Caroline SANDRE BALLESTER PhD

Scientific Coordinator

Summary

1. Peroxydases

2. OSCN Production

3. OSCN Action

4. OSCN Target Microorganisms

5. Bibliography

Peroxidases are a group of enzymes that catalyze oxidation-reduction reactions. As such, they are classified as oxidoreductases. They are given the official EC number 1.11.1.

Superoxide anion (O₂^.-) and hydroxyl radicals (^.OH), toxic molecules, can be found in cells due to the presence of oxygen. These are by-products of aerobic respiration. They are eliminated by a number of enzymes present inside the cell. Superoxide, for example, is destroyed by superoxide dismutase (SOD). The degradation, however, produces more hydrogen peroxide (H₂O₂), which is, in turn, destroyed by peroxidase. Peroxidases reduce H₂O₂ to water while oxidizing a variety of substrates. Thus, peroxidases are oxidoreductases which use H₂O₂ as electron acceptor for catalyzing different oxidative reactions. The overall reaction is as follows:

Donor + H₂O₂ <=> oxidized donor + 2 H₂O

You find numerous peroxidases in biological fluids, such as:

Salivary peroxidase (also known as lactoperoxidase)

Myeloperoxidase

Eosinophil peroxidase

Tears peroxidase

Thyroid peroxidase

Lactoperoxidase

Lactoperoxidase, Eosinophil peroxidase, tears peroxidase, salivary peroxidase use principally SCN^- as pseudo halide for the reaction & combine with H₂O₂ in order to produce (OSCN)^-

The structure and function of lactoperoxidase in creation antimicrobial molecules have been reviewed by de Wit and Hooydonk (1996). Next to xanthine oxidase, lactoperoxidase is the most abundant enzyme in milk and is found in the whey after cheese making.

Biological activity:

Pruitt. et al. (1982) showed that the lactoperoxidase-catalysed reactions yield short lived intermediary oxidation products of SCN^-, providing antibacterial activity. The major intermediary oxidation product is hypothiocyanite (OSCN)^-, which is produced in an amount of about 1 mol per mol of hydrogen peroxide. At the pH optimum of 5.3, the (OSCN)^- is in equilibrium with HOSCN. The uncharged HOSCN is considered to be more bactericidal of the two forms (Thomas. et al. 1983).

The action of (OSCN)^- against bacteria is reported to be caused by sulfhydryls (SH) oxidation (Aune and Thomas, 1978; Ekstrand. et al. 1985). The oxidation of -SH groups in the bacterial cytoplasmic membrane results in loss of the ability to transport glucose and also in leaking of potassium ions, amino acids and peptide.

(OSCN)^- has also been identified as an antimicrobial agent in milk, saliva, tears, mucus,...

To summarize, peroxidase are able to produce the antimicrobial component (H)OSCN which is now recognised as playing an important and leading role in the human host defense system.

(OSCN)^- &/or HOSCN are able to kill a wide range of pathogens.

2 OSCN Production

Catalyzed by a peroxidase under suitable conditions, potassium or sodium thiocyanate and hydrogen peroxide generate the antimicrobial ion, (OSCN)^- in water. The water containing the (OSCN)^- is free of enzymes and hydrogen peroxide.

(OSCN)^- can be produced up to 2000 µM in water using specially designed equipment or kits comprising the essential components.

The method, equipments, reactives and the antimicrobials produced (eg (OSCN)^-), are currently protected by patents.

3 OSCN Action

Hypothiocyanite (OSCN)^-/HOSCN it would be incorporated in airways and lungs (inhalation) or diffuses into the gastro-intestinal tracts (ingestion). It is noteworthy that the salivary peroxidase (SPO) or lactoperoxidase (LPO)/SCN^-/H₂O₂ system is endogenous.

Airway lactoperoxidase (LPO) activity was shown to increase the rate of bacterial clearance from sheep airways (Conner GE. et al. Am. J. Respir. Crit. Care Med., 2002; 166: S57-S61). In the same way, human airways contain a complete and functional antibacterial system that is effective against a variety of respiratory pathogens and thus may offer new approaches to boosting airway host defense in individuals with increased exposure or susceptibility to respiratory infection (Wijkstrom-Frei C. et al. Am. J. Respir. Crit. Care Med, 2003; 29: 206-12).

The Na⁺ - I^- symporter (NIS) may play an important role in moving SCN^- in epithelial cells and the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) channel may regulate its transport into the airway lumen (Fragoso MA. et al. J. Physiol., 2004; 561: 183-94).

Three mechanisms for SCN^- transport were identified in bronchial epithelial cells:

- Cl^- channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), which is cAMP-activated anion channel,

- Ca²⁺ dependent anion channels (Cl^- channels or oxidases),

- Anion transporter “pendrin”(Pedemonte N. et al. Journal of Immunology, 2007; 178: 5144-53).

Chronic respiratory infections in cystic fibrosis result from mutations of the CFTR channel used to concentrate SCN^- at the apical surface. Thus, impaired SCN^- transport to the airway lumen could potentially cause significant alteration of both the epithelial-derived lactoperoxidase (LPO) system as well as the neutrophil-derived myeloperoxidase (MPO) antibacterial activity (Conner GE. et al. FEBS Letters, 2007; 581: 271-18).

The GSH-SCN^- adducts are also carried by CFTR channel and by detoxication proteins (multi-drug resistance: MDR). SCN^- is a more preferred CFTR substrate than chloride anions (Childers M. et al. Medical hypotheses, 2007; 68: 101-12).

Hypothiocyanite-mediated bacterial killing, a novel innate defense system of airways, is defective in the Cystic Fibrosis (CF) airway epithelium due to the diminished SCN^- transport across the CFTR mutated channel. Normally, the production of (OSCN)^- eliminates Staphylococcus aureus and Pseudomonas aeruginosa, two frequent CF pathogens, on airway mucosal surfaces whereas it is nontoxic for the host (Moskwa P. Banfi B et al. Am. J. Respir. Crit. Care Med, 2007; 175:174-83).

Lactoperoxidase/SCN^-/H₂O₂ system has specific bacteriostatic and bactericidal impacts on invading microorganisms which can be explained by outer membrane permeability of bacteria for hydrophilic ^–OSCN ion. The membrane of the streptococcus is less affected by (OSCN)^- than that of E. coli certainly because of the different composition and physical structure of the cell wall and cell membrane (Marshall VME. et al. J. Gen. Microbiol., 1980; 120 : 513-16).

Porins (ompC and ompF) are proteins incorporated in the outer bacterial membrane. These channels, allowing the passive diffusion of small hydrophilic molecules, are involved in hypothiocyanite uptake (De Spiegeleer P. et al. Appl. Env. Microbiol, 2005; 71: 3512-18).

Gastric peroxidase (GPO) is an endogenous protein of the gastric mucosa. SCN^- appears to be the major physiological electron donor for GPO. (OSCN)^- cannot accumulate in the stomach because it reduced back to SCN^- by a high level of GSH which is oxidized to GSSG. In order to operate an efficient scavenging system for H₂O₂, GSSG should be reduced back to GSH by cellular glutathione reductase (GR) in the presence of NADPH (Das D. et al. Biochem. J., 1995; 305: 59-64).

Thiocyanate increases defense mechanism in the stomach of acidified nitrite, whereas in contrast glutathione impaired gastric antimicrobial activity (Fite A. et al. Antimicrobiol. Agents Chemother., 2004; 48: 655-58).

Orally administered lactoperoxidase (LPO) induces a significantly lower number of infiltrated leukocytes recovered from bronchoalveolar fluid (BALF) and pro-inflammatory cytokine, IL-6, levels in the BALF and serum of influenza-virus-infected mice after 6 days. These results suggest the potential of oral administration of LPO to attenuate pneumonia through the suppression of infiltration of inflammatory cells in the lung. Further studies are required to more clearly understand the mechanism behind the immunomodulatory action of LPO (Shin K. et al. J. Med. Microbiol., 2005; 54: 717-23).

4. OSCN Target Microorganisms

(Non Exhaustive list)

5 Bibliography

(Non Exhaustive List)

Agence Française de Sécurité Sanitaire des Aliments (AFSSA). Catallix Technology & hypothiocyanite ion Full assessment (Toxicology, Efficacy, Safety). Saisine n°2003-SA_0015 June 2^nd 2003

Banfi B, A novel host defense system of airways is defective in cystic fibrosis: update. Am J Respir Crit Care Med 2007 May 1;175(9):967

Childers M, Eckel G, Himmel A, Caldwell J.A new model of cystic fibrosis pathology: lack of transport of glutathione and its thiocyanate conjugates, Med Hypotheses. 2007;68(1):101-12. Epub 2006 Aug 24.

Conner GE, Wijkstrom-Frei C, Randell SH, Fernandez VE, Salathe M., The lactoperoxidase system links anion transport to host defense in cystic fibrosis, FEBS Lett. 2007 Jan 23;581(2):271-8. Epub 2006 Dec 19.

Conner GE, Salathe M, Forteza R. Lactoperoxidase and hydrogen peroxide metabolism in the airway, Am J Respir Crit Care Med. 2002 Dec 15;166(12 Pt 2):S57-61. Review.

Das D, De PK, Banerjee RK. Thiocyanate, a plausible physiological electron donor of gastric peroxidase. Biochem J. 1995 Jan 1;305 ( Pt 1):59-64.

De Spiegeleer P, Sermon J, Vanoirbeek K, Aertsen A, Michiels CW. Role of porins in sensitivity of Escherichia coli to antibacterial activity of the lactoperoxidase enzyme system. Appl Environ Microbiol. 2005 Jul;71(7):3512-8.

Farrag, S.A. and Marth, E.H. Escherichia coli O157:H7, Yersinia enterocolitica and their control in milk by the lactoperoxidase system: a review. Lebensm.-Wiss. u.-Technol., (1992), 25, 201-211.

Fite A, Dykhuizen R, Litterick A, Golden M, Leifert C. Effects of ascorbic acid, glutathione, thiocyanate, and iodide on antimicrobial activity of acidified nitrite. Antimicrob Agents Chemother. 2004 Feb;48(2):655-8.

Fragoso MA, Fernandez V, Forteza R, Randell SH, Salathe M, Conner GE. Transcellular thiocyanate transport by human airway epithelia. J Physiol. 2004 Nov 15;561(Pt 1):183-94. Epub 2004 Sep 2.

Furtmuller PG, Zederbauer M, Jantschko W, Helm J, Bogner M, Jakopitsch C, Obinger C., Active site structure and catalytic mechanisms of human peroxidases, Arch Biochem Biophys. 2006 Jan 15;445(2):199-213. Epub 2005 Oct 26. Review.

Gerson C, Sabater J, Scuri M, Torbati A, Coffey R, Abraham J, Lauredo I, Forteza R, Wanner A, Salathe M, Abraham W, Conner G. The lactoperoxidase system functions in bacterial clearance of airways. Am J Resp Cell Mol Biol (2000) Jun;22(6):665-71

Haukioja A, Ihalin R, Loimaranta V, Lenander M, Tenovuo J Related Articles, Sensitivity of Helicobacter pylori to an innate defence mechanism, the lactoperoxidase system, in buffer and in human whole saliva, J Med Microbiol. 2004 Sep;53(Pt 9):855-60.

Kussendrager, K.D. and van Hooijdonk, A.C.M. Lactoperoxidase: physico-chemical properties, occurrence, mechanism of action and applications. Br. J. Nutr., (2000), 84, suppl. 1, S19-S25.

Lenander-Lumikari M. , Inhibition of Candida albicans by the Peroxidase/SCN^-/H₂O₂ system. Oral Microbiol Immunol. 1992 Oct;7(5):315-20.

Lovaas E. Free radical generation and coupled thiol oxidation by lactoperoxidase/SCN^-/H₂O₂. Free Radic Biol Med. 1992 Sep;13(3):187-95.

Marshall VME, Reiter B., Comparison of the antibacterial activity of the hypothiocyanite anion towards Streptococcus lactis and E.coli. J. Gen. Microbiol., p 120:513 (1980).

Mikola H, Waris M, Tenovuo J. Inhibition of Herpes Simplex Virus type 1, Respiratory Syncytial Virus and Echovirus type 11 by peroxidase-generated hypothiocyanite. Antiviral Res. 1995 Mar;26(2):161-71.

Moskwa P, Lorentzen D, Excoffon KJ, Zabner J, McCray PB, Nauseef WM, Dupuy C, Bánfi B, A novel host defense system of airways is defective in cystic fibrosis. Am J Respir Crit Care Med. 2007 Jan 15;175(2):174-83. Epub 2006 Nov 2.

Pedemonte N, Caci E, Sondo E, Caputo A, Rhoden K, Pfeffer U, Di Candia M, Bandettini R, Ravazzolo R, Zegarra-Moran O, Galietta L. Thiocyanate transport in resting & IL-4-stimulated human bronchial epithelial cells: role of pendrin and anion channels. J Immunol. 2007 Apr 15;178(8):5144-53.

Pourtois M, Binet C, Van Tieghem N, Courtois PR, Vandenabbeele A, Thirty L. Saliva can contribute in quick inhibition of HIV infectivity. AIDS. (1991) May;5(5):598-600.

Pruitt, K.M., Tenovuo, J., Andrews, R.W., and Mc Kane, T. Lactoperoxidase-catalyzed oxidation of thiocyanate: polarographic study of the oxidation products. Biochemistry, (1982), 21, 562-567.

Reiter B, Härnulv G : Lactoperoxidase antibacterial system: Natural occurrence, Biological functions and Practical applications. J. Food Protect. 47 n°9, p 724-732. (1984).

Shin, K., Hayasawa, H., Lönnerdal, B. Inhibition of Escherichia coli respiratory enzymes by the lactoperoxidase-hydrogen peroxide thiocyanate antimicrobial system. J.Appl Microbiol, (2001), 90, 489-493.

Shin K, Wakabayashi H, Yamauchi K, Teraguchi S, Tamura Y, Kurokawa M, Shiraki K, Effects of orally administered bovine lactoferrin and lactoperoxidase on influenza virus infection in mice. J Med Microbiol. 2005 Aug;54(Pt 8):717-23.

Still J, Delahaut P, Coppe P, Kaeckenbeeck A, Perraudin JP., Treatment of induced enterotoxigenic colibacillosis in calves by the lactoperoxidase system & lactoferrin, Ann Rech Vet.. 1990;21(2):143-52

Tsuge K, Kataoka M, Seto Y. , Cyanide and thiocyanate levels in blood, saliva of healthy adult volunteers. J Health Science, 2000 46(5) 343-350

Valimaa H, Waris M, Hukkanen V, Blankenvoorde MF, Nieuw Amerongen AV, Tenovuo J. Salivary defense factors in herpes simplex virus infection. J Dent Res. 2002 Jun;81(6):416-21..

Wijkstrom-Frei C, El-Chemaly S, Ali-Rachedi R, Gerson C, Cobas MA, Forteza R, Salathe M, Conner GE. Lactoperoxidase and human airway host defense, Am J Respir Cell Mol Biol (2003) Aug;29(2):206-12.

William E. White, Jr., Kenneth M. Pruitt and Britta Mansson-Rahemtulla, Peroxidase-Thiocyanate-Peroxide Antibacterial System Does Not Damage DNA, Antimicrob. Agents Chemother. 1983 February; 23(2): 267–272.