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Lysosomes and the dental pulp
Lysosomesand the dental pulp Parts I and II Roger J. Spott, D.D.S.,’ Philadelphia, Pa. TEMPLE
UNIVERSITY
and Theodore Rosett, Ph.D.,**
SCHOOL
OF DENTISTRY
These lysosome studies represent an attempt to explain the action of drugs used in the treatment of dental pulp and to set up a model system for their evaluation.
M
ore than 20 years have elapsed since the postulation, discovery, and localization of lysosomesl Although the role of lysosomes in many intracellular and extracellular functions has been widely researched and carefully documented, little information is currently available on the relationship of the lysosome to pulpal disease. However, the role of the lysosome in the oral cavity is being researched. Oral lysosomal membrane stability has been examined during local anesthesia,2 and the role of lysosomes in periodontal disease has been studied.3-5 Therefore, the purpose of this study was threefold: 1. To elucidate, via literary comparison, the diverse roles that the lysosome might play in pulpal disease. 2. To isolate and identify the lysosomes from bovine dental pulp. 3. To investigate how these subcellular, cytoplasmic particles react to various drugs commonly used in dentistry. When deDuve and associates nicknamed the lysosome the “suicide sac” because of the associated acid hydrolases, it was not fully known that this function played only a small role in the lysosome’s total activity. To explain more adequately the wide physiologic and pathologic implications, Weissmann’ referred to this struct,ure as the many-faceted lysosome. Although the lysosome Submitted by the senior author in partial fulfillment of the requirements for the certificate of advanced training in endodontics, Temple University School of Dentistry. *Graduate Department of Endodontics. Present address: 13975 Connecticut Ave., Wheaton, Md. **Department of Biochemistry.
569
570 Table
Spotf md
Kosett
I. Enzymes
and other
substances
Acid phosphatase Arid ribonuclease Acid desoxyribnuclease Cathepsins B,C,D Phosphoprotein phosphatasc Phosphatidic. acid phosphatase Organophosphate-resistant esterases Beta-glucuronidasc Beta-gala.ctosidase Beta-N-acetylglucosaminidase Alpha-L fucosidase Alpha-l-4 glueosidasc Alpha mannosidxse Alpha-N acetylglucosaminidase Alpha-N ncetylgalctosaminidase (From Weissmann, G.: The Role Med. 18:97-112, 1967.)
Table
II.
Agents stabilizing
associatetl with lysosomcs Hpaluronidase Lysozymc Collngenase Aryl sulfatases A and B Phospholipase Acid lipxse Phagocytin Endogcnous pyrogen Chcmotaxis factor TJnidentified basic proteins Cationic inflammatory protein Permeability-inducing protease Plasminogen activator Hemolysins Mucopolysaccharides and glycoproteins
of Lysosomes
or labilizing StaGlizing
Cortisone-cortisol Prednisone Beta-methasone Chloroquine Labilizing Vitamin A Endotoxin Streptolysin 0 and S Staphylococcal alpha and gamma toxin 2,4-Dinitrophenol Carbon tetrachloride Progesterones and testosterone Deoxycorticosterone Etiocholanolone, 5 Beta-H steroids 5 Beta-H bile acids Icterogenin Cantharidin (From Chemother.
Allison, A. : The 3 :253-302, 1968.)
Role
of Lysosomes
in
Inflammation
and
Disease,
Ann.
Rev.
lysosomes in vitro agents : Benadryl Salicylate Chlorpromazine Colchicine agents: Chloropromazine (high concentration) Hyperoxia and anoxia Shock Ultraviolet radiation x-radiation Antigen-antibody reactions Triton WR.-1339 Silica Carrageenan Asbestos Photosensitizing agents 7.Hydroxymethyl-12-methyl benzanthracene in the
Action
of Drugs
and
Hormones,
Adv.
functions in many cellular processes, it appears to act essentially in two ways. First, it is involved in intracellular digestion.7, 8 In interacting with phagocytized material, the lysosomal enzymes perform their catalytic actions and allow the resultant materials to enter the cytoplasm or to be eliminated (Table I) .9* I0 This ability to ingest and digest various materials has been studied and quantitated by Sawant, Desai, and Tappel.ll, l3 Autophagy is the ability to engulf fragments of intracellular material. This apparently allows the cell to reuse its own components for energy and rebuilding.13 Second, the lysosome has the potential of becoming a mechanism of cell destruction. By an uncontrolled or pathologic rupture, the lysosome can destroy the surrounding and adjacent protoplasm. This destruction is limited only by inactivation of the liberated enzymes.15 The lysosome ruptures for many reasons, among them the actions of drugs, immune reactions, and infections.16* 1r
Volume Number
36 4
Separation 50.0 500
Lysosomes
I Combine, Centrifuge
571
scheme:
g pulp tissue make up to ml. with 0.25M sucrose
Supernatant Supernatant
and dental pulp
Homogenize with polytron >2.0 minutes at highest
f f
filter
through
15,000
xg
glass
l
wool
B
min.-
speed.
1 Centrifuge 15,000 Ppt. rehomogenize sucrose centrifuge
xg min. in 200 ml. 15,000 xg
1 Ppt.-
Discard
Ppt.A
Discard
Supernatant+
Discard
Supernatantj
Discard
l
l
min.
Supernatant 1 Combine,
filter
Centrifuge
4.3
through X lo6
glass xg
l
wool
min.-
1 Ppt. suspend in 24 ml 0.25M Sucrose centrifuge 5.4 X lo6 xg min.l
Pellet, to suspend density gradient round-bottomed Fig. Biophys.
Physiologic
on top of in 100 ml. centrifuge tube
2. (From Rosett, Acta 222: 5-14,
T., and 1970.)
associates:
Studies
in
the
Biochemistry
of
Skin,
Biochim.
responses
The physiologic category of lysosomal activities includes tooth development, bone resorption, and phagocytic response of cells to toxic materials. Lysosomelike bodies have been seen microscopically in odontoblasts.18-21These structures have also been associated with tooth formation.22-24 An extensive lysosomal system has been shown in the cells of the ameloblastic layer. The Tomes process contains very high lysosome concentrations; however, confirmation of their role extracellularly still is unreported. 25x26 Thus, lysosome enzymes have been correlated with secretory activity and their distribution correlated with amelogenesis and dentinogenesis. It was suggested by deDuve and Wattiaux13 that the secretory functions of cells may be subjected to regulatory inactivation by the lysosomes. Bone resorption, including both mineral and organic components, was studied in tissue culture by VaesZ7 who found that the resorption was accompanied by release of six lysosomal enzymes, This release could be a consequence of bone resorption, but it was concluded to be a cause of the resorption. Electron microscopic studies have shown resorption to be initially extracellular. The organic fibers are first dissociated, apparently by disruption of the ground substance. A loss of collagen fiber cross banding then follows. ScherfP postulated that the osteoclasts, or similar cells, excrete agents which could accomplish
572
8pott
awl
Olxl October,
Roseti Lysosome
prep.
t Test
Surg. 1973
material
4, Incubate
30
1 Centrifuge
min.
4,000
37”
C.
R.P.M.
20 min. ) Ppt +
4, Supernatant
Discard
1 Test
amount
t Enzyme
substrate
1 Incubate Add
30 min.
J enzyme
1 Centrifuge
assay 2,000
37’
C. buffer
R.P.M.
10 min. ) Ppt +
Discard
Supernotant
& Read
540~ Fig.
spectrophotometer 2. Test
sequence.
both processes.It is unlikely that this release of agents is due to overt disruption of the lysosomal membrane; rather, it is probably due to a mechanism similar to that found in secretory cells.” The phagocytic responsesof cells to toxic materials differ from the secretorylike functions, but thep are still physiologic reactions, since all particulate materials appear to be concentrated in lysosomes. Plastics and certain metals, such as iron, silver, lead, copper, and mercury, are also concentrated in lysosomes.2s-31 Intracellular accumulation of these materials might be significant with reference +,otoxicology and carcinogenicity. Pathologic
responses
Pathologic release of lysosomal enzymes is seenin diseaseand inflammation.13 The uncontrolled enzyme release may be due to normal but total lysosome membrane rupture or to pathologic conditions which give rise to, or cause formation of, more fragile lysosomal membranes.lJ Lysosomal rupture has been produced by a variety of agents. When streptolysin 0, a bacterial toxin, was added to living cells, the lysosomes were seen to rupture.32 Antilysosome antibodies, with complement, when added to living cells, produced lysosome membrane breakdown.33 When photosensitizing agents, such as neutral red and acridine orange, were allowed to accumulate in lysosomes and the latter were then exposed to ultraviolet light, rupture occurred.34’ 35 Lysosomal breakage produces damage to surrounding tissues, as evidenced by loss of cartilage matrix in hypervitaminosis A. There appears to be a direct action of Vitamin A on the lysosome.36 Lysosomal rupture is implicated in certain diseases. The Chediak-Higaski syndrome of children shows abnormal leukocyte lysosomes. This seemsto result
Volume Number
36 4
Lysosomes
and dental
pulp
573
EUGENOL I)miMExRAsoNE ULTRASOUND PREDNISOLONE BENADRYL CPMCP FORMCZREWL GODIUM
FLUORmE PRFDNISONE
DIMETmL
SU~XIDE TRITONX ASPIRIN
% CONTROL STABILIZE
a&
+
LARILIZE
I Fig.
3.
in a higher susceptibility to infeetion.~ ?‘I, 38 During the course of hepatitis, lysosomes were found to be labilizedS9 Certain connective tissue disorders, including rheumatoid arthritis and lupus erythematosus, involve the presence of more fragile lysosomes.4nT 41 There is evidence that agents used to control these disorders may act somewhere in this sequence.l” A listing of these agents is given in Table II. The foregoing is a cursory examination of the pathologic implications of lysosomes which may be of particular interest. Research on lysosomal activity of the dental pulp, however, is practically nonexistent. Inasmuch as the lysosomes were implicated in both the physiologic and pathologic processes, this research project had these aims : I. To isolate and identify lysosomes from bovine dental pulp, thereby ascribing to the pulp the physiologic lysosomal activities present in other tissues. II. To examine, in vitro, membrane stability of lysosomes subjected to drugs commonly used in dentistry, thereby determining the presence of any pathologic lysosomal response. PART Methods
I. ISOLATION and
OF
LYSOSOMES
materials
Bovine dental pulp was chosen as the material to be examined because of its large size and easy availablity. According to Rosett and associates, the bovine
574
Spott
Oral October,
and Kosett
Surg. 1973
RESULTS BETR-GLUCURDNIDASE
EUGENDL -NE ULTRASOUND PRmNIsDLDNE
k I
CPM2P
-I
FOR)rlOCRESOL SODIUM
-I
c
FLUDRIDE
I
mNIscNE DIMEI’RYL
I
SULFDXIDE TRITON-X
4
ASPIRIN
I I I 203104050~
I
I I 7080901
I
3 ’
lb
’
I 140
I ’ 160
% CDrnL STABILIZE
+
+
LABILIZE
I Fig.
4.
mandibles are chilled and ready for transport within 5 minutes after animal death. The first cheek tooth was fractured horizontally at the height of the alveolar bone, and pulps were removed by severing the canal contents near the apex. The intact pulp masses about 1.5 grams. ** The resultant pulps represented a pooled cellular sample. The extracted bovine pulps were combined and suspended in a 0 to 4’ C. 0.25M sucrose solution at pH 7.4 (50.0 grams of pulp to 500 ml. of sucrose solution). 43 This was then homogenized at top speed for 2 minutes in a Willems Polytron homogenizer (Brinkman Co.). The homogenized cellular components were separated by centrifugation. There is a large variety of separation schemes, each having been devised by a different researcher.44-46 Some improved methods have adapted zonal centrifugation for separating large quantities rapidly and with a high degree of purity.47-50 This research employed a modification of the method of Rosett and associates.43 Fig. 1 illustrates the scheme for this differential centrifugation procedure. Identification of the lysosomal fraction was essentially biochemical. AS previously shown (Table I) many enzymes have been isolated or identified in association with lysosomes.Most authorities have utilized more than one enzyme in demonstrating lysosomal activity. In this study, analysis therefore included two enzymes. Acid phosphatase was assayed by an adaptation of the method of Huggins and Talalay.51 Beta-glucuronidase was assayed by an adaptation of the method of Fishman and associates.52Cytochrome oxidase activity was also
Volume Number
Lysosomes
36 4
Table III. Materials
and dental
pulp
575
tested 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Aspirin Triton-X Dimethyl sulfoxide Prednisone Sodium fluoride Formocresol Camphorated paramonochlorophenol Benadryl Prednisolone Ultrasound Dexamethasone Eugenol
assayed as a marker for mitochondria to assure that they were not present in high concentrations in the lysosome fractions. This method used cytochrome C as substrate and measured the decrease of the intensity of the substrate’s spectrophotometric absorption as it was oxidized by the enzyme.53-55 PART Il. EFFECTS OF DRUGS ON LYSOSOMES After it had been shown that lysosomal alterations accompanied cell damage, techniques arose to utilize this fragility of lysosomes as a detection modality for cell injuryas Also, the ability of certain materials to stabilize this fragility was studied.57-5g The effects of vitamins, hormones, and other compounds have been investigated.60$ 61 Part II examined the effect on isolated bovine pulp lysosomes of drugs commonly used in dentistry. Methods
and materials
Lysosomes were subjected to the drugs listed in Table III. The lysosome fraction from Part I of this study was resuspended in a solution containing 50 per cent by volume of 0.25M sucrose and 50 per cent acetate buffer (pH 5.0). One part of test substance to 20 parts of lysosome suspension was incubated at 37O C. for 30 minutes.62* 63 After incubation, the lysosomal fraction was centrifuged to the bottom and removed. The supernatant was assayed for acid phosphatase and beta-glucuronidase in the manner previously described. The data form was spectrophotometer readings, and the samples were compared to controls to give the per cent of stabilization or labilization. Results The combined pulp pool was homogenized and separated according to the separation scheme (Fig. 1). The resultant pellet was resuspended in 0.25M sucrose and assayed with the test material as indicated in Fig. 2. The spectrophotometer results are compared and expressed as per cent of control (Figs. 3 and 4). Controls of the lysosomal preparation and the test reactions are shown in Fig. 5. DISCUSSION Lysosome preparations were localized in fractions of bovine dental pulp. Purification by gradient separation and electron microscopic study of the pellet specimens are currently being completed. The results as indicated in Figs. 3 and
576
Spotf
and
fiosett
Controls Free
enzyme
t Test
material-
Assay Should equal
Free Known
enzyme amount
__j of enzyme
Assay
t Test material-
be
> Assay Should be equal and correct
Known
amount
-
of enzyme Fig.
Assay
>
5.
4 reveal two major factors: First, results obtained in this study parallel results obtained with the sametest materials on lysosome preparations from other tissue sources.30-40, 56-63This would ascribe to the dental pulp a similarity, at an ultrastructural level, to the lysosome properties carefully worked out for other sites.“6-63Second, results indicate that some materials used in dentistry cause labilization of the lysosomal fractions. Dilution studies and correlated histologic studies might make a very good method of evaluating present materials and comparing their properties. Research into new materials would be aided by histologic and quantitative lysosomal enzyme assays. One interesting result, among many others, was that of sodium fluoride. Fig. 6 shows the results which indicate that the material acts as a stabilizer of both enzymes, The control reactions, however, show that sodium fluoride also inhibits the free enzymes, which means that it did not affect the lysosomal membrane per se but, rather, acted on the enzymes. The potential importance of the results of this study depends on application. First, isolation and identification of lysosomesfrom dental pulp may ascribe to the dental pulp lysosomes the physiologic activities of lysosomes seen in other tissues. This might aid in applying more widespread medical stabilization and labilization study data to the treatment of pulpal disease. Second, testing of lysosomal membrane stability can determine the presence of any pathologic lysosomal response to dental medications and drugs. The finding of grossly injurious materials might indicate the necessity for further study prior to general usage. Proposals for extension of this study should include other test materials, human in vitro studies, and animal and human in vivo pulpal studies. SUMMARY
Intact dental pulps from the mandibular cheek teeth of freshly killed cattle were removed, suspended in 0.25M sucrose, homogenized, and placed in a separation schemeto produce lysosomc-rich preparations. The lysosomeswere incubated with various substances known to labilizc or stabilize lysosomes, as well as with some drugs commonly used in medicine and dentistry. The controls were both no drug or drug- and supernatant-free enzymes. The enzyme assays were for acid phosphatase and Beta-glucuronidase. Drugs which stabilized included
Volume Number
36 4
Lysosomes
and dental pulp
577
GLKXJRDNIDASE ACID FHCBFHATASE 20 30 40 50 60 70 so 90 100 STABILIZE + +LABILIZE I ~CFCcwMaL FREEENZYME Fig.
6.
dexamethasone, prednisolone, benadryl, prednisone, dimethyl-sulfoxide, and aspirin. Drugs which labilized included euginol, ultrasound, camphorated paramonochlorophenol, formocresol, and Triton-X. Sodium fluoride appeared to inhibit enzymatic activity. The chemistry, Dentistry,
authors wish to acknowledge Electron Microscopy, and Philadelphia, Pennsylvania.
the valuable assistance of all members Endodontic Departments at Temple University
of
the BioSchool of
REFERENCES
1. deDuve, C., Pressman, B., Gianetto, R., Wattiaux, R., and Appelmans, F.: Tissue Fractionation Studies, Biochem. J. 60: 604-617, 1955. 2. Smith, C. J., and Cimasoni, G.: Stability of Oral Epithelial Lysosomes During Local Anaesthesia, Helv. Odontol. Acta 9: 95-100, 1965. 3. Squier, C. A., and Waterhouse, J. P.: Lysosomes in Oral Epithelium, Arch. Oral Biol. 15: X3-168, 1970. 4. Waterhouse, J. P.: The Gingival Part of Human Periodontium, Dent. Pratt. 15: 409-415, 1965. 5. Hamp, S., and Polk, L.: The Lysosomes and Their Possible Role in Periodontal Disease, Odontol. Tidskr. 76: 353-375, 1968. 6. Weissmann, 0. : The Many-Faceted Lysosome, Hosp. Pratt. 3: 31-39, 1968. 7. deDuve, C. : The Lysosome, Sci. Am. 208: 64-72, 1967. 8. Novikoff, A. B.: Lysosomes in the Physiology and Pathology of Cells, Ciba Foundation Symposium on Lysosomes, Boston, 1963, Little, Brown 8; Company, pp. 36-77. 9. R.uebner, B. H., Hirano, T., Slusser, R., Osborn, J., and Medearis, D. N.: Cytomegalovirus Infection, Am. J. Pathol. 48: 971-989, 1966. 10. deDuve, C.: General Properties of Lysosomes, Ciba Foundation Symposium on Lysosomes, Boston, 1963, Little, Brown & Company, pp. l-35. 11. Sawant, P. L., Desai, I. D., and Tappel, A. L.: Digestive Capacity of Purified Lysosomes, Biochim. Biophys. Acta 85: 93-102, 1964. 12. Tappel, A. L.: Lysosomes, Distributions in Animals, Hydrolytic Capacity and Other Properties, Ciba Foundation Symposium on Lysosomes, Boston, 1963, Little, Brown & Company, pp. 78-113. 13. Idg&$ve, C., and Wattiaux, R.: Functions of Lysosomes, Ann. Rev. Physiol. 28: 435-492, 14. Thomas, L.: The Role of Lysosomes in Tissue Injury, The Inflammatory Process, New York, 1965, Academic Press, Inc., pp. 449-463. 15. Weissmann, G.: The Role of Lysosomes in Inflammation and Disease, Ann, Rev. Med. 18: 97-112. 1967. 16. Allison, A.: The Role of Lysosomes in the Actions of Drugs and Hormones, Adv. Chemother. 3: 253-302, 1968. 17. Golub, E. S., and Spitznagel, J. K.: The Role of Lysosomes in Hypersensitivity Reactions, J. Immunol. 95: 1060-1066, 1966.
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and Kosett
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Surg. 1973
18. Garant, P. R., Szabo, Cr., and Nalbandian, J.: The Fine Structure of the Mouse Odontoblast. Arch. Oral Biol. 13: 857-876. 1968. 19. Jessen, H. : ‘The Ultrastructure of Odontoblast in Perfusion Fixed, Demineralized Incisors of Adult Rat, Acta Odontol. Stand. 25: 491523, 1967. 20. Katchburian, E., and Halt, S. J.: Ultrastructure Studies on Lysosomes and Acid Phosphatase in Odontoblasts. 1% Dentin and Pulp: Their Structure and Reactions, Dundee, 1968, D. C. Thomson Company, pp. 43-57. 21. Nag&i, N.: Electron Microscopy of the Cytoplasmic Bodies in the Odontoblasts of Young Rat Incisors. Bull. Tokyo Dent. Coll. 11: 47-83. 1970. 22. Reith, E. J.: Ultra&uctural Aspects of Dentinogenesis. In Dentin and Pulp: Their Structure and Reactions, Dundee, 1968, D. C. Thomson Company, pp. 19-41. 23. Reith, E. J.: Collagen Formation in Developing Teeth of Rats, J. Ultrastrue. Res. 21: 383-414. 1968. 24. Takuma, S.: Ultrastructure of Dentinogenesis. In, Miles, A. E. W. and Greulich, R. C. (editors) : Structural and Chemical Organization of Teeth, New York, 1967, Academic Press, Inc., vol. I, pp. 325-370’. 25. Katchburian, E., and Holt, 5.: Role of Lysosomes in Amelogenesis, Nature 223: 1367-1368, 1969.
26. Katchburian, E.: Histochemistry of Lysosomal Enzymes in Developing Teeth of Alhino Rats, J. Anat. 101: 783-792, 1967. 27. Vaes, G.: The Action of Parathyroid Hormone on the Excretion and Synthesis of Lysosomal Enzymes and on the Extracellular Release of Acid by Bone Cells, J. Cell. Biol. 39: 676-697, 1968. 28. Scherft, J. P.: The Resorption of the Organic Matrix of Calcified Cartilage as Seen With the Electron Microscope, Calcif. Tissue Res. 2: Supp. 96-96B, 1968. 29. Koenig, H. J.: Epileptogenic Action of Substances That Interact With Gangliosides; In Vivo Release of Lysosomal Enzymes as a Disease Mechanism, J. Histochem. Cytochem. 11: 120-121, 1963. 30. Little, K.: Reactions of the Body to Implanted Materials, Jaarb. Kankeronderz Nederl. 14: 75-81, 1964. 31. Allison. A.: Lvsosomes and the Resoonses of Cells to Toxic Materials. Sci. Basis Med. Anna. Rev. 18: 18-30, 1968. 32. Hirsch, J. G., Bernheimer, A. W., and Weissmann, G.: Motion Picture Study of the Toxic A&ion of Streotolvsins on Leukocvtes. J. EXD. Med. 118: 223-228. 1963. 33. Quie, P. G., a?nd “Hirsch, J. G.: “Antiserum to Leukocyte Lysosomes, J. Exp. Med. 120: 149-160, 1964. 34. Allison, A. C., and Paton, G. R.: Chromosome Damage in Human Diploid Cells Following Activation of Lysosomal Enzymes, Nature 207: 1170-1173, 1965. 35. Allison, A. C., Magnus, I. A., and Young, M. R.: Role of Lysosomes and of Cell Membranes in Photosensitization, Nature 209: 874-878, 1966. 36. Dingle, J.: Action of Vitamin A on the Stability of Lysosomes In Vivo and In Vitro, Ciba Foundation Symposium on Lysosomes, Boston, 1963, Little, Brown & Company, pp. 384-398. 37. Windhorst, D. B., Zelickson, A. S., and Good, R. A.: Chediak-Higashi Syndrome: Hereditary Gigantism of Cytoplasmic Organelles, Science 151: 81-83, 1966. 38. White, J. G.: The Chediak-Higashi Syndrome, a Possible Lysosomal Disease, Blood 28: 143-156, 1966. and Acid 39. Pagliaro, L., Giglio, F., LeMoli, S., and Citarrela, P.: Beta-Glucuronidase Phosphatase Activities of Lysosomal Preparations From Human Liver Tissue Obtained by Needle Biopsy From Subjects With Acute Hepatitis and Cirrhosis, J. Clin. Invest. 63: 977-985, 1964. 40. Barland, P., Novikoff, A,, and Hamerman, D.: Fine Structure and Cytochemistry of the Rheumatoid Synovial Membrane With Special Reference to Lysosomes, Am. J. Pathol. 44: 853-866, 1964. 41. Weissmann, G., and Rothfield, N.: Structure and Function of Connective Tissue, Oxford, 1964, Blackwell Sm., pp. 466-483. and Cofactor Effects on Respiration of 42. Pollack, R., Green, D., and Rosett, T.: Substrate Bovine Dental Pulp, International Association for Dental Research, 49th General Session Program and Abstracts of Papers, p. 184, March, 1971. D., McDonald, T., and Brown, K.: Studies in 43. Rosett, ‘I., Matsuo, I., Bailey, A., Smith, the Biochemistry of Skin, V. The Particulate Origin of Adenylate Kinase in the Skm Of Neonatal Rats, Biochim. Biophys. Acta 222: 5-14, 1970. 44. deDuve, C.: The Separation and Characterization of Subcellular Particles, Harvey Lect. 59: 49-87, 1965. 45. Sawant, P. L., Shibko, S., Kumta, U., and Tappel, A.: Isolation of Rat Liver Lysosomes and Their General Properties, Biochim. Biophys. Acta 85: 82-92, 1964. 46. Ragab, II., Beck, C., Dillard, C., and Tappel, A.: Preparation of Rat Liver Lysosomea, Biochrm. Biophys. Acta 148: 501-505, 1964. I
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47. Schuel, H., Schuel, R., and Unakar, N.: Separation of Rat Liver Lysosomes and Mitochondria in the Z-XII Zonal Centrifuge. Anal. Bioehem. 25: 146-163. 1968. 48. Brown, D. H. : Separation of Mitochondrra,’ Peroxisomes and Lysosomes by’ Zonal Centrifugation in a Ficoll Gradient, Biochim. Biophys. Acta 162: 152-153, 1968. 49. Leighton, F., Poole, B., Beaufay, H., Baudhuin, P., Cocffey, J. W., Fower, S. and deDuve, C.: The Large Scale Separation of Peroxisomes, Mitochondria and Lysosomes From the Livers of Rats Injected With Triton WR-1339, J. Cell. Biol. 37: 482-513, 1968. 50. Withers, B., Davies, I. A., and Wynn, C. H.: The Purification of Lysosomes by Isopycnic Zonal Ultracentrifugation, Biochem. Biophys. Res. Commun. 30: 227-231, 1968. 51. Huggins, C., and Talalay, P.: Sodium Phenolphalein Phosphate as a Substrate for Phosuhate Tests. J. Biol. Chem. 159: 399-410. 1945. 52. Fish&an, W. H.; Springer, B., and Br&etti,‘R.:--Application of an Improved Glucuronidase Assay Method to the Study of Human Blood Beta-Glucuronidase. J. Biol. Chem. 173: 449-456, 1948. 53. deDuve, C:, Wattiaux, R., and Baudhuin, P.: Distribution of Enzymes Between Subcellular Fractions m Animal Tissues, Adv. Enzymol. 24: 291-358, 1962. 54. Cooperstein, S. J., and Lazarow, A.: A Microspectrophotometric Method for the Determination of Cytochrome Oxidase, J. Biol. Chem. 189: 665-670, 1951. 55. Smith, L.: Spectrophotometric Assay of Cytochrome C Oxidase, Methods Biochem. Anal. 2: 427-434, 1955. 56. Bitensky, L.: The Reversible Activation of Lysosomes in Normal Cells and the Effects of Pathologic Conditions, Lysosomes, London, 1963, J. & A. Churchill, Ltd., pp. 362-383. 57. Popov, C. 5.: Study on the Stability of Lysosomal Membranes, Blochem. Pharmacol. 18: 1778-1779, 1969. 58. Weissmann. G.: Lvsosomes and Joint Disease. Arthritis Rheum. 9: 834-840. 1966. 59. Tamaka, K., and “Iizuka? Y.: Supression of ‘Enzyme Release From Isolated Rat Liver Lysosomes by Non-sterordal Anti-inflammatory Drugs, B&hem. Pharmacol. 17: 20232032, 1968. 60. Perumal, A. S., Lnkshmanan, M. R., and Cama, H. R.: Effects of Vitamins A and K on Colon Lysosomes, Bioehim. Biophys. Acta 170: 399-408, 1968. 61. Brown. J. H.. and Schwartz. N.: Interaction of Lvsosomes and Anti-inflammatorv ” Drues.Y I * Proc. Sot. Exp. Biol. Med. lgl: 614-620, 1969. 62. Anderson, A. J.: The Effect of Anti-inflammatory Drugs on the Enzymatic Activity of a Rat Liver Granular Fraction which Increases Vascular Permeability, Biochem. Pharmacol. 17: 2253-2264, 1968. 63. Ennis, R. S., Ganda, J. L., and Posner, A. S.: Effect of Gold Salts and Other Drugs on the Release and Activity of Lysosomal Hydrolases, Arthritis Rheum. 11: 756-764, 1968. Reprint requests to : Dr. Roger J. Spott 13975 Connecticut Ave. Wheaton, Md. 26906 or Dr. Theodore Rosett Department of Biochemistry Temple University School Philadelphia, Pa. 19140
of Dentistry
UNIVERSITY
and Theodore Rosett, Ph.D.,**
SCHOOL
OF DENTISTRY
These lysosome studies represent an attempt to explain the action of drugs used in the treatment of dental pulp and to set up a model system for their evaluation.
M
ore than 20 years have elapsed since the postulation, discovery, and localization of lysosomesl Although the role of lysosomes in many intracellular and extracellular functions has been widely researched and carefully documented, little information is currently available on the relationship of the lysosome to pulpal disease. However, the role of the lysosome in the oral cavity is being researched. Oral lysosomal membrane stability has been examined during local anesthesia,2 and the role of lysosomes in periodontal disease has been studied.3-5 Therefore, the purpose of this study was threefold: 1. To elucidate, via literary comparison, the diverse roles that the lysosome might play in pulpal disease. 2. To isolate and identify the lysosomes from bovine dental pulp. 3. To investigate how these subcellular, cytoplasmic particles react to various drugs commonly used in dentistry. When deDuve and associates nicknamed the lysosome the “suicide sac” because of the associated acid hydrolases, it was not fully known that this function played only a small role in the lysosome’s total activity. To explain more adequately the wide physiologic and pathologic implications, Weissmann’ referred to this struct,ure as the many-faceted lysosome. Although the lysosome Submitted by the senior author in partial fulfillment of the requirements for the certificate of advanced training in endodontics, Temple University School of Dentistry. *Graduate Department of Endodontics. Present address: 13975 Connecticut Ave., Wheaton, Md. **Department of Biochemistry.
569
570 Table
Spotf md
Kosett
I. Enzymes
and other
substances
Acid phosphatase Arid ribonuclease Acid desoxyribnuclease Cathepsins B,C,D Phosphoprotein phosphatasc Phosphatidic. acid phosphatase Organophosphate-resistant esterases Beta-glucuronidasc Beta-gala.ctosidase Beta-N-acetylglucosaminidase Alpha-L fucosidase Alpha-l-4 glueosidasc Alpha mannosidxse Alpha-N acetylglucosaminidase Alpha-N ncetylgalctosaminidase (From Weissmann, G.: The Role Med. 18:97-112, 1967.)
Table
II.
Agents stabilizing
associatetl with lysosomcs Hpaluronidase Lysozymc Collngenase Aryl sulfatases A and B Phospholipase Acid lipxse Phagocytin Endogcnous pyrogen Chcmotaxis factor TJnidentified basic proteins Cationic inflammatory protein Permeability-inducing protease Plasminogen activator Hemolysins Mucopolysaccharides and glycoproteins
of Lysosomes
or labilizing StaGlizing
Cortisone-cortisol Prednisone Beta-methasone Chloroquine Labilizing Vitamin A Endotoxin Streptolysin 0 and S Staphylococcal alpha and gamma toxin 2,4-Dinitrophenol Carbon tetrachloride Progesterones and testosterone Deoxycorticosterone Etiocholanolone, 5 Beta-H steroids 5 Beta-H bile acids Icterogenin Cantharidin (From Chemother.
Allison, A. : The 3 :253-302, 1968.)
Role
of Lysosomes
in
Inflammation
and
Disease,
Ann.
Rev.
lysosomes in vitro agents : Benadryl Salicylate Chlorpromazine Colchicine agents: Chloropromazine (high concentration) Hyperoxia and anoxia Shock Ultraviolet radiation x-radiation Antigen-antibody reactions Triton WR.-1339 Silica Carrageenan Asbestos Photosensitizing agents 7.Hydroxymethyl-12-methyl benzanthracene in the
Action
of Drugs
and
Hormones,
Adv.
functions in many cellular processes, it appears to act essentially in two ways. First, it is involved in intracellular digestion.7, 8 In interacting with phagocytized material, the lysosomal enzymes perform their catalytic actions and allow the resultant materials to enter the cytoplasm or to be eliminated (Table I) .9* I0 This ability to ingest and digest various materials has been studied and quantitated by Sawant, Desai, and Tappel.ll, l3 Autophagy is the ability to engulf fragments of intracellular material. This apparently allows the cell to reuse its own components for energy and rebuilding.13 Second, the lysosome has the potential of becoming a mechanism of cell destruction. By an uncontrolled or pathologic rupture, the lysosome can destroy the surrounding and adjacent protoplasm. This destruction is limited only by inactivation of the liberated enzymes.15 The lysosome ruptures for many reasons, among them the actions of drugs, immune reactions, and infections.16* 1r
Volume Number
36 4
Separation 50.0 500
Lysosomes
I Combine, Centrifuge
571
scheme:
g pulp tissue make up to ml. with 0.25M sucrose
Supernatant Supernatant
and dental pulp
Homogenize with polytron >2.0 minutes at highest
f f
filter
through
15,000
xg
glass
l
wool
B
min.-
speed.
1 Centrifuge 15,000 Ppt. rehomogenize sucrose centrifuge
xg min. in 200 ml. 15,000 xg
1 Ppt.-
Discard
Ppt.A
Discard
Supernatant+
Discard
Supernatantj
Discard
l
l
min.
Supernatant 1 Combine,
filter
Centrifuge
4.3
through X lo6
glass xg
l
wool
min.-
1 Ppt. suspend in 24 ml 0.25M Sucrose centrifuge 5.4 X lo6 xg min.l
Pellet, to suspend density gradient round-bottomed Fig. Biophys.
Physiologic
on top of in 100 ml. centrifuge tube
2. (From Rosett, Acta 222: 5-14,
T., and 1970.)
associates:
Studies
in
the
Biochemistry
of
Skin,
Biochim.
responses
The physiologic category of lysosomal activities includes tooth development, bone resorption, and phagocytic response of cells to toxic materials. Lysosomelike bodies have been seen microscopically in odontoblasts.18-21These structures have also been associated with tooth formation.22-24 An extensive lysosomal system has been shown in the cells of the ameloblastic layer. The Tomes process contains very high lysosome concentrations; however, confirmation of their role extracellularly still is unreported. 25x26 Thus, lysosome enzymes have been correlated with secretory activity and their distribution correlated with amelogenesis and dentinogenesis. It was suggested by deDuve and Wattiaux13 that the secretory functions of cells may be subjected to regulatory inactivation by the lysosomes. Bone resorption, including both mineral and organic components, was studied in tissue culture by VaesZ7 who found that the resorption was accompanied by release of six lysosomal enzymes, This release could be a consequence of bone resorption, but it was concluded to be a cause of the resorption. Electron microscopic studies have shown resorption to be initially extracellular. The organic fibers are first dissociated, apparently by disruption of the ground substance. A loss of collagen fiber cross banding then follows. ScherfP postulated that the osteoclasts, or similar cells, excrete agents which could accomplish
572
8pott
awl
Olxl October,
Roseti Lysosome
prep.
t Test
Surg. 1973
material
4, Incubate
30
1 Centrifuge
min.
4,000
37”
C.
R.P.M.
20 min. ) Ppt +
4, Supernatant
Discard
1 Test
amount
t Enzyme
substrate
1 Incubate Add
30 min.
J enzyme
1 Centrifuge
assay 2,000
37’
C. buffer
R.P.M.
10 min. ) Ppt +
Discard
Supernotant
& Read
540~ Fig.
spectrophotometer 2. Test
sequence.
both processes.It is unlikely that this release of agents is due to overt disruption of the lysosomal membrane; rather, it is probably due to a mechanism similar to that found in secretory cells.” The phagocytic responsesof cells to toxic materials differ from the secretorylike functions, but thep are still physiologic reactions, since all particulate materials appear to be concentrated in lysosomes. Plastics and certain metals, such as iron, silver, lead, copper, and mercury, are also concentrated in lysosomes.2s-31 Intracellular accumulation of these materials might be significant with reference +,otoxicology and carcinogenicity. Pathologic
responses
Pathologic release of lysosomal enzymes is seenin diseaseand inflammation.13 The uncontrolled enzyme release may be due to normal but total lysosome membrane rupture or to pathologic conditions which give rise to, or cause formation of, more fragile lysosomal membranes.lJ Lysosomal rupture has been produced by a variety of agents. When streptolysin 0, a bacterial toxin, was added to living cells, the lysosomes were seen to rupture.32 Antilysosome antibodies, with complement, when added to living cells, produced lysosome membrane breakdown.33 When photosensitizing agents, such as neutral red and acridine orange, were allowed to accumulate in lysosomes and the latter were then exposed to ultraviolet light, rupture occurred.34’ 35 Lysosomal breakage produces damage to surrounding tissues, as evidenced by loss of cartilage matrix in hypervitaminosis A. There appears to be a direct action of Vitamin A on the lysosome.36 Lysosomal rupture is implicated in certain diseases. The Chediak-Higaski syndrome of children shows abnormal leukocyte lysosomes. This seemsto result
Volume Number
36 4
Lysosomes
and dental
pulp
573
EUGENOL I)miMExRAsoNE ULTRASOUND PREDNISOLONE BENADRYL CPMCP FORMCZREWL GODIUM
FLUORmE PRFDNISONE
DIMETmL
SU~XIDE TRITONX ASPIRIN
% CONTROL STABILIZE
a&
+
LARILIZE
I Fig.
3.
in a higher susceptibility to infeetion.~ ?‘I, 38 During the course of hepatitis, lysosomes were found to be labilizedS9 Certain connective tissue disorders, including rheumatoid arthritis and lupus erythematosus, involve the presence of more fragile lysosomes.4nT 41 There is evidence that agents used to control these disorders may act somewhere in this sequence.l” A listing of these agents is given in Table II. The foregoing is a cursory examination of the pathologic implications of lysosomes which may be of particular interest. Research on lysosomal activity of the dental pulp, however, is practically nonexistent. Inasmuch as the lysosomes were implicated in both the physiologic and pathologic processes, this research project had these aims : I. To isolate and identify lysosomes from bovine dental pulp, thereby ascribing to the pulp the physiologic lysosomal activities present in other tissues. II. To examine, in vitro, membrane stability of lysosomes subjected to drugs commonly used in dentistry, thereby determining the presence of any pathologic lysosomal response. PART Methods
I. ISOLATION and
OF
LYSOSOMES
materials
Bovine dental pulp was chosen as the material to be examined because of its large size and easy availablity. According to Rosett and associates, the bovine
574
Spott
Oral October,
and Kosett
Surg. 1973
RESULTS BETR-GLUCURDNIDASE
EUGENDL -NE ULTRASOUND PRmNIsDLDNE
k I
CPM2P
-I
FOR)rlOCRESOL SODIUM
-I
c
FLUDRIDE
I
mNIscNE DIMEI’RYL
I
SULFDXIDE TRITON-X
4
ASPIRIN
I I I 203104050~
I
I I 7080901
I
3 ’
lb
’
I 140
I ’ 160
% CDrnL STABILIZE
+
+
LABILIZE
I Fig.
4.
mandibles are chilled and ready for transport within 5 minutes after animal death. The first cheek tooth was fractured horizontally at the height of the alveolar bone, and pulps were removed by severing the canal contents near the apex. The intact pulp masses about 1.5 grams. ** The resultant pulps represented a pooled cellular sample. The extracted bovine pulps were combined and suspended in a 0 to 4’ C. 0.25M sucrose solution at pH 7.4 (50.0 grams of pulp to 500 ml. of sucrose solution). 43 This was then homogenized at top speed for 2 minutes in a Willems Polytron homogenizer (Brinkman Co.). The homogenized cellular components were separated by centrifugation. There is a large variety of separation schemes, each having been devised by a different researcher.44-46 Some improved methods have adapted zonal centrifugation for separating large quantities rapidly and with a high degree of purity.47-50 This research employed a modification of the method of Rosett and associates.43 Fig. 1 illustrates the scheme for this differential centrifugation procedure. Identification of the lysosomal fraction was essentially biochemical. AS previously shown (Table I) many enzymes have been isolated or identified in association with lysosomes.Most authorities have utilized more than one enzyme in demonstrating lysosomal activity. In this study, analysis therefore included two enzymes. Acid phosphatase was assayed by an adaptation of the method of Huggins and Talalay.51 Beta-glucuronidase was assayed by an adaptation of the method of Fishman and associates.52Cytochrome oxidase activity was also
Volume Number
Lysosomes
36 4
Table III. Materials
and dental
pulp
575
tested 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Aspirin Triton-X Dimethyl sulfoxide Prednisone Sodium fluoride Formocresol Camphorated paramonochlorophenol Benadryl Prednisolone Ultrasound Dexamethasone Eugenol
assayed as a marker for mitochondria to assure that they were not present in high concentrations in the lysosome fractions. This method used cytochrome C as substrate and measured the decrease of the intensity of the substrate’s spectrophotometric absorption as it was oxidized by the enzyme.53-55 PART Il. EFFECTS OF DRUGS ON LYSOSOMES After it had been shown that lysosomal alterations accompanied cell damage, techniques arose to utilize this fragility of lysosomes as a detection modality for cell injuryas Also, the ability of certain materials to stabilize this fragility was studied.57-5g The effects of vitamins, hormones, and other compounds have been investigated.60$ 61 Part II examined the effect on isolated bovine pulp lysosomes of drugs commonly used in dentistry. Methods
and materials
Lysosomes were subjected to the drugs listed in Table III. The lysosome fraction from Part I of this study was resuspended in a solution containing 50 per cent by volume of 0.25M sucrose and 50 per cent acetate buffer (pH 5.0). One part of test substance to 20 parts of lysosome suspension was incubated at 37O C. for 30 minutes.62* 63 After incubation, the lysosomal fraction was centrifuged to the bottom and removed. The supernatant was assayed for acid phosphatase and beta-glucuronidase in the manner previously described. The data form was spectrophotometer readings, and the samples were compared to controls to give the per cent of stabilization or labilization. Results The combined pulp pool was homogenized and separated according to the separation scheme (Fig. 1). The resultant pellet was resuspended in 0.25M sucrose and assayed with the test material as indicated in Fig. 2. The spectrophotometer results are compared and expressed as per cent of control (Figs. 3 and 4). Controls of the lysosomal preparation and the test reactions are shown in Fig. 5. DISCUSSION Lysosome preparations were localized in fractions of bovine dental pulp. Purification by gradient separation and electron microscopic study of the pellet specimens are currently being completed. The results as indicated in Figs. 3 and
576
Spotf
and
fiosett
Controls Free
enzyme
t Test
material-
Assay Should equal
Free Known
enzyme amount
__j of enzyme
Assay
t Test material-
be
> Assay Should be equal and correct
Known
amount
-
of enzyme Fig.
Assay
>
5.
4 reveal two major factors: First, results obtained in this study parallel results obtained with the sametest materials on lysosome preparations from other tissue sources.30-40, 56-63This would ascribe to the dental pulp a similarity, at an ultrastructural level, to the lysosome properties carefully worked out for other sites.“6-63Second, results indicate that some materials used in dentistry cause labilization of the lysosomal fractions. Dilution studies and correlated histologic studies might make a very good method of evaluating present materials and comparing their properties. Research into new materials would be aided by histologic and quantitative lysosomal enzyme assays. One interesting result, among many others, was that of sodium fluoride. Fig. 6 shows the results which indicate that the material acts as a stabilizer of both enzymes, The control reactions, however, show that sodium fluoride also inhibits the free enzymes, which means that it did not affect the lysosomal membrane per se but, rather, acted on the enzymes. The potential importance of the results of this study depends on application. First, isolation and identification of lysosomesfrom dental pulp may ascribe to the dental pulp lysosomes the physiologic activities of lysosomes seen in other tissues. This might aid in applying more widespread medical stabilization and labilization study data to the treatment of pulpal disease. Second, testing of lysosomal membrane stability can determine the presence of any pathologic lysosomal response to dental medications and drugs. The finding of grossly injurious materials might indicate the necessity for further study prior to general usage. Proposals for extension of this study should include other test materials, human in vitro studies, and animal and human in vivo pulpal studies. SUMMARY
Intact dental pulps from the mandibular cheek teeth of freshly killed cattle were removed, suspended in 0.25M sucrose, homogenized, and placed in a separation schemeto produce lysosomc-rich preparations. The lysosomeswere incubated with various substances known to labilizc or stabilize lysosomes, as well as with some drugs commonly used in medicine and dentistry. The controls were both no drug or drug- and supernatant-free enzymes. The enzyme assays were for acid phosphatase and Beta-glucuronidase. Drugs which stabilized included
Volume Number
36 4
Lysosomes
and dental pulp
577
GLKXJRDNIDASE ACID FHCBFHATASE 20 30 40 50 60 70 so 90 100 STABILIZE + +LABILIZE I ~CFCcwMaL FREEENZYME Fig.
6.
dexamethasone, prednisolone, benadryl, prednisone, dimethyl-sulfoxide, and aspirin. Drugs which labilized included euginol, ultrasound, camphorated paramonochlorophenol, formocresol, and Triton-X. Sodium fluoride appeared to inhibit enzymatic activity. The chemistry, Dentistry,
authors wish to acknowledge Electron Microscopy, and Philadelphia, Pennsylvania.
the valuable assistance of all members Endodontic Departments at Temple University
of
the BioSchool of
REFERENCES
1. deDuve, C., Pressman, B., Gianetto, R., Wattiaux, R., and Appelmans, F.: Tissue Fractionation Studies, Biochem. J. 60: 604-617, 1955. 2. Smith, C. J., and Cimasoni, G.: Stability of Oral Epithelial Lysosomes During Local Anaesthesia, Helv. Odontol. Acta 9: 95-100, 1965. 3. Squier, C. A., and Waterhouse, J. P.: Lysosomes in Oral Epithelium, Arch. Oral Biol. 15: X3-168, 1970. 4. Waterhouse, J. P.: The Gingival Part of Human Periodontium, Dent. Pratt. 15: 409-415, 1965. 5. Hamp, S., and Polk, L.: The Lysosomes and Their Possible Role in Periodontal Disease, Odontol. Tidskr. 76: 353-375, 1968. 6. Weissmann, 0. : The Many-Faceted Lysosome, Hosp. Pratt. 3: 31-39, 1968. 7. deDuve, C. : The Lysosome, Sci. Am. 208: 64-72, 1967. 8. Novikoff, A. B.: Lysosomes in the Physiology and Pathology of Cells, Ciba Foundation Symposium on Lysosomes, Boston, 1963, Little, Brown 8; Company, pp. 36-77. 9. R.uebner, B. H., Hirano, T., Slusser, R., Osborn, J., and Medearis, D. N.: Cytomegalovirus Infection, Am. J. Pathol. 48: 971-989, 1966. 10. deDuve, C.: General Properties of Lysosomes, Ciba Foundation Symposium on Lysosomes, Boston, 1963, Little, Brown & Company, pp. l-35. 11. Sawant, P. L., Desai, I. D., and Tappel, A. L.: Digestive Capacity of Purified Lysosomes, Biochim. Biophys. Acta 85: 93-102, 1964. 12. Tappel, A. L.: Lysosomes, Distributions in Animals, Hydrolytic Capacity and Other Properties, Ciba Foundation Symposium on Lysosomes, Boston, 1963, Little, Brown & Company, pp. 78-113. 13. Idg&$ve, C., and Wattiaux, R.: Functions of Lysosomes, Ann. Rev. Physiol. 28: 435-492, 14. Thomas, L.: The Role of Lysosomes in Tissue Injury, The Inflammatory Process, New York, 1965, Academic Press, Inc., pp. 449-463. 15. Weissmann, G.: The Role of Lysosomes in Inflammation and Disease, Ann, Rev. Med. 18: 97-112. 1967. 16. Allison, A.: The Role of Lysosomes in the Actions of Drugs and Hormones, Adv. Chemother. 3: 253-302, 1968. 17. Golub, E. S., and Spitznagel, J. K.: The Role of Lysosomes in Hypersensitivity Reactions, J. Immunol. 95: 1060-1066, 1966.
570
Spott
and Kosett
Oral October,
Surg. 1973
18. Garant, P. R., Szabo, Cr., and Nalbandian, J.: The Fine Structure of the Mouse Odontoblast. Arch. Oral Biol. 13: 857-876. 1968. 19. Jessen, H. : ‘The Ultrastructure of Odontoblast in Perfusion Fixed, Demineralized Incisors of Adult Rat, Acta Odontol. Stand. 25: 491523, 1967. 20. Katchburian, E., and Halt, S. J.: Ultrastructure Studies on Lysosomes and Acid Phosphatase in Odontoblasts. 1% Dentin and Pulp: Their Structure and Reactions, Dundee, 1968, D. C. Thomson Company, pp. 43-57. 21. Nag&i, N.: Electron Microscopy of the Cytoplasmic Bodies in the Odontoblasts of Young Rat Incisors. Bull. Tokyo Dent. Coll. 11: 47-83. 1970. 22. Reith, E. J.: Ultra&uctural Aspects of Dentinogenesis. In Dentin and Pulp: Their Structure and Reactions, Dundee, 1968, D. C. Thomson Company, pp. 19-41. 23. Reith, E. J.: Collagen Formation in Developing Teeth of Rats, J. Ultrastrue. Res. 21: 383-414. 1968. 24. Takuma, S.: Ultrastructure of Dentinogenesis. In, Miles, A. E. W. and Greulich, R. C. (editors) : Structural and Chemical Organization of Teeth, New York, 1967, Academic Press, Inc., vol. I, pp. 325-370’. 25. Katchburian, E., and Holt, 5.: Role of Lysosomes in Amelogenesis, Nature 223: 1367-1368, 1969.
26. Katchburian, E.: Histochemistry of Lysosomal Enzymes in Developing Teeth of Alhino Rats, J. Anat. 101: 783-792, 1967. 27. Vaes, G.: The Action of Parathyroid Hormone on the Excretion and Synthesis of Lysosomal Enzymes and on the Extracellular Release of Acid by Bone Cells, J. Cell. Biol. 39: 676-697, 1968. 28. Scherft, J. P.: The Resorption of the Organic Matrix of Calcified Cartilage as Seen With the Electron Microscope, Calcif. Tissue Res. 2: Supp. 96-96B, 1968. 29. Koenig, H. J.: Epileptogenic Action of Substances That Interact With Gangliosides; In Vivo Release of Lysosomal Enzymes as a Disease Mechanism, J. Histochem. Cytochem. 11: 120-121, 1963. 30. Little, K.: Reactions of the Body to Implanted Materials, Jaarb. Kankeronderz Nederl. 14: 75-81, 1964. 31. Allison. A.: Lvsosomes and the Resoonses of Cells to Toxic Materials. Sci. Basis Med. Anna. Rev. 18: 18-30, 1968. 32. Hirsch, J. G., Bernheimer, A. W., and Weissmann, G.: Motion Picture Study of the Toxic A&ion of Streotolvsins on Leukocvtes. J. EXD. Med. 118: 223-228. 1963. 33. Quie, P. G., a?nd “Hirsch, J. G.: “Antiserum to Leukocyte Lysosomes, J. Exp. Med. 120: 149-160, 1964. 34. Allison, A. C., and Paton, G. R.: Chromosome Damage in Human Diploid Cells Following Activation of Lysosomal Enzymes, Nature 207: 1170-1173, 1965. 35. Allison, A. C., Magnus, I. A., and Young, M. R.: Role of Lysosomes and of Cell Membranes in Photosensitization, Nature 209: 874-878, 1966. 36. Dingle, J.: Action of Vitamin A on the Stability of Lysosomes In Vivo and In Vitro, Ciba Foundation Symposium on Lysosomes, Boston, 1963, Little, Brown & Company, pp. 384-398. 37. Windhorst, D. B., Zelickson, A. S., and Good, R. A.: Chediak-Higashi Syndrome: Hereditary Gigantism of Cytoplasmic Organelles, Science 151: 81-83, 1966. 38. White, J. G.: The Chediak-Higashi Syndrome, a Possible Lysosomal Disease, Blood 28: 143-156, 1966. and Acid 39. Pagliaro, L., Giglio, F., LeMoli, S., and Citarrela, P.: Beta-Glucuronidase Phosphatase Activities of Lysosomal Preparations From Human Liver Tissue Obtained by Needle Biopsy From Subjects With Acute Hepatitis and Cirrhosis, J. Clin. Invest. 63: 977-985, 1964. 40. Barland, P., Novikoff, A,, and Hamerman, D.: Fine Structure and Cytochemistry of the Rheumatoid Synovial Membrane With Special Reference to Lysosomes, Am. J. Pathol. 44: 853-866, 1964. 41. Weissmann, G., and Rothfield, N.: Structure and Function of Connective Tissue, Oxford, 1964, Blackwell Sm., pp. 466-483. and Cofactor Effects on Respiration of 42. Pollack, R., Green, D., and Rosett, T.: Substrate Bovine Dental Pulp, International Association for Dental Research, 49th General Session Program and Abstracts of Papers, p. 184, March, 1971. D., McDonald, T., and Brown, K.: Studies in 43. Rosett, ‘I., Matsuo, I., Bailey, A., Smith, the Biochemistry of Skin, V. The Particulate Origin of Adenylate Kinase in the Skm Of Neonatal Rats, Biochim. Biophys. Acta 222: 5-14, 1970. 44. deDuve, C.: The Separation and Characterization of Subcellular Particles, Harvey Lect. 59: 49-87, 1965. 45. Sawant, P. L., Shibko, S., Kumta, U., and Tappel, A.: Isolation of Rat Liver Lysosomes and Their General Properties, Biochim. Biophys. Acta 85: 82-92, 1964. 46. Ragab, II., Beck, C., Dillard, C., and Tappel, A.: Preparation of Rat Liver Lysosomea, Biochrm. Biophys. Acta 148: 501-505, 1964. I
Volume Number
36 4
Lysosomes
and
dental pulp
579
47. Schuel, H., Schuel, R., and Unakar, N.: Separation of Rat Liver Lysosomes and Mitochondria in the Z-XII Zonal Centrifuge. Anal. Bioehem. 25: 146-163. 1968. 48. Brown, D. H. : Separation of Mitochondrra,’ Peroxisomes and Lysosomes by’ Zonal Centrifugation in a Ficoll Gradient, Biochim. Biophys. Acta 162: 152-153, 1968. 49. Leighton, F., Poole, B., Beaufay, H., Baudhuin, P., Cocffey, J. W., Fower, S. and deDuve, C.: The Large Scale Separation of Peroxisomes, Mitochondria and Lysosomes From the Livers of Rats Injected With Triton WR-1339, J. Cell. Biol. 37: 482-513, 1968. 50. Withers, B., Davies, I. A., and Wynn, C. H.: The Purification of Lysosomes by Isopycnic Zonal Ultracentrifugation, Biochem. Biophys. Res. Commun. 30: 227-231, 1968. 51. Huggins, C., and Talalay, P.: Sodium Phenolphalein Phosphate as a Substrate for Phosuhate Tests. J. Biol. Chem. 159: 399-410. 1945. 52. Fish&an, W. H.; Springer, B., and Br&etti,‘R.:--Application of an Improved Glucuronidase Assay Method to the Study of Human Blood Beta-Glucuronidase. J. Biol. Chem. 173: 449-456, 1948. 53. deDuve, C:, Wattiaux, R., and Baudhuin, P.: Distribution of Enzymes Between Subcellular Fractions m Animal Tissues, Adv. Enzymol. 24: 291-358, 1962. 54. Cooperstein, S. J., and Lazarow, A.: A Microspectrophotometric Method for the Determination of Cytochrome Oxidase, J. Biol. Chem. 189: 665-670, 1951. 55. Smith, L.: Spectrophotometric Assay of Cytochrome C Oxidase, Methods Biochem. Anal. 2: 427-434, 1955. 56. Bitensky, L.: The Reversible Activation of Lysosomes in Normal Cells and the Effects of Pathologic Conditions, Lysosomes, London, 1963, J. & A. Churchill, Ltd., pp. 362-383. 57. Popov, C. 5.: Study on the Stability of Lysosomal Membranes, Blochem. Pharmacol. 18: 1778-1779, 1969. 58. Weissmann. G.: Lvsosomes and Joint Disease. Arthritis Rheum. 9: 834-840. 1966. 59. Tamaka, K., and “Iizuka? Y.: Supression of ‘Enzyme Release From Isolated Rat Liver Lysosomes by Non-sterordal Anti-inflammatory Drugs, B&hem. Pharmacol. 17: 20232032, 1968. 60. Perumal, A. S., Lnkshmanan, M. R., and Cama, H. R.: Effects of Vitamins A and K on Colon Lysosomes, Bioehim. Biophys. Acta 170: 399-408, 1968. 61. Brown. J. H.. and Schwartz. N.: Interaction of Lvsosomes and Anti-inflammatorv ” Drues.Y I * Proc. Sot. Exp. Biol. Med. lgl: 614-620, 1969. 62. Anderson, A. J.: The Effect of Anti-inflammatory Drugs on the Enzymatic Activity of a Rat Liver Granular Fraction which Increases Vascular Permeability, Biochem. Pharmacol. 17: 2253-2264, 1968. 63. Ennis, R. S., Ganda, J. L., and Posner, A. S.: Effect of Gold Salts and Other Drugs on the Release and Activity of Lysosomal Hydrolases, Arthritis Rheum. 11: 756-764, 1968. Reprint requests to : Dr. Roger J. Spott 13975 Connecticut Ave. Wheaton, Md. 26906 or Dr. Theodore Rosett Department of Biochemistry Temple University School Philadelphia, Pa. 19140
of Dentistry