</sec> <sec> <title>Introduction

DRJ

Dent Res J

Dental Research Journal

1735-3327 2008-0255

Medknow Publications Pvt Ltd

India

DRJ-12-389

10.4103/1735-3327.166169

Review Article

The antioxidant master glutathione and periodontal health

Bains

Vivek K

Bains

Rhythm

Department of Periodontology, Sarsawati Dental College and Hospital, Lucknow, Uttar Pradesh, India Department of Conservative Dentistry and Endodontics, Faculty of Dental Sciences, King George′s Medical University, Lucknow, Uttar Pradesh, India

Address for correspondence:Rhythm Bains, House Number 1317, Sector 16, Indiranagar, Lucknow - 226 016, Uttar Pradesh, India docrhythm77@gmail.com

Sep–Oct 2015

12 5 389 405

2015

This is an open access article distributed under the terms of the Creative Commons Attribution NonCommercial ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Glutathione, considered to be the master antioxidant (AO), is the most-important redox regulator that controls inflammatory processes, and thus damage to the periodontium. Periodontitis patients have reduced total AO capacity in whole saliva, and lower concentrations of reduced glutathione (GSH) in serum and gingival crevicular fluid, and periodontal therapy restores the redox balance. Therapeutic considerations for the adjunctive use of glutathione in management of periodontitis, in limiting the tissue damage associated with oxidative stress, and enhancing wound healing cannot be underestimated, but need to be evaluated further through multi-centered randomized controlled trials.

Antioxidants gingival crevicular fluids glutathione periodontitis saliva

</sec> <sec> <title>Introduction

Oxidation is defined as a "chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent." Although, it is essential for survival of an organism, oxidation can produce reactive oxygen species (ROS). These include both true or oxygen derived free radicals such as superoxide radical (O ₂ ⁻), ¹hydroxyl radical (OH) and nitric oxide (NO) radical (NO ⁻) as well as non-oxygen derived radicals such as hydrogen peroxide (H ₂O ₂) and hypochlorous acid (HOCl); ²and can start chain reactions resulting in cell damage or death.

"Antioxidants (AOs) are molecules, which when present at low concentrations compared to those of an oxidizable substrate, will significantly delay or inhibit oxidation of that substance." ³, ⁴They cease destructive chain reactions started by ROS. ¹Living organism cells have developed numerous AO defense systems to contain ROS mediated damage, viz enzymatic AOs such as peroxidases, ⁵thiol-containing peptides such as glutathione and thioredoxine, and sophisticated transcriptional programs such as ones controlled by Forkhead box O. ⁶Melatonin, which is N-acetyl-5-methoxy-tryptamine, mainly produced by pineal gland, has also been shown to encompass powerful free-radical scavenger activity. ⁷, ⁸Currently, AOs have been categorized according to their location of action (intracellular, extracellular or membrane associated); solubility (water soluble or fat soluble); origin (exogenous, endogenous or synthetic); structures they protect (DNA protective, protein-protective or lipid protective); mode of action (preventive or scavenging by chain breaking); ⁴Major exogenous sources of AOs are naturally obtained from the diet such as phytonutrients. Recently, manganese-based superoxide dismutase like synthetic AO synzyme (M40403), and azymes ⁹have also been added to synthetic AOs group of N-acetylcysteine, pencillinamine and tetracyclines. ⁴

Oxidative stress (OS) leads to molecular damage resulting from disturbances in redox signaling and control due to an imbalance between oxidants and AOs oxidants ROS. ¹⁰, ¹¹, ¹²OS is considered to play an important role in the pathogenesis of chronic inflammatory conditions like non-insulin dependent diabetes mellitus, cardiovascular disease, metabolic syndrome, ¹¹, ¹³, ¹⁴, ¹⁵acute respiratory distress syndrome, ⁵, ¹⁶and periodontal disease. ⁵, ¹⁷, ¹⁸, ¹⁹, ²⁰, ²¹, ²²Recently, it has been proposed as a central link between systemic diseases and periodontitis. ²³

Ubiquitous AO master, glutathione is a low-molecular weight, major non-protein cellular thiol, present in both eukaryotic as well as prokaryotic cells, ²⁴, ²⁵, ²⁶and exist in every single cell of the human body. ²⁴It is a potent AO that prevents ROS mediated damage to essential cellular components and acts as a cofactor for enzymes in the destruction of ROS. It serves as a reservoir for cysteine, participates in detoxification reactions for xenobiotics and metabolism of numerous cellular compounds (e.g., NO), and is required for synthesis of some prostaglandins (PGs) and thermotolerance. ²⁵It also has a significant role in nutrient metabolism and regulating cellular events as cell apoptosis, cell proliferation, cytokine production, immune response, gene expression, DNA and protein synthesis, signal transduction and protein glutathionylation. ²⁷

Periodontitis is a chronic inflammatory condition initiated in response to plaque biofilm, characterized by exaggerated inflammation and loss of periodontal and bone support, with the excessive production of ROS and proteolytic enzymes. ²⁸Periodontitis patients reported having reduced total AO capacity in whole saliva, ⁵and lower concentrations of reduced glutathione (GSH) in serum and gingival crevicular fluid (GCF). ²⁹, ³⁰This paper thus aims to review and discuss the basic mechanism and properties of glutathione homeostasis for the clinicians and researchers along with updating the current knowledge and investigations implicit in periodontal health.

Materials and Methods

Related literature was searched using the MedLine/PubMed database, online Cochrane library and Google scholar, with an emphasis on peer-reviewed dental journal until October 2013. Appropriate information was also obtained from Kyoto Encyclopedia of Genes and Genomes and BioProject online database. The medical subject headings (MeSH) terms used for search were "glutathione and periodontal disease," "glutathione and periodontitis" and "glutathione and periapical health." Search engine MedLine/PubMed when searched for MeSH word "glutathione" revealed 109509 papers, and search when was further filtered for MeSH word "glutathione and periodontal disease," and "glutathione and periodontitis" shown 85, and 71 papers respectively. Online Cochrane library database revealed one controlled clinical trial when searched for "glutathione and periodontal." Pertinent articles in English language on the topic and abstracts of relevant papers were scrutinized thoroughly, and finally papers pertaining to the topic were included after excluding the duplicates Table 1, Table 2, Table 3, Table 4and Table 5. ⁴, ⁵, ²⁸, ²⁹, ³¹, ³³, ³⁴, ³⁵, ³⁶, ³⁷, ³⁸, ³⁹, ⁴⁰, ⁴¹, ⁴², ⁴³, ⁴⁴, ⁴⁵, ⁴⁶, ⁴⁷, ⁴⁸, ⁴⁹, ⁵⁰, ⁵¹, ⁵², ⁵³, ⁵⁴, ⁵⁵, ⁵⁶, ⁵⁷, ⁵⁸, ⁵⁹, ⁶⁰, ⁶¹, ⁶², ⁶³, ⁶⁴, ⁶⁵, ⁶⁶, ⁶⁷, ⁶⁸, ⁶⁹, ⁷⁰, ⁷¹, ⁷², ⁷³, ⁷⁴, ⁷⁵, ⁷⁶, ⁷⁷, ⁷⁸, ⁷⁹, ⁸⁰, ⁸¹, ⁸², ⁸³, ⁸⁴, ⁸⁵, ⁸⁶, ⁸⁷, ⁸⁸, ⁸⁹, ⁹⁰, ⁹¹, ⁹², ⁹³, ⁹⁴, ⁹⁵, ⁹⁶, ⁹⁷, ⁹⁸, ⁹⁹, ¹⁰⁰, ¹⁰¹, ¹⁰², ¹⁰³, ¹⁰⁴, ¹⁰⁵, ¹⁰⁶, ¹⁰⁷, ¹⁰⁸, ¹⁰⁹, ¹¹⁰, ¹¹¹Relevant literature in common textbooks, bibliographies of papers and review article together with appropriate peer-reviewed print journals, were also analyzed for additional information.{Table 1}{Table 2}{Table 3}{Table 4}{Table 5}

Literature Review

In 1888, de Ray Pailhade gave the first hint of "organic stuff" related to the metabolism of sulfur. ¹¹²In 1922, Sir Frederick Gowland Hopkins ¹¹³discovered and characterized glutathione, and erroneously described it as dipeptide of glutamic acid and cysteine in a classic paper published in Journal of Biological Chemistry. In 1927, Hunter and Eagles doubted Hopkin′s hypothesis, and reported tripeptide structure of glutathione; ¹¹⁴which was later on accepted by Hopkins ¹¹⁵in 1929, who then indeed described it as a tripeptide of glutamic acid, glycine and cysteine (Glu-Cys-Gly). ¹¹⁶

Biochemistry

Tripeptide, glutathione (chemical formula, C ₁₀H ₁₇N ₃O ₆S; structural formula, γ-L-glutamyl-L-cysteinylglycine, International Union of Pure and Applied Chemistry name 2-amino-5-{[2-carboxymethyl)-amino]-1-(mercaptomethyl)-2-oxoethyl]-amino}-5-oxopentanoic acid) consists of a unique gamma (γ) peptide linkage among the amine group of cysteine with the carboxyl group of the glutamate side-chain. ¹¹⁷It is water soluble in nature, having molecular mass of 307.3235 g/mol and melting point of 383°F/195°C. Glutathione is the predominant cellular thiol present in mammalian cells at concentration 0.5-10 mmol/L. Eighty-five to ninety percent of cellular glutathione is present in the cytosol, and rest 10-15% is distributed in many intracellular organelles like mitochondria, nuclear matrix, endoplasmic reticulum (ER), peroximes and nucleus. ²⁶, ²⁷, ¹¹⁸Concentration of mitochondrial glutathione is similar to that of the cytosol (10-14 mM) by the volume of the mitochondrial matrix. ¹¹⁸Its concentration in the human liver tissue is evaluated as 6,400 mmol/kg. ¹¹⁷Relatively low (2-20 μmol/L in plasma) concentration of glutathione is observed in the extracellular environment, whereas, bile acids may contain up to 10 mmol/L glutathione. ²⁷Glutathione concentration is found to be greatest in the liver, the organ involved in detoxification and elimination of toxins, and in the lung epithelial lining fluid (ELF) (of about 400 μM), which is raised in the case of chronic smokers and lung cancer patients, ³⁴, ¹¹⁷and decreased in patients with pulmonary fibrosis and acute respiratory distress syndrome. ³⁴

The AO properties of glutathione are mediated by redox-active thiol group that becomes oxidised when glutathione reduces target molecules, which may be attributed to the fact that glutathione displays a low redox potential (E′0 = −240 mV), and is found in high concentration in the cells (approximately 10-15 Mm). ¹¹⁸In the presence of electrophilic substances, e.g., free radicals and ROS/nitrogen sps, glutathione readily oxidizes non-enzymatically to glutathione disulfide (GSSG). ²⁷As glutathione acts as electron carriers, so when electrons are lost by oxidation, glutathione becomes oxidized (GS ⁻), and two such molecules dimerized by a disulfide bridge to form disulfide glutathione or oxidized glutathione (GSSG). Total glutathione pool consist of more than 90% of "the reduced-form (GSH)", and <10% exists in "the disulfide or oxidized form (GSSG)" in healthy cells and tissues, ¹¹⁷and a significant amount (15%) of total glutathione of cell (GSH + 2GSSH) concentration may be bound to protein. ²⁷Under normal physiological conditions, the GSH: GSSH ratio (that is an indicator of cellular redox state) is >10. Antioxidative capacity of cells is determined majorly by redox couple GSH/GSSG, however, other redox couples as nicotinamide adenine dinucleotide phosphate (NADPH)/NADP ⁺and thioredoxinred/thioredoxinox could also affect its value. Many pathological conditions, OS, protein malnutrition, can markedly reduce the cellular glutathione concentrations. ²⁷The process of regulation of relative levels of glutathione and GSSG, constitute glutathione peroxidase (GSHPX) reaction by which glutathione reduces H ₂O ₂or lipid peroxides producing GSSG. NADPH is used to reduce the GSSG to glutathione, which is catalyzed through glutathione reductase (GR), a flavoprotein enzyme. ¹¹⁹NADPH is resupplied by a reduction of NADP ⁺via pentose-phosphate pathway Figure 1. ¹¹⁸In cases of rapid production of GSSG, or impaired GR activities, GSSG accumulation occurs, that is either transported from the cells or reacts with protein sulfhydryls (SHs), via a mixed disulfide reaction, potentially resulting in impaired protein function, and net loss of intracellular glutathione. ²⁵ Figure 1

Oxidation and reduction of glutathione. ¹¹⁸

Figure 1

Intracellular compartmentalization of glutathione includes separate redox pools that are distinct from the cytoplasmic pools in terms of GSH/GSSG ratio, their redox potential, and their control of cellular activities. In the nucleus, critical protein sulfhydryl necessary for DNA repair and expression is maintained by glutathione. Further, it plays a contributory role in DNA synthesis by donating hydrogen in ribonucleotide reductase-catalyzed reduction of ribonucleotides to deoxyribonucleotides. In the ER, glutathione mainly exist as GSSG, which is the main source of oxidizing equivalents to provide a suitable environment for favoring disulfide bond formation and proper folding of nascent proteins. However, in mitochondria, glutathione is found mainly in reduced form (GSH) and represents a minor fraction of the total glutathione pool (10-15%). Because of the lack of catalase in mitochondria, H ₂O ₂metabolism is mediated through glutathione, with participation of GSHPX or peroxiredoxin. In mitochondria matrix, cytosolic GSHPX (cGSHPX or GSHX-1), which is the major isoform of GSHPX localized in cytoplasm, is found only in a small fraction. ¹¹⁸

Glutathione biosynthesis

Glutathione is synthesized intracellularly, by two enzymes namely glutamate-cysteine ligase (GCL) or γ-glutamate-cysteinyl-synthetase (GCS), which is the rate-limiting step and glutathione synthetase, and is regenerated by six enzyme-catalyzed reactions known as γ-glutamyl cycle. ³⁴Of the three building blocks of glutathione, viz., cysteine, glutamic acid and glycine, the sulphydryl or thiol (SH) group in cysteine serves as a proton donor, and acts as a limiting factor in glutathione synthesis. In the absence of reduced cysteine, the addition of an acetyl group to cysteine (N-acetyl-cysteine [NAC]) provides the ability for a molecule to cross the cell membrane and promotes intracellular glutathione synthesis. ¹¹⁷

It is synthesized in cytosol outside the ribosomes that requires ATP, but does not require the participation of RNA. L-glutamate condenses with L-cysteine to form γ-L-glutamyl-L-cysteine, which requires GCS in presence of ATP, K ⁺and Mg ⁺⁺with the formation of the enzyme bound γ-glutamyl-(P) as an intermediate. ⁹γ-carboxyl group of glutamate form a peptide γ-linkage after reaction with the amino group of cysteine, which protects glutathione from hydrolysis by intracellular peptidase. γ-L-glutamyl-L-cysteine can be a substrate for γ-glutamylcyclotransferase (GCT), however, due to higher affinity and activity of glutathione synthetase (GS), glutathione synthesis is favored in animal cells. ¹¹⁸In the presence of GS and ATP, γ-L-glutamyl-L-cysteine is phosphorylated to form enzyme bound γ-glutamyl-cysteinyl-(P), which reacts with Gly to liberate free glutathione and Pi. ⁹This pathway occurs virtually in all cell types (except epithelial cells), with liver being the major producer and exporter of glutathione.

γ-glutamate-cysteinyl-synthetase or GCL is in part regulated by glutathione feedback inhibition, ²⁶which can be partially prevented by an excess of glutamic acid that blocks the regulatory site on the enzyme. ²⁵Thus, if glutathione is exhausted due to OS, exposure to xenobiotics or inflammation, de novo synthesis of glutathione is up-regulated. ²⁶Inhibition of GS results in the accumulation of γ-L-glutamyl-L-cysteine that is converted to cysteine and oxoproline by GCT, and then oxoproline is converted to glutamic acid via 5-oxoprolinase reaction by oxoproplinase. Life-threatening acidosis occurs due to the accumulation of oxoproline in the absence of GS Figure 2. ²⁵Two amino acid transport systems namely; sodium-dependent system for cysteine transports, and sodium-independent cysteine and glutamate transport system, participate in primary regulation of substrates for glutathione synthesis. Transpeptidase activity at the cell surface is accountable for recovering amino acids from circulating glutathione for reuse in the intracellular glutathione synthesis. ²⁵ Figure 2

Glutathione Synthesis (γ-glutamyl cycle). Dotted line show mechanism of life threatening acidosis in case of inhibition of glutathione synthetase. ²⁵

Figure 2

After synthesis, intracellular glutathione is exported from most cells. Outside the cell, γ-glutamyl-cysteine bond of glutathione may be hydrolyzed by γ-glutamyl transpeptidase (GGT) ²⁴, ²⁶and transfers a γ-glutamyl moiety from extracellular glutathione to an amino acid. ²⁵The activity of GGT is important in glutathione cycle and serves as a salvage pathway for cellular glutathione. ²⁶Glutathione and other γ-glutamyl containing compounds (e.g. GSSG and γ-glutamyl glutathione) react with the transpeptidase at the outer cell surface. The γ-glutamyl moiety is transferred to a suitable amino acid acceptor, and both the γ-glutamyl amino acid (dipeptide) and the cysteinylglycine are transported into the cell, and reused for glutathione synthesis. Peptidase cleaves cysteineglycine, and if the dipeptide is also γ-glutamylcysteine, they can be utilized directly for glutathione synthesis; otherwise dipeptide is converted to glutamate by GCT and oxoprolinase. ²⁵

Glutathione functions in systemic health

Glutathione has been considered as a true protagonist in cellular regulation. ¹¹²Interrelations linking the antitoxic, AO, prooxidant, and modulator roles of glutathione in cellular homeostasis have been outlined by Pompella et al., in 2003. ¹¹²Glutathione is involved in the formation and maintenance of protein disulfide bonds, as well as in the amino acids transport across cell membranes. Glutathione efficiently scavenges free radicals and other ROS (OH, lipid peroxyl radical, peroxynitrites and H ₂O ₂), directly or indirectly, while oxidizing glutathione to GSSG. ²⁷By donating H ₂, it helps to destroy H ₂O ₂and other peroxides in cells, which are catalyzed by containing GSHPX. ⁹, ²⁷The reaction given below is catalyzed by GSHPX as follows: ¹²⁰, ¹²¹

2GSH + H ₂O ₂→ 2GSSH + 2H ₂O

Rotruck et al. ¹²²recognized that the selenium (as an active site selenocysteine residue) ¹¹⁹is a structural constituent of the active center of the animal enzyme GSHPX-1. Analysis of selenoproteome characterized the functions and sequence of six types of GSHPX in mammals as; cytosolic (cGSHPX or GSHPX-1), gastrointestinal (GI-GSHPX or GSHPX-2), plasma (pGSHPX or GSHPX3), phospholipid hydroperoxide (phGSHPX or GSHPX-4), epididymal androgen related protein or secretory (GSHPX-5) and olfactory (GSHPX-6). ¹²³

Glutathione peroxidase-1 prevents cytotoxic peroxide-induced oxidative damage, lipid peroxidation and protein degeneration. GSHPX-1 counteracts hydroperoxide-modulated events, such as cytokine signaling and cluster of differentiation-95-triggered apoptosis, to eliminate neoplastic cells and plays an important role in HIV infection. Studies report that reduction in glutathione and GSHPX-1 activity prior to infection can result in a decrease in virus spread because of apoptosis initiated by oxidative microenvironment. In HIV patients, the glutathione levels are the sole predictor of death, and there is also a hypothesis that, "the mechanism responsible for AIDS could be reversed by the administration of reducing agents, especially those containing SH groups". ¹¹⁷GSHPX-2 might be an anti-inflammatory and anti-carcinogenic enzyme. GSHPX-4 is required for embryogenesis and male fertility. ¹²³

For AO actions, glutathione acts as a cofactor for GSHPX and other enzymes (as glutathione-dependent dehydroascorbate reductase) active in cell defense against prooxidants. Further, glutathione-dependent dehydroascorbate reductase is involved in the reduction of the oxidized product, dehydroascorbate to ascorbic acid by using glutathione as an electron donor (ascorbate recycling). It is thus a primary cellular tool in order to maintain steady state concentrations of ascorbic acid (a perfect AO) in accelerated oxidation conditions. ¹¹²

It acts as a coenzyme with liver enzyme glutathione-insulin transhydrolase which helps in the catabolism and degradation of the protein hormone inulin. ⁹Targeting endogenous NO, intracellular glutathione, conjugates with NO to form an S-nitrosoglutathione (GSNO) adduct that in-turn is cleaved by the thioredoxin system to produce glutathione and NO. Both glutathione and NO are necessary for the hepatic action of insulin-sensitizing agents, thus have a critical role in regulating glucose, amino acid and lipid utilization. ²⁷Glutathione and the enzyme glutathione transhydrogenase help to cause reductive cleavage of S-S linkages in thyroglobulin glycoprotein. Many-SH group contacting enzymes (e.g. glyceraldehydes-3-P-dehydrogenase) are also protected by glutathione against their - SH groups oxidation. ⁹

Glutathione plays an important role for vital functions as it is required for the proliferation of cells (lymphocytes and interstitial epithelial cells); spermatogenesis and sperm maturation; activation of T-lymphocytes and polymorphonuclear leukocytes; for cytokine production and for inhibiting influenza virus infection as reported by in vivo studies. ²⁷It is required as coenzyme/cofactor with prostaglandin-synthetase system (cyclooxegenase) used for formation of endoperoxides from arachidonate in prostaglandin synthesis, ⁹and for the conversion of prostaglandin H ₂into prostaglandin D ₂and E ₂by endoperoxide isomerase. ²⁷It is required as a coenzyme with maleylacetoacetate isomerase that catalyzes Cis-trans isomerization of the maleylacetoacetate to fumarylacetoacetate, and for the enzyme glyoxylase which converts methylglyxol to lactic acid through intramolecular oxidation-reduction. ⁹, ²⁷It also takes part in γ-glutamyl cycle for absorption of amino acid from the gut. ⁹

It function as a coenzyme with formaldehyde dehydrogenase which catalyzes the oxidation of formaldehyde, a carcinogen, produced during metabolism of methionine, choline, methanol, sarcosine and xenobiotics (by the cytochrome P450-dependent monooxygenase system of the ER), to formic acid. ⁹, ²⁷Glutathione-S-transferase (GST), a family of phase II detoxification enzymes, initiates reaction of glutathione with various electrophiles, physiological metabolites (e.g. estrogen, PGs, melanins and leukotrienes), and xenobiotics (e.g. drugs [bromobenzenes and acetaminophen], pollutants, carcinogens) to form mercaptures. ²⁷Beside conjugation of xenobiotics, GST-Pi act as Jun N-terminal kinase (JNK)-inhibitor, thus preventing c-Jun phosphorylation, by forming macromolecular complexes with JNK in vitro. ¹²⁴

It is capable of forming disulfide bonds with cysteine residues of protein known as S-glutathionylation. ¹¹²S-glutathionylation of proteins (ubiquitin-conjugating enzyme, cytochrome c oxidase and thioredoxin) plays an important role in cell physiology, ²⁷affecting members of signal transduction chains involved in cell proliferation. ¹¹²Increase in apoptosis and reduction in cellular proliferation by the activation of several signaling pathways like protein kinase B, H-ras, T-cell p59, c-JNK, apoptosis signal-regulated kinase 1, protein phosphatase 1 and 2A, calcineurin, nuclear factor-kB (NF-kB)/p50, and mitogen-activated protein kinase, is evident by the reduction in GSH/GSSG ratio. ²⁷, ¹¹²

Glutathione is needed for the conversion of Dopa-quinone to glutathione-Dopa synthesis of pheomelanin and trichochromes, as well as a coenzyme for liver enzyme for activation of methionine to form active form "S-adenosyl methionine." ⁹GSSG is harmful to red blood cell (RBC) membranes and lens proteins and is converted to reduced glutathione, which is required for the integrity of RBCs membrane and lens proteins. ⁹

Recent studies have shown the pro-oxidant properties of glutathione in which glutathione and its catabolites (e.g. Cys-Gly) promote oxidative process, by participating in metal ion-mediated reactions membrane-bound by GGT activity, eventually leading to formation of ROS and free radicals. ¹¹²Extracellular breakdown of glutathione by GGT, and the consequent release of the highly reactive dipeptide thiol Cys-Gly is likely to be a major determinant of protection exhibited by glutathione cisplastin nephrotoxicity. ¹²⁵Studies have further identified membrane-bound GGT activity as a factor of drug resistance, in normal and cancer cells. ¹²⁶Cells expressing sufficient GGT activity at their surface might be able to effect ′extracellular detoxification′ of electrophilic drugs. ¹¹²

Transcription factor "NF (erythroid-derived 2)-like 2 (NFE2-L2 or NFE2-related-factor-2 [Nrf2])" is a master regulator of the AO response that modulate the expression of hundreds of genes controlling various immune and inflammatory responses, carcinogenesis and metastasis, tissue remodeling and fibrosis, and cognitive dysfunction and addictive behavior. ¹²⁶Nrf2, a positive regulator of the human AO response element (ARE), develops expression of AO enzyme such as NADPH: Quinone oxidoreductase 1. ¹²⁷Itoh et al. ¹²⁸described the mechanism of Nrf2 activation that involve a protein Keap1 which is a suppressor protein anchored in the cytoplasm that physically binds Nrf2, thus preventing its translocation to the nucleus and its access to ARE containing promoters. Wild et al. ¹²⁹have validated that Nrf2 is positive controller of GCS gene expression, involved in glutathione synthesis. Fan et al. ¹³⁰hypothesized that "HIV-1-related proteins inhibit Nrf2 mediated AOs defences and thereby disrupt the normally tight alveolar epithelial barrier". They suggested that "activating Nrf2/ARE pathway with the dietary supplement sulforaphane (SFN) could augment AO defenses and lung health in HIV-1 infected individuals." ¹³⁰Dietary supplement Protandim, a potent composition of highly synergistic phytochemical Nrf2 activators, shows significant modulation not only of AO enzymes, but also those related to colon cancer, cardiovascular disease and Alzheimer disease. ¹²⁶

Glutathione in periodontal health and disease

Role of reduced glutathione in the regulation of pro-inflammatory cytokines is of great importance in periodontal disease. Studies revealed that by increasing the concentrations of the cytosolic cysteine, and thus glutathione of monocytes and macrophages using a synthetic form of cysteine called NAC, blocks H ₂O ₂mediated activation of NF-kB, and thus pro-inflammatory cytokines (tumor necrosis factors [TNF]-α, interleukin [IL]-1 β and IL-6) by this route. TNF-α, IL-1 β and IL-6 are associated with activation of bone-resorbing processes, and IL-8 is reported to polymorphonuclear leukocyte activity. ⁵, ³⁴

More recently, Dias et al., ⁹⁹first time revealed that significant redox disturbances exist in neutrophils of patients with periodontitis, which are associated with deregulation of anti-inflammatory transcription factor Nrf2 pathway resulting in neutrophil hyperactivity/hyper-reactivity. Further, activation of the redox-sensitive protein acid sphingomyelinase to promote lipid raft formation, and therefore assembly of the NADPH oxidase enzyme was also mediated by a disturbance in redox balance. Both mechanisms, result in persistent hyperactivity in neutrophils of periodontitis patients. ⁹⁹

Activities of certain pathogens and cigarette smoking have shown to lower glutathione levels within periodontal cells, and OS depletes glutathione, resulting in activation of redox-sensitive transcription factors and thus in the creation of a pro-inflammatory state. ⁴Certain putative periodontopathogens, e.g., Fusobacterium nucleatum vincentii, F. nucleatum polymorphum, F. nucleatum nucleatum, F. nucleatum fusiforme, Treponema denticola, Bacteroides Loescheii, Porphyromonas gingivalis, Bacteroides inermedius and Peptostreptococcus micros, are capable of metabolizing L-cysteine or degrading glutathione to form hydrogen sulfide (H ₂S) within the periodontal pocket, which is toxic to mammalian cells by inactivating cytochrome oxidase, and is also reported to inhibit the catalase. ⁵, ³⁴

Mδkinen and Mδkinen ¹³¹reported the presence of enzyme GGT (that breaks down glutathione to Cys-Gly in human cell membrane) in the outer cell envelope of T. denticola that may play an important role in its propagation in inflamed periodontal tissues. Three-step pathways have been proposed for the glutathione metabolism in T. denticola. In the first step, glutathione is cleaved into glutamate or glutamine and dipeptide Cys-Gly, catalyzed by GGT. Second step include cleavage of Cys-Gly into Gly and L-cysteine, in the presence of key enzyme cysteinyl-glycinase (52-kDa leucyl aminopeptidase); ⁶⁶and finally, L-cysteine is degraded into pyruvate, ammonia and H ₂S, in the presence of L-cysteine desulfhydrase (cystalysin, 46-kDa protein). ⁴², ⁴⁵, ⁶⁶, ¹³²Chu et al., ⁴², ⁴⁵ascribed virulence activity of T. denticola to the synthesis of H ₂S and puryvate from glutathione. Former is crucial for hemoxidative, hemolytic and other toxic activities that could occur in vivo, and pyruvate promotes bacterial growth.

Two sub-species of P. micros and five Fusobacteria species have also been reported to utilize glutathione to produce H ₂S. ³¹, ³⁴Within the periodontal pocket, H ₂S formation is toxic to mammalian cells that inactivate cytochrome oxidase and inhibit the catalase. The latter activity could positively feedback into NF-kB mediated transcription of proinflammatory cytokines. ²⁹, ¹³³Peptostreptococcus, Eubacterium, Selenomonas, Centipede, Bacteriodes, and Fusobacterium also metabolized cysteine to produce volatile sulfur compounds. ¹³⁴Moreover, Chapple ⁵, ³⁴advocated that degradation of cysteine by oral microorganisms may also prevent the inhibition of NF-kB mediated production of pro-inflammatory cytokines in the periodontal environment, and hence cytokine related tissue destruction.

A series of studies have shown association linking cigarette smoking and nicotine toxicity to glutathione depletion within periodontal tissues. ⁴Studies of betel nut chewing, and periodontitis have shown that arcea nut alkaloid, arecoline-induced thiol depletion in periodontal ligament fibroblasts may render them more susceptible to the effects of nicotine; ⁵⁷, ⁵⁸glutathione protection negated nicotine toxicity; ⁴¹and that cigarette smoke decreased periodontal ligament fibroblast glutathione levels in a dose-dependent manner and stimulated stress-specific genes. ⁴⁴

As already discussed, GST is xenobiotic metabolizing enzyme required for the detoxification reactions and utilizes free glutathione. Thus, inducers of GST gene expression generate ROS by metabolic processes resulting in depletion of free intracellular glutathione. Kim et al., ⁴⁷reported that subjects with polymorphic GST-M1 allele have significantly increased risk of periodontitis.

Glutathione and saliva

Periodontal diseases are associated with disturbances in the balance between the oxidants and AOs, in favor of oxidants due to both an increase in ROS as well as decreased AO activity in saliva. Salivary glutathione levels, evidence of the neutralization (scavenging) of ROS were low in patients from periodontal diseases in contrast to individuals with healthy gums. ¹¹¹Studies have demonstrated reduced amounts of salivary glutathione, GSH peroxides and thiol levels in the gingivitis and periodontitis patients as compared to the healthy group Table 3. ⁷², ¹⁰⁷, ¹¹¹

Studies reveal that the periodontal treatment result in improvement in salivary concentrations of glutathione and thiol levels in gingivitis and periodontitis subgroups, which was significant within groups, but not between groups. ⁵⁰, ¹⁰², ¹⁰⁷Diab et al., ¹¹¹reported improvement in salivary glutathione concentrations from 3.12 ± 0.21 μM/L to 3.6 ± 0.22 μM/L, collected in the patients with periodontal disease before and after treatment with mouthwash with extracts of Solanum melongena (egg-plant) peduncles against placebo during 3 months. They further reported that S. melongena extracts may protect the oral cavity by enhancing its AO properties, and consequently the adverse effect of smoking was attenuated. ¹¹¹

Glutathione and gingival crevicular fluid

Data from the high-performance liquid chromatography analyses indicate that glutathione present within the GCF is responsible for the stepped AO response. In GCF concentrations of glutathione (range 0.5-2.5 mM) were reported to be 1000 times greater than that normally detected within extracellular tissue compartments (0.5-5 μM in human plasma). ²⁹The high levels of glutathione within GCF may be the result of increased synthesis by cells of the periodontal tissues, or of active release mechanisms or passive release secondary to protease activity on gingival epithelial cells. ⁴Neutrophils and/or junctional/crevicular epithelium (JE) are significant contributors for the high concentrations of glutathione in GCF. Glutathione may be added to the list of defense factors in gingival crevice and lung known to be epithelial products (e.g., β-defensins and migration inhibiting factor-8). The relative position and function of JE of the gingival crevice are similar to the alveolar epithelium in the lungs; however, in contrast, lung epithelium is bathed and protected by alveolar ELF rather than GCF. Neutrophilic inflammation occurs in both lung and GCF, where activating cytokines are produced by the underlying fibroblasts, lining the epithelium and inflammatory cells in response to microbial insult.

The high concentrations of glutathione in GCF in health and extracellularly in the lung are similar, and therefore may represent an important AO and anti-inflammatory (anti-proinflammatory cytokines) defense strategy common to exposed epithelial cells. ²⁹Both sites consist of fluid containing high concentrations of glutathione, and in chronic inflammatory conditions, glutathione concentrations are reduced in both sites, owing to the failure of normal tissue homeostatic mechanisms to protect against the host-mediated tissue damage. Chapple et al., ²⁹were first to investigate the possible differences in GCF AO capacity between periodontal health and disease. They observed that glutathione concentrations are reduced in chronic periodontal diseases to about 1183.1 ± 580.3 μM as compared to 1899.8 ± 494 μM in control GCF. The lower concentrations of glutathione in GCF of periodontitis patients could be because of various factors resulting in decreased synthesis and/or enhanced local degradation. It may involve an inborn defect in γ-glutamyl pathway of glutathione synthesis (controlled by cellular factor) or other exogenous or endogenous agents such as the local microbes (already explained). ²⁹More recently, Grant et al., ⁸²reported improvement in glutathione concentrations in GCF following periodontal therapy from 1182 ± 711 μM to 1424 ± 554 μM Table 4.

Glutathione levels in serum, plasma, gingival tissues and other cells

Gingival tissue analysis revealed that the AO activity in patients with periodontitis was decreased when compared to control the group. Various studies reported significant increase in GSHPX and GST, whereas decrease in GSH and total glutathione levels in gingival tissues and serum from an experimental group (periodontitis patients) when compared to the control group (periodontally healthy patients) Table 5. ²⁸, ⁵⁷, ⁹⁰, ⁹², ¹⁰³Peripheral blood neutrophils from chronic periodontitis patients have more GSSG level relative to GSH as compared to neutrophils from healthy control subjects. Normally, the kelch-like erythroid-cell-derived protein with CNC homology (ECH)-Associated protein-1-Nrf2-(Keap-1-Nrf2-ARE) signaling pathway elicits an adaptive response for cell survival under OS, but they did not appear to operate effectively in periodontitis patients. ⁹⁹

Therapeutic considerations of glutathione

Glutathione supplementation may help to prevent or treat many conditions associated with impaired immune function due to elevated OS or aging [Table 6]. ¹¹⁷It has been postulated that raising glutathione levels in cells have reduced cancer growth by suppressing the activity of certain chemically-reactive oxygen molecules in the test tubes. Although, glutathione is readily available in fresh fruits, vegetables and meat (e.g. apple, carrot, grapes, spinach, tomato, asparagus, avocado and purslane), but consuming them would transfer only small amount of reduced glutathione into the blood stream because most of it is lost in the digestive tract. However, studies revealed that blood glutathione levels increased by supplementing its precursors or other dietary products. ¹¹⁷{Table 6}

Hence, with the growing evidences that link reduced glutathione levels with periodontal inflammation, Chapple and Matthews ⁴postulated that increasing or preserving intracellular glutathione levels within the cells of the periodontal tissues is likely to provide a novel adjunctive AO and anti-inflammatory strategy to traditional periodontal therapies. P. gingivalis induces excessive activation of the innate immune response and induces melanization which leads to the generation of ROS, and apoptotic cell death in the host tissue. AOs (N-acetyl-L-cysteine, glutathione and catalase) have therapeutic effects in delaying P. gingivalis induced death of silk-worms. ⁸⁰Further, the stress-inducible protein heme oxygenase-1 expressed in human gingival fibroblasts exposed to nicotine has been reported to be overcome by the addition of the glutathione precursor N-acetyl-L-cysteine. ⁴⁹, ⁸⁸Minor antioxidative effects have been documented after the application of exogenous glutathione and cysteine, however, whether exogenously applied substances elevated the intracellular glutathione concentrations, still need to be investigated. ¹³⁵

Improvement in the rate of wound healing has been observed with topical NO donor, GSNO. Improved rates of decreased inflammation, re-epithelialization, wound contraction, increased collagen fiber density and organization as well as decreased neovascularization when applied to ischemic wounds have been observed as compared with control groups. Thus, topical GSNO containing hydrogel has therapeutic potential to improve wound healing in an environment driven by OS by re-establishment of matrix metalloproteinase-1/ tissue inhibitor of matrix metalloproteinase-1 (MMP-1/TIMP-1) ratio, resulted in extracellular matrix production and re-epithelialization, which could be beneficial to periodontitis patients. ⁸⁸Both exercise particularly moderate exercise (vs. vigorous exercise) and dietary interventions such as consumption of cruciferous vegetables have also been shown to increase glutathione levels. ²⁶

Recent evidence of the increase in glutathione level and reduction in activity of GSHPX has been observed in experimental-periodontitis rats treated with proanthocyanidins (PC), a type of flavanoid extracted generally from grape seeds, suggests enhanced host resistance and inhibited OS by the dietary supplementation of PC. ⁸¹SFN, a natural product found in cruciferous vegetables, induces Nrf2 dependent AO gene expression by binding to cys151 on Keap-1. SFN have been reported to reinstate the intracellular redox state (GSH:GSSG) in neutrophils from periodontitis. Recent evidences highlighted the use of SFN to control Nrf2 dysregulation to restore neutrophil redox balance along with reducing hyper-reactivity. ⁹⁹Chapple et al. ¹³⁶also reported statistically significant clinical benefits of daily dietary supplements of capsules containing AO juice powder phytonutrients juice plus fruit-vegetable and fruit-vegetable-berry as adjunct to conventional scaling root planing in a double-blind, placebo-controlled randomized clinical trial.

Discussion

Oxidative stress in the pathogenesis of periodontitis is the result of an alteration in redox balance of the cells and tissues of innate immunity system. Glutathione, considered to be the master AO, is the most important redox regulator that controls inflammatory processes, and thus damage to the periodontium. Glutathione acts directly as a generic ROS scavenger or as a cofactor of GSHPX and GST, either by the conjugation/excretion processes of the Phase II reactions or by catalyzing the reduction of lipid hydroperoxides and H ₂O ₂.

Review of literature reported the utilization of different AO diagnostic biomarkers for periodontal diseases associated with glutathione. These include levels of reduced (GSH) and oxidized (GSSH) glutathione level, ratio of GSH: GSSH, ⁸²thio levels, glutaredoxin or N-acetyl cysteine, ¹⁰⁷GST, ¹⁰⁹GSHPX, GR, ⁵⁷and cysteine level, in patients′ GCF, saliva, serum or plasma, erythrocytes and gingival tissue. Studies have demonstrated high concentrations of glutathione in GCF, saliva, serum and gingival tissues which are compromised in the diseased state, due to abnormal redox balance secondary to OS resulting from periodontal inflammation. Periodontal pathogens may deplete glutathione and shift GSH: GSSG ratio toward oxidants, representing a clear biomarker of OS in periodontitis. Further, successful periodontal therapy has proven to restore the redox balance. ⁸²

Increased GSHPX in the gingival tissue in periodontal diseases has been reported by many. ⁵⁷This increase may signify possible AO recompense in detoxification reactions of organic peroxides created due to OS in gingival tissues. Similarly, the significant increase in GST concentrations in periodontitis patients is probably related to their catalyzing role in detoxification of xenobiotics derived from periopathogens, and neutralization of hydroperoxides derived from the lipoperoxidation process related to OS caused by the periodontal inflammatory process, in conjunction with glutathione. GR has a significant auxiliary AO function related to GSHPX and GST. It intervenes continuously to regenerate glutathione from GSSG in the presence of NADPH, therefore, prevents cellular loss of glutathione. ⁵⁷Patel et al., ⁹⁰reported statistically significant reduction in GSHPX concentrations of 19.41 ng/μl and 85.21 ng/μl in GCF and serum after non-surgical periodontal therapy, respectively.

Antioxidant as an adjunct to root surface debridement have improved the periodontal therapy outcome in periodontitis patients by overcoming delayed wound healing induced by imbalance in redox status. Zhang et al. ¹³⁷in a clinical survey demonstrated that lower dietary AOs intake leads to destruction of the periodontium and a higher incidence of periodontal diseases. AO epigallocatechin-3-gallate, a most abundant catechin in green tea, reduces gingival inflammation, prevent bone resorption and limit the growth of periodontal pathogens, thus promoting periodontal health. ¹³⁸Organosulfur-compound diallyl sulfide enhances AO activities by scavenging superoxide ions and enhancing GSHPX levels, thus unveiling a significant antimicrobial effect against the Aggregatibacter actinomycetemcomitans. ¹³⁹Recent reports suggest that the topical use of esterified glutathione could potentially minimize effects of OS with raised intracellular levels of glutathione and acceleration of wound healing by improving the capacity of fibroblasts and keratinocytes. ⁸⁸

Oxidative stress is one of the underlying factors explaining the systemic pathophysiological mechanisms associated with the chronic inflammatory conditions as periodontitis. ⁹⁹A decrease in salivary reduced-glutathione levels in type 1 diabetes mellitus patients may have a role in the destruction of the periodontium by predisposing the periodontal tissues to OS. ⁷⁰It can be postulated that patients with reduced or depleted glutathione content, as in systemic disorders with compromised AO defenses, ⁸²may be more prone to further increase in OS, and worsening of the condition when superimposed with periodontal infection. Hence, future studies exploring this interrelationship of worsening of redox balance in systemically compromised patients and periodontitis, are further needed.

Conclusion

Although emerging evidences mostly in the form of case-control and animal studies have shown the role of glutathione in both periodontal pathogenesis as well as controlling periodontal disease, however limited number of randomized clinical trials have been published. Nevertheless, therapeutic considerations for the adjunctive use of glutathione in management of periodontitis, in limiting the tissue damage associated with OS and enhancing wound healing cannot be underestimated; however, it needs to be evaluated further through multi-centered randomized controlled trials.

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