Successful implementation of community water fluoridation via the community diagnosis process.

OBJECTIVES: This paper describes the community diagnosis process and how it was used to implement community water fluoridation in Tennessee. METHODS: Public health dental staff developed a survey instrument to collect community-specific data on the oral health status of schoolchildren. Key survey findings were presented to county health councils who were determining and prioritizing the health needs of their communities. RESULTS: Community-specific data showed higher caries levels in children without access to an optimally fluoridated community water supply. Presentation of local survey findings to county health councils resulted in fluoridation being a high-priority health issue in several counties. With health council support, opposition to fluoridation by utility district officials was overcome when decision makers were challenged with local survey findings. The community diagnosis process resulted in the successful fluoridation of six community water systems serving a total of 33,000 residents. CONCLUSIONS: The community diagnosis approach was successful in implementing community water fluoridation in geographic areas historically opposed to this public health measure. The success of these fluoridation initiatives was attributed to: (1) current, community-specific assessments of children's oral health; (2) identification of communities with disparate oral health needs, problems, and resources; and (3) effective presentation of community-specific oral health survey data to community leaders, stakeholders, and decision makers.

Polyhydroxybenzoates inhibit ascorbic acid activation of mitochondrial glycerol-3-phosphate dehydrogenase: implications for glucose metabolism and insulin secretion.

Glycerol-3-phosphate dehydrogenase from pig brain mitochondria was stimulated 2.2-fold by the addition of 50 microm l-ascorbic acid. Enzyme activity, dependent upon the presence of l-ascorbic acid, was inhibited by lauryl gallate, propyl gallate, protocatechuic acid ethyl ester, and salicylhydroxamic acid. Homogeneous pig brain mitochondrial glycerol-3-phosphate dehydrogenase was activated by either 150 microm L-ascorbic acid (56%) or 300 microm iron (Fe(2+) or Fe(3+) (62%)) and 2.6-fold by the addition of both L-ascorbic acid and iron. The addition of L-ascorbic acid and iron resulted in a significant increase of k(cat) from 21.1 to 64.1 s(-1), without significantly increasing the K(m) of L-glycerol-3-phosphate (10.0-14.5 mm). The activation of pure glycerol-3-phosphate dehydrogenase by either L-ascorbic acid or iron or its combination could be totally inhibited by 200 microm propyl gallate. The metabolism of [5-(3)H]glucose and the glucose-stimulated insulin secretion from rat insulinoma cells, INS-1, were effectively inhibited by 500 microm or 1 mm propyl gallate and to a lesser extent by 5 mm aminooxyacetate, a potent malate-aspartate shuttle inhibitor. The combined data support the conclusion that l-ascorbic acid is a physiological activator of mitochondrial glycerol-3-phosphate dehydrogenase, that the enzyme is potently inhibited by agents that specifically inhibit certain classes of di-iron metalloenzymes, and that the enzyme is chiefly responsible for the proximal signal events in INS-1 cell glucose-stimulated insulin release.

Thioltransferase overexpression increases resistance of MCF-7 cells to adriamycin.

Thioltransferase, a small redox protein with thiol-disulfide oxidoreductase and dehydroascorbate reductase activities, has been reported to be expressed at higher levels in Adriamycin-resistant MCF-7 human breast tumor cells (MCF-7 ADR(R)) when compared with Adriamycin sensitive MCF-7 WT (MCF-7 WT) cells. The present study examined the effects of stably transfecting MCF-7 WT cells with the cDNA for human thioltransferase and the effects of subsequent Adriamycin cytotoxicity in the MCF-7 WT transfected cells. All transfected cell lines overexpressing thioltransferase activity were more resistant to Adriamycin than untransfected MCF-7 WT cells, supporting the hypothesis that increases in thioltransferase expression are related to Adriamycin resistance. This resistance was independent of the ability of thioltransferase to catalyze reduction of dehydroascorbic acid to ascorbic acid, as the addition of an ascorbate generating derivative, L-ascorbic acid-2-phosphate, to the media did not additionally increase Adriamycin resistance.

Identification of the dehydroascorbic acid reductase and thioltransferase (Glutaredoxin) activities of bovine erythrocyte glutathione peroxidase.

Bovine erythrocyte glutathione (GSH) peroxidase (GPX, EC 1.11.1.9) was examined for GSH-dependent dehydroascorbate (DHA) reductase (EC 1.8.5.1) and thioltransferase (EC 1.8.4.1) activities. Using the direct assay method for GSH-dependent DHA reductase activity, GPX had a kcat (app) of 140 +/- 9 min-1 and specificity constants (kcat/Km(app)) of 5.74 +/- 0.78 x 10(2) M-1s-1 for DHA and 1.18 +/- 0.17 x 10(3) M-1s-1 for GSH based on the monomer Mr of 22,612. Using the coupled assay method for thioltransferase activity, GPX had a kcat (app) of 186 +/- 9 min-1 and specificity constants (app) of 1. 49 +/- 0.14 x 10(3) M-1s-1 for S-sulfocysteine and 1.51 +/- 0.18 x 10(3) M-1s-1 for GSH based on the GPX monomer molecular weight. GPX has a higher specificity constant for S-sulfocysteine than DHA, and both assay systems gave nearly identical specificity constants for GSH. The DHA reductase and thioltransferase activities of GPX adds to the repertoire of functions of this enzyme as an important protector against cellular oxidative stress.

The catalytic mechanism of the glutathione-dependent dehydroascorbate reductase activity of thioltransferase (glutaredoxin).

The catalytic mechanism of the glutathione (GSH)-dependent dehydroascorbic acid (DHA) reductase activity of recombinant pig liver thioltransferase (RPLTT) was investigated. RPLTT and the C25S mutant protein had equivalent specificity constants (kcat/Km) for both DHA and GSH. Iodoacetamide (IAM) inactivated the DHA reductase activities of RPLTT and C25S, confirming the essential role of cysteine in the reaction mechanism. When preincubated with DHA, RPLTT but not C25S was protected against IAM inactivation, suggesting that RPLTT has the ability to chemically reduce DHA forming ascorbic acid (AA) and the intramolecular disulfide form of the enzyme. Electrochemical detection of AA demonstrated the ability of both reduced RPLTT and C25S to chemically reduce DHA to AA in the absence of GSH. However, RPLTT had an initial rate of DHA reduction which was 4-fold greater than that of C25S, and after 10 min, RPLTT resulted in an AA concentration 11-fold greater than that of C25S. Isoelectric focusing analysis revealed that the product of reaction of reduced RPLTT but not C25S with DHA was consistent with the oxidized form of the enzyme. This result suggested that even though both RPLTT and the C25S mutant had equivalent specificity constants for DHA and GSH, they may have different catalytic mechanisms. On the basis of the experimental results, a catalytic mechanism for the DHA reductase activity of RPLTT is proposed. This is the first description of a catalytic mechanism of a glutathione:dehydroascorbate oxidoreductase (EC 1.8.5.1).

Spontaneous conversion of L-dehydroascorbic acid to L-ascorbic acid and L-erythroascorbic acid.

Dehydroascorbic acid, an oxidation product of ascorbic acid (vitamin C), spontaneously decomposed at neutral and higher pH levels to form three products that could be quantitated by HPLC-electrochemical analysis. One of the products was ascorbic acid, suggesting that dehydroascorbic acid was reduced to ascorbic acid without adding an exogenous reductant. The major newly produced compound was almost identical to ascorbic acid by UV spectroscopy, which therefore potentially interfered in the study of ascorbic acid metabolism. The ascorbic acid-like compound was isolated by reversed-phase HPLC and identified as L-erythroascorbic acid by mass spectrometry. Fe(II) and Cu(I) increased, whereas desferrioxamine, a potent iron chelator, inhibited L-erythroascorbic acid production. Phosphate, used as buffer, and cyanide greatly enhanced dehydroascorbic acid conversion to L-erythroascorbic acid. The identification of L-erythroascorbic acid and its quantitation by an electrochemical method provides a useful tool for future study of dehydroascorbic acid metabolism.

Ascorbic acid is a stimulatory cofactor for mitochondrial glycerol-3-phosphate dehydrogenase.

Mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) from control and scorbutic guinea pig brain, liver and skeletal muscle and from normal rat liver was stimulated several fold by l-ascorbic acid (AA). The amount of AA that gave half-maximal stimulation of guinea pig brain mGPDH was 7.1 microM. At concentrations of AA higher than 500 microM, mGPDH activity decreased to nearly the same activity as in the absence of AA. D-Ascorbic acid, erythorbic acid, was equally potent in the activation of washed mitochondrial mGPDH activity. The AA activation of mGPDH was completely inhibited by 50 microM EGTA, but could be fully restored by the sequential addition of 100 microM Ca2+. The AA activation of mGPDH was likewise completely inhibited by iron specific chelators, bathophenanthrolinedisulfonic acid, desferrioxamine, and 1,10-phenanthroline, but the activation could not be restored by the addition of excess Ca2+. In the absence of AA, mGPDH activity was not inhibited by either EGTA or the iron chelators and Ca2+ addition had no effect on the activity. The iron-sulfur protein, succinic dehydrogenase (complex II), was not significantly different in brain mitochondria from control or scorbutic guinea pigs, and was not activated by the subsequent addition of AA. In the presence of AA, succinic dehydrogenase activity was not affected by either bathophenanthrolinedisulfonic acid or EGTA. The results suggest that mGPDH is a probable site of action of AA in the related glucose-coupled insulin release from pancreatic islets.

Studies on the essential role of ascorbic acid in the energy dependent release of insulin from pancreatic islets.

Pancreatic islets from young normal and scorbutic male guinea pigs were examined for their ability to release insulin when stimulated with depolarizing levels of KCl (45 mM) and by 20 mM D-glyceraldehyde. Islets from normal guinea pigs released insulin in a K+ and D-glyceraldehyde dependent manner showing a rapid initial secretion phase followed by secondary waves of insulin release during a 120 min period. Islets from scorbutic guinea pigs were able to respond to elevated K+ in a manner identical to that of the control islets. In contrast, insulin release from ascorbic acid deficient islets in response to the secretagogue, D-glyceraldehyde, was significantly delayed and decreased responses were observed during the 120 min period after D-glyceraldehyde stimulation. The results are consistent with the site of action of ascorbic acid on energy-dependent insulin release lying between the triose-phosphate level of glycolysis and the generation of ATP by oxidative phosphorylation.

Glutathione dependent reduction of alloxan to dialuric acid catalyzed by thioltransferase (glutaredoxin): a possible role for thioltransferase in alloxan toxicity.

Recombinant pig liver thioltransferase (rPLTT) catalyzes the reduction of alloxan to dialuric acid by glutathione (GSH). This is the second non-disulfide substrate, after dehydroascorbic acid, described for thioltransferase. The reaction kinetics, measured by a coupled assay including glutathione disulfide reductase and NADPH yielded a Km = 82 microM for alloxan, a k(cat) = 37 s(-1), and a k(cat)/Km = 4.5 x 10(5) M(-1) s(-1). The presence of rPLTT suppressed the competitive formation of compound 305, an alloxan-GSH conjugate of unknown structure, and at GSH concentrations between 0.05 mM and 1.5 mM, oxygen consumption was greater than that recorded in the uncatalyzed reaction. Both superoxide dismutase and catalase inhibited oxygen consumption in 1.0 mM GSH and 0.2 mM alloxan in the presence of rPLTT. This study suggests that thioltransferase (glutaredoxin) plays a significant role in the cytotoxicity of alloxan in vulnerable tissues.

Purification and characterization of a glutathione dependent dehydroascorbate reductase from human erythrocytes.

A GSH-dependent dehydroascorbate reductase (EC 1.8.5.1) was purified to homogeneity from human erythrocytes. The enzyme was a monomer of 32 kDa and was purified 133-fold from a crude DEAE-Sepharose fraction with a 25% yield. The reduced protein had a pI of 5.1 as judged by isoelectric focusing. Kinetic analysis gave a Kcat of 316 min-1, a Km of 0.21 mM for DHA with a Kcat/Km of 2.47 x 10(4) M-1 sec-1, and a Km of 3.5 mM for GSH with a Kcat/Km of 1.51 x 10(3) M-1 sec-1. This is the second DHA reductase (after thioltransferase) isolated from human erythrocytes, but unlike thioltransferase, it has no thiol-disulfide oxido-reductase activity.

alpha-Lipoic acid dependent regeneration of ascorbic acid from dehydroascorbic acid in rat liver mitochondria.

Rat liver mitochondria were examined for their ability to reduce dehydroascorbic acid to ascorbic acid in an alpha-lipoic acid dependent or independent manner. The alpha-lipoic acid dependent reduction was stimulated by factors that increased the NADH dependent reduction of alpha-lipoic acid to dihydrolipoic acid in coupled reactions. Optimal conditions for dehydroascorbic acid reduction to ascorbic acid were achieved in the presence of pyruvate, alpha-lipoic acid, and ATP. Electron transport inhibitors, rotenone and antimycin A, further enhanced the dehydroascorbic acid reduction. The reactions were strongly inhibited by 1 mM iodoacetamide or sodium arsenite. Mitoplasts were qualitatively similar to intact mitochondria in dehydroascorbate reduction activity. Pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase reduced dehydroascorbic acid to ascorbic acid in an alpha-lipoic acid, coenzyme A, and pyruvate or alpha-ketoglutarate dependent fashion. Dehydroascorbic acid was also catalytically reduced to ascorbic acid by purified lipoamide dehydrogenase in an alpha-lipoic acid (K0.5 = 1.4 +/- 0.8 mM) and lipoamide (K0.5 = 0.9 +/- 0.3 mM) dependent manner.

Ascorbic acid is essential for the release of insulin from scorbutic guinea pig pancreatic islets.

Pancreatic islets from young normal and scorbutic male guinea pigs were examined for their ability to release insulin when stimulated with elevated D-glucose. Islets from normal guinea pigs released insulin in a D-glucose-dependent manner showing a rapid initial secretion phase and three secondary secretion waves during a 120-min period. Islets from scorbutic guinea pigs failed to release insulin during the immediate period, and only delayed and decreased responses were observed over the 40-60 min after D-glucose elevation. Insulin release from scorbutic islets was greatly elevated if 5 mM L-ascorbic acid 2-phosphate was supplemented in the perifusion medium during the last 60 min of perifusion. When 5 mM L-ascorbic acid 2-phosphate was added to the perifusion medium concurrently with elevation of medium D-glucose, islets from scorbutic guinea pigs released insulin as rapidly as control guinea pig islets and to a somewhat greater extent. L-Ascorbic acid 2-phosphate without elevated D-glucose had no effect on insulin release by islets from normal or scorbutic guinea pigs. The pancreas from scorbutic guinea pigs contained 2.4 times more insulin than that from control guinea pigs, suggesting that the decreased insulin release from the scorbutic islets was not due to decreased insulin synthesis but due to abnormal insulin secretion.

Crystal structure of thioltransferase at 2.2 A resolution.

We report here the first three-dimensional structure of a mammalian thioltransferase as determined by single crystal X-ray crystallography at 2.2 A resolution. The protein is known for its thiol-redox properties and dehydroascorbate reductase activity. Recombinant pig liver thioltransferase expressed in Escherichia coli was crystallized in its oxidized form by vapor diffusion technique. The structure was determined by multiple isomorphous replacement method using four heavy-atom derivatives. The protein folds into an alpha/beta structure with a four-stranded mixed beta-sheet in the core, flanked on either side by helices. The fold is similar to that found in other thiol-redox proteins, viz. E. coli thioredoxin and bacteriophage T4 glutaredoxin, and thus seems to be conserved in these functionally related proteins. The active site disulfide (Cys 22-Cys 25) is located on a protrusion on the molecular surface. Cys 22, which is known to have an abnormally low pKa of 3.8, is accessible from the exterior of the molecule. Pro 70, which is in close proximity to the disulfide bridge, assumes a conserved cis-peptide configuration. Mutational data available on the protein are in agreement with the three-dimensional structure.

Ascorbic acid and cell survival of adriamycin resistant and sensitive MCF-7 breast tumor cells.

The ability of human cells to regenerate ascorbic acid from dehydroascorbate is partially dependent on the glutathione redox status of the cell and the relative activity of dehydroascorbate reductases. Mammalian dehydroascorbate reductase activity is associated with two proteins known as thioltransferase (glutaredoxin) and protein disulfide isomerase. We compared the specific activity of thioltransferase, protein disulfide isomerase, and other GSH-related enzymes in Adriamycin-resistant human breast tumor cells, MCF-7 ADRR, and Adriamycin-sensitive, MCF-7 WT, tumor cells. MCF-7 ADRR cells had higher activities of glutathione peroxidase (34.7 fold), nonseleno-glutathione peroxidase (glutathione S-transferases; 5.3 fold), thioredoxin (2.3 fold), and thioltransferase (4.0 fold) compared with the WT Adriamycin-sensitive cell line. Thioltransferase was detected in Western blots in extracts of ADRR MCF-7 cells but not in WT MCF-7 cells. alpha-Tocopherol in the membrane and cytosolic fractions was 2.8 and 3.0 fold higher, respectively, in Adriamycin-resistant compared with Adriamycin-sensitive cells. Supplementation of MCF-7 cells with L-ascorbic acid 2-phosphate (2 and 10 mM) had no effect on WT cell viability after 5 days incubation with up to 0.33 microM Adriamycin. In contrast, supplementation of ADRR MCF-7 cells with L-ascorbic acid 2-phosphate resulted in enhanced resistance up to 3.4 microM Adriamycin over a 5-day incubation. Both lines of MCF-7 cells demonstrated the ability to utilize ascorbic acid as the 2-phosphate derivative. After 48 h incubation with 8.6 microM Adriamycin, the resistant cells maintained normal viability and ascorbate-dehydroascorbate levels, whereas drug-sensitive cells had significantly lower ascorbate with a higher percent dehydroascorbate and increased cell death as judged by cell protein levels (52% of controls).

Dehydroascorbate reduction.

Dehydroascorbic acid is generated in plants and animal cells by oxidation of ascorbic acid. The reaction is believed to occur by the one-electron oxidation of ascorbic acid to semidehydroascorbate radical followed by disproportionation to dehydroascorbic acid and ascorbic acid. Semidehydroascorbic acid may recycle to ascorbic acid catalyzed by membrane-bound NADH-semidehydroscorbate reductase. However, disproportionation of the free radical occurs at a rapid rate, 10(5) M-1 s-1, accounting for measurable cellular levels of dehydroascorbate. Dehydroascorbate reductase, studied earlier and more extensively in plants, is now recognized as the intrinsic activity of thioltransferases (glutaredoxins) and protein disulfide isomerase in animal cells. These enzymes catalyze the glutathione-dependent two-electron regeneration of ascorbic acid. The importance of the latter route of ascorbic acid renewal was seen in studies of GSH-deficient rodents (Meister, A. (1992) Biochem. Pharmacol. 44, 1905-1915). GSH deficiency in newborn animals resulted in decreased tissue ascorbic acid and increased dehydroascorbate-to-ascorbate ratios. Administration of ascorbic acid daily to GSH-deficient animals decreased animal mortality and cell damage from oxygen stress. A cellular role is proposed for dehydroascorbate in the oxidation of nascent protein dithiols to disulfides catalyzed in the endoplasmic reticulum compartment by protein disulfide isomerase.

Thioltransferases.

A family of small molecular weight proteins with thiol-disulfide exchange activity have been discovered, widely distributed from E. coli to mammalian systems, called thioltransferases or glutaredoxins. There are no substantiated reports of thioltransferases-glutaredoxins in plants; however, partially purified dehydroascorbate reductase from peas had thiol-disulfide exchange catalytic activity using glutathione as reductant and S-sulfocysteine as thiosulfate cosubstrate (unpublished data). Thus, this class of proteins is universally distributed. Based on mutagenesis studies, a sequence of Cys-Pro-Tyr(Phe)-Cys- followed by Arg-Lys- or Lys alone is critical for both the thiol-disulfide exchange reaction and the dehydroascorbate reductase activity. The dithiol-disulfide loop represented by this structure is unique since the cystine closer to the N-terminus has a highly acidic thiol pKa (3.8 as determined for the pig liver enzyme) that contributes to the protein's high S- nucleophilicity. Compared with the microbial enzyme, the mammalian thioltransferases (glutaredoxins) are extended at both N and C termini by 10-12 amino acid residues, including a second pair of cysteines toward the C-terminus with no known special function. Yeast thioltransferase is more like mammalian enzymes in length (106 amino acids) but more like E. coli glutaredoxin in being unblocked at the N-terminus and having only one set of cysteines; that is, at the active center. The three mammalian enzymes, for which sequences are available, are blocked at the N-terminus by an acetyl group linked to alanine with no known special function other than possibly to impart greater cellular turnover stability. A report of carbohydrate (8.6%) content in rat liver thioltransferase has not been verified by more sensitive methods of carbohydrate analysis, nor has carbohydrate been identified in samples of purified glutaredoxin from any source. Thiol transferase and glutaredoxin are two names for the same protein based on similarity of amino acid sequence, immunochemical cross-reactivity, and other enzyme properties. The inability of thioltransferase from some mammalian sources to act as an electron carrier in ribonucleotide reductase systems, whether homologous or heterologous in origin, remains to be explained in future studies.

Interactions of platinum complexes with thioltransferase(glutaredoxin), in vitro.

Under anaerobic conditions, recombinant pig liver thioltransferase (glutaredoxin)(TT, GRX) (EC 1.8.4.1) was strongly inhibited by cis and carbo-platin and somewhat less sensitive to trans-platin, in vitro. By extrapolation to total inhibition, the ratio of platinum drug/thioltransferase was approximately 1.0 for cis and carbo-platin, but greater than 1.0 for trans-platin. When thioltransferase was not reduced, inhibition by preincubation with the platinum complexes required molar excesses of 1,300 and 675 to one for cis-platin and trans-platin, respectively or 400-500 microM for 50% inhibition. The inhibition of thioltransferase at high drug concentrations in the presence of oxygen was associated with cross-linking of monomers into dimers within 5 min and, in the case of cis-platin treatment, to trimers in 20 min incubation.

Identification and characterization of the functional amino acids at the active center of pig liver thioltransferase by site-directed mutagenesis.

By using site-directed mutagenesis techniques, the essential amino acids at the catalytic center of porcine thioltransferase (glutaredoxin) were determined. Seven oligonucleotides were designed, synthesized, and used to construct mutants, ETT-C22S, ETT-C25S, ETT-C25A, ETT-R26V, ETT-K27Q, ETT-R26V: K27Q, and ETT-C78S:C82S, by altering their codons in pig liver thioltransferase cDNA/M13mp18 clones. Each of the thioltransferases was purified to homogeneity and its dithiol-disulfide exchange, and dehydroascorbate reductase activities were compared with those of the wild-type (ETT). Evidence was obtained that Cys22 was essential for catalytic activity, and the extremely low pKa value of its sulfhydryl group was facilitated primarily by Arg26. The role of Lys27 at the active center was different from that of Arg26 and may be important in stabilizing the E.S intermediate by electrostatic forces. The second pair of cysteines, Cys78 and Cys82, nearer the C terminus, were not directly involved in the active center, but may play a role in defining the native protein structure. The replacement of the original Cys with a Ser at position 25 increased rather than decreased the enzyme activity, suggesting that the proposed intramolecular disulfide bond between Cys22 and Cys25 is not necessary for the catalytic mechanism of the Ser25 mutant, but does not rule out such a mechanism for the wild-type enzyme.

Catalytic mechanism of thioltransferase.

To evaluate potential catalytic mechanism for thioltransferase thiol-disulfide exchange reactions, seven pig liver mutants were constructed by site-directed mutagenesis. All the expressed enzymes, including wild-type and mutants with the exception of the inactive mutant, ETT-C22S, were variably inhibited by iodoacetamide, and similar results were obtained when these enzymes were preincubated with GSH. However, when preincubated with S-sulfocysteine or hydroxyethyl disulfide, the activity of the enzymes was totally or partially protected against inhibition by iodoacetamide, with the exception of the mutants, ETT-C25S and ETT-C25A. When simultaneously pretreated with GSH and S-sulfocysteine, all enzymes were highly protected. Isoelectric focusing analysis of the above preincubation mixtures showed that different enzyme-substrate intermediates occurred. Using radioactively labeled substrates, [U-14C]cystine and [glycine-2-3H] GSH, enzyme-substrate intermediates were detected. These data indicate that reduced thioltransferase reacts first with disulfide substrates, then with a thiol substrate, e.g. GSH. The formation of either enzyme-substrate mixed disulfide or protein intramolecular disulfide protected the enzyme from inactivation by iodoacetamide. Based on the experimental results, alternative methods of the catalytic mechanism for thioltransferases are proposed.