergoing hematopoietic stem cell transplantation (HSCT) are exposed to various types of drugs, and understanding how these drugs interact is of the utmost importance. The pharmacokinetics of busulfan, melphalan, and cyclophosphamide, drugs commonly used for HSCT, are known to be affected by a variety of other drugs with differing molecular structures. We hypothesized that these structurally unrelated drugs affect the transport of DNA-alkylating agents. To test this hypothesis, we developed a flow cytometry assay that used 5-carboxyfluorescein diacetate acetoxymethyl ester, which is cleaved by nonspecific intracellular esterases to 5-carboxyfluorescein (5-CF), a fluorescent ligand for the drug transporter MRP1. A decreased 5-CF efflux in the presence of a test compound suggests competitive inhibition. We demonstrated that chlorambucil, 4-hydroperoxycyclophosphamide, ketoconazole, ethacrynic acid, everolimus, and sirolimus strongly inhibited 5-CF efflux in lymphoma and leukemia cell lines. The efflux of these drugs partially depends on the glutathione (GSH) level, and their cytotoxicity is synergistic with inhibited GSH synthesis. This is consistent with the hypothesis that their GSH-conjugated products are ligands of a common cellular drug transporter. Our results may explain clinical observations on the effects of various drugs on the pharmacokinetics and pharmacodynamics of alkylating agents, and the assay may be used to deduce interaction mechanisms of drugs transported by a common system.

The glutathione synthesis inhibitor buthionine sulfoximine synergistically enhanced melphalan activity against preclinical models of multiple myeloma.

Melphalan (L-PAM) has been an integral part of multiple myeloma (MM) treatment as a conditioning regimen before stem cell transplant (SCT). After initial response, most treated patients experience relapse with an aggressive phenotype. Increased glutathione (GSH) in MM may mediate resistance to L-PAM. We demonstrated that the GSH synthesis inhibitor buthionine sulfoximine (BSO) synergistically enhanced L-PAM activity (inducing 2–4 logs of cell kill) against nine MM cell lines (also in the presence of marrow stroma or cytokines) and in seven primary MM samples (combination indices <1.0). In MM cell lines, BSO significantly (P<0.05) depleted GSH, increased L-PAM-induced single-strand DNA breaks, mitochondrial depolarization, caspase cleavage and apoptosis. L-PAM depleted GSH, but GSH rapidly recovered in a L-PAM-resistant MM cell line unless also treated with BSO. Treatment with N-acetylcysteine antagonized BSO+L-PAM cytotoxicity without increasing GSH. In human MM xenografted into beige-nude-xid mice, BSO significantly depleted MM intracellular GSH and significantly increased apoptosis compared with L-PAM alone. BSO+L-PAM achieved complete responses (CRs) in three MM xenograft models including maintained CRs >100 days, and significantly increased the median event-free survival relative to L-PAM alone. Combining BSO with L-PAM warrants clinical testing in advanced MM.

Metformin induces an intracellular reductive state that protects oesophageal squamous cell carcinoma cells against cisplatin but not copper-bis(thiosemicarbazones).

BackgroundOesophageal squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a poor survival rate. One of the most commonly used chemotherapeutic drugs, cisplatin, displays varied and often poor efficacy in vivo. Therefore, alternative, cost-effective and more efficacious treatments are required. Metformin has been previously shown to reduce proliferative rates in various carcinoma cell lines. We report for the first time, the effect of metformin on OSCC cell proliferation and show that it antagonises cisplatin-induced but not copper-bis(thiosemicarbazone)-induced cytotoxicity in OSCC cells.MethodsCell proliferation and stage of the cell cycle were quantified by trypan blue counts and flow cytometry, respectively. All cytotoxicity measurements were made using the tetrazolium based MTT assay. Metabolic alterations to cells were determined as follows: glycolysis via a lactate dehydrogenase assay, reducing equivalents by MTT reduction and reduced intracellular thiols by monobromobimane-thiol fluorescence, and glutathione depletion using buthionine sulfoximine. Inductively coupled plasma mass spectrometry was used to quantify cisplatin-DNA adduct formation.ResultsMetformin was found to reduce cell proliferation significantly in all OSCC cell lines, with an accumulation of cells in G0/G1 phase of the cell cycle. However, metformin significantly protected OSCC cells against cisplatin toxicity. Our results indicate that a major mechanism of metformin-induced cisplatin resistance results from a significant increase in glycolysis, intracellular NAD(P)H levels with a concomitant increase in reduced intracellular thiols, leading to decreased cisplatin-DNA adduct formation. The glutathione synthesis inhibitor buthionine sulfoximine significantly ablated the protective effect of metformin. We subsequently show that the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM, which are trapped in cells under reducing conditions, cause significant OSCC cytotoxicity, both alone and in combination with metformin.ConclusionsThis is the first study showing that metformin can be used to decrease cell proliferation in OSCC cells. However, metformin protects against cisplatin cytotoxicity by inducing a reducing intracellular environment leading to lower cisplatin-DNA adduct formation. As such, we advise that caution be used when administering cisplatin to diabetic patients treated with metformin. Furthermore, we propose a novel combination therapy approach for OSCC that utilises metformin with metformin-compatible cytotoxic agents, such as the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM.

Tumor Xenograft Response to Redox-Active Therapies Assessed by Magnetic Resonance Imaging Using a Thiol-Bearing DOTA Complex of Gadolinium

Gd-LC6-SH is a thiol-bearing DOTA complex of gadolinium designed to bind plasma albumin at the conserved Cys34 site. The binding of Gd-LC6-SH shows sensitivity to the presence of competing thiols. We hypothesized that Gd-LC6-SH could provide magnetic resonance imaging (MRI) enhancement that is sensitive to tumor redox state and that the prolonged retention of albumin-bound Gd-LC6-SH in vivo can be exploited to identify a saturating dose above which the shortening of MRI longitudinal relaxation time (T1) of tissue is insensitive to the injected gadolinium dose. In the Mia-PaCa-2 pancreatic tumor xenograft model in SCID mice, both the small-molecule Gd-DTPA-BMA and the macromolecule Galbumin MRI contrast agents produced dose-dependent decreases in tumor T1. By contrast, the decreases in tumor T1 provided by Gd-LC6-SH at 0.05 and 0.1 mmol/kg were not significantly different at longer times after injection. SCID mice bearing Mia-PaCa-2 or NCI-N87 tumor xenografts were treated with either the glutathione synthesis inhibitor buthionine sulfoximine or the thiol-oxidizing anticancer drug Imexon, respectively. In both models, there was a significantly greater increase in tumor R1 (=1/T1) 60 minutes after injection of Gd-LC6-SH in drug-treated animals relative to saline-treated controls. In addition, Mercury Orange staining for nonprotein sulfhydryls was significantly decreased by drug treatment relative to controls in both tumor models. In summary, these studies show that thiol-bearing complexes of gadolinium such as Gd-LC6-SH can serve as redox-sensitive MRI contrast agents for detecting differences in tumor redox status and can be used to evaluate the effects of redox-active drugs.

Insulin-like Growth Factor 1 and Transforming Growth Factor-β Stimulate Cystine/Glutamate Exchange Activity in Dental Pulp Cells

Introduction The growth factors insulin-like growth factor (IGF-1) and transforming growth factor-β (TGF-β) are protective to dental pulp cells in culture against the toxicity of the composite materials Durafill VS and Flow Line (Henry Schein Inc, New York, NY). Because the toxicity of these materials is mediated by oxidative stress, it seemed possible that the protective effects of IGF-1 and TGF-β were through the enhancement of an endogenous antioxidant mechanism. Methods We used cultured dental pulp cells to determine the mechanism of the protective effects of IGF-1 and TGF-β, focusing on the glutathione system and the role of cystine/glutamate exchange (system xc-). Results We found that the toxicity of Durafill VS and Flow Line was attenuated by the addition of glutathione monoethylester, suggesting a specific role for the cellular antioxidant glutathione. Supporting this hypothesis, we found that IGF-1 and TGF-β were protective against the toxicity of the glutathione synthesis inhibitor buthionine sulfoximine. Because levels of cellular cystine are the limiting factor in the production of glutathione, we tested the effects of IGF-1 and TGF-β on cystine uptake. Both growth factors stimulated system xc–mediated cystine uptake. Furthermore, they attenuated the glutathione depletion induced by Durafill VS and Flow Line. Conclusions The results suggest that IGF-1 and TGF-β are protective through the stimulation of system xc–mediated cystine uptake, leading to maintenance of cellular glutathione. This novel action of growth factors on dental pulp cells has implications not only for preventing toxicity of dental materials but also for the general function of these cells.

Characterization of apical and basal thiol-disulfide redox regulation in human colonic epithelial cells

Control of extracellular thiol-disulfide redox potential (E(h)) is necessary to protect cell surface proteins from external oxidative and reductive stresses. Previous studies show that human colonic epithelial Caco-2 cells, which grow in cell culture with the apical surface exposed to the medium, regulate extracellular cysteine/cystine E(h) to physiological values (approximately -80 mV) observed in vivo. The present study tested whether extracellular E(h) regulation occurs on the basal surface of Caco-2 cells and investigated relevant mechanisms. Experiments were performed with confluent, differentiated cells grown on a permeable membrane surface. Cells were exposed to an oxidizing potential (0 mV) using a fixed cysteine-to-cystine ratio, and culture medium was sampled over time for change in E(h). Regulation of extracellular thiol-disulfide E(h) on the basal domain was faster, and the extent of change at 24 h was greater than on the apical surface. Mechanistic studies showed that redox regulation on the basal surface was partially sodium dependent and inhibited by extracellular lysine, a competitive inhibitor of cystine transport by the y(+)L system and by quisqualic acid, an inhibitor of the x(c)(-) system. Studies using the thiol-reactive alkylating agent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid and the glutathione synthesis inhibitor buthionine sulfoximine showed that extracellular redox regulation was not attributable to plasma membrane cysteine/cystine interconversion or intracellular glutathione, respectively. Thus the data show that redox regulation occurs at different rates on the apical and basal surfaces of the polarized Caco-2 epithelial cell line and that the y(+)L and x(c)(-) systems function in extracellular cysteine/cystine redox regulation on the basal surface.

"Shock and kill" effects of class I-selective histone deacetylase inhibitors in combination with the glutathione synthesis inhibitor buthionine sulfoximine in cell line models for HIV-1 quiescence

Latently infected, resting memory CD4+ T cells and macrophages represent a major obstacle to the eradication of HIV-1. For this purpose, "shock and kill" strategies have been proposed (activation of HIV-1 followed by stimuli leading to cell death). Histone deacetylase inhibitors (HDACIs) induce HIV-1 activation from quiescence, yet class/isoform-selective HDACIs are needed to specifically target HIV-1 latency. We tested 32 small molecule HDACIs for their ability to induce HIV-1 activation in the ACH-2 and U1 cell line models. In general, potent activators of HIV-1 replication were found among non-class selective and class I-selective HDACIs. However, class I selectivity did not reduce the toxicity of most of the molecules for uninfected cells, which is a major concern for possible HDACI-based therapies. To overcome this problem, complementary strategies using lower HDACI concentrations have been explored. We added to class I HDACIs the glutathione-synthesis inhibitor buthionine sulfoximine (BSO), in an attempt to create an intracellular environment that would facilitate HIV-1 activation. The basis for this strategy was that HIV-1 replication decreases the intracellular levels of reduced glutathione, creating a pro-oxidant environment which in turn stimulates HIV-1 transcription. We found that BSO increased the ability of class I HDACIs to activate HIV-1. This interaction allowed the use of both types of drugs at concentrations that were non-toxic for uninfected cells, whereas the infected cell cultures succumbed more readily to the drug combination. These effects were associated with BSO-induced recruitment of HDACI-insensitive cells into the responding cell population, as shown in Jurkat cell models for HIV-1 quiescence. The results of the present study may contribute to the future design of class I HDACIs for treating HIV-1. Moreover, the combined effects of class I-selective HDACIs and the glutathione synthesis inhibitor BSO suggest the existence of an Achilles' heel that could be manipulated in order to facilitate the "kill" phase of experimental HIV-1 eradication strategies.

Cyclophosphamide‐evoked heart failure involves pronounced co‐suppression of cytoplasmic thioredoxin reductase activity and non‐protein free thiol level

Heart failure is a life-threatening complication of high-dose cyclophosphamide (CTX) chemotherapy, and the present study aimed at identifying the mechanism involved in mice. CTX at 800 mg/kg resulted in heart failure, in which cytoplasmic thioredoxin reductase (TrxR1) activity and non-protein free thiol (NPFT) level were suppressed by 90 and 62%, respectively. The combination of 350 mg/kg CTX and the glutathione synthesis inhibitor buthionine sulfoximine (BSO) also evoked heart failure, in which TrxR1 activity and NPFT level were suppressed by 66 and 62%, respectively. NPFT depletion alone by BSO did not cause cardiac toxicity. CTX at 350 mg/kg alone also did not cause cardiac toxicity, even though it suppressed TrxR1 activity by 68%. Previous studies have shown that half inactivation of TrxR1 in tumour, bladder, and kidneys was associated with toxicological consequences. Cardiac TrxR1 is dispensable, but cardiac cytoplasmic thioredoxin (Trx1) is essential. The potential uncoupling between TrxR1 and Trx1 may explain why there is no cardiac toxicity following TrxR1 inhibition. However, TrxR1 inactivation may still play a role in CTX-evoked heart failure because inactivated TrxR1 gains cytotoxic function, which may engender noticeable toxicity when massive NPFT is deleted. CTX-evoked heart failure involves pronounced co-suppression of TrxR1 activity and NPFT level.

Endogenous glutathione and catalase protect cultured rat astrocytes from the iron-mediated toxicity of hydrogen peroxide

Primary astrocyte cultures from rat brain were exposed to hydrogen peroxide (H 2O 2) to investigate peroxide toxicity and clearance by astrocytes. After bolus application of H 2O 2 (100 μM), the peroxide was eliminated from the incubation medium following first-order kinetics with a half-time of approximately 4 min. The rate of peroxide detoxification was significantly slowed by pre-incubating the cells with the glutathione synthesis inhibitor buthionine sulfoximine (BSO), or the catalase inhibitor 3-amino-1,2,4-triazole (3AT), and was retarded further when both treatments were combined. H 2O 2 application killed a small proportion of cells, as indicated by the levels of the cytosolic enzyme lactate dehydrogenase in the media 1 and 24 h later. In contrast, cell viability was strongly compromised when the cells were pre-incubated with 3AT and/or BSO before peroxide application. The iron chelator deferoxamine completely prevented this cell loss. These results demonstrate that chelatable iron is involved in the toxicity of H 2O 2 and that both the glutathione system and catalase protect astrocytes from this toxicity.

Novel Role of Vitamin K in Preventing Oxidative Injury to Developing Oligodendrocytes and Neurons

Oxidative stress is believed to be the cause of cell death in multiple disorders of the brain, including perinatal hypoxia/ischemia. Glutamate, cystine deprivation, homocysteic acid, and the glutathione synthesis inhibitor buthionine sulfoximine all cause oxidative injury to immature neurons and oligodendrocytes by depleting intracellular glutathione. Although vitamin K is not a classical antioxidant, we report here the novel finding that vitamin K1 and K2 (menaquinone-4) potently inhibit glutathione depletion-mediated oxidative cell death in primary cultures of oligodendrocyte precursors and immature fetal cortical neurons with EC50 values of 30 nm and 2 nm, respectively. The mechanism by which vitamin K blocks oxidative injury is independent of its only known biological function as a cofactor for gamma-glutamylcarboxylase, an enzyme responsible for posttranslational modification of specific proteins. Neither oligodendrocytes nor neurons possess significant vitamin K-dependent carboxylase or epoxidase activity. Furthermore, the vitamin K antagonists warfarin and dicoumarol and the direct carboxylase inhibitor 2-chloro-vitamin K1 have no effect on the protective function of vitamin K against oxidative injury. Vitamin K does not prevent the depletion of intracellular glutathione caused by cystine deprivation but completely blocks free radical accumulation and cell death. The protective and potent efficacy of this naturally occurring vitamin, with no established clinical side effects, suggests a potential therapeutic application in preventing oxidative damage to undifferentiated oligodendrocytes in perinatal hypoxic/ischemic brain injury.

Signals of Oxidant-Induced Cardiomyocyte Hypertrophy: Key Activation of p70 S6 Kinase-1 and Phosphoinositide 3-Kinase

Cardiomyocytes in culture can survive low or mild doses of oxidants but later increase cell volume and protein content. To understand the mechanism, we determined the early signaling events of oxidative stress. With 200 microM H2O2, the activity of p70 S6 kinase-1 (p70S6K1) increased at 30 min and reached a plateau at 90 min. Dose-response studies at the 60 min time point show that p70S6K1 activity reached its highest level with 150 microM H2O2. Increased p70S6K1 activity correlated with phosphorylation of Thr389 and Thr421/Ser424 residues, suggesting the involvement of an upstream kinase. Phosphoinositide 3-kinase (PI3K) activity was elevated by 5 min, reached a plateau at 10 min, and remained more than 6-fold induced for at least 60 min after 200 microM H2O2 exposure. The dose-response studies at 10 min found that 150 microM H2O2 induced the highest PI3K activity. Increased PI3K activity correlated with tyrosine phosphorylation of the 85-kDa regulatory subunit. Inactivating PI3K with wortmannin prevented H2O2 from inducing Thr389 phosphorylation and p70S6K1 activation. Wortmannin and rapamycin prevented H2O2 from inducing increases in cell volume and protein content. The antineoplastic drugs doxorubicin and daunorubicin also induced significant enlargement of cardiomyocytes at 10 to 100 nM dose range. Although the glutathione synthesis inhibitor buthionine sulfoximine potentiated the effect of doxorubicin and H2O2, the antioxidant N-acetylcysteine prevented induction of cell enlargement. Our data suggest that oxidative stress induces activation of PI3K, which leads to p70S6K1 activation and enlargement of cell size.

Glutathiolation of the Proteasome Is Enhanced by Proteolytic Inhibitors

The proteasome inhibitors lactacystin, clastro lactacystin β-lactone, or tri-leucine vinyl sulfone (NLVS), in the presence of [35S]cysteine/methionine, caused increased incorporation of 35S into cellular proteins, even when protein synthesis was inhibited by cycloheximide. This effect was blocked by incubation with the glutathione synthesis inhibitor buthionine sulfoximine. Proteasome inhibitors also enhanced total glutathione levels, increased reduced/oxidized glutathione ratio (GSH/GSSG) and upregulated γ-glutamylcysteine synthetase (rate-limiting in glutathione synthesis). Micromolar concentrations of GSH, GSSG, or cysteine stimulated the chymotrypsin-like activity of purified 20S proteasome, but millimolar GSH or GSSG was inhibitory. Interestingly, GSH did not affect 20S proteasome's trypsin-like activity. Enhanced proteasome glutathiolation was verified when purified preparations of the 20S core enzyme complex were incubated with [35S]GSH after pre-incubation with any of the inhibitors. NLVS, lactacystin or clastro lactacystin β-lactone may promote structural modification of the 20S core proteasome, with increased exposure of cysteine residues, which are prone to S-thiolation. Three main conclusions can be drawn from the present work. First, proteasome inhibitors alter cellular glutathione metabolism. Second, proteasome glutathiolation is enhanced by inhibitors but still occurs in their absence, at physiological GSH and GSSG levels. Third, proteasome glutathiolation seems to be a previously unknown mechanism of proteasome regulation in vivo.

Induction of oxidative stress and apoptosis in myeloma cells by the aziridine-containing agent imexon

Imexon is an iminopyrrolidone derivative that has selective antitumor activity in multiple myeloma. The exact mechanism of imexon action is unknown. In human 8226 myeloma cells, the cytotoxicity of imexon was schedule-dependent, and long exposures (≥48 hr) to low concentrations of imexon were most effective at inducing cytotoxicity. Our data suggest that imexon does not affect DNA, but it can alkylate thiols by binding to the sulfhydryl group. We have also demonstrated by HPLC studies that in human 8226 myeloma cells, imexon depletes cellular stores of cysteine and glutathione. Oxidative stress in 8226 cells exposed to imexon was detected by immunohistochemical staining with a monoclonal antibody to 8-hydroxydeoxyguanosine (8-OHdG), followed by confocal microscopy. These images showed increased levels of 8-OHdG in the cytoplasm of cells treated with different concentrations of imexon at 8, 16, and 48 hr. Interestingly, 8-OHdG staining was not observed in the nuclei of imexon-treated cells, in contrast to the diffuse staining seen with t-butyl hydroperoxide. Myeloma cells exposed to imexon showed classic morphologic features of apoptosis upon electron microscopy, and increased levels of phosphatidylserine exposure, detected as Annexin-V binding, on the cell surface. To prevent depletion of thiols, 8226 myeloma cells exposed to imexon were treated with N-acetylcysteine (NAC). Simultaneous, as well as sequential, treatment with NAC before imexon exposure resulted in protection of myeloma cells against imexon-induced cytotoxicity. Conversely, the glutathione synthesis inhibitor buthionine sulfoximine increased imexon cytotoxicity. These data suggest that imexon perturbs cellular thiols and induces oxidative stress leading to apoptosis in human myeloma cells.

Anethole dithiolethione prevents oxidative damage in glutathione-depleted astrocytes

Astrocytes protect neurons against reactive oxygen species such as hydrogen peroxide, a capacity which reportedly is abolished following loss of the antioxidant glutathione. Anethole dithiolethione, a sulfur-containing compound which is used in humans, is known to increase cellular glutathione levels and thought thereby to protect against oxidative damage. In the present study we found that anethole dithiolethione increased the glutathione content of cultured rat striatal astrocytes. This effect was abolished by coincubation with the glutathione synthesis inhibitor buthionine sulfoximine. Nevertheless, in the presence of buthionine sulfoximine, despite the lack of an increase in the lowered glutathione level, anethole dithiolethione fully protected the astrocytes against the enhanced toxicity of hydrogen peroxide. Thus, apparently other mechanisms than stimulation of glutathione synthesis are involved in the compound's protective action in astrocytes. Considering the occurrence of lowered glutathione levels in neurodegenerative syndromes, we conclude that further evaluation of the therapeutic potential of anethole dithiolethione is warranted.

Anethole dithiolethione prevents oxidative damage in glutathione-depleted astrocytes

Astrocytes protect neurons against reactive oxygen species such as hydrogen peroxide, a capacity which reportedly is abolished following loss of the antioxidant glutathione. Anethole dithiolethione, a sulfur-containing compound which is used in humans, is known to increase cellular glutathione levels and thought thereby to protect against oxidative damage. In the present study we found that anethole dithiolethione increased the glutathione content of cultured rat striatal astrocytes. This effect was abolished by coincubation with the glutathione synthesis inhibitor buthionine sulfoximine. Nevertheless, in the presence of buthionine sulfoximine, despite the lack of an increase in the lowered glutathione level, anethole dithiolethione fully protected the astrocytes against the enhanced toxicity of hydrogen peroxide. Thus, apparently other mechanisms than stimulation of glutathione synthesis are involved in the compound's protective action in astrocytes. Considering the occurrence of lowered glutathione levels in neurodegenerative syndromes, we conclude that further evaluation of the therapeutic potential of anethole dithiolethione is warranted.

The Leukotriene LTD 4 Receptor Antagonist Mk571 Specifically Modulates MRP Associated Multidrug Resistance

The microltidrug resistant cell lines HL60/AR and GLC4/ADR show high overexpression of the gene encoding the microltidrug resistance associated protein MRP compared to their drug sensitive parental counterparts. This and the virtual absence of mdr1/P-glycoprotein gene expression was proven by a complementary DNA polymerase chain reaction (cDNA-PCR) approach. Applying a 72-hour tetrazolium based colorimetric MTT-assay we demonstrate on both MDR sublines a dose-dependent modulation of drug resistances by the leukotriene LTD 4 receptor antagonist MK571. A complete reversal of vincristine resistances was achieved at final MK571 concentrations of 30 μM (HL60/AR) or 50 μM (GLC4/ADR) which by itself did not disturb cellular proliferation. The drug resistance of a mdr1/P-gp overexpressing microltidrug-resistant HL60 subline, in contrast, was not significantly affected by MK571. Similar effects were seen using the glutathione (GSH) synthesis inhibitor buthionine sulfoximine (BSO). Our results point to a relationship between MRP and a conjugate transporter and identify MK571 as a new tool structure for developing modulators specific for a MRP associated microltidrug resistance.

Phenylpropanolamine Potentiation of Acetaminophen-Induced Hepatotoxicity: Evidence for a Glutathione-Dependent Mechanism

Pretreatment of male ICR mice with the adrenergic agonist phenylpropanolamine (200 mg/kg, ip) resulted in a marked potentiation of hepatotoxicity produced by acetaminophen (400 mg/kg, ip). Enhanced liver necrosis with phenylpropanolamine pretreatment was evident both by measurement of serum aminotransferase activity and by histopathologic examination. Several lines of experimental evidence suggest this interaction is a result of the hepatic glutathione depression produced by a-adrenergic compounds, which adds to the glutathione depression caused by toxic, or nearly toxic, doses of acetaminophen. First, the potentiation of acetaminophen hepatotoxicity was time-dependent, being observed only when phenyipropanolamine was administered as a 3-hr pretreatment and not when given 1 hr before, with, or 3 hr after acetaminophen. The 3-hr interval between phenyipropanolamine and acetaminophen doses corresponds to the characteristic lag period required for α-adrenergic agents (including phenylpropanolamine) to produce significant and maximal effects on hepatic glutathione content. Second, dose-response relationships for phenylpropanolamine and acetaminophen were such that increased toxicity was observed only when the interaction was sufficient to lower hepatic glutathione concentrations below a level regarded as critical in preventing acetaminophen-induced hepatotoxicity. Third, when animals were pretreated with two nonadrenergic depletors of hepatic glutathione, diethylmaleate (125 mg/kg, ip) or the glutathione synthesis inhibitor buthionine sulfoximine (222 mg/kg, ip), at doses producing glutathione depletion approximating that observed with the adrenergic agents, acetaminophen hepatotoxicity was potentiated to the same extent. From these observations it is postulated that a variety of adrenergic compounds known to deplete hepatic glutathione by a moderate 30-50% may potentiate the hepatotoxicity of acetaminophen and possibly other hepatotoxic compounds for which glutathione conjugation is an important detoxification pathway.

The mechanism of Hg 2+ toxicity in cultured human oral fibroblasts: The involvement of cellular thiols

To study amalgam-related toxicity in a primary target cell type, human oral fibroblasts were grown in a low-serum medium containing 1.25% fetal bovine serum and exposed to Hg 2+, a corrosion product of amalgam. A 1-h exposure to various concentrations of Hg 2+ resulted in a dose-dependent loss of colony forming efficiency. Removal of the low-molecular-weight thiol cysteine from the medium increased the toxicity of Hg 2+ almost 50-fold in comparison with complete medium or medium without fetal bovine serum. Accordingly, fetal bovine serum was not found to contain detectable levels of low-molecular-weight thiols. The levels of cellular free protein thiols were shown to be depleted by Hg 2+ at significantly lower concentrations of the metal ion than those required to decrease the levels of the major cellular low-molecular weight thiol glutathione. These decreases were dependent on the exposure conditions, i.e. the presence of serum and thiols, in a manner similar to the effect on colony forming efficiency. Other functions commonly related to cell viability, including the accumulation of the vital dye neutral red, the cytosolic retention of deoxyglucose and the mitochondrial reduction of tetrazolium were also inhibited by Hg 2+, albeit at higher concentrations. Finally, the depletion of cellular glutathione, by pre-exposure of the cells to the glutathione synthesis inhibitor buthionine sulfoximine, somewhat increased the toxicity of Hg 2+ and potentiated the depletion of protein thiols. Taken together, the toxicity of Hg 2+ in human oral fibroblasts was demonstrated in several assays of which colony forming efficiency was the most sensitive, cell killing by this agent was related to its high affinity for protein thiols, whereas glutathione showed a significant, but limited, ability to protect the cells from Hg 2+ toxicity.

On the expression and regulation of 5-lipoxygenase in human lymphocytes.

The expression of arachidonate 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) and the 5-lipoxygenase-activating protein (FLAP) genes in human tonsillar B cells and lymphoblastoid B-cell lines was demonstrated at the transcriptional level by reverse transcription-PCR analysis. Also, five lymphoblastoid T-cell lines were investigated and found to express the FLAP gene but not the 5-lipoxygenase gene, suggesting that the transcriptional regulation of these two genes is different. Western blot analysis of the cytosolic proteins from a lymphoblastoid B-cell line with an antiserum raised against purified human leukocyte 5-lipoxygenase revealed an immunoreactive band that comigrated with recombinant human 5-lipoxygenase. Intact B cells produced very low amounts of leukotriene B4 and 5-hydroxyeicosatetraenoic acid upon stimulation with the calcium ionophore A23187 and arachidonic acid, in comparison to the amounts formed by sonicates of these cells. However, preincubation of intact lymphoblastoid B cells with the glutathione-depleting agents azodicarboxylic acid bis(dimethylamide) or 1-chloro-2,4-dinitrobenzene prior to the addition of the calcium ionophore A23187 and arachidonic acid led to similar amounts of leukotriene B4 as were formed by sonicated cells. In contrast, the glutathione synthesis inhibitor buthionine sulfoximine diminished the cellular level of glutathione by greater than 90% but did not influence the production of leukotriene B4 or 5-hydroxyeicosatetraenoic acid in intact cells. These results demonstrate that certain drugs affecting the redox status can stimulate the cryptic 5-lipoxygenase activity in intact lymphoblastoid B cells but that the mechanism of this activation is unclear and appears not to be directly related to intracellular glutathione levels.