Surfactant-induced alterations of permeability of rabbit oral mucosa in vitro

44, 132-137 (1986)

EXPERIMENTALANDMOLECLJLARPATHOLOGY

Surfactant-Induced

Alterations Mucosa

IVENS A. SIEGEL’

of Permeability in Vitro1

AND HERBERT

of Rabbit

Oral

P GORDON

Department of Oral Biology and Center for Research in Oral Biology. Uni,*ersity of Washington. Seattle, Washington 98195. and University of Illinois College of Medicine, Urbana, Illinois 61801 Received March 2.5, 1985. and in revised form

Nol>ernber 6, 1985

The permeability of rabbit oral mucosa to eight nonelectrolytes was measured in vitro in the absence and in the presence of 0.025, 0.1, and 1.0% concentrations of anionic, cationic. and nonionic surfactants. The anionic surfactant sodium lauryl sulfate and the cationic surfactants cetyltrimethylammonium bromide and cetylpyridinium chloride caused greater increases in permeability than polysorbate 80, a nonionic surfactant. The increases in permeability brought about by the surfactants were concentration dependent. Q 1986 Academic Press. Inc.

INTRODUCTION The oral epithelium is considered to be an important physiological barrier to the passage of potentially harmful substances-through it to the underlying connective tissue. Thus the integrity of this barrier can be important to the organism and changes in the ability of this barrier to perform its function could result in deleterious effects. Although at least one group has questioned whether visually observable damage occurs when surfactants are applied to human oral mucosa (Rothenstein et al., 197X), surfactant-induced damage to several epithelial systems, including human epidermis (Wood and Bettley, 197 1; Dugard and Scheuplein, 1973), human cornea (Marsh and Maurice, 1971), human gastrointestinal tract (Kalafallah et al., 197.5), and bovine cornea (Carter et al., 1973) have been reported. Despite the evidence suggesting that the surfactant may have harmful effects, surface-active substances are included in the formulation of many products used in the oral cavity. They are contained, for example, in toothpastes, mouthwashes, and are incorporated into topical anesthetics. Also, in recent years they have been suggested for use as dental anti-plaque agents (Ciancio et al., 1978). In this present study we have examined the effects of surfactants on the barrier function through studies of their effects on the permeability of oral mucosa. METHODS The lingual frena used in this study were removed from adult male New Zealand rabbits anesthetized with 30 mglkg of sodium pentobarbital administered into a marginal ear vein. The excised tissue was immediately placed in Krebs-Ringer phosphate (KRP) solution bubbled with 100% oxygen. The composition of the KRP solution was 5 mM K, 148 mM Na, 1.33 mM Mg, 2.0 mM Ca, 154 mM Cl, 1.33 mM S04, 0.1% glucose, phosphate buffer 8.6 mM, pH 7.4. The cut edges of the frenulum were carefully spread and the inner (blood) side of the tissue was cleaned free of extraneous tissue with aid of a lo-power dissecting microscope. The resulting thin membrane was cut into up to four pieces. Mucosal pieces were i This investigation was supported by Grant DE0 2600 from the National Institutes of Health. 2 Current address to which reprint requests should be sent: Dr. Ivens A. Siegel. University of Illinios College of Medicine, 190 Medical Sciences Building, 506 South Mathews Avenue, Urbana, Illinois 61801. 132 0014-4800/86 $3.00 Copyright Q 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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mounted in modified Ussing (1949) chambers as described in an earlier publication (Siegel et al., 1976). After mounting, the chambers were filled with KRP solution, continuously bubbled with 100% oxygen, and the tissue was allowed to equilibrate for 1 hr. During this 1-hr equilibration the KRP solution was changed at 5-min intervals. Following the equilibration period the half-chamber facing the inner side of the membrane was filled with fresh KRP and bubbled with oxygen. The half-chamber facing outside (oral side) of the tissue was filled with oxygenated KRP containing 1 t.&i/ml of solute and the surfactant under study. Ten-microliter samples were removed from each half-chamber at 30-min intervals from the first to the fourth hour of incubation. We have previously shown that transport of substances from the outside to the inside of the membrane is linear over this time period (Siegel, 1981). Permeability coefficients were calculated from the Fick formula as described in an earlier publication (Siegel and Izutsu, 1980). [methoxy-JH]Dextrans, [ 1,2-*4C]ethylene glycol, [U-i4C]glucose, [carboxyl- WIinulin, and [r”C]urea were purchased from New England Nuclear, and [ 1,7-14Clheptanediol and [ I-*4C]n-propanol were purchased from ICN Chemical and Radioisotope Division. Radioactive compounds were stored under conditions recommended by the manufacturer and were used within 3 weeks of receipt. Surfactants were purchased from the Sigma Chemical Company. RESULTS The calculated permeability coefficients in the absence of surfactant and in the presence of the cationic surfactants cetyltrimethylammonium bromide and cetylpyridinium chloride, the anionic surfactant sodium lauryl sulfate and the nonionic polysorbate 80 are given in Table I. Each concentration of cationic surfactant tested significantly increased the calculated permeability coefficient to all solutes except those with molecular weights of 4500 (inulin) and higher. The permeability coefficients to these higher molecular weight compounds were increased only by the two higher concentrations of cationic surfactant. Except for inulin at the lowest surfactant concentration, sodium lauryl sulfate, the anionic surfactant, caused significant increases in the permeability coefficient of all the test compounds. As with the cationic surfactants the increase in permeability with sodium lauryl sulfate was concentration dependent. In most instances the percentage increase in permeability induced by sodium lauryl sulfate was greater than that caused by either of the cationic surfactants when compared at equal concentrations. The nonionic surfactant polysorbate 80 did not increase permeability to any compound at the two lower concentrations tested, and caused significant increases to only three of the eight solutes at the highest concentration used. DISCUSSION The results of these experiments indicate that cationic and anionic surfactants can cause concentration-dependent increases in the permeability of oral mucosa to three types of penetrants. The types of organic nonelectrolyte molecules used as oral mucosal penetrants include two (heptanediol and propanol) which have higher oil to water distribution coefficients than water and are believed to diffuse across the epithelium via solvation in cell membranes; two with oil to water distribution coefficients less than that of water and with molecular volumes less than 80 cc/mole (urea and ethylene glycol) which are thought to diffuse via pores in the membrane; and four (sucrose, insulin, dextran 20,000 molecular weight, and dex-

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tran 75,000 molecular weight) with oil to water distribution coefficients less than that of water and molecular volumes greater than 80 cc/mole which the available evidence suggests diffuse through extracellular routes (Siegel, 198 1; Alvares and Siegel, 1981). The fact that both the cationic and anionic surfactants caused increases in permeability to all three types of penetrants suggests that their effect is so general that it affects each of these routes of penetration or, alternatively, the surfactant could affect a rate-limiting barrier either prior to or after the molecule’s movement through its route of penetration. Barriers to penetration across the oral mucosa have been variously described as the salivary mucins which coat the epithelium (Adams, 1974a, b, c), the keratin coating of the epithelium (Kaaber, 1974; Adams, 1974b), the released contents of the membrane-coating granules (Hill and Squire, 1979) and the basal lamina (Brandtzaeg and Tolo, 1977; Alfano et al., 1977). It seems unlikely that saliva itself in our in vitro experiments provided a significant barrier because most saliva would undoubtedly be washed away during the initial I-hr equilibration. However, the presence of residual mucins of salivaryy origin (Adams, 1975; McMillan, 1980) which could act as a barrier to penetration is a distinct possibility, and these mucins may have been altered by the surfactants. The ability of the ionic surfactants to affect the keratinized surface of oral tissue has been demonstrated (Siegel and Gordon, 1985), and the magnitude of the damage correlated well with increases in permeability. If the keratinized layer is an important barrier in oral epithelium as it is in skin (Tregear, 1966), then the type and extent of injury caused by the cationic and anionic surfactants could account for the observed increases in permeability. If either the membrane-coating granule contents or the basal lamina are the site of action of the surtactants, then these substances must be able to penetrate the oral epithelium. Evidence from in vivo experiments (Siegel and Gordon, 1985) indicated that sodium lauryl sulfate can penetrate the oral mucosa and be measured in the blood following the application to the surface of the oral epithehum. The mechanism by which these compounds increase permeability is not well understood. Chronic application of surfactants has been reported to produce an increased rate of biosynthesis of epidermal phospholipids, nucleic acids, and acid soluble material (Mezai and Lee, 1970; Mezai, 1975). However, these are effects that are manifested after several days of application and are unlikely to be of major importance in our 4-hr in vitro experiments. The mechanism by which permeability is increased is probably not related to simple detergency or reacting with and dissolving cellular lipid components. According to Schulman et al. (1955), a substance must reduce the surfact tension of water from 720 to 380 pN/cm~ to induce damage by simple detergency. Each of the surfactants used in this study was at concentrations higher than those required to reduce the surface tension by this amount yet there were vast differences in permeability increases. Further, at the concentrations used, all were capable of removing surface lipids from epithelium (Landsdown and Grasso, 1972). It seems more likely that those surfactants which increase permeability do so through reactions with epithelial proteins. Measurements employing a spin label technique (Kirkpatrick and Sandberg, 1973) have shown that anionic, but not nonionic, surfactants cause changes in the conformation of solubilized membrane proteins. Further, application to human epidermis of an iodoacetamide method for measuring thiol groups has demontrated that cationic and anionic, but not

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nonionic surfactants have the ability to expose thiol groups (Wood and Bettley, 1971) and that in most instances the degree of protein denaturation correlated with effects on permeability. The fact that permeability of oral mucosa can be increased several fold by ionic surfactants may make them less than ideal as dental plaque-reducing substances. Even though they may reduce plaque and thus decrease the concentration or total amount of substances produced by the oral flora, the surfactants could increase the rate of absorption of the diminished amount. The net effect would then depend upon the balance between a reduced quantity of material produced by the bacteria of plaque and the increased rate of penetration induced by the surfactant. REFERENCES ADAMS, D. (1974a). Surface coatings of cells in the oral epithelium of the human fetus. .I. Ancct. 118, 61-75. ADAMS, D. (1974b). The effect of saliva on the penetration of fluorescent dyes into the oral mucosa of the rat and rabbit. Arch Oral Biol. 19, 505-510. ADAMS, D. (1974c). Penetration of water through human and rabbit oral mucosa in i?tr.o. Arch Or01 Biol. 19, 865-872. ADAMS, D. (1975).

The mucosa barrier and absorption through the oral mucosa. /. Dent. Res. (spec. Issue B) 54, Bl9-B26. ALFANO, M. C.. CHASENS, A. I., and MASI. C. W. (1977). Autoradiographic study of the penetration of radiolabeled dextrans and inulin through nonkeratinized oral mucosa in \sitro. J. Periodonrul Res. 12, 368-377. ALVARES, 0. and SIEGEL. I. (1981). Permeability of gingival sulcular epithelium in the development of scorbutic gingivitis. J. Oral Putho/. 10, 40-48. BRANDTZAEG. P., and TOLO. K. (1977). Mucosal penetrability enhanced by serum-derived antibodies. Nature CARTER,

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L. M., DUNCAN, G., and RENNIE,G. K. (1973). Effects of detergents on the ionic balance and permeability of isolated bovine cornea. Exp. Eye Res. 17, 409-416. CIANCIO, S. G., MATHER, M. L., and BRUNNELL, H. L. (1978). The effect of a quaternary ammoniumcontaining mouthwash on formed plaque. Phurmuco/. Ther. Dent. 3, 1-6. DUCARD, P. H.. and SCHEUPLEIN. R. J. (1973). Effects of ionic surfactants on the permeability of human epidermis: An electrometric study. J. Inresr. Dermutol. 60, 263-269. HILL, M. W.. and SQUIRE, C. A. (1979). The permeability of rat palatal mucosa maintained in organ culture. J. Anar. 128, 169- 178. IZUTSU, K. T.. TRUELOVE, E. L., BLEYER. W. A.. ANDERSON, W. M., SCHUBERT, M. M., and RICE. J. C. (1981). Whole saliva albumin as an indicator of stomatitis in cancer therapy patients. Cancer 48, 1450- 1454. KAABER, S. (1974). The permeability and barrier functions of the oral mucosa with respect to water and electrolytes (Thesis). Actu Odontol. Stand. (Suppl. 66) 32. KHALAFALLAH. N., GOUDA, M. W.. and KHALIL, S. A. (1975). Effects of surfactants on absorption through membranes. IV: Effects of dioctyl sodium sulfosuccinate on abssorption of a poorly absorbable drug, phenolsulfonphthalein, in humans. J. Pharm. Sci. 64, 991-994. KIRKPATRICK, R. H., and SANDBERG, H. E. (1973). Effect of anionic surfactants, nonionic surfactants. and neutral salts on the conformation of spin-labeled erythrocyte membrane protein. Biochem. Biophys. Acfa 298, 209-218. LANDSDOWN, A. B. G., and GRASSO, P. (1972). Physiol-chemical factors influencing epidermal damage by surface active agents. Brit. J. Dermarol. 86, 361-373. MARSH, R. J.. and MAURICE, D. M. (1971). The influence of non-ionic detergents and other surfactants on human cornea1 permeability. Exp. Eyes Res. 11, 43-48. MCMILLAN, M. D. (1980). Transmission and scanning electron microscopic studies on the surface coat of the oral mucosa in the rat. J. Periodontal Res. 15, 288-296. MEZEI, M. (1975). Effect of polysorbate 85 on human skin. J. Invest. Dermatol. 64, 165-168. MEZEI, M., and LEE, A. (1970). Dermatitic effect of nonionic surfactants: Phospholipid composition of normal and surfactant-treated rabbit skin. J. Pharm. Sci. 59, 858-861.

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A. S., Picozzr, A., DOYLE, J. L., CANCRO, L. P., and SINGER, E. J. (1978). Soft tissue responses to high frequency use of commercial mouthwashes. Pharmacol. Ther. Dent. 3, 25-29. SCHLJLMAN, J. H., PETHICA, B. A., FEW, A. V., and SALTON, M. R. J. (1955). The physical chemistry of haemolysis and bacteriolysis by surface active agents and antibiotics. Pvog. Biophys. Biopys. Chem. 5, 41-71. SIEGEL, I. A. (1981). Effect of chemical structure on nonelectrolyte penetration of oral mucosa. J. Invest. Dermatol. 76, 137-140. SIEGEL, I. A., and GORDON, H. P. (1985). Surfactant-induced increases of permeability of rat oral mucosa to non-electrolytes in vitro. Arch. Oral Biol. 30, 43-47. SIEGEL, I. A., and IZUTSU. K. T. (1980). Permeability of oral mucosa to organic compounds. J. Dent. Res. 59, 1604-1605. SIEGEL, I. A., IZUTSU, K. T., and BURKHART, J. (1976). Transfer of alcohols and ureas across the oral mucosa using streaming potentials and radioisotopes. J. Pharm. Sci. 65, 129- 131. TREGEAR, R. T. (1966). “Physical Functions of Skin.” Academic Press, New York. USSING, H. H. (1949). Active ion transport through the isolated frog skin in the light of tracer studies. Acta Physiol. Stand. 17, l-37. WOOD, D. C. F., and BETTLEY, F. R. (1971). The effect of various detergents on human epidermis. Brit. J. Dermatol. 84, 320-325. ROTHENSTEIN,