Review article

Leukotrienes: their possible role in pulpal and periapical diseases

Torabinejad M, Cotti E, Lessard G. Leukotrienes: their possible role in pulpal and periapical diseases. Endod Dent Traumatol 1991; 7: 233-241. Abstract - Eeukotrienes play an important role in inflammation and its sequelae such as pain, swelling, and bone resorption. This review covers, their history, structure, synthesis, metabolism, biological effects, inhibitors, antagonists, and their possible role in pathogenesis of pulpal and periapical disease. Mahmoud Torabinejad', Eiisabetta Cotti^ George Lessard^
Departments of ^Conservative Dentistry, University of Cagliari, Italy, ^Endodontics, ^Biochemistry, Loma Linda University, California, USA.

Key words: leukotrienes; pulp; inflammation; periapical disease Accepted June 11, 1991

Arachidonic acid is an important precursor to a number of local messengers implicated in the control of numerous biologic activities. Upon cellular stimulation and activation of phospholipases a large amount of arachidonic acid is released from phospholipids of the cell membrane. Enzymatic oxidation of arachidonic acid results in formation of a group of biologically important compounds, sometimes referred to as eicosanoids, including the prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs) and lipoxins. (Fig. 1). These products have a wide spectrum of physiologic and sometimes pathologic effects and play an important role in inflammation and its sequelae including pain, swelling, and bone resorption. The possible role of prostaglandins in the pathogenesis of pulpal and periapical diseases was addressed in two review papers a decade ago (1, 2). This current paper reviews the history, chemical structure and nomenclature, biosynthesis and metabolism, biological effects, pharmacologic inhibitors and antagonists of leukotrienes and their possible role in pathogenesis of pulpal and periapical disease. History Sixty-one years ago Burr & Burr identified linoleic acid as an essential fatty acid for growth and health in mammals (3). This fatty acid is converted by mammals to C-20 polyunsaturated fatty acids such as dihomo-5-linolenic and arachidonic acids. Arachidonic acid, a very abundant fatty acid, is located predominantly at the 2-position of membrane glycero-phospholipids (46) and can be transformed into various leukotrienes. Evidence for the existence

of LTs came from an experiment by Harkavy who applied aqueous solutions of ethanolic extracts of the sputum of asthmatic patients to smooth muscle preparations from cat jejunum and rabbit duodenum. Application of the extracts to the muscle caused contraction of the muscle strips when the extracts were collected during an asthmatic attack. However, sputum extracts obtained prior to an asthmatic attack failed to produce the same effects (7). Feldberg & Kellaway (8) and later Kellaway & Trethewie (9) reported that perfusion of the lungs of ovalbumin-immunized guinea pigs with solutions containing the same antigen resulted in the release of a substance capable of causing contraction of jejunum. Because of the slow onset of this reaction, the substance was named slow-reacting substance (SRS) by the cited investigators. In 1960, Brocklehurst obtained the term "slow reacting substance of anaphylaxis" or SRS-A to describe a substance which caused a slow and long-lasting contraction of the guinea pig ileum and resisted the antihistaminic drugs (10). The term SRS-A refers to any slow reacting substance released during an antigen-antibody reaction. The biochemical and pharmacological characteristics of SRS-A and the mechanisms involved have been studied by Orange et al. (11 15). The discovery by Orange & Chang that pre-incubation of human lung fragments with cysteine resulted in a dose-dependent release of SRSA suggested that cysteine might be a precursor of this molecule (14). At the same time that other investigators were examining the role of arachidonic acid in the formation of ionophore-induced slow SRS, (16, 17). Borgeat & Samuelsson found a new metabolite produced upon incubation of arachidon233

Torabinejad et al. ase, the enzyme which specifically acts on arachidonic acid to produce prostaglandins, the lipoxygenases can oxidize other fatty acids such as eicosatrienoic and eicosapentaenoic acids as well. Almost all mammalian cells (except erythrocytes) contain cyclooxygenases. However, the 5-lipoxygenase appears to be present only in inflammatory cells such as neutrophils, eosinophils, mast cells, basophils, macrophages, and monocytes (2124). Goetzl has suggested lymphocytes as another source of LTs (25). Lipoxygenases are dioxygenases which catalyze the insertion of molecular oxygen into the 1,4-pentadiene structure of polyunsaturated fatty acids (26). Cells which possess a 5-lipoxygenase also contain cyclooxygenase as well. Therefore, the activated enzymes are in competition for the arachidonic acid according to both their Kms (affinity constant) and their catalytic activity (27). When the 5-lipoxygenase acts on arachidonic acid, it produces 5-hydroperoxyeicosatetraenoic acid (5-HPFTE) through loss of water and forms the unstable epoxide intermediate, 5,6-oxido-7,9,l 1,14eicosatetraenoic acid (LTA4) (27). LTA4 is the precursor for the formation of other LTs. It can either be hydrolyzed by the epoxide hydrolase, LTA4 hydrolase, to form 5(S), 12R-dihydroxy-6,14-cis8,10-trans-eicosatetraenoic acid (LTB4), or can be altered by addition of glutathione to 5-hydroxy-6glutathionyl derivative (LTC4) via LTC4 synthetase. Studies have shown that various cells produce different LTs. Human pulmonary mast cells (28) and eosinophils (29) produce mainly LTC4. In contrast, human pulmonary macrophages (30) and PMNL generate mainly LTB4. It appears that the human monocyte is capable of producing both LTB4 and sulfidopeptide LTs such as LTC4 and LTD4 (31). Further metabolism of LTC4 occurs via two different pathways: an oxidative pathway and peptidolytic pathway. The peptidolytic pathway leads to the

PROSTAGIANOINS THROMBOXANE PGI2 PGF2a PGE2 TxA2 TxB2

LEUKOTRIENES LTA4 LTB4 LTC4 LTD4 LTE4 LTF4

LIPOXINS LPA LPB

Fig. I. Pathways of formation of various arachidonic acid metabolites and their inhibitors.

ic acid with polymorphonuclear leukocytes (PMNL) (18). They incubated rabbit PMNLs with ''^C-labelled arachidonic acid and found a family of dihydroxy acids containing three conjugated double bonds. Nomenclature Because of the fact that these molecules were first discovered in leukocytes and have at least three conjugated double bonds, they were called leukotrienes (19). To simplify references to the various types of LTs, the long chemical names (table) are customarily avoided, and the letter designations (A-F), e.g. LTA, are given indicating the order in which they were discovered. The numerical subscripts 3, 4 or 5, (e.g. LTA4,) following the letter designations indicate the degree of unsaturation and correspond to the number of double bonds in each molecule. The molecules derived from dihomo-5-linolenic acid carry the subscript " 3 " , while substances formed from arachidonic acid carry the subscript "4" (Fig. 2). Products of eicosapentaenoic acid 20:5 (A5,8,11,14,17) carry the subscript "5". Biosynthesis and metaboiism Like other arachidonic acid metabolites, LTs are not stored in tissues but are synthesized from fatty acid precursors upon cell stimulation and activation of enzymes called lipoxygenase. There are several lipoxygenases in both humans and plants which catalyze this reaction (20). Unlike the cyclooxygen-

Table. Chemical names and abreviations for various leukotrienes 1. 2. 3. 4. 5. 5,6-oxido-7,9 trans-11-cis . 5(S),12(R)-Dihydroxy-6-cis-8,10 Trans 5(S)-Hydroxy-6(R)-S- -Glutamyl-cystelnyl-Glycyl-7,9-Trans-11-cis 5{S)-Hydroxy-6(R)-S-Cysteinylglycyl-7,9-Trans-11-cis 5(S)-Hydroxy-6(R)-S-Cysteinyl-7,9-Trans-11-cis eicosapolyenoic acid LTA LTB LTC LTD LTE

Fig. 2. Chemical structure of leukotriene B4.

234

Possibie role in puipai and periapicai diseases production of LTD4 through the enzymatic action of the 6-Glutamyl-transpeptidase (32). LTD4 is transformed subsequently to LTE4 in a further peptidolytic reaction which involves the release of cysteinyl-glycine by the dipeptidase (33). LT C4, D4, and E4 are the substances which were referred to as SRS-A (10). Another LT has been recently identified as LTF4 which has cysteinyl-glutamate at C-6 (20). Inactivation of LTC4, LTD4 and LTE4, occurs through the oxidative pathway. This usually takes place in the extracellular environment of activated PMNLs and eosinophils. It is a complex mechanism and involves the production of hydrogen peroxide, chloride ions and hypochlorous acid. LTE4, which is excreted in the urine, has so far been shown to be the major catabolite of LTC4, and LTD4 (34, 35). More detailed descriptions on the chemical structure, biosynthesis, and metabolism of LTs are obtainable from other reviews (19, 30, 36-40). Biological effects of leukotrienes LTs have several important biological actions. LTB4 is a potent chemotactic agent for PMNLs and eosinophils. It causes plasma exudation in the presence of PMNLs. Thiol-ether LTs (LTC4, LTD4, LTE4) cause arteriolar constriction, dilation of venules and plasma exudation. In addition, they are coronary vasoconstrictors, heart arrhythmogenic agents, and can cause the constriction of bronchi as well as causes an increase in mucus secretion (41). Based on the multiple actions of LTs, it appears that specific LT receptors must occur in different organs and tissues. Most of the known biological responses to these substances appear to be receptor mediated. The first evidences for the presence of receptors for LTB4 was reported by Goldman & Goetzl (42), as well as Kreisle & Parker (43). Further studies by Goldman & Goetzl showed existence of both low and high affinity receptors for LTB4 on PMNLs (44). These studies clearly show specific binding on LTB4 to human PMNLs and existence of marked species differences for LT receptors. In addition to PMNLs T-lymphocytes have also been shown to have receptors for LTB4 (45). In this study, the binding sites for sulfidopeptide LTs appear to be different from those for LTB4. Receptors for LTG4, LTD4, and LTE4 have been reported in the lung tissue of experimental.animals (46-48). LTs have a wide range of biological activities and participate in pathophysiological conditions such as infiammation, anaphylaxis and asthma, as well as the normal neuroendocrine functions and immune responses (9, 30, 49-56). Infiammation plays a critical role in the pathogenesis of pulpal and periapical lesions. In this review we will discuss only the role of LTs in infiammation. For more detailed descriptions of the many other functions of LTs readers are referred to comprehensive reviews by Piper (57) and others (30, 41). Roie of ieukotrienes in infiammation Tissue injury results in release of a group of substances which affect the host microcirculation and development of infiammation. Gells such as platelets, leukocytes and endothehal cells can mobilize arachidonic acid and metabohze it to form substances such as the PGs and LTs, each with its specific biological activity. The first indication that lipoxygenase products play a role in infiammation came from the observation that 12-HETE was present in the epidermis of patients with psoriasis (58). The 15-HPETE and 15-HETE found in the tissue had no effect on human PMNLs (59). 5,12 diHETE (LTB4) was the most potent lipoxygenase product among the three types of LT studied. LTB4 is a potent chemotactic and degranulating agent for PMNLs and causes the accumulation of PMNLs at the site of infiammation (60, 61). LTB4 has been also detected in rheumatoid arthritis, gout and psoriasis (62, 63). LTB4 plays a significant role in the infiammatory process. It affects the leukocyte functions and its presence has been demonstrated in infiammatory exudates experimentally induced by implantation of sponges containing 1 % uric acid, zymosan, or carrageenin (64). Turner et al. incubated the soluble fraction of human blood platelets which contained the lipoxygenase activity with pure arachidonic acid and found that HETE (12-L-Hydroxy-5,8,10,14eicosatetraenoic acid) is a potent mediator of neutrophil chemotaxis (65). LTB4 is considered as one of the most potent chemotactic agents for PMNLs. Palmer and co-workers investigated the chemotactic potency of some 5-lipoxygenases products for human, rat and rabbit PMNLs. They showed that in all three species, LTB4 was 100-fold more potent than any other compound studied. (59). In 1980, Goetzl & Pickett (66) evaluated the in vitro effect of the 5-lipoxygenase metabolites on human PMNLs. They found that LTB4 was by far the most potent agent. LTB4 evoked a chemotactic response in neutrophils at a concentration as low as 3 ng/ml while other lipoxygenase products such as LTC4 did not elicit any response until higher concentrations were rendered. At 30 ng/ml, an intense chemotaxis was observed, which was similar to that evoked by optimal concentrations of G5a (66). Follow up studies have confirmed the validity of these findings, (67, 68) and the in vitro effects of LTB4 on PMNLs have been confirmed in in vivo (69, 70). The chemotactic potential of LTB4 has been studied both in the hamster cheek pouch and the 235

Torabinejad et ai. rabbit mesentery models. Histologic examination of sections revealed a LTB4-mediated adhesion of PMNLs to the vascular endothelium of the venules. An intravenous injection of LTB4 caused a profound but transient neutropenia in the rabbit, while an intradermal injection of LTB4 caused accumulation of PMNLs. The same results were observed after topical application of LTB4 over abrasions of human forearms (69). In vivo leukocytes accumulation subsequent to injection or topical application of LTB4, has been observed in both rabbit eye and guinea pig peritoneal cavity (70). Recently, chemotactic effects of LTB4 and G5a on isolated human neutrophils have been compared by using three micron-pore cellulose nitrate filters. The results suggest that LTB4 primarily has as its major role that of mediating cell adhesion and penetration into the endothelium, whereas G5a has as its major role that of cell migration (71). Although LTB4 has its primary effect on PMNLs, it may also promote migration of human monocytes, (72) macrophages, (73) and to a lesser extent, fibroblasts, (74). The chemotactic effect of LTB4 on eosinophils is controversial: Goetzl has reported that guinea pig eosinophils migrated more rapidly than PMNLs in response to presence of LTB4, (75) whereas Uden et al. found no chemotactic response by human eosinophils to LTB4 (76). The results of the study by Uden et al. (76) also contrasts with the findings obtained by Gzarnetzki et al. who showed that LTB4 and its metabolites have a greater chemotaxotic effect on eosinophils than on neutrophils (77). LTB4 does not appear to possess vasodilator activity. However, it does contribute to bradykinin-induced plasma leakage when injected subcutaneously in rabbits, rats, or pigs. These effects are significantly increased when used in combination with PGE2 (78, 79). Throughout the infiammatory process, when nerve fiber terminals are sensitized by mediators, hyperalgesia is created. LTB4 has a role in induction of pain. Rackham et al. injected LTB4 into the rat paw and reported hyperalgesia similar to that induced by PGE,; however no synergism was seen between the two mediators tested (80). In the same study, LTB4 reduced the latency of development of yeast-induced hyperalgesia in the rat paw. LTD4 had no demonstrable role in these events (80). Levine et al. have reported that the intradermal injection of LTB4 in the rat paw produced hyperalgesia similar, in magnitude, to that of bradykinin injection (81). The failure or indomethacin (a cyclooxygenase inhibitor) to antagonize LTB4 action suggests that the pathway by which LTB4 induces pain is independent of that for PGs. The depletion of circulating PMNLs altered the hyperalgesic function of
236

LTB4 without affecting PG-induced pain. The authors suggested a possible relationship between hyperalgesia and presence of LTB4 and its release from PMNLs (81). The vascular response to cysteinyl-LTs (LTC4 LTD4 LTE4) is more complex and varies in different species. Although the administration of synthetic cysteinyl-LTs causes vasoconstriction in guinea pig skin, (82) in human skin they cause vasodilation. Intradermal injections of LTG4, LTD4, and LTE4 in man produce increased blood fiow as well as erythema and wheal formation (83-85). Ghan and associates have shown vasodilatory effects in human and pig skins following the application of these LTs (79). The vasodilatory response in the pig occurred at considerably lower doses than that seen in humans. When even higher doses of LTG4 and LTD4, similar to those used in humans, were tested in the pigs, vasoconstrictor responses were observed. Based on the results of these studies, it appears that at low concentrations cysteinyl-LTs are vasodilators in pigs and humans; while they cause vasoconstriction at very high doses (79). The dilatory action of LTs in skin is in marked contrast to the contraction seen in the coronary artery (86). The cysteinyl-LTs increase microvascular permeability in airways and other tissues. They are thought to act specifically on the endothelial lining of the postcapillary venule. The leakage of plasma induced by LTG4 and its metabolites is generally preceded by an arteriolar constriction (30). The cysteinyl-LTs are potent bronchoconstrictors, they increase vascular permeability in postcapillary venules and stimulate mucus secretion (19). Inbibitors and antagonists of leukotrienes A great deal of interest has been focused on agents which can inhibit or antagonize the action of the LTs. There are two reasons for this interest; first, LTs appear to be involved in lymphocytic chemotaxis as well as vasodilation and second, the specific role of them in many infiammatory events is unknown. Three general classes of substances have been utilized to explore LT metabolism and action (30, 87). The first group consists of agents which block arachidonic acid availability. The second group are the inhibitors of 5-lipoxygenase, the enzyme responsible for the synthesis of 5-HPETE and thus all LTs (87). (Figure 1) The third group are substances which antagonize LT receptor binding. A. Inhibitors of arachidonic acid release Availability of arachidonic acid, released from components of membrane-associated phospholipids can

Possible roie in pulpal and periapicai diseases be inhibited by the action of glucocorticoids (Fig.). A reduction in the availability of arachidonic acid reduces the substrate for both the cyclooxygenase and lipoxygenases. Glucocorticoids appear to function by synthesizing proteins, the so-called lipomodulins or lipocortins. These molecules act to stimulate synthesis of cytoskeletal calcium-binding proteins called calpactins which in turn control the hydrolytic stability of the membrane-associated phosphoHpids (88). An interesting corollary of this mechanism is the difficulty of isolating corticoid effects because of competition for arachidonic acid. This competition has been shown to cause problems when a combination of glucocorticoids and nonsteroid anti-infiammatory drugs have been used concurrently. than systematic names. However, they appear to fall in the limited number of categories. Gertain agents have been shown to affect both the cyclooxygenase and lipoxygenases, for this reason they are classified as dual inhibitors. In vitro dual inhibitors include: timegadine and GBS-1108 (94). One of the few compounds which has been shown to be an in-vivo inhibitor of eicosanoid synthesis is the phenylpyrazoline, BW755G. This compound is known to be an inhibitor of both the cyclooxygenase and the 5-, 12- and 15-lipoxygenases. Because BW755G is a multiple inhibitor, it has similar antiedema, analgesic and antipyrietic activity to indomethacin, but is more effective in reducing leukocyte accumulation when used in therapeutic doses. The difficulty with such a multiple inhibitor is that it is difficult to isolate the role of the inhibitor on the 5-lipoxygenase. The fact that the 5-lipoxygenase is an iron-containing enzyme led to the development of a series of acetohydroxamic acid derivatives. Phenoxycinnamyl-, benzyoxybenzyl-, and tetrahydronapthylpropenyl acetohydroxamic acids (BWA4G, A137G and BWA797G) inhibit calcium-mediated LTB4 synthesis both in vitro and in vivo.

B. Inhibition of 5-iipoxygenase in leukotriene synthesis The action of the 5-lipoxygenase and thus LTA4 synthesis is specifically stimulated by calcium in a concentration-dependent manner similar to that caused by a number of cytokines and other mediators of infiammation (89). The synthesized LTA4 is converted by one route through LTA4 hydrolase to LTB4 (5,12-di-HETE). This has provided a means by which the activity of the lipoxygenase may be directly assayed. Addition of the calcium ionophore, A23187 selectively stimulates the production of LTB4 in PMNLs (41, 90). Thus inhibition of A23187-induced LTB4 synthesis has been considered characteristic of 5-lipoxygenase inhibition. A number of pharmacological agents have been developed which either specifically or non-specifically inhibit the 5-lipoxygenase. Early non-specific inhibitors included the antioxidants; baicalein, esculetin, caffeic acid, and nordihydroguaiaretic acid (NDGA), and the competitively binding inhibitor, ETYA (eicosotetraynoic acid). The benzoquinone, AA-861 is a specific antioxidant inhibitor of the 5lipoxygenase. In most cases these inhibitors have been either ineffective or non-specific in vivo. Other inhibitors thought to be specific include: Diethylcarbamazine, (91) Lonapalene (RS-43179), TZ141127 and REV-5901 and the phenothiazenone (L-651, 392) (92). These have been shown in various systems to have therapeutic effects. Benoxaprofen, although shown to be effective blocking A23187-induced LTB4 synthesis in control of psoriasis, has been shown to be non-specific and ineffective in certain in vivo applications (93). These findings emphasize the need fbr correlation between in vitro and in vivo results prior to identifying an agent as a specific inhibitor. A majority of the specific and non-specific inhibitors of 5-lipoxygenase are proprietary compounds and are thus identified by number rather

Piriprost (U60,275), a PGI,, derivative is an inhibitor of sulfoleukotriene or cysteinyl-LT synthesis but has also been reported to be an inhibitor of the 5-lipoxygenase (95). Another class of inhibitors are the Azodisalicylates such as Sulfasalazine and L651392. Both of these drugs act via their common metabolite, 5-aminosalicylate to inhibit the production of LTB4 and 5-HETE, both chemotactic infiammatory mediators (96, 97). MK886, a translocational inhibitor of the 5-lipoxygenase has been found to be effective in the inhibition of LTB4, synthesis (97). Receptor antagonists in inflammation Gellular receptors for leukotrienes have been characterized in a number of cell types. Receptors for LTB4 have been detected in human PMNLs (42, 90). The time course of uptake of LTB4 into PMNLs parallels the time frame for physiologic effects, suggesting the involvement of an LTB4 receptor. Only one study on LTB4 antagonists has been reported. Fretland utilized an antagonist, SG41930 to antagonize LTB4 uptake (98). Most studies on LT receptors have focused on the cysteinyl-LT receptors. Antagonists of LTD4 binding alter cellular and tissue contraction without affecting the LTB4-induced PMNL-chemotactic responses. Early studies with the receptor antagonist FPL55712 indicated antagonism to LTD4, and possibly LTG4 (92). Rokach & Young synthesized specific LTD4 antagonists which have an increased inhibitory effect on smooth 237

Torabinejad et al. muscle contraction as compared to other 4-alkylphenylketobutenoic acid antagonists (92). Role of leukotrienes in puipai and periapicai diseases Bacterial, mechanical, and chemical irritation of the pulp or periapicai tissues causes infiammation. Apart from their anatomic location, infiammatory responses in these tissues are probably very similar to other connective tissues in the body. Elevated levels of various prostanoids metabolites of arachidonic acid have been reported in infiamed pulps and periapicai tissues of human and experimental animals (99-104). The 5-lipoxygenase metabolites of arachidonic acid are proinfiammatory. LTB4 causes chemotaxis of PMNLs both in vitro (59, 67) and in vivo (69). It also promotes adhesion of PMNLs to endothelium, (106, 107) stimulates aggregation, degranulation and generation of superoxide in PMNL (67, 108, 109). In addition, LTB4 has been implicated for infiltration of PMNLs in psoriasis (63) and rheumatoid arthritis (110) and immune complex induced pleurisies (111). The cysteinyl-leukotrienes are also pro-infiammatory (19). Despite the presence of numerous reports on the role of lipoxygenase metabolites of arachidonic acid in inflammation there are few reports in the dental literature focusing on their role in oral pathosis. A number of investigators by using various techniques have found high concentrations of lipoxygenase products of arachidonic acid in infiamed human gingival tissues. Most of these studies report a greater elevation of lipoxygenase products than cyclooxygenase metabolites in the same specimens (112-118). Lessard et al. (100) examined the metabolism of arachidonic acid in normal and infiamed canine tooth pulps and demonstrated that in the infiamed pulp there are elevated levels of LTC4 as well as PGEr, and 6 keto PGFj^ as compared to levels found in the normal pulp. In 1984 Matejka et al. reported presence of PGE2 and PGI2 in the dental squamous cell cysts (119). They later investigated the possible role of LTG4 and LTD4 in human dental cysts. They found that incremental additions of LTG4 and LTB4 to the samples caused a definite increase in the rates of synthesis of PGI2. They concluded that LTB4 and LTD4 may elicit osteolysis in chronic periapicai infiammatory processes by stimulating the synthesis of PGI2 (120). Okiji et al. induced infiammation by application of lipopolysaccharide (LPS) to rat pulps and found elevated levels of PGEg and PGI2 (101). In a recent study using the same model they reported release of LTB4 into the medium and accumulation of PMNL
238

in infiamed pulps. When a dual inhibitor of cyclooxygenase and lipoxygenase was administered prior to the application of LPS, the production of LTB4 and the number of PMNLs were significantly decreased. Administration of indomethacin had no effect on the production of this metabolite (121). In a recent study, we determined the concentrations of LTB4 and LTG4 in human periradicular lesions (122). Periapicai specimens from asymptomatic and symptomatic patients and pulpal tissues from unerupted third molars as well as chronically infiamed gingival tissues were obtained and frozen. The concentrations of LTB4 and LTG4 were determined by reverse phase high pressure liquid chromatography. Representative samples from each group were also examined microscopically. Low levels of LTB4 and LTG4 were detected in the uninfiamed pulpal samples in contrast to the elevated levels found in chronically infiamed gingival tissues as well as asymptomatic and symptomatic periradicular lesions. A significant statistical difference was noted between the concentration of LTB4 in symptomatic lesions compared to those found in asymptomatic lesions and chronically infiamed gingival tissues (P<0.05). In addition, a positive correlation was found between the presence of symptoms, the concentrations o^ LTB4 and presence of infiltrating PMNLs in symptomatic patients. No significant statistical difference was found between the levels of LTG4 in periradicular lesions of symptomatic and asymptomatic patients and those detected in chronically infiamed gingival tissues. These results suggest that LTB4 may play a significant role in pathogenesis of symptomatic periradicular lesions. Since high levels of LTB4 have been found in symptomatic periapicai lesions and are capable of attracting PMN leukocytes to the infiammatory site, leukotriene antagonists and inhibitors alone or in conjunction with other inhibitors or antagonists of arachidonic acid metabolites could be considered as antiinfiammatory substances in symptomatic patients. References
1. Prostaglandins: their possible role in the pathogenesis of pulpal and periapicai diseases, Part 1. J Endod 1980; 6: 733-39. TORABINEJAD M , BAKLAND LK. Prostaglandins: their possible role in the pathogenesis of pulpal and periapicai diseases, Part. 2. J Endod 1980; 6: 769-76. BURR GO, BURR MM. On the nature and role of the fatty acids essential in nutrition. J Biol Chem 1930; 83: 587-621. VAN DEN BOSCH H . Intracellular phospholipases A. Biochem Biophjs Acta 1980; 604: 191-246. IRVINE R F . HOW is the level of free arachidonic acid controlled in mammalian cells? Biochem J 1982; 204: 3-16. BLACKWELL G J , FLOWER R J . Inhibition of phospholipase. Br Med Bull 1983; 39: 260-64.
TORABINEJAD M , BAKLAND L K .

2.

3. 4. 5. 6.

Possible rele in puipai and periapicai diseases

7. Spasm-producing substance in the sputum of patients with bronchial asthma. Arch Intern Med. 1930; 45: 641-46. FELDBERG W, KELLAWAY C H . Liberation of histamine and formation of lysocithin-like substances by cobra venom. J Physiol 1938; 94: 187-227. KELLAWAY C V H , TRETHEWIE E R . Liberation of a slow reacting smooth muscle stimulating substance in anaphylaxis. (IJ Fxp Physiol 1940; 30: 121-45. BROCKLEHURST W E . The release of histamine and formation of slow-reacting substance (SRS-A) during anaphylactic shock. J Physiol (London) 1960; 151: 416-35. ORANGE R P , AUSTEN K F . Slow reacting substance of anaphylaxis. Adv Immunol 1969; 10: 105-144. ORANGE RP, VALENTINE M D , AUSTEN KF. Release of slow reacting substance of anaphylaxis in the rat: Polymorphonuclear leukocyte. Science 1967; 157: 318-319. ORANGE RP, MURPHY R C , AUSTEN KF. Inactivation of slow reacting substance of anaphylaxis (SRS-A) by arylsulfatases. J Immunol 1974; 113: 316-22. ORANGE RP, CHANG PL. The effect of thiols on immunologic release of slow reacting substance of anaphylaxis. I. Human lung. J Immunol 1975; 115: 1072-77. ORANGE RP, AUSTEN KF. Pharmacologic dissociation of immunologic release of histamine and slow reacting substance of anaphylaxis in rats. Proc Soc Exp Biol Med 1968; 129: 836-41. JAKSCHIK BA, FALKENHEIN S, PARKER C W . Precursor role of arachidonic acid in release of slow reacting substance from rat basophilic leukemia cells. Proc Natl Acad Sci 1977; 74: 4577-81. BACH M K , BRASHLER J R , GORMAN R R . On the structure of slow-reacting substance of anaphylaxis: evidence of biosynthesis from arachidonic acid. Prostaglandin 1977; 14: 2'l-28. BoRGEAT P, SAMUELSSON B. Transformation of arachidonic acid by rabbit polymorphonuclear leukocytes: formation of a novel dihydroxyeicosatetraenoic acid. J Biol Chem 1979; 254: 2643-2646. SAMUELSSON B. Leukotrienes: Mediators of immediate hypersensitivity reactions and inflammation. Science 1983; 220: 568-75. SALMON JA, HIGGS G A . Prostaglandins and leukotrienes as inflammatory mediators. Br Med Bull 1987; 43: 285-96. LEWIS R A , AUSTEN KF. The biologically active leukotrienes: biosynthesis, metabolism, receptors, functions, and pharmacology. J Clin Invest 1984; 73: 889-97. MACGLASHAN D W , SCHLEIMER RP, PETERS SP, et al. Generation of leukotrienes by purified human lung mast cells. J Clin Invest 1982; 70: 747-51.
HARKAVY J . DW JR., PETERS SP, WARNER J, et al.

29.

8.

30.

9.

31.

10.

32.

11. 12.

33.

13.

34.

14.

35.

15.

36. 37. 38.

16.

17.

39.

18.

40. 41. 42.

19.

20. 21.

43.

22.

44.

23. MACGLASHAN

24.

25.

26. 27.

Characteristics of human basophil sulfidopeptide leukotriene release: releasability defined as the ability of the basophil to respond to dimeric cross-links. J Immunol 1986; 136: 2231-39. MARTIN T R , ALTMAN L C , ALBERT R K , et al. Leukotriene B4 production by the human alveolar macrophage: a potential mechanism for amplifying inflammation in the lung. Am Rev Respir Dis 1984; 129: 106-11. GOETZL E J . Selective feedback inhibition of the 5-lipoxygenation of arachidonic acid in human T-lymphocytes. Biochem Biophys Res Commun 1981; 101: 344-50. SAMUELSSON B, FUNK CD. Enzymes involved in the biosynthesis of leukotriene B^. J Biol Chem 1989; 264: 19469-19472. SHIMIZU T. Enzymes functional in the synthesis of leukotrienes and related compounds. Int J Biochem 1988; 20: 66166.
al.

45.

46.

47.

48.

28. PETERS SP, MACGLASHAN DW J R . . SCHULMAN E S , et

49.

Arachidonic acid metabolism in purified human lung mast cells. J Immunol 1984; 132: 1972-1979.

OWEN W F , SOBERMAN RJ, YOSHIMOTO T, et al. Synthesis and release of leukotriene C4 by human eosinophils. J Immunol 1987; 138: 532-38. SAMUELSSON B, DAHLEN S E , LINDGREN J, et al. Leukotrienes and lipoxins: structures, biosynthesis and biological effects. Science 1987; 237: 1171-75. WILLIAMS JD, ROBIN JL, LEWIS RA, et al. Generation of leukotrienes by human monocytes pretreated with cythochalasin B and stimulated with formyl-methiomyl-leucyl-phenylalanine. J Immunol 1986; 136: 642-48. HAMMERSTROM S, ORNING L, BERNSTROM M , et al. Metabolism of leukotriene C4 in rats and humans. Adv Prostaglandin Thromboxane Leukotriene Res 1985; 15: 185-88. LEE CW, LEWIS RA, COREY EJ, et al. Conversion of leukotriene D4 to leukotriene E4 by a dipeptidase released from the specific granule of human polymorphonuclear leukocytes. Immunology 1983; 48: 27-35. LEE CW, LEWIS RA, COREY EJ, et al. Oxidative inactivation of leukotriene C4 by stimulated human polymorphonuclear leukocytes. Proc Natl Acad Sci 1982; 79: 4166-70. LEE CW, LEWIS RA, TAUBER AI, et al. The myeloperoxidase-dependent metabolism of leukotrienes C4, D4, and E4, to 6-trans-leukotriene B4 diastereoisomers and the subclassspecific S-Diastereoisomeric sulfoxides. J Biol Chem 1983; 268: 15004-10. HAMMERSTROM S. Leukotrienes. Ann Rev Biochem 1983; 52.355-77. HANSSON G , LINDGREN JA, DAHLEN SE, et al. FEBS Lett 1981; 130: 107-12. SHAK S, GOLDSTEIN I M . Carbon monoxide inhibits w-oxidation of leukotriene B4 by human polymorphonuclear leukocytes: Evidence that catabolism of leukotriene B4 is mediated by a cytochrome p-450 enzyme. Biochem Biophys Res Commun 1984; 123: 475-81. SuMiMOTO H, TAKESHIGE K , SAKAI H , et al. A cell-free preparation of human neutrophils catalyzing NADPH-dependent conversion of leukotriene B4. Biochem Biophys Res Commun 1984; 125: 615-21. SERAFIN W E , OATES JA, HUBBARD W C . Prostaglandins 1984; 27: 899-911. NEEDLEMAN P, TURK J , JAKSCHIK BA, et al. Arachidonic acid metabolism. Ann Rev Biochem 1986; 55: 69-102. GOLDMAN DW, GOETZL EJ. Specific binding of leukotriene B4 to receptors on human polymorphonuclear leukocytes. J Immunol 1982; 129: 1600-4. KREISLE RA, PARKER CW. Specific binding of leukotriene B4 to a receptor on human polymorphonuclear leukocytes. J Exp Med 1983; 157: 628-41. GOLDMAN DW, GOETZL EJ. Heterogeneity of human polymorphonuclear leukocytes receptors for leukotriene B4: identification of a subset of high affinity receptors that transduce the chemotactic response. J Exp Med 1984; 159: 1027-1041. PAYAN D G , MISSIRIAN-BASTIAN A, GOETZL EJ. Human Tlymphocyte subset specificity of the regulatory effects of leukotriene B4. Proc Natl Acad Sci 1984; 81: 3501-3505. PONG S S , DEHAVEN R N , KUEHL FA JR., et al. Leukotriene C4 binding to rat lung membranes. J Biol Chem 1983; 258: 9616-19. MoNG S, Wu HL, HOGABOOM G K , et al. Characterization of the leukotriene D4 receptor in guinea-pig lung. Eur J Pharmacol 1984; 102: 1-11. CHENG J B , TOWNLEY R G . Evidence for a similar receptor site binding (^H)-leukotriene E4 and (^H)-leukotriene D4 to the guinea pig crude lunge membrane. Biochem Biophys Res Commun 1984; 122: 949-54. SAMUELSSON B, BORGEAT P, HAMMARSTROM S, et al. Introduction of a nomenclature: leukotrienes. Prostaglandins 1979; 17: 785-87.

239

Toraiiinejad et ai.
50.
DAHLEN SE, HEDQUIST P, HAMMERSTROM S, et al. Leukotrienes are potent constrictors of human bronchi. Nature 1980; 288: 484-86. HEDqvisT P, DAHLEN SE, GUSTAFSSON S, et al. Biological profile of leukotrienes C4 and D4. Acta Physiol Scand 1980; 110: 331-33. SAMHOUN M N , PIPER P J . Leukotriene F4: comparison of its pharmacological profile with that of the other cysteinylcontaining leukotrienes in guinea pig ileum and lung parenchyma in vitro. Prostaglandins 1984; 28: 623-38. SMEDEGAARD G , HEDQVIST P, DAHLEN SE, et al. Leukotriene C4 affects pulmonary and cardiovascular dynamics in monkeys. Nature 1982; 295: 327-29. WEISS J W , DRAZEN J, COLES N . Bronchoconstrictor effects of leukotriene C in humans. Science 1982; 216: 196-98. ROLA-PLESZCZYNSKI M , BORGEAT P, SIROIS P. Leukotriene B4 induces human suppressor lymphocytes. Biochem Biophys Res Commun 1982; 108: 1531-37. ROLA-PLESZCZYNSKY M , GAGNON L , SIROIS P. Leukotriene B4 augments human natural cytotoxic cell activity. Biochem Biophys Res Commun 1983; 113: 531-37. PIPER PJ. Formation and actions of leukotrienes. Physiol Rev 1984; 64: 744-61. In-

51. 52.

73. 74. 75. 76.

53.

54. 55.

77.

56. 57.

leukotriene B4 and platelet activating factor in patients with inflammatory dermatoses. Clin Exp Immunol 1983; 54486-88. ZiCARi A, LiPARi M, LENTI L, et al. Chemotactic response of rat macrophages is enhanced by diastereoisomers of LTB4. Int J Immunopharmacol 1989; 11: 589-96. MENSING H , CZARNETZKI B M . Leukotriene B4 induces in vitro fibroblast chemotaxis. J Invest Dermatol 1984; 82: 9-13. GOETZL EJ. Mediators of immediate hypersensitivity derived from arachidonic acid. New Engl J Med 1980; 303822-26. UDEN AM, PALMBLAD J, LINDGREN JA, et al. Effects of novel lipoxygenase products on migration of eosinophils and neutrophils, in vitro. Int Arch Allergy Appl Immunol 1983; 72: 91-93. CZARNETZKI B, ROSENBACH T Chemotaxis of human neutrophils and eosinophils towards leukotriene B4 and its 20w-oxidation products in vitro. Prostaglandins 1986; 31851-58. Leukotriene B4: a mediator of vascular permeability. Br J Pharmacol 1981; 72: 483-86. CHAN C C , FORD-HUTCHINSON AW. Effects of synthetic leukotrienes on local blood flow and vascular permeability in porcine skin. J Invest Dermatol 1985; 84: 154-57. RACKHAM A, FORD-HUTCHINSON AW. Inflammation and pain sensitivity: effects of leukotrienes D4, B4 and prostaglandin E, in the rat paw. Prostaglandins 1983; 25: 193-203. LEVINE JD, LAU W, KWIAT G. Leukotriene B4 produces hyperalgesia that is dependent on polymorphonuclear leukocytes. Science 1984; 225: 743-45. PECK M J , PIPER PJ, WILLIAMS T J . The effect of leukotrienes C4 and D4 on the microvasculature of the guinea-pig skin. Prostaglandins 1981; 2/.- 315-21. DRAZEN J M , AUSTEN KF, LEWIS RA, et al. Comparative airway and vascular activities of leukotrienes C, and D in vivo and in vitro. Proc Natl Acad Sci USA 1980; 77: 4354-58. BisGAARD H, KRISTENSEN J, SoNDERGAARD J. The effects of leukotriene C4 and D4 on cutaneous blood flow in humans. Prostaglandins 1982; 23: 797-801. SoTER NA, LEWIS RA, COREY EJ, et al. Local effects of synthetic leukotrienes (LTC4, LTD4, LTE4, and LTB4) in human skin. J Invest Dermatol 1983; 80: 115-19. LETTS L G , PIPER PJ, NEWMAN D L . Leukotrienes and their action in the coronary circulation, leukotrienes and other lipoxygenase products. New York: Wiley, 1983: 94-107. SMITH L . The eicosanoids and their biochemical mechanisms of action. Biochem J 1989; 259: 315-324. ROBINSON DR. Eicosanoids, inflammation, and anti-inflammatory drugs. Clin Exp Rheumatol 1989; 7/S-3: 155-161. LEWIS RA, AUSTEN KF, SOBERMAN RJ. Leukotrienes and other products of the 5-hpoxygenase pathway. New Engl J Med 1990; 323: 645-655. LEWIS RA, AUSTEN KF. Leukotrienes. In: Gallinet et al., eds. Inflammation: basic principles and clinical correlates. New York: Raven, 1988. MATTHEWS W R , MURPHY RC. Inhibition of leukotriene biosynthesis in mastocytoma cells by diethycarbamazine. Biochem Pharmacol 1982; 31: 2129-32. ROKACH J, YOUNG RN. The^ development of new antileukotriene drugs: specific leukotriene D4 antagonists and 5hpoxygenase inhibitors. Adv Ex Med Biol 1989; 259: 75-108.

78. BRAY MA, CUNNINGHAM F M , FORD-HUTCHINSON AW, et al.

79.

58. HAMMARSTROM S, HAMBERG M , SAMUELSSON B, et al.

59.

60. 61.

62. 63. 64.

65.

66.

67.

68.

69.

70.

71.

72.

creased concentrations of non-esterified arachidonic acid, 12L-hydroxy-5,8,10,14-eicosatetraenoic acid, prostaglandin E2 and prostaglandin Y.,-^ in epidermis of psoriasis. Proc Natl Acad Sci 1975; 72: 5130-34. PALMER R M J , STEPNEY RJ, HIGGS GA, et al. Chemokinetic activity of arachidonic acid lipoxygenase products on leukocytes from different species. Prostaglandins 1980; 20: 411-418. BRAY MA. The pharmacology and pathophysiology of leukotriene B4. Br Med Bull 1983; 39: 249-54. HIGGS GA, SALMON JA, SPAYNE JA. The inflammatory effects of hydroperoxy and hydroxy acid products of arachidonate hpoxygenase in rabbit skin. Br J Pharmacol 1981; 74: 429-33. RAE S A , DAVIDSON EM, SMITH M J H . Leukotriene B4, an inflammatory mediator in gout. Lancet 1982; ii: 1122-24. BRAIN SD, CAMP R D R , DOWD P M , et al. Psoriasis and leukotriene B4. Lancet 1982; 11: 762-63. FoRD-HuTCHiNSON AW, BRUNET G, SAVARD P, et al. Leukotriene B4, polymorphonuclear leukocytes and inflammatory exudates in the rat. Prostaglandins 1984; 28: 13-27. TURNER S R , TAINER JA, LYNN W S . Biogenesis of chemotactic molecules by the arachidonate lipoxygenase system of platelets. Nature 1975; 257: 680-1. GOETZL EJ, PICKETT WC. The human PMN chemotactic activity of complex hydroxy-eicosatetraenoic acid (HETEs). J Immunol 1980; 125: 1789-91. FoRD-HuTCHiNsoN AW, BRAY MA, DOIG M V , et al. Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 1980; 286: 264-65. SMITH MJH, FORD-HUTCHINSON AW; BRAY MA. Leukotriene B: a potential mediator of inflammation. J Pharm Pharmacol 1980; 32: 517-18. BRAY MA, FORD-HUTCHINSON AW, SMITH MJH. Leukotriene B4: an inflammatory mediator in vivo. Prostaglandins 1981; 22: 213-22. BHATTACHERJEE P, EAKINS K E , HAMMOND B. Chemotactic activity of arachodonic acid lipoxygenase products in the rabbit eye. Br J Pharma 1981; 73: 254-55. PsYCHOYOS S, UziEL-Fusi S. Comparison of LTB4- and C5a-stimulated chemotaxis of isolated human neutrophils: difference revealed by cell migration in thick filters using the multi-well cap procedure. Agents Actions 1989; 27: 380-84. CzARNETZKi B. Increased monocyte chemotaxis towards

80.

81.

82.

83.

84.

85.

86.

87. 88. 89.

90.

91.

92.

93. BHATTACHERJEE P, BOUGHTON-SMITH N K , FOLLENFANT R L ,

et al. The effects of a novel series of selective inhibitors of arachidonate 5-lipoxygenase on anaphylactic and inflammatory responses. Ann Ny Acad Sci 1988; 524: 307-320. 94. KANTOR T G , KETOPROFEN: a review of its pharmacologic and cHnical properties. Pharmacotherapy 1986; 6".- 93-103. 95. BACH MK. Inhibitor of leukotriene synthesis and action.

240

Possibie roie in puipai and periapicai diseases

In: Chakrin LW, Bailey DM, eds. The leukotrienes. New York: Academic Press, 1984. STENSON WF, LOBOS E . Sulfasaiazine inhibits the synthesis of chemotactic lipids by neutrophils. J Clin Invest 1982; 69: 494-97. WALLACE JL. 5-hpoxygenase: a rational target for therapy of inflammatory bowel disease. Trends Pharmacol Sci 1990; ;;.- 51-53. FRETLAND D J , LEVIN S, TSAI BS. The effect of leukotriene B4 receptor antagonist SC-41930 on acetic acid induced colonic inflammation. Agent Actions 1989; 27: 395-97. COHEN J S , READER A, FERTEL R , et al. A radioimmunoassay determination of the concentrations of prostaglandins E2 and F^a in painful and asymptomatic human dental pulps. J Endod 1985; 11: 330-35. LESSARD G M , TORABINEJAD M , SWOPE D . Arachidonic acid metabolism in canine tooth pulps and the effects of nonsteroidal anti-inflammatory drugs. J Endod 1986; 12: 146-149. OKIJI T, MORITA I, KOBAYASHI C , et al. Arachidonicacid metabolism in normal and experimentally-inflamed rat dental pulp. Arch Oral Biol 1987; 32: 723-27. HASHIMOTO S, UCHIYAMA K , A4AEDA M , et al. In vivo and in vitro effects of zinc oxide-eugenol (ZOE) on biosynthesis of cyclo-oxygenase products in rat dental pulp. J Dent Res 1988; 67: 1092-96. OKIJI T, MORITA I, SUNADA I, et al. Involvement of arachidonic acid metabolites in increases in vascular permeability in experimental dental pulpal inflammation in the rat. Arch Oral Biol 1989; 34: 523-28. McNiCHOLAS S, TORABINEJAD M , BLANKENSHIP J, et al. The concentration of prostaglandin E, in human periradicular lesions. J E?idod 1991; 17: 97-100. CAMP RDR, COUTTS A A , GREAVES M W ; et al. Responses of human skin to intradermal injection of leukotriene C4, D4, and B4. Br J Pharmacol 1983; 80: 497-502. DAHLEN SE, BJORK J, HED^VIST P, et al. Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules: in vivo effects with relevance to the acute inflammatory response. Proc Natl Acad Sci 1981; 75.' 3887-91. BJORK J, DAHLEN SE, HED^VIST P, et al. Leukotrienes B4 and C4 have distinct microcirculatory actions in vivo. Adv Pg Tx Lt Res 1983; 12: 1-6. BoKOCH GM, REED P W Effect of various lipoxygenase metabolites of arachidonic acid on degranulation of polymorphonuclear leukocytes. J Biol Chem 1981; 256: 5317-20. 109. SuMiMOTO H, TAKESHIGE K , MINAKAMI S. Superoxide production of human polymorphonuclear leukocytes stimulated by leukotriene B4. Biochem Biophys Acta 1984; 803: 271-77. 110. KLICKSTEIN LB, SHAPLEIGH C , GOETZL EJ. Lipoxygenation of arachidonic acid as a source of polymorphonuclear leukocyte chemotactic factors in synovial fluid and tissue in rheumatoid arthritis and spondyarthritis. J Clin Invest 1980; 66: 1166-70. 111. TANAKA K , UENO A, KATORI M . Roles of leukotrienes in two rat allergic inflammatory models; IgE-mediated and IgG-antigen complex-induced pleurisies. Prostaglandins 1986; 31: 1099-1116. 112. SiDHAGEN B, HAMBER M , FREDHOLM BB. Formation of 12hydroxyeicosatetraenoic acid (12-HETE) by gingival tissue. J Dent Res 1982; 61: 761-63. 113. EL ATTAR TM, LIN HS. Relative conversion of arachidonic acid through lipoxygenase and cycloxygenase pathways by homogenates of diseased periodontal tissues. J Oral Pathol 1983; 12: 7-10. 114. EL ATTAR TM, LIN HS, TIRA DE. The effect of nonsteroidal anti-inflammatory drugs on the metabolism of'*Carachidonic acid by human gingival tissue. J Dent Res 1983; 62: 975-79. 115. EL ATTAR TM, LIN HS, TIRA DE. Arachidonic acid metabolism in inflamed gingiva and its inhibition by anti-inflammatory drugs. J Period 1984; 55: 536-39. 116. EL ATTAR TM, LIN HS, KILLOY V \ J , et al. Hydroxy fatty acids and prostaglandin formation in diseased human periodontal pocket tissue. J Period Res 1986; 21: 169-76. 117. OFFENBAKER S, VANDYKE TE, OLDE BM, et al. Role of leukotriene B4 in localized juvenile periodontitis. J Dent Res 1984; 63 (special issue) #313. 118. SCOTT S, ODLE B, OFFENBACKER S. Inflamed periodontal tissues containing high levels of leukotriene B4. J Dent Res 1985; 64: (special issue) #1812. 119. MATEJKA M , PORTEDER H , ULRICH W, et al. Prostaglandin synthesis in dental cysts. Br J Oral Surg 1985; 23: 190-94. 120. MATEJKA M , PORTEDER H , KLEINERT, et al. Evidence that PGL-generation in human dental cysts is stimulated by leukotrienes C4 and D4. J Max-Fac Surg 1985; 13: 93-96. 121. OKIJI T, MORITA I, SUNADA I, et al. The role of leukotriene B4 in neutrophil infiltration in experimentally-induced inflammation of rat tooth pulp. J Dent Res 1991; 70: 34-37. 122. TORABINEJAD M , COTTI E, JUNG TTK. Concentrations of leukotriene B4 in symptomatic and asymptomatic periapicai lesions. J Endodon (submitted).

96. 97.

98. 99.

100.

101.

102.

103.

104.

105. 106.

107.

108.

241