Free Thiol Form Research Articles

Crystallographic and cryogenic electron microscopic structures and enzymatic characterization of sulfur oxygenase reductase from Sulfurisphaera tokodaii

Sulfur oxygenase reductases (SORs) are present in thermophilic and mesophilic archaea and bacteria, and catalyze oxygen-dependent oxygenation and disproportionation of elemental sulfur. SOR has a hollow, spherical homo-24-mer structure and reactions take place at active sites inside the chamber. The crystal structures of SORs from Acidianus species have been reported. However, the states of the active site components (mononuclear iron and cysteines) and the entry and exit paths of the substrate and products are still in dispute. Here, we report the biochemical and structural characterizations of SORs from the thermoacidophilic archaeon Sulfurisphaera tokodaii (StSOR) and present high-resolution structures determined by X-ray crystallography and cryogenic electron microscopy (cryo-EM). The crystal structure of StSOR was determined at 1.73 Å resolution. At the catalytic center, iron is ligated to His86, His90, Glu114, and two water molecules. Three conserved cysteines in the cavity are located 9.5–13 Å from the iron and were observed as free thiol forms. A mutational analysis indicated that the iron and one of the cysteines (Cys31) were essential for both activities. The cryo-EM structure was determined at 2.24 Å resolution using an instrument operating at 200 kV. The two structures determined by different methodologies showed similar main chain traces, but the maps exhibited different features at catalytically important components. A possible role of StSOR in the sulfur metabolism of S. tokodaii (an obligate aerobe) is discussed based on this study. Given the high resolution achieved in this study, StSOR was shown to be a good benchmark sample for cryo-EM.

Quantitation of Total and Free Thiol Angiotensinogen as a Prognostic Marker for Preeclampsia.

Angiotensinogen mediates an important role in the pathophysiology of preeclampsia, a disorder of pregnancy characterized by hypertension and proteinuria usually after 20weeks of gestation. Angiotensinogen is found in two distinct posttranslational forms in the plasma, an oxidized and a reduced (free thiol) form. Higher levels of the oxidized form are associated with an increased risk of preeclampsia. We have developed novel ELISA assays to quantitate the levels of total and free thiol angiotensinogen allowing for calculation of the amount of oxidized angiotensinogen species. We describe the methodology for performing these assays.

Dual roles of different redox forms of complement factor H in protecting against age related macular degeneration

Complement Factor H (CFH) is an important inhibitor of the alternate complement pathway in Bruch's membrane (BM), located between the choriocapillaris and the retinal pigment epithelium. Furthermore dysfunction of its activity as occurs with certain polymorphisms is associated with an increased risk of age related macular degeneration (AMD).The retina is a site of high generation of reactive oxygen species (ROS) and dysfunction of redox homeostasis in this milieu also contributes to AMD pathogenesis. In this study we wanted to explore if CFH exists in distinct redox forms and whether these species have unique protective biological functions. CFH can be reduced by the naturally occurring thioredoxin − 1 in CFH domains 1–4, 17–20. We found a duality of function between the oxidised and reduced forms of CFH. The oxidised form was more efficient in binding to C3b and lipid peroxidation by-products that are known to accumulate in the retinae and activate the alternate complement pathway.Oxidised CFH enhances Factor I mediated cleavage of C3 and C3b whereas the reduced form loses this activity. In the setting of oxidative stress (hydrogen peroxide)-mediated death of human retinal pigment epithelial cells as can occur in AMD, the free thiol form of CFH offers a protective function compared to the oxidised form. We found for the first time using a novel ELISA system we have developed for free thiol CFH, that both redox forms of CFH are found in the human plasma. Furthermore there is a distinct ratio of these redox forms in plasma depending if an individual has early or late AMD, with individuals with early AMD having higher levels of the free thiol form compared to late AMD.

Transmembrane redox control and proteolysis of PdeC, a novel type of c‐di‐GMP phosphodiesterase

The nucleotide second messenger c‐di‐GMP nearly ubiquitously promotes bacterial biofilm formation, with enzymes that synthesize and degrade c‐di‐GMP being controlled by diverse N‐terminal sensor domains. Here, we describe a novel class of widely occurring c‐di‐GMP phosphodiesterases (PDE) that feature a periplasmic “CSS domain” with two highly conserved cysteines that is flanked by two transmembrane regions (TM1 and TM2) and followed by a cytoplasmic EAL domain with PDE activity. Using PdeC, one of the five CSS domain PDEs of Escherichia coli K‐12, we show that DsbA/DsbB‐promoted disulfide bond formation in the CSS domain reduces PDE activity. By contrast, the free thiol form is enzymatically highly active, with the TM2 region promoting dimerization. Moreover, this form is processed by periplasmic proteases DegP and DegQ, yielding a highly active TM2 + EAL fragment that is slowly removed by further proteolysis. Similar redox control and proteolysis was also observed for a second CSS domain PDE, PdeB. At the physiological level, CSS domain PDEs modulate production and supracellular architecture of extracellular matrix polymers in the deeper layers of mature E. coli biofilms.

Posttranslational forms of beta 2-glycoprotein I in the pathogenesis of the antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is an autoimmune disease characterised by a procoagulant state that predisposes to recurrent thrombosis and miscarriages. Two major discoveries have advanced our understanding of the underlying complex pathogenesis of the APS. The first was the discovery that beta-2 glycoprotein-1 (β2GPI) is the major auto antigen in APS. The second was the discovery in more recent years that β2GPI contains allosteric disulphide bonds susceptible to posttranslational modification that may be involved in the development of autoantibodies in APS. The main allosteric disulphide bond in the fifth domain of β2GPI can exist in two redox states: free thiol or oxidised. It is the conformational transformation of β2GPI from its free thiol form to its more immunogenic oxidised form that exposes neo-epitopes on the first and fifth domains. The purpose of this review is to highlight the recent findings on the posttranslational forms of β2GPI in the pathogenesis of APS. We suggest that novel assays quantitating the different redox forms of β2GPI in plasma or serum may be used to supplement existing clinical and laboratory assays to more accurately stratify risk of thrombosis or miscarriage in APS patients.

Free Thiol of Transthyretin in Human Plasma Most Accessible to Modification/Oxidation.

Free-thiol(s) in proteins, especially, when located on the surface of the molecule, are susceptible to oxidation/modification, which may cause loss of function or an alteration in the ternary structure. This suggests that the status of thiol group(s) of cysteine residue(s) in a protein, i.e., free-thiol versus an oxidized/modified form, in vivo, could reflect the physiological state of the molecule with respect to susceptibility to oxidative stress. To address this issue, we established an efficient method for isolating proteins that contain free thiol groups from a complex mixture, which permits the amount of free-thiol form(s) to modified/oxidized forms to be estimated. Albumin, which accounts for 55% of the total plasma proteins and has such a free thiol and has been reported to scavenge various reactive oxygen species (ROS) in vivo. The developed method was used to isolate the free form of albumin from fresh plasma. However, contrary to our expectations, transthyretin (TTR), which also has a single free thiol, was found to be the major protein that was the most susceptible to modification/oxidation. In addition, the free-thiol form could be separated from oxidized or modified molecules, permitting the relative abundance of the free-thiol form to be estimated. The findings show that the levels of the free-thiol form of TTR in plasma was significantly lowered after a hydrogen peroxide treatment, even at low concentrations (0.1 mM), suggesting that TTR could be a useful biomarker for monitoring a ROS imbalance in relation to various oxidative stress conditions.

Do Beta 2-Glycoprotein I Disulfide Bonds Protect the Human Retina in the Setting of Age-Related Macular Degeneration?

Age-related macular degeneration (AMD) affects the region of the retina that is responsible for high-resolution vision. It is a major cause of blindness in the aging population. This is the first study that examines the association of redox-modified, cysteine-based, post-translational forms of beta 2-glycoprotein I (β2GPI) in the plasma of individuals with early and late stages of patients with AMD compared with controls. Exploration is also undertaken to assess whether the free thiol form of β2GPI versus the oxidized disulfide form have distinct functional properties in the setting of hydrogen peroxide (H(2)O(2))-mediated cell death of an immortalized human retinal pigment epithelium (RPE) cell line. We demonstrate β2GPI in the retina and choroid of patients with AMD. Free thiol β2GPI is shown to protect the immortalized human RPE cell line against H(2)O(2)-induced cell death, whereas the oxidized form of β2GPI and free thiol bovine serum albumin were not protective. Free thiol β2GPI levels were significantly decreased in patients with late AMD compared with early AMD and healthy controls. Our observations lead to the hypothesis that free thiol β2GPI may protect against oxidative stress injury to RPE cells in the early stages of AMD.

Tissue Factor De-encryption, Thrombus Formation, and Thiol-disulfide Exchange

Tissue factor (TF) by forming a complex with factor VIIa (FVIIa) initiates blood coagulation. It was traditionally believed that the separation of FVIIa in circulation from subendothelial TF was the main control that was preventing spontaneous initiation of thrombosis and that circulating cells and endothelium did not express TF protein at rest in healthy individuals. However, TF has been detected in healthy human plasma and animal models of thrombosis, which indicate that TF in circulation can contribute to thrombin generation and fibrin formation after an activation event. Circulating TF-and indeed, most of the TF on the cell surface-is "encrypted" or coagulation inactive. The de-encryption step involves exposure of phosphatidylserine (PS), but PS exposure alone is insufficient for full TF activity. Allosteric disulfide bonds control protein function by mediating conformal change through the formation and breaking of disulfide bonds. TF contains a typical surface exposed allosteric bond in the membrane proximal fibronectin type III domain. Thiol-disulfide exchange involving this disulfide is implicated in TF activation with the formation of the disulfide bond corresponding with the active conformation of TF and free thiol or thiol-modified forms corresponding with encryption. Although the exact mechanism by which TF de-encryption occurs remains a subject of debate, thiol blockade and inhibition of oxidoreductases show an important role for thiol-disulfide reactions in platelet-independent pathways of coagulation in vitro and in vivo. In particular, redox active extracellular protein disulfide isomerase is involved in the earliest stages of thrombus initiation and has proven to be a potential target for antithrombotic drug development.

A Manganese Superoxide Dismutase (SOD2)-Mediated Adaptive Response

Very low doses of ionizing radiation, 5 to 100 mGy, can induce adaptive responses characterized by elevation in cell survival and reduction in micronuclei formation. Utilizing these end points, RKO human colon carcinoma and transformed mouse embryo fibroblasts (MEF), wild-type or knockout cells missing TNF receptors 1 and 2 (TNFR1(-)R2(-)), and C57BL/6 and TNFR1(-)R2(-) knockout mice, we demonstrate that intact TNF signaling is required for induction of elevated manganese superoxide dismutase (SOD2) activity (P < 0.001) and the subsequent expression of these SOD2-mediated adaptive responses when cells are challenged at a later time with 2 Gy. In contrast, amifostine's free thiol form WR1065 can directly activate NF-κB giving rise to elevated SOD2 activity 24 h later and induce an adaptive response in both MEF wild-type and TNF signaling defective TNFR1(-)R2(-) cells. Transfection of cells with SOD2 siRNA completely abolishes both the elevation in SOD2 activity and expression of the adaptive responses. These results were confirmed in vivo using a micronucleus assay in splenocytes derived from C57BL/6 and TNFR1(-)R2(-) knockout mice that were exposed to 100 mGy or 400 mg/kg amifostine 24 h prior to exposure to a 2 Gy whole-body dose. A dose of 100 mGy also conferred enhanced protection to C57BL/6 mice exposed 24 h later to 100 mg/kg of N-Ethyl-N-nitrosourea (ENU). While very low radiation doses require an intact TNF signaling process to induce a SOD2-mediated adaptive response, amifostine can induce a similar adaptive response in both TNF receptor competent and knockout cells, respectively.

High-Affinity VEGF Antagonists by Oligomerization of a Minimal Sequence VEGF-Binding Domain

Vascular endothelial growth factor (VEGF) neutralizing antagonists including antibodies or receptor extracellular domain Fc fusions have been applied clinically to control angiogenesis in cancer, wet age-related macular degeneration, and edema. We report here the generation of high-affinity VEGF-binding domains by chemical linkage of the second domain of the VEGF receptor Flt-1 (D2) in several configurations. Recombinant D2 was expressed with a 13 a.a. C-terminal tag, including a C-terminal cysteine to enable its dimerization by disulfide bond formation or by attachment to divalent PEGs and oligomerization by coupling to multivalent PEGs. Disulfide-linked dimers produced by Cu(2+) oxidation of the free-thiol form of the protein demonstrated picomolar affinity for VEGF in solution, comparable to that of a D2-Fc fusion (sFLT01) and ~50-fold higher than monomeric D2, suggesting the 26 a.a. tag length between the two D2 domains permits simultaneous interaction of both faces of the VEGF homodimer. Extending the separation between the D2 domains by short PEG spacers from 0.35 kD to 5 kD produced a modest ~2-fold increase in affinity over the disulfide, thus defining the optimal distance between the two D2 domains for maximum affinity. By surface plasmon resonance (SPR), a larger (~5-fold) increase in affinity was observed by conjugation of the D2 monomer to the termini of 4-arm PEG, and yielding a product with a larger hydrodynamic radius than sFLT01. The higher affinity displayed by these D2 PEG tetramers than either D2 dimer or sFLT01 was largely a consequence of a slower rate of dissociation, suggesting the simultaneous binding by these tetramers to neighboring surface-bound VEGF. Finally, disulfide-linked D2 dimers showed a greater resistance to autocatalytic fragmentation than sFLT01 under elevated temperature stress, indicating such minimum-sequence constructs may be better suited for sustained-release formulations. Therefore, these constructs represent novel Fc-independent VEGF antagonists with ultrahigh affinity, high stability, and a range of hydrodynamic radii for application to multiple therapeutic targets.

Novel assays of thrombogenic pathogenicity in the antiphospholipid syndrome based on the detection of molecular oxidative modification of the major autoantigen β2‐glycoprotein I

ObjectiveBeta-2-glycoprotein I (β2GPI) constitutes the major autoantigen in the antiphospholipid syndrome (APS), a common acquired cause of arterial and venous thrombosis. We recently described the novel observation that β2GPI may exist in healthy individuals in a free thiol (biochemically reduced) form. The present study was undertaken to quantify the levels of total, reduced, and posttranslationally modified oxidized β2GPI in APS patients compared to various control groups.MethodsIn a retrospective multicenter analysis, the proportion of β2GPI with free thiols in serum from healthy volunteers was quantified. Assays for measurement of reduced as well as total circulating β2GPI were developed and tested in the following groups: APS (with thrombosis) (n = 139), autoimmune disease with or without persistent antiphospholipid antibodies (aPL) but without APS (n = 188), vascular thrombosis without APS or aPL (n = 38), and healthy volunteers (n = 91).ResultsTotal β2GPI was significantly elevated in patients with APS (median 216.2 μg/ml [interquartile range 173.3–263.8]) as compared to healthy subjects (median 178.4 μg/ml [interquartile range 149.4–227.5] [P < 0.0002]) or control patients with autoimmune disease or vascular thrombosis (both P < 0.0001). The proportion of total β2GPI in an oxidized form (i.e., lacking free thiols) was significantly greater in the APS group than in each of the 3 control groups (all P < 0.0001).ConclusionThis large retrospective multicenter study shows that posttranslational modification of β2GPI via thiol-exchange reactions is a highly specific phenomenon in the setting of APS thrombosis. Quantification of posttranslational modifications of β2GPI in conjunction with standard laboratory tests for APS may offer the potential to more accurately predict the risk of occurrence of a thrombotic event in the setting of APS.

Molecular pathophysiology of the antiphospholipid syndrome: the role of oxidative post-translational modification of beta 2 glycoprotein I.

It has been well established that antiphospholipid antibodies and specifically those directed against beta 2 glycoprotein I (β2GPI) are pathogenic for the development of thrombosis in the antiphospholipid syndrome (APS). Several groups have shown that anti-β2GPI antibodies, in complex with β2GPI, elicit effects on blood cells and coagulation-fibrinolysis proteins, which prime the arterial and venous vasculature for the development of thrombosis. However, much less is known about the mechanism initiating the production of autoantibodies against β2GPI, a physiological abundant protein of blood. In the current review, novel findings are presented regarding the structure and oxidative post-translational modifications of β2GPI, which trigger the immune response. The majority of circulating β2GPI exists in a form containing unpaired cysteines (free thiols), which constitutes the reduced form of β2GPI. The free thiols exposed on β2GPI are involved in the interaction with platelets and endothelial cells. We propose that this abundant pool of free thiols may serve as an antioxidant reservoir protecting cells or critical molecules from oxidative stress. Oxidation of β2GPI confers an increase in its immunogenicity through a Th1 immunological mechanism. The clinical significance of these observations is that serum from patients with APS, assessed by a novel ELISA assay, have a significant increase in oxidised β2GPI. These findings hold promise, not only for the delineation of the role of β2GPI as an immunological target, but also for the development of improved diagnostic and prognostic assays for APS.

Study of the Thiol/Disulfide Redox Systems of the Anaerobe Desulfovibrio vulgaris Points Out Pyruvate:Ferredoxin Oxidoreductase as a New Target for Thioredoxin 1

Sulfate reducers have developed a multifaceted adaptative strategy to survive against oxidative stresses. Along with this oxidative stress response, we recently characterized an elegant reversible disulfide bond-dependent protective mechanism in the pyruvate:ferredoxin oxidoreductase (PFOR) of various Desulfovibrio species. Here, we searched for thiol redox systems involved in this mechanism. Using thiol fluorescent labeling, we show that glutathione is not the major thiol/disulfide balance-controlling compound in four different Desulfovibrio species and that no other plentiful low molecular weight thiol can be detected. Enzymatic analyses of two thioredoxins (Trxs) and three thioredoxin reductases allow us to propose the existence of two independent Trx systems in Desulfovibrio vulgaris Hildenborough (DvH). The TR1/Trx1 system corresponds to the typical bacterial Trx system. We measured a TR1 apparent K(m) value for Trx1 of 8.9 μM. Moreover, our results showed that activity of TR1 was NADPH-dependent. The second system named TR3/Trx3 corresponds to an unconventional Trx system as TR3 used preferentially NADH (K(m) for NADPH, 743 μM; K(m) for NADH, 5.6 μM), and Trx3 was unable to reduce insulin. The K(m) value of TR3 for Trx3 was 1.12 μM. In vitro experiments demonstrated that the TR1/Trx1 system was the only one able to reactivate the oxygen-protected form of Desulfovibrio africanus PFOR. Moreover, ex vivo pulldown assays using the mutant Trx1(C33S) as bait allowed us to capture PFOR from the DvH extract. Altogether, these data demonstrate that PFOR is a new target for Trx1, which is probably involved in the protective switch mechanism of the enzyme.

Mechanism of Inhibition of Aliphatic Epoxide Carboxylation by the Coenzyme M Analog 2-Bromoethanesulfonate

The bacterial metabolism of epoxypropane formed from propylene oxidation uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) as the nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation to form acetoacetate in a three-step metabolic pathway. 2-Ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of this pathway, is the only known member of the disulfide oxidoreductase family of enzymes that is a carboxylase. In the present work, the CoM analog 2-bromoethanesulfonate (BES) is shown to be a reversible inhibitor of 2-KPCC and hydroxypropyl-CoM dehydrogenase but not of epoxyalkane:CoM transferase. Further investigations revealed that BES is a time-dependent inactivator of dithiothreitol-reduced 2-KPCC, where the redox active cysteines are in the free thiol forms. BES did not inactivate air-oxidized 2-KPCC, where the redox active cysteine pair is in the disulfide form. The inactivation of 2-KPCC exhibited saturation kinetics, and CoM slowed the rate of inactivation. Mass spectral analysis demonstrated that BES inactivation of reduced 2-KPCC occurs with covalent modification of the interchange thiol (Cys(82)) by a group with a molecular mass identical to that of ethylsulfonate. The flavin thiol Cys(87) was not alkylated by BES under reducing conditions, and no amino acid residues were modified by BES in the oxidized enzyme. The UV-visible spectrum of BES-modifed 2-KPCC showed the characteristic charge transfer absorbance expected with alkylation at Cys(82). These results identify BES as a reactive CoM analog that specifically alkylates the interchange thiol that facilitates thioether bond cleavage and enolacetone formation during catalysis.

Site-Selective Fragmentation of Peptides and Proteins at Quinone-Modified Cysteine Residues Investigated by ESI-MS

Described herein are several unique analytical applications utilizing mass spectrometry and the selective modification of the free thiol form of cysteine in both peptides and proteins by various quinones. This simple modification can be used to quantify the number of free or disulfide bound cysteines in a protein. In addition, quinone modification can also be used to easily probe the solvent accessibility of cysteine residues, which provides information about protein structure or folding state. Furthermore, the chromophoric properties of the quinone moiety can be leveraged for site specific photodissociation of the backbone. The photodissociation reveals both the presence and location of modified cysteine residues. For example, cleavage of the protein backbone of alpha-hemoglobin is observed selectively at a single cysteine out of 140 residues in the whole protein. This selective backbone fragmentation is accompanied by a parent ion mass loss, which is unique to the modifying quinone. When combined, this information can be used to determine both the presence and site of modification generated by naturally occurring molecules, such as dopamine, which can harness quinone chemistry to modify proteins.

Abstract 507: Elevated manganese superoxide dismutase (SOD2) activity as a mechanism for the low dose dadiation- and thiol-induced adaptive responses in human RKO36 colon carcinoma cells and BFS2C mouse fibrosarcoma cells

Abstract Exposure of cells to low non-lethal doses of ionizing radiation (≤ 10 cGy) or WR1065, the active free thiol form of amifostine, can induce pro-survival pathways that result in protection against the damaging effects of a 2 Gy dose of ionizing radiation. One such signaling pathway involves the elevation of active manganese superoxide dismutase (SOD2). SOD2 is a mitochondrial matrix protein that serves as the primary mitochondrial defense against superoxide formation. Its primary function is to facilitate the dismutation of two molecules of superoxide anion (O2−) produced by normal respiratory processes or following exposure to ionizing radiation into water and hydrogen peroxide. To characterize the role of SOD2 in the radiation- and thiol-induced adaptive responses, RKO36 human colon carcinoma cells and BFS2C mouse fibrosarcoma cells were exposed to 10 cGy x-rays and 40 μM or 4 mM WR1065 and SOD2 activity measured 24 h later. Significant increases in SOD2 activity in RKO36 cells (3.7-fold, P &amp;lt; 0.001) and BFS2C cells (3.0-fold, P = 0.005) were observed 24 h after exposure to 10 cGy. SOD2 activity was also observed to be significantly elevated in RKO36 cells (3.5-fold, P = 0.014 and 3.4-fold, P = 0.017) and BFS2C cells (3.7-fold, P = 0.007 and 2.5 fold, P = 0.021) 24 h after treatment with 40 μM or 4 mM WR1065, respectively. The protective effect of the low dose radiation- and thiol-induced elevation in active SOD2 was examined using the endpoint of micronuclei formation as a measure of chromosomal damage. Exposure of RKO36 and BFS2C cells to 2 Gy resulted in a significant increase in the mean number of micronuclei observed relative to unirradiated control cells (8.8 versus 2.2, P &amp;lt; 0.001; 5.4 versus 2.1, P &amp;lt; 0.001, respectively). The frequency of micronuclei formation was significantly reduced in both RKO36 cells (P = 0.003) and BFS2C cells (P = 0.011) exposed to 10 cGy 24 h prior to the 2 Gy dose. A significant reduction in micronuclei formation was also observed in RKO36 and BFS2C cells treated with 40 μM or 4 mM WR1065 30 min or 24 h prior to irradiation with 2 Gy. Treatment of both cell lines with SOD2 siRNA reduced SOD2 activity relative to mock-transfected control cells which resulted in the abrogation of both the radiation- and thiol-induced protective effect. This work was supported by NIH/NCI grant R01 CA132998 and DOE grant DE- PS02-08ER08-21 (D.J.G.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 507.

Maintenance of Manganese Superoxide Dismutase (SOD2)-Mediated Delayed Radioprotection Induced by Repeated Administration of the Free Thiol Form of Amifostine

Thiol-containing drugs such as WR1065, the free thiol form of amifostine, have been shown to induce a delayed radioprotective effect in both malignant and non-malignant cells. In mammalian cells exposed to a dose as low as 40 microM WR1065, the redox-sensitive nuclear transcription factor kappaB (NFkappaB) is activated, leading to an elevation in the expression of the antioxidant gene manganese superoxide dismutase (SOD2) and a concomitant increase in active SOD2 enzyme levels that peaks 24 to 32 h later. Exposure of cells to ionizing radiation during the period of elevated SOD2 enzymatic activity results in an enhanced radiation resistance. This is seen as an increase in surviving fraction as determined by standard colony formation assays. To determine whether this delayed radioprotection can be maintained over a prolonged period in cells of either malignant or non-malignant origin, both human microvascular endothelial cells (HMEC) and SA-NH mouse sarcoma cells were grown to confluence and exposed to 40 muM WR1065 using three administration protocols: (1) daily drug exposure for 10 days followed each day by irradiation with 2 Gy; (2) drug exposure once every 48 h followed by irradiation with 2 Gy 48 h later for 14 days; and (3) drug exposure every 72 h followed by irradiation with 2 Gy 72 h later for 12 days. As a function of each experimental condition, cell numbers and associated SOD2 enzymatic activities were measured at the time of each irradiation. None of the treatment conditions were toxic to either HMEC or SA-NH cells. SOD2 activity was elevated 5.3- and 1.8-fold over background on average for HMEC exposed to 40 microM WR1065 every 24 or 48 h, respectively. Likewise, SOD2 activity was elevated in SA-NH mouse sarcoma cells 7.8- and 4.9-fold after daily exposure to WR1065 or exposure to WR1065 once every 48 h, respectively. Both HMEC and SA-NH cells exhibited enhanced radiation resistance that correlated with the increase in SOD2 activity. The average respective increases in cell survival were 1.33 +/- 0.01 (SEM), 1.23 +/- 0.01 and 1.04 +/- 0.01 for HMEC exposed to WR1065 every 24, 48 and 72 h, respectively, and 1.27 +/- 0.01, 1.18 +/- 0.02 and 1.02 +/- 0.02 for SA-NH cells exposed to WR1065 every 24, 48 and 72 h, respectively. Both the elevation in WR1065-induced SOD2 enzymatic activity and the corresponding increase in radiation resistance were completely inhibited in HMEC and SA-NH cells transfected with human or mouse SOD2 siRNA oligomers and irradiated 24 h later. These data demonstrate that a delayed radioprotective effect can be induced and maintained over a prolonged period in both non-malignant and malignant cells exposed to thiol-containing drugs such as WR1065. For non-malignant cells this represents a novel paradigm for radiation protection. The ability of WR1065 to induce a persistent elevated radiation resistance in malignant cells, however, suggests a new potential concern regarding the issue of tumor protection in patients exposed to thiol-containing drugs.

Manganese Superoxide Dismutase (SOD2)-Mediated Delayed Radioprotection Induced by the Free Thiol Form of Amifostine and Tumor Necrosis Factor α

RKO36 cells, a subclone of RKO colorectal carcinoma cells that have been stably transfected with the pCMV-EGFP2Xho vector, were grown to confluence and then exposed to either the radioprotector WR-1065, i.e. the active thiol form of amifostine, for 30 min at doses of 40 microM and 4 mM or the cytokine tumor necrosis factor alpha (TNFalpha, TNFA) for 30 min at a concentration of 10 ng/ml and then washed. Total protein was isolated as a function of time up to 32 h after these treatments. Both doses of WR-1065 as well as the concentration of TNFalpha used were effective in elevating intracellular levels of the antioxidant protein SOD2 (also known as MnSOD) at least 15-fold over background levels as determined by Western blot analysis, while measured SOD2 activity was elevated between 5.5- and 6.9-fold. SOD2 reached a maximal level 24 h and 20 h after WR-1065 and TNFalpha treatments, respectively. The antioxidant proteins catalase and glutathione peroxidase (GPX) were also monitored over the 32-h period. In contrast to the robust changes observed in intracellular levels of SOD2 as a function of time after exposure of cells to WR-1065, catalase levels were elevated only 2.6-fold over background as determined by Western blot analysis, while GPX activity was unaffected by WR-1065 exposure. GPX protein levels were extremely low in cells, and analysis of GPX activity using a spectrophotometric method based on the consumption of reduced NADPH also revealed no measurable change as a function of WR-1065 or TNFalpha exposure. RKO36 cells either were irradiated with X rays in the presence of either 40 microM or 4 mM WR-1065 or 10 ng/ml TNFalpha or were irradiated 24 or 20 h later, respectively, when SOD2 protein levels were most elevated. The concentrations and exposure conditions used for WR-1065 and TNFalpha were not cytotoxic and had no effect on plating efficiencies or cell survival compared to untreated controls. No protection or sensitization was observed for cells irradiated in the presence of 40 microM WR-1065 or TNFalpha. Survival was elevated 1.90-fold for cells irradiated in the presence of 4 mM WR-1065. When RKO36 cells were irradiated with 2 Gy 24 h after 40 microM or 4 mM WR-1065 and 20 h after TNFalpha treatments when SOD2 levels were the most increased, survival was elevated 1.42-, 1.48- and 1.36-fold, respectively. This increased survival represents a SOD2-mediated delayed radioprotective effect. SOD2 appears to be an important antioxidant gene whose inducible expression is an important element in adaptive cellular responses in general, and the delayed radioprotective effect in particular. It can be induced by a range of agents including cytoprotective nonprotein thiols such as WR-1065 and pleiotropic cytokines such as TNFalpha.

Differential activation of nuclear transcription factor κB, gene expression, and proteins by amifostine's free thiol in human microvascular endothelial and glioma cells

The effects of WR1065 (SH), the free thiol form of amifostine, on nuclear transcription factor kappaB (NFkappaB) activation, manganese superoxide dismutase (MnSOD) gene expression, and secretion of human vascular endothelial cell growth factor (hVEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-alpha (TNF-alpha), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, P-selectin, and interleukins IL-1alpha, IL-6, and IL-8 were investigated and compared in human microvascular endothelial (HMEC) and human glioma cells. WR1065 was evaluated at 2 concentrations, 4 mmol/L, ie, its most effective cytoprotective dose, and 40 micromol/L, a noncytoprotective but highly effective dose capable of preventing radiation and chemotherapeutic drug-induced mutations in exposed cells. A 30-minute exposure of HMEC and glioma cell lines U87 and U251 to WR1065 at either of the concentrations resulted in a marked activation of NFkappaB as determined by a gel shift assay, with the maximum effect observed between 30 minutes and 1 hour after treatment. Using a supershift assay, WR1065 exposure was observed to affect only the p50-p65 heterodimer, and not the homodimers or heterodimers containing p52 or c-Rel subunits of NFkappaB. WR1065 was also found to enhance MnSOD gene expression in both HMEC and glioma cells. Gene expression was enhanced 1.8-fold over control levels in HMEC over a period ranging from 12 to 24 hours after the time of maximum activation of NFkappaB. In contrast, MnSOD gene expression in U87 cells rose 3.5 times above control levels over this same period. WR1065 had no effect on the levels of adhesion molecules, cytokines, and growth factors secreted by cells exposed for up to 24 hours as measured by enzyme-linked immunosorbent assay.

Bioanalysis of captopril: two sensitive high-performance liquid chromatographic methods with pre- or postcolumn fluorescent labeling.

This study describes the development and comparison of two HPLC methods for the analysis of the antihypertensive drug captopril. The first method is based on a precolumn derivatization of captopril with the fluorescent label monobromobimane (MBB). The second method is based on a postcolumn reaction with the fluorescent reagent o-phthaldialdehyde (OPA). Since the disulfide metabolites of captopril can be reconverted to the active drug in vivo, the bioanalysis of captopril should involve both the determination of its free thiol form (free captopril) and the total amount of free thiol and reducible disulfides (total captopril). For total captopril analysis, disulfides were reduced with tributylphosphine (TBP) prior to protein precipitation. Since the reducing agent interfered with the MBB derivatization reaction, this method was not suitable for total captopril analysis. Both methods were validated for the bioanalysis of free captopril in human plasma. After removal of plasma proteins, samples were analyzed without an additional extraction procedure. The limit of quantitation in plasma was 12.5 ng/ml for the MBB method (limit of detection 30 pg) and 25 ng/ml for the OPA method (limit of detection 50 pg). The OPA method was also validated for total captopril analysis in human plasma and urine. The limit of quantitation was 25 ng/ml in plasma and 250 ng/ml in urine (limit of detection 50 pg). We conclude that for the analysis of free captopril the precolumn MBB method is superior to the OPA method since only the derivatization reaction has to be carried out immediately. The postcolumn OPA method is especially suitable for the analysis of total captopril since reducing reagents and high concentrations of endogenous thiols do not interfere with the derivatization reaction.