PARP1- and CTCF-Mediated Interactions between Active and Repressed Chromatin at the Lamina Promote Oscillating Transcription.

Transcriptionally active and inactive chromatin domains tend to segregate into separate sub-nuclear compartments to maintain stable expression patterns. However, here we uncovered an inter-chromosomal network connecting active loci enriched in circadian genes to repressed lamina-associated domains (LADs). The interactome is regulated by PARP1 and its co-factor CTCF. They not only mediate chromatin fiber interactions but also promote the recruitment of circadian genes to the lamina. Synchronization of the circadian rhythm by serum shock induces oscillations in PARP1-CTCF interactions, which is accompanied by oscillating recruitment of circadian loci to the lamina, followed by the acquisition of repressive H3K9me2 marks and transcriptional attenuation. Furthermore, depletion of H3K9me2/3, inhibition of PARP activity by olaparib, or downregulation of PARP1 or CTCF expression counteracts both recruitment to the envelope and circadian transcription. PARP1- and CTCF-regulated contacts between circadian loci and the repressive chromatin environment at the lamina therefore mediate circadian transcriptional plasticity.

Complex Formation of Resorufin and Resazurin with Β-Cyclodextrins: Can Cyclodextrins Interfere with a Resazurin Cell Viability Assay?

Resazurin (or Alamar Blue) is a poorly fluorescent dye. During the cellular reduction of resazurin, its highly fluorescent product resorufin is formed. Resazurin assay is a commonly applied method to investigate viability of bacterial and mammalian cells. In this study, the interaction of resazurin and resorufin with ß-cyclodextrins was investigated employing spectroscopic and molecular modeling studies. Furthermore, the influence of ß-cyclodextrins on resazurin-based cell viability assay was also tested. Both resazurin and resorufin form stable complexes with the examined ß-cyclodextrins (2.0-3.1 × 10³ and 1.3-1.8 × 10³ L/mol were determined as binding constants, respectively). Cells were incubated for 30 and 120 min and treated with resazurin and/or ß-cyclodextrins. Our results suggest that cyclodextrins are able to interfere with the resazurin-based cell viability assay that presumably results from the following mechanisms: (1) inhibition of the cellular uptake of resazurin and (2) enhancement of the fluorescence signal of the formed resorufin.

A high-performance liquid chromatography-based assay of glutathione transferase omega 1 supported by glutathione or non-physiological reductants.

The unusual glutathione S-transferase GSTO1 reduces, rather than conjugates, endo- and xenobiotics, and its role in diverse cellular processes has been proposed. GSTO1 has been assayed spectrophotometrically by measuring the disappearance of its substrate, S-(4-nitrophenacyl)glutathione (4-NPG), in the presence of 2-mercaptoethanol that regenerates GSTO1 from its mixed disulfide. To assay GSTO1 in rat liver cytosol, we have developed a high-performance liquid chromatography (HPLC)-based procedure with two main advantages: (i) it measures the formation of the 4-NPG reduction product 4-nitroacetophenone, thereby offering improved sensitivity and accuracy, and (ii) it can use glutathione, the physiological reductant of GSTO1, which is impossible to do with the spectrophotometric procedure. Using the new assay, we show that (i) the GSTO1-catalyzed reduction of 4-NPG in rat liver cytosol also yields 1-(4-nitrophenyl)ethanol, whose formation from 4-nitroacetophenone requires NAD(P)H; (ii) the two assays measure comparable activities with 2-mercaptoethanol or tris(2-carboxyethyl)phosphine used as reductant; (iii) the cytosolic reduction of 4-NPG is inhibited by GSTO1 inhibitors (KT53, 5-chloromethylfluorescein diacetate, and zinc), although the inhibitory effect is strikingly influenced by the type of reductant in the assay and by the sequence of reductant and inhibitor addition. Characterization of GSTO1 inhibitors with the improved assay provides better understanding of interaction of these chemicals with the enzyme.

Reduction of the Pentavalent Arsenical Dimethylarsinic Acid and the GSTO1 Substrate S-(4-Nitrophenacyl)glutathione by Rat Liver Cytosol: Analyzing the Role of GSTO1 in Arsenic Reduction.

Dimethylarsinic acid (DMAs(V)) is the major urinary metabolite of inorganic arsenic. The relatively atoxic DMAs(V) is reduced in the body to the much more toxic and thiol-reactive dimethylarsinous acid (DMAs(III)). Glutathione S-transferase omega 1 (GSTO1) can catalyze this toxification step; however, its role in the reduction of DMAs(V) in vivo or by tissue extracts is unclear. Therefore, we assessed the role of GSTO1 in the reduction of DMAs(V) to DMAs(III) by rat liver cytosol. The experiments revealed that glutathione (GSH) supported the cytosolic DMAs(V) reduction specifically and that GSH analogues and GSH conjugates, such as S-alkylglutathiones and S-(4-nitrophenacyl)glutathione (4-NPG; a GSTO1 specific substrate), inhibited the formation of DMAs(III). Observations in line with the view that GSTO1 catalyzes the cytosolic reduction of DMAs(V) include (i) findings pointing to the presence of a GSH-binding site on the DMAs(V)-reducing cytosolic enzyme, (ii) identical responsiveness of the DMAs(V)- and 4-NPG-reducing activities in rat liver cytosol to the GSTO1 specific inhibitors KT53 and chloromethylfluorescein diacetate, and (iii) perfect coelution of the two activities during affinity and anion exchange chromatography of cytosolic proteins. Other observations appear ambiguous as to the role of GSTO1 in the cytosolic reduction of DMAs(V). These include the different sensitivities of the DMAs(V)-reducing and GSTO1 activities to aurothioglucose, trivalent antimony, and zinc ions, as well as the preserved GSTO1 activity in cytosols whose DMAs(V)-reducing activity was lost due to spontaneous thiol oxidation. These disparate findings may be reconciled by assuming that GSTO1 catalyzes the reduction of both DMAs(V) and 4-NPG in rat liver cytosol; however, the enzyme employs different sites and/or mechanisms when reducing these substrates.

Reduction of dimethylarsinic acid to the highly toxic dimethylarsinous acid by rats and rat liver cytosol.

Dimethylarsinic acid (DMAs(V)), the major urinary metabolite of inorganic arsenic, is weakly cytotoxic, whereas its reduced form, dimethylarsinous acid (DMAs(III)), is highly toxic. Although glutathione S-transferase omega 1 (GSTO1) and arsenic methyltransferase have been shown or thought to catalyze DMAs(V) reduction, their role in DMAs(V) reduction in vivo, or in cell extracts is uncertain. Therefore, the reduction of DMAs(V) to DMAs(III) in rats and in rat liver cytosol was studied to better understand its mechanism. To assess DMAs(V) reduction in rats, a novel procedure was devised based on following the accumulation of red blood cell (RBC)-bound dimethylarsenic (DMAs), which represents DMAs(III), in the blood of DMAs(V)-injected anesthetized rats. These studies indicated that rats reduced DMAs(V) to DMAs(III) to a significant extent, as in 90 min 31% of the injected 50 µmol/kg DMAs(V) dose was converted to DMAs(III) that was sequestered by the circulating erythrocytes. Pretreatment of rats with glutathione (GSH) depletors (phorone or BSO) delayed the elimination of DMAs(V) and the accumulation of RBC-bound DMAs, whereas the indirect methyltransferase inhibitor periodate-oxidized adenosine was without effect. Assessment of DMAs(V)-reducing activity of rat liver cytosol revealed that reduction of DMAs(V) required cytosolic protein and GSH and was inhibited by thiol reagents, GSSG and dehydroascorbate. Although thioredoxin reductase (TRR) inhibitors (aurothioglucose and Sb(III)) inhibited cytosolic DMAs(V) reduction, recombinant rat TRR plus NADPH, alone or when added to the cytosol, failed to support DMAs(V) reduction. On ultrafiltration of the cytosol through a 3 kDa filter, the reducing activity in the retentate was lost but was largely restored by NADPH. Such experiments also suggested that the reducing enzyme was larger than 100 kDa and was not GSTO1. In summary, reduction of DMAs(V) to the highly toxic DMAs(III) in rats and rat liver cytosol is a GSH-dependent enzymatic process, yet its mechanism remains uncertain.

Glutathione-supported arsenate reduction coupled to arsenolysis catalyzed by ornithine carbamoyl transferase.

Three cytosolic phosphorolytic/arsenolytic enzymes, (purine nucleoside phosphorylase [PNP], glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase) have been shown to mediate reduction of arsenate (AsV) to the more toxic arsenite (AsIII) in a thiol-dependent manner. With unknown mechanism, hepatic mitochondria also reduce AsV. Mitochondria possess ornithine carbamoyl transferase (OCT), which catalyzes phosphorolytic or arsenolytic citrulline cleavage; therefore, we examined if mitochondrial OCT facilitated AsV reduction in presence of glutathione. Isolated rat liver mitochondria were incubated with AsV, and AsIII formed was quantified. Glutathione-supplemented permeabilized or solubilized mitochondria reduced AsV. Citrulline (substrate for OCT-catalyzed arsenolysis) increased AsV reduction. The citrulline-stimulated AsV reduction was abolished by ornithine (OCT substrate inhibiting citrulline cleavage), phosphate (OCT substrate competing with AsV), and the OCT inhibitor norvaline or PALO, indicating that AsV reduction is coupled to OCT-catalyzed arsenolysis of citrulline. Corroborating this conclusion, purified bacterial OCT mediated AsV reduction in presence of citrulline and glutathione with similar responsiveness to these agents. In contrast, AsIII formation by intact mitochondria was unaffected by PALO and slightly stimulated by citrulline, ornithine, and norvaline, suggesting minimal role for OCT in AsV reduction in intact mitochondria. In addition to OCT, mitochondrial PNP can also mediate AsIII formation; however, its role in AsV reduction appears severely limited by purine nucleoside supply. Collectively, mitochondrial and bacterial OCT promote glutathione-dependent AsV reduction with coupled arsenolysis of citrulline, supporting the hypothesis that AsV reduction is mediated by phosphorolytic/arsenolytic enzymes. Nevertheless, because citrulline cleavage is disfavored physiologically, OCT may have little role in AsV reduction in vivo.

Glutathione-dependent reduction of arsenate by glycogen phosphorylase responsiveness to endogenous and xenobiotic inhibitors.

Rabbit muscle glycogen phosphorylase-a (GPa) reduces arsenate (As(V)) to the more toxic arsenite (As(III)) in a glutathione (GSH)-dependent fashion. To determine whether reduction of As(V) by GPa is countered by compounds known to inhibit GP-catalyzed glycogenolysis, the effects of thiol reagents, endogenous compounds (glucose, ATP, ADP) as well as nonspecific glycogen phosphorylase inhibitors (GPIs; caffeine, quercetin, flavopiridol [FP]), and specific GPIs (1,4-dideoxy-1,4-imino-D-arabinitol [DAB], BAY U6751, CP320626) were tested on reduction of As(V) by rabbit muscle GPa in the presence of glycogen (substrate), AMP (activator), and GSH, and the As(III) formed from As(V) was quantified by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry. The As(V)-reducing activity of GPa was moderately sensitive to thiol reagents. Glucose above 5mM and ADP or ATP at physiological levels diminished GPa-catalyzed As(V) reduction. All GPIs inhibited As(V) reduction by GPa in a concentration-dependent fashion; however, their effects were differentially affected by glucose (10mM) or AMP (200microM instead of 25microM), known modulators of the action of some GPIs on the GP-catalyzed glycogenolysis. Inhibition of As(V) reduction by DAB and quercetin was not influenced by glucose or AMP. Glucose that potentiates the inhibitory effects of caffeine, BAY U6751, and CP320626 on the glycogenolytic activity of GPa also enhanced the inhibitory effects of these GPIs on GPa-catalyzed As(V) reduction. AMP at high concentration alleviated the inhibition by BAY U6751 and CP320626 (whose antagonistic effect on GP-catalyzed glycogen breakdown is also AMP sensitive), whereas the inhibition in As(V) reduction by FP or caffeine was little affected by AMP. Thus, GPIs inhibit both the glycogenolytic and As(V)-reducing activities of GP, supporting that the latter is coupled to glycogenolysis. It was also shown that a GPa-rich extract of rat liver contained GSH-dependent As(V)-reducing activity that was inhibited by specific GPIs, suggesting that the liver-type GPa can also catalyze reduction of As(V).

Glutathione-dependent reduction of arsenate by glycogen phosphorylase a reaction coupled to glycogenolysis.

Arsenate (As(V)) is reduced in the body to the more toxic arsenite (As(III)). We have shown that two enzymes catalyzing phosphorolytic cleavage of their substrates, namely purine nucleoside phosphorylase and glyceraldehyde-3-phosphate dehydrogenase, can reduce As(V) in presence of an appropriate thiol and their substrates. Another phosphorolytic enzyme that may also reduce As(V) is glycogen phosphorylase (GP). With inorganic phosphate (P(i)), GP catalyzes the breakdown of glycogen to glucose-1-phosphate; however, it also accepts As(V). Testing the hypothesis that GP can reduce As(V), we incubated As(V) with the phosphorylated GPa or the dephosphorylated GPb purified from rabbit muscle and quantified the As(III) formed from As(V) by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry. In the presence of adenosine monophosphate (AMP), glycogen, and glutathione (GSH), both GP forms reduced As(V) at rates increasing with enzyme and As(V) concentrations. The As(V) reductase activity of GPa was 10-fold higher than that of GPb. However, incubating GPb with GP kinase and ATP (that converts GPb to GPa) increased As(V) reduction by phosphorylase up to the rate produced by GPa incubated under the same conditions. High concentration of inorganic sulfate, which activates GPb like phosphorylation, also promoted reduction of As(V) by GPb. As(V) reduction by GPa (like As(V) reduction in rats) required GSH. It also required glycogen (substrate for GP) and was stimulated by AMP (allosteric activator of GP) even at low micromolar concentrations. P(i), substrate for GP competing with As(V), inhibited As(III) formation moderately at physiological concentrations. Glucose-1-phosphate, the product of GP-catalyzed glycogenolysis, also decreased As(V) reduction. Summarizing, GP is the third phosphorolytic enzyme identified capable of reducing As(V) in vitro. For reducing As(V) by GP, GSH and glycogen are indispensable, suggesting that the reduction is linked to glycogenolysis. While its in vivo significance remains to be tested, further characterization of the GP-catalyzed As(V) reduction is presented in the adjoining paper.

Effects of phosphate binders on the gastrointestinal absorption of arsenate and of an SGLT2 inhibitor drug on the urinary excretion of arsenite in mice.

Arsenate (AsV) and arsenite (AsIII) are typical sources of acute and chronic arsenic poisoning. Therefore, reducing inner exposure to these arsenicals is a rational objective. Because AsV mimics phosphate, phosphate binder drugs may decrease the intestinal AsV absorption. Indeed, lanthanum and aluminium salts and sevelamer removed AsV from solution in vitro, especially at acidic pH. In mice gavaged with AsV, lanthanum chloride, lanthanum carbonate and aluminium hydroxide given orally also lowered the urinary excretion and tissue levels of AsV and its metabolites, indicating that they decreased the gastrointestinal AsV absorption. As some glucose transporters may carry AsIII, the effect of the SGLT2 inhibitor dapagliflozin was investigated in AsIII-injected mice. While producing extreme glucosuria, dapagliflozin barely affected the urinary excretion and tissue concentrations of AsIII and its metabolites. Thus, phosphate binders (especially lanthanum compounds) can reduce the gastrointestinal absorption of AsV; however, SGLT2 inhibition cannot diminish the renal reabsorption of AsIII.

Effect of an inactivator of glyceraldehyde-3-phosphate dehydrogenase, a fortuitous arsenate reductase, on disposition of arsenate in rats.

The environmentally prevalent arsenate (AsV) is reduced in the body to the much more toxic arsenite (AsIII). Recently, we have demonstrated that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reduction of AsV in the presence of glutathione, yet the role of GAPDH in AsV reduction in vivo is unknown. Therefore, we examined the effect of (S)-alpha-cholorhydrin (ACH), which forms a GAPDH-inhibitory metabolite, on the reduction of AsV in rats. These studies confirmed the in vitro role of GAPDH as an AsV reductase, inasmuch as 3 h after administration of ACH (100 or 200 mg/kg, ip) to rats both the cytosolic GAPDH activity and the AsV-reducing activity dramatically fell in the liver, moderately decreased in the kidneys, and remained unchanged in the muscle. Moreover, the AsV-reducing activity closely correlated with the GAPDH activity in the hepatic cytosols of control and ACH-treated rats. Two confounding effects of ACH (i.e., a slight fall in hepatic glutathione levels and a rise in urinary AsV excretion) prompted us to examine its influence on the disposition of injected AsV (50 micromol/kg, iv) in rats with ligated bile duct as well as in rats with ligated bile duct and renal pedicles. These experiments demonstrated that the hepatic retention of AsV significantly increased, and the combined levels of AsV metabolites (i.e., AsIII plus methylated arsenicals) in the liver decreased in response to ACH; however, ACH failed to delay the disappearance of AsV from the blood of rats with blocked excretory routes. Thus, the GAPDH inactivator ACH inhibits AsV reduction by the liver, but not by the whole body, probably because the impaired hepatic reduction is compensated for by hepatic and extrahepatic AsV-reducing mechanisms spared by ACH. It is most likely that ACH inhibits hepatic AsV reduction predominantly by inactivating GAPDH in the liver; however, a slight ACH-induced glutathione depletion may also contribute. While this study seems to support the conclusion that GAPDH in the liver is involved in AsV reduction in rats, confirmation of the in vivo role of GAPDH as an AsV reductase is desirable.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase works as an arsenate reductase in human red blood cells and rat liver cytosol.

The mammalian enzymes responsible for reduction of the environmentally prevalent arsenate (AsV) to the much more toxic arsenite (AsIII) are unknown. In the previous paper (Nemeti and Gregus, 2005), we proposed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and/or phosphoglycerate kinase (PGK) may catalyze reduction of AsV in human red blood cells (RBC), hemolysate, or rat liver cytosol. In testing this hypothesis, we show here that, if supplied with glutathione (GSH), NAD, and glycolytic substrate, the mixture of purified GAPDH and PGK indeed catalyzes the reduction of AsV. Further analysis revealed that GAPDH is endowed with AsV reductase activity, whereas PGK serves as an auxiliary enzyme, when 3-phosphoglycerate is the glycolytic substrate. The GAPDH-catalyzed AsV reduction required GSH, NAD, and glyceraldehyde-3-phosphate. ADP and ATP moderately, whereas NADH strongly inhibited the AsV reductase activity of the enzyme even in the presence of NAD. Koningic acid (KA), a specific and irreversible inhibitor of GAPDH, inhibited both the classical enzymatic and the AsV-reducing activities of the enzyme in a concentration-dependent fashion. To assess the contribution of GAPDH to the reduction of AsV carried out by hemolysate, rat liver cytosol, or intact erythrocytes, we determined the concentration-dependent effect of KA on AsV reduction by these cells and extracts. Inactivation of GAPDH by KA abolished AsV reduction in intact RBC as well as in the hemolysate and the liver cytosol, when GAPDH in the latter extracts was abundantly supplied with exogenous NAD and glycolytic substrate. However, despite complete inactivation of GAPDH by KA, the hepatic cytosol exhibited significant residual AsV-reducing activity in the absence of exogenous NAD and glycolytic substrate, suggesting that besides GAPDH, other cytosolic enzyme(s) may contribute to AsV reduction in the liver. In conclusion, the key glycolytic enzyme GAPDH can fortuitously catalyze the reduction of AsV to AsIII, if GSH, NAD, and glycolytic substrate are available. AsV reduction may take place during, or as a consequence of, the arsenolytic cleavage of the thioester bond formed between the enzyme's Cys149 and the 3-phosphoglyceroyl moiety of the substrate. Although GAPDH is exclusively responsible for reduction of AsV in human erythrocytes, its role in AsV reduction in vivo remains to be determined.

Reduction of arsenate to arsenite by human erythrocyte lysate and rat liver cytosol - characterization of a glutathione- and NAD-dependent arsenate reduction linked to glycolysis.

Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is of high toxicological importance, yet in vivo relevant enzymes involved have not been identified. Purine nucleoside phosphorylase (PNP) is an efficient AsV reductase in vitro, but its role in AsV reduction is irrelevant in vivo. Intact human red blood cells (RBC) possess an AsV reductase activity that is PNP-independent, diminished by depletion of glutathione (GSH), enhanced by oxidants of erythrocytic NAD(P)H, and possibly linked to the lower part of the glycolytic pathway. In order to characterize this PNP-independent AsV reductase activity further, we examined the effects of GSH, inorganic phosphate, some inhibitors of glucose metabolism, glycolytic substrates, and pyridine, as well as adenine nucleotides on AsV reduction in lysed RBC and rat liver cytosol in the presence of BCX-1777, a PNP inhibitor. In hemolysate, GSH enhanced AsV reduction in a concentration-dependent manner, whereas phosphate inhibited it. Glycolytic substrates, especially fructose-1,6-bisphosphate and phosphoglyceric acids, improved AsV reductase activity. NAD, especially together with these substrates, strongly increased AsIII formation, whereas NADH strongly inhibited it. NADP and adenine nucleotides diminished, while 2-phosphoglycollate, which increases the breakdown of the RBC-specific compound 2,3-bisphosphoglycerate to 3-phosphoglycerate, doubled the AsV reductase activity. Although AsV reduction by the liver cytosol responded similarly to GSH, NAD, and glycolytic substrates as in the hemolysate, it was barely influenced by NADH, was diminished by 2-phosphoglycollate, and was stimulated by NADP. Collectively, hemolysate and rat liver cytosol possess a PNP-independent AsV reductase activity. This enzymatic activity requires GSH, NAD, and glycolytic substrates, and purportedly involves one or both of the two functionally linked glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. In addition, the data presented here suggest that yet another PNP-independent AsV reductase resides in the hepatic cytosol. Although this latter enzyme remains unknown, identification of the AsV reductase depending on GSH, NAD, and glycolytic substrates is presented in the following paper.

Glutathione-dependent reduction of arsenate in human erythrocytes--a process independent of purine nucleoside phosphorylase.

Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is toxicologically important, yet its mechanism is unknown. To clarify this, AsV reduction was investigated in human red blood cells (RBC), as they possess a simple metabolism. RBC were incubated with AsV in gluconate buffer, and the formed AsIII was quantified by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). The observations are compatible with the following conclusions. (1) Human RBC reduce AsV intracellularly, because 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, inhibitor of the chloride-bicarbonate exchanger, which also mediates phosphate and AsV uptake), as well as chloride and phosphate, countered AsIII formation. (2) Purine nucleoside phosphorylase (PNP), whose AsV reductase activity has been directly demonstrated, cannot be a physiologically relevant AsV reductase, because its inhibitor (BCX-1777) failed to decrease the basal erythrocytic AsV reduction, although it prevented the increase in AsIII formation caused by artificial activation of PNP with inosine and dithiothreitol. (3) The basal (PNP-independent) AsV reduction requires glutathione (GSH), because the GSH depletor diethylmaleate strongly diminished AsIII formation. (4) The erythrocytic AsV reduction apparently depends on NAD(P) supply, because oxidants of NAD(P)H (i.e., pyruvate, ferricyanide, methylene blue, nitrite, tert-butylhydroperoxide, dehydroascorbate, 4-dimethylaminophenol) enhanced AsIII formation from AsV. The oxidant-stimulated AsV reduction is PNP-independent, because BCX-1777 failed to affect it, but is GSH-dependent, because diethylmaleate impaired it. (5) Pyruvate-induced glucose depletion, which causes NAD enrichment in the erythrocytes at the expense of NADH, enhanced AsV reduction. This suggests that the erythrocytic AsV reduction requires both NAD supply and operation of the lower part of the glycolytic pathway starting from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that, unlike the upper part, remains fed with substrates originating from the degradation of 2,3-bisphosphoglycerate in RBC depleted of glucose by pyruvate. (6) Fluoride, which arrests glycolysis at enolase and thus prevents NAD formation, inhibited AsV reduction in glucose-sufficient RBC, but increased it in glucose-deficient (NAD-enriched) cells, suggesting that the section of glycolysis coupled to AsV reduction lies between GAPDH and enolase. In conclusion, besides the artificial PNP-dependent AsV reduction, human RBC contain a PNP-independent AsV-reducing mechanism. This appears to require the supply of GSH, NAD, and substrate to one or more of the glycolytic enzymes localized between GAPDH and enolase.

Reduction of arsenate to arsenite in hepatic cytosol.

After finding that rat liver mitochondria reduce arsenate (AsV) to the more toxic arsenite (AsIII), it was of interest to know if other cell fractions also carried out this process. Postmitochondrial supernatant (PMSN), isolated from rat liver, reduced AsV to AsIII only in the presence of a thiol. Dithiothreitol (DTT) supported the reduction much more effectively than glutathione. Separation of PMSN into microsome and cytosol revealed that the AsV reducing activity resided in the cytosol. AsV-like oxyanions, e.g., phosphate (P(i)) and vanadate, as well as mercurial thiol reagents inhibited the cytosolic AsV reducing activity, indicating the involvement of a P(i)-utilizing SH enzyme. On searching for a reduction partner, it was found unexpectedly that oxidized pyridine nucleotides (NAD(+) or NADP(+)), but not their reduced forms, increased AsIII formation. Some other purine nucleotide derivatives (e.g., AMP, GMP), but not pyrimidine nucleotides, also increased the rate 2-3-fold. Examination of the effect of nucleosides and nucleobases on AsV reduction yielded dramatic results: purine nucleosides (inosine or guanosine) increased the reduction 80-100-fold, whereas purine bases (hypoxanthine or guanine) decreased it 80-90%. Although the retentate obtained by ultrafiltration of cytosol was almost inactive, its AsV reductase activity could be regained by adding the filtrate or inosine or guanosine to the retentate, indicating that endogenous purine nucleosides were essential for AsV reduction by the cytosol. The hepatic cytosol of mice, hamsters, guinea pigs, and rabbits also exhibited AsV reductase activities in the presence of DTT, which were dramatically enhanced by inosine. Thus, the hepatic cytosol of all tested species can reduce AsV to AsIII. The reduction requires the presence of an appropriate thiol as well as a purine nucleoside (inosine or guanosine) and is inhibited by thiol reagents, the AsV analogue phosphate, and purine bases. Characterization of this AsV reductase activity led us to identification of a cytosolic AsV reductase, which is presented in the accompanying paper.

Purine nucleoside phosphorylase as a cytosolic arsenate reductase.

The findings of the accompanying paper (Németi and Gregus, Toxicol: Sci. 70, 4-12) indicate that the arsenate (AsV) reductase activity of rat liver cytosol is due to an SH enzyme that uses phosphate (or its analogue, arsenate, AsV) and a purine nucleoside (guanosine or inosine) as substrates. Purine nucleoside phosphorylase (PNP) is such an enzyme. It catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides according to the following scheme: guanosine (or inosine) + phosphate <--> guanine (or hypoxanthine) + ribose-1-phosphate. Therefore, we have tested the hypothesis that PNP is responsible for the thiol- and purine nucleoside-dependent reduction of AsV to AsIII by rat liver cytosol. AsIII formed from AsV was quantified by HPLC-hydride generation-atomic fluorescence spectrometry analysis of the deproteinized incubates. The following findings support the conclusion that PNP reduces AsV to AsIII, using AsV instead of phosphate in the reaction above: (1) Specific PNP inhibitors (CI-1000, BCX-1777) at a concentration of 1 microM completely inhibited cytosolic AsV reductase activity. (2) During anion-exchange chromatography of cytosolic proteins, PNP activity perfectly coeluted with the AsV reductase activity, suggesting that both activities belong to the same protein. (3) PNP purified from calf spleen catalyzed reduction of AsV to AsIII in the presence of dithiothreitol (DTT) and a 6-oxopurine nucleoside (guanosine or inosine). (4) AsV reductase activity of purified PNP, like the cytosolic AsV reductase activity, was inhibited by phosphate (a substrate of PNP alternative to AsV), guanine and hypoxanthine (products of PNP favoring the reverse reaction), mercurial thiol reagents (nonspecific inhibitors of PNP), as well as CI-1000 and BCX-1777 (specific PNP inhibitors). Thus, PNP appears to be responsible for the AsV reductase activity of rat liver cytosol in the presence of DTT. Further research should clarify the mechanism and the in vivo significance of PNP-catalyzed reduction of AsV to AsIII.

Arsenate reduction in human erythrocytes and rats--testing the role of purine nucleoside phosphorylase.

Reduction of the pentavalent arsenate (AsV) to the thiol-reactive arsenite (AsIII) toxifies this environmentally prevalent form of arsenic, yet its biochemical mechanism in mammals is incompletely understood. Purine nucleoside phosphorylase (PNP) has been shown recently to function as an AsV reductase in vitro, provided its substrate (inosine or guanosine) and an appropriate dithiol (e.g., dithiothreitol, DTT) were present. It was of interest to know if this ubiquitous enzyme played a significant role in reduction of AsV to AsIII in vivo. Two approaches were used to test this. First, it was determined if compounds that influenced AsV reduction by purified PNP (i.e., nucleosides, thiols, and PNP inhibitors) would similarly affect reduction of AsV by human erythrocytes. Erythrocytes were incubated with AsV, and the formed AsIII was quantified by HPLC-hydride generation-atomic fluorescence spectrometry. The red blood cells reduced AsV at a considerable rate, which could be enhanced by inosine or inosine plus DTT. These stimulated AsIII formation rates were PNP-dependent, as PNP inhibitors strongly inhibited them. In contrast, PNP inhibitors had little if any inhibitory effect on AsIII formation in the absence of exogenous inosine, indicating that this basal rate of AsV reduction is PNP-independent. Second, the role of PNP in reduction of AsV in vivo was also assessed by investigating the effect of the PNP inhibitor BCX-1777 on the biotransformation of AsV in control and DTT-treated rats with cannulated bile duct and ligated renal pedicles. Although it abolished hepatic PNP activity, BCX-1777 influenced neither the biliary excretion of AsIII and monomethylarsonous acid, nor the tissue concentration of AsV and its metabolites in either group of AsV-injected rats. Thus, despite its in vitro activity, PNP does not appear to play a significant role in AsV reduction in human erythrocytes and in rats in vivo. Further research should clarify the in vivo relevant mechanisms of AsV reduction in mammals.

Dose-dependent biotransformation of arsenite in rats--not S-adenosylmethionine depletion impairs arsenic methylation at high dose.

Arsenite (AsIII) is eliminated via excretion and methylation. Monomethylarsonous acid (MMAsIII) is a super toxic metabolite of AsIII, while dimethylarsinic acid produced in the next metabolic step is relatively atoxic. Since the role of methylation in the acute toxicity and elimination of AsIII in vivo is unclear, we have examined the excretion and tissue retention of AsIII and its metabolites in rats exposed to increasing AsIII doses. Rats were injected i.v. with 20, 50 and 125 micromol/kg AsIII and arsenic metabolites in bile, urine and tissues were analysed. The excretion of AsIII increased almost proportionately to the dose, while its concentration in tissues rose more than proportionately. In contrast, the excretion and tissue concentrations of methylated metabolites increased less than the dosage, or they even decreased after injection of the largest dose of AsIII. To elucidate the mechanism of the dose-dependent decrease of methylation, we quantified S-adenosylmethionine (SAME), glutathione (GSH), and adenine nucleotides in the liver of AsIII-injected rats. AsIII decreased the hepatic concentrations of GSH and adenosine 5'-triphosphate (ATP) and the energy charge in a dose-dependent manner, but increased the level of SAME. Thus, impaired methylation after AsIII overdose is not due to SAME shortage, but probably to methyltransferase inhibition. It appears that exhausted elimination capacity of AsIII, rather than MMAsIII produced from AsIII, contributes significantly to the acute toxicity of AsIII. After GSH depletion the retained AsIII can increasingly inhibit SH-enzymes, thus causing ATP depletion and energetic disorder.

Glutathione synthetase promotes the reduction of arsenate via arsenolysis of glutathione.

The environmentally prevalent arsenate (As(V)) undergoes reduction in the body to the much more toxic arsenite (As(III)). Phosphorolytic enzymes and ATP synthase can promote the reduction As(V) by converting it into arsenylated products in which the pentavalent arsenic is more reducible by glutathione (GSH) to As(III) than in inorganic As(V). Glutathione synthetase (GS) can catalyze the arsenolysis of GSH (γ-Glu-Cys-Gly) yielding two arsenylated products, i.e. γ-Glu-Cys-arsenate and ADP-arsenate. Thus, GS may also promote the reduction of As(V) by GSH. This hypothesis was tested with human recombinant GS, a Mg(2+) dependent enzyme. GS markedly increased As(III) formation when incubated with As(V), GSH, Mg(2+) and ADP, but not when GSH, Mg(2+) or ADP were separately omitted. Phosphate, a substrate competitive with As(V) in the arsenolysis of GSH, as well as the products of GSH arsenolysis or their analogs, e.g. glycine and γ-Glu-aminobutyrate, decreased As(V) reduction. Replacement of ADP with ATP or an analog that cannot be phosphorylated or arsenylated abolished As(V) reduction, indicating that GS-supported As(V) reduction requires formation of ADP-arsenate. In the presence of ADP, however, ATP (but not its metabolically inert analog) tripled As(V) reduction because ATP permits GS to remove the arsenolysis inhibitory glycine and γ-Glu-Cys by converting them into GSH. GS failed to promote As(V) reduction when GSH was replaced with ophthalmic acid, a GSH analog substrate of GS containing no SH group (although ophthalmic acid did undergo GS-catalyzed arsenolysis), indicating that the SH group of GSH is important for As(V) reduction. Our findings support the conclusion that GS promotes reduction of As(V) by catalyzing the arsenolysis of GSH, thus producing ADP-arsenate, which upon being released from the enzyme is readily reduced by GSH to As(III).

The mechanism of the polynucleotide phosphorylase-catalyzed arsenolysis of ADP.

Using ADP and arsenate (AsV), polynucleotide phosphorylase (PNPase) catalyzes the apparent arsenolysis of ADP to AMP-arsenate and inorganic phosphate, with the former hydrolyzing rapidly into AMP and AsV. However, in the presence of glutathione, AMP-arsenate may also undergo reductive decomposition, yielding AMP and arsenite (AsIII). In order to clarify the mechanism of ADP arsenolysis mediated by Escherichia coli PNPase, we analyzed the time course of the reaction in the presence of increasing concentrations of ADP, with or without polyadenylate (poly-A) supplementation. These studies revealed that increasing supply of ADP enhanced the consumption of ADP but inhibited the production of both AMP and AsIII. Formation of these products was amplified by adding trace amount of poly-A. Furthermore, AMP and AsIII production accelerated with time, whereas ADP consumption slowed down. These observations collectively suggest that PNPase does not catalyze the arsenolysis of ADP directly (in a single step), but in two separate consecutive steps: the enzyme first converts ADP into poly-A, then it cleaves the newly synthesized poly-A by arsenolysis. It is inferred that one active site of PNPase can catalyze only one of these reactions at a time and that high ADP concentrations favor poly-A synthesis, thereby inhibiting the arsenolysis.

Polynucleotide phosphorylase and mitochondrial ATP synthase mediate reduction of arsenate to the more toxic arsenite by forming arsenylated analogues of ADP and ATP.

We have demonstrated that phosphorolytic-arsenolytic enzymes can promote reduction of arsenate (AsV) into the more toxic arsenite (AsIII) because they convert AsV into an arsenylated product in which the arsenic is more reducible by glutathione (GSH) or other thiols to AsIII than in inorganic AsV. We have also shown that mitochondria can rapidly reduce AsV in a process requiring intact oxidative phosphorylation and intramitochondrial GSH. Thus, these organelles might reduce AsV because mitochondrial ATP synthase, using AsV instead of phosphate, arsenylates ADP to ADP-AsV, which in turn is readily reduced by GSH. To test this hypothesis, we first examined whether the RNA-cleaving enzyme polynucleotide phosphorylase (PNPase), which can split poly-adenylate (poly-A) by arsenolysis into units of AMP-AsV (a homologue of ADP-AsV), could also promote reduction of AsV to AsIII in presence of thiols. Indeed, bacterial PNPase markedly facilitated formation of AsIII when incubated with poly-A, AsV, and GSH. PNPase-mediated AsV reduction depended on arsenolysis of poly-A and presence of a thiol. PNPase can also form AMP-AsV from ADP and AsV (termed arsenolysis of ADP). In presence of GSH, this reaction also facilitated AsV reduction in proportion to AMP-AsV production. Although various thiols did not influence the arsenolytic yield of AMP-AsV, they differentially promoted the PNPase-mediated reduction of AsV, with GSH being the most effective. Circumstantial evidence indicated that AMP-AsV formed by PNPase is more reducible to AsIII by GSH than inorganic AsV. Then, we demonstrated that AsV reduction by isolated mitochondria was markedly inhibited by an ADP analogue that enters mitochondria but is not phosphorylated or arsenylated. Furthermore, inhibitors of the export of ATP or ADP-AsV from the mitochondria diminished the increment in AsV reduction caused by adding GSH externally to these organelles whose intramitochondrial GSH had been depleted. Thus, whereas PNPase promotes reduction of AsV by incorporating it into AMP-AsV, the mitochondrial ATP synthase facilitates AsV reduction by forming ADP-AsV; then GSH can easily reduce these arsenylated nucleotides to AsIII.