Volume 76 • Number 6

956

* Department of Physiology and Pharmacology, Faculty of Medicine, Federal University ofCeará, Ceará, Brazil.

† Department of Clinical Medicine, Faculty of Medicine, Federal University of Ceará.‡ Department of Morphology, Faculty of Medicine, Federal University of Ceará.

Nitric oxide (NO) is a free radicalassociated with a multitude ofphysiological functions. These

functions include modulation of cardio-vascular tone and integrity,1 regulationof platelet aggregation,2 neurotransmis-sion,3 and strong oxidative activity thatcontributes to the killing of microorgan-isms.4 NO is synthesized from L-arginineby a group of isoenzymes collectivelytermed NO synthases (NOS). NOS existas three distinct isoforms, the constitu-tive endothelial NOS (eNOS) and neu-ronal NOS (nNOS) isoforms and theinducible NOS (iNOS). The eNOS andnNOS are constitutively expressed andrelease small amounts of NO for a shortperiod of time. In contrast, iNOS pathwayis principally regulated at the transcrip-tional level and its activation lead to theproduction of a large amount of NO forsustained periods of time.1 Proinflam-matory cytokines, such as tumor necro-sis factor alpha (TNF-α) and interferon(IFN-γ)5 as well as endotoxin are potentinducers of iNOS in a wide variety of celltypes, whereas glucocorticoids and theanti-inflammatory cytokines interleukin-4(IL-4), IL-10, and the transforming growthfactor-β (TGF-β) supress NO produc-tion.1,6 The production of large amountof NO by iNOS has been shown to playa major role in immune reactions andin many inflammatory processes, includ-ing joint diseases such as rheumatoidarthrits.7,8

The physiologic role of NO can beestablished by the inhibition of NOS bysome agents such as L-NAME and

Nitric Oxide Synthase Inhibition PreventsAlveolar Bone Resorption in ExperimentalPeriodontitis in RatsR.F.C. Leitão,* R.A. Ribeiro,* H.V. Chaves,* F.A.C. Rocha,† V. Lima,* and G.A.C. Brito‡

Background: Periodontitis is the most frequent cause of toothloss in adults. Nitric oxide (NO) has been linked to bone resorp-tion mechanisms during inflammation processes. The aim ofthis study was to investigate the effect of NOS (NO synthase)inhibitors in the alveolar bone loss in an experimental perio-dontitis disease (EPD) model.

Methods: Wistar rats were subjected to a ligature placementaround the second upper left molars and were sacrificed at 11days. Alveolar bone loss was evaluated by the sum of distancesbetween the cusp tips and the alveolar bone along the axis ofeach molar root, subtracting from the contralateral side. His-topathological analysis was based on cell influx, alveolar bone,and cementum integrity. Leukogram was performed at 6 hoursand 1, 7, and 11 days after the EPD induction. Groups weretreated with the NOS inhibitors, aminoguanidine (AG) (2.5 to10 mg/kg/d), or L-arginine methyl ester (L-NAME, 5 to 20 mg/kg/d) intraperitoneally (i.p.), 1 hour before the EPD inductionand daily for 11 days. Controls received only saline (EPD group).As controls for L-NAME specificity, groups were co-treated witheither L-arginine (150 to 600 mg/kg/d) or D-arginine (600 mg/kg/d) and L-NAME (20 mg/kg/d). Different groups were usedfor morphometric and histopathological analysis.

Results: Both L-NAME and AG significantly and dose-depen-dently inhibited the alveolar bone loss as compared to EPDgroup. L-NAME (20 mg/kg/d) reduced the alveolar bone lossby 50%, whereas AG (5 mg/kg/d) reduced it by 47% comparedto EPD. This result was coupled to a significant reduction of cellinflux to the periodontium, as well as to the preservation of alve-olar bone and cementum, seen at histopathology, for both com-pounds. The co-administration of L-arginine, but not of D-argininereversed L-NAME effects.

Conclusion: These data provide evidence that NOS inhibitorsprevent inflammatory bone resorption in experimental perio-dontitis. J Periodontol 2005;76:956-963.

KEY WORDSAlveolar bone loss/etiology; animal studies; dental models;models, animal; nitric oxide synthase inhibitors; periodontitis.

40171.qxd 6/7/05 12:46 PM Page 956

957

J Periodontol • June 2005 Leitão, Ribeiro, Chaves, Rocha, Lima, Brito

aminoguanidine. L-NAME is a competitive and non-seletive inhibitor of the NO synthase.1 Aminoguani-dine inhibits NOS, particularly the inducible NOSisoform.9 The inhibition of NOS by these agents canbe reversed by simultaneous application of L-arginine,but not by its enantiomer D-arginine, consistent withtheir competitive blockade of the enzymatic active site.10

Periodontitis is characterized by a local inflammatoryreaction, which results in the destruction of the dentalattachment apparatus thus provoking tooth loss. Therole of bacteria in periodontitis has been establishedand it has been also shown that microbial productsare potent inducers of NO synthesis.11,12 NO synthesisinhibition has been shown to reduce bone resorptionin animals.13 On the other hand, we have shown thatlow (basal) levels of NO appear to be crucial to humanosteoblasts metabolism in vitro.14 Although bacterialinduction of iNOS has been demonstrated in vitro,12

the role of NO in the pathogenesis of periodontitis hasnot been fully elucidated.

Whereas low NO levels may help homeostasis locally,increased NO levels during inflammatory reactions maycontribute to local tissue damage.15 Some reports havedemonstrated that ligation-induced periodontitis resultedin marked plasma extravasation in the gingivomucosaltissue that was associated with the activation of nuclearpoly (ADP-ribose) polymerase, a mediator of down-stream nitric oxide toxicity. The pharmacological inhi-bition of iNOS diminished plasma extravasation andbone destruction.16,17 Thus, the literature indicates thatin the process of periodontal destruction, NO playsmany important roles, but the mechanism throughwhich NO induced bone resorption has yet to be clar-ified. Based on these assumptions, we investigated theeffect of non-selective or selective NOS inhibitors in dif-ferent aspects of experimental periodontal disease inrats, including bone and cementum destruction, inflam-matory cell influx, and leukogram in order to examinewhich NOS isoform is involved in these parameters.

MATERIALS AND METHODSAnimalsOne hundred fifty-six Wistar rats weighing 160 to 200 gfrom the Federal University of Ceará, were housed intemperature-controlled rooms and received water andfood ad libitum. Surgical procedures and animal treat-ments were conducted in accordance with the guidelinesof Institutional Animal Care and Use of Ceara FederalUniversity.

Induction of Experimental Periodontal Disease (EPD)A sterilized nylon (000) thread ligature was placedaround the cervix of the second left upper molar of ratsanesthetized with 10% chloral hydrated (285 mg/kg,i.p.), as described elsewhere.18 The ligature was knottedon the buccal side of the tooth, resulting in subgingival

position palatinally and in supragingival position buc-cally. The contralateral right side was used as the unli-gated control. Animals were weighed daily. Twenty µlof blood was taken from the rat tails and added to380 µl Turk solution immediately before and 6 hoursand 1, 7, and 11 days after periodontitis induction.Total white blood cell counts were performed using aNeubauer chamber and differential counts were per-formed using smears stained with Wright giemsa.

Drug TreatmentsThere were six animals in each experimental group. Foreach treatment administered, there was one groupused for analysis of alveolar bone resorption andanother for histopathology. Animals received eitherL-NAME (5, 10, or 20 mg/kg) or aminoguanidine (2.5,5, or 10 mg/kg), (i.p.) 1 hour before the surgical pro-cedure and daily until the sacrifice on the eleventh day.In another set of experiments, L-arginine (150, 300, or600 mg/kg, i.p., a 45-fold molar excess of L-arginine)or D-arginine (600 mg/kg, i.p.) was co-administeredwith L-NAME (20 mg/kg) 1 hour before periodontitisinduction and daily until sacrifice, on the eleventh day.Control groups consisted of animals subjected to thesurgical procedure that received only saline solution(EPD), i.p., and a naive group that was not subjectedto manipulation (bilaterally unligated). Three EPDgroups were used for measurement of alveolar boneloss (one for each experiment). For histopathologicalanalysis, only one EPD group was used.

Measurements of Alveolar Bone LossThe animals were sacrificed after 11 days of perio-dontitis and maxillae were excised and fixed in 10%neutral formalin. Both maxillary halves were thendefleshed and stained with methylene blue (1%) in orderto differentiate bone from teeth. The bone loss wasanalyzed using a stereoscope loupe (4× magnification)to measure the distance between the cusp tip and thealveolar bone crest, based on the method described bySallay et al18 and Samejima et al.19 In the present study,measurements were made along the long axis of thevestibular root surfaces of all molar teeth. Three record-ings for the first molar (three roots) and two recordingsfor the second and third molar teeth (two roots each)were made. Alveolar bone loss was obtained by sub-tracting the values of the right maxilla (unligated con-trol) from the left one. The sum of these seven values(mm) was used as a measure of the total alveolar boneloss on the vestibular surface for each animal. Then, itwas made the mean of six animals per group.

Histopathological AnalysisAfter sacrifice, under anesthesia, animals had theirmaxillae excised. The specimens were fixed in 10%neutral buffered formalin and demineralized in 7% nitric

40171.qxd 6/7/05 12:46 PM Page 957

958

Nitric Oxide Synthase Prevents Alveolar Bone Resorption Volume 76 • Number 6

Figure 1.Effect of L-NAME and its reversion by L-arginine, but not by D-arginineon ABL on EPD. Data represent the mean ± SEM of six rats for eachgroup. *P <0.05 was considered significantly different compared tothe rats subjected to periodontitis which received only saline solution.†P <0.05 was considered significantly different compared to the ratssubjected to periodontitis treated with L-NAME. (ANOVA; Bonferroni’stest).

Figure 2.Effect of AG on ABL in EPD. Data represent the mean ± SEM of sixrats for each group. *P <0.05 was considered significantly differentcompared to the rats subjected to periodontitis which received onlysaline solution (EPD) (ANOVA; Bonferroni’s test).

acid. These specimens were then dehydrated, embed-ded in paraffin, and sectioned along the molars in amesio-distal plane, for hematoxylin and eosin staining.Three sections of 6 µm each, which included two rootsof the first and two roots of second molar were used.The areas between the first and second molars, wherethe ligature was placed, were analyzed under lightmicroscopy using on a 0 to 3 score grade, consideringthe inflammatory cell influx, and alveolar bone andcementum integrity, as described previously:20 Score 0:absence of or only discrete cellular infiltration (inflam-matory cell infiltration is sparse and restricted to theregion of the marginal gingival), preserved alveolarprocess and cementum. Score 1: moderate cellularinfiltration (inflammatory cellular infiltration present allover the insert gingival), some but minor alveolarprocess resorption and intact cementum. Score 2:accentuated cellular infiltration (inflammatory cellularinfiltration present in both gingival and periodontal lig-ament), accentuated degradation of the alveolar pro-cess, and partial destruction of cementum. Score 3:accentuated cellular infiltrate, complete resorption of thealveolar process and severe destruction of cementum.

Statistical AnalysisThe data are presented as the mean ± SEM or as medi-ans, where appropriate. Univariate analysis of variance(ANOVA) followed by Bonferroni’s test was used tocompare means and Kruskal Wallis and Mann-Whitneytests were used to compare medians. A probabilityvalue of P <0.05 was considered to indicate significantdifferences.

RESULTSEffect of L-NAME and Aminoguanidine in EPDThe treatment of animals subjected to 11 days of experi-mental periodontal disease with L-NAME (20 mg/kg) oraminoguanidine (5 and 10 mg/kg) significantly reducedthe alveolar bone loss. These changes reached statisti-cal significance (P <0.05), as compared to the untreatedanimals subjected to EPD (Figs. 1 and 2). The inhibitoryeffect of L-NAME on bone loss was reversed by the co-administration of L-arginine (600 mg/kg; a 45-fold molarexcess of L-arginine) but not of D-arginine (Fig. 1).These data can be clearly seen in Figure 3A that showsthe macroscopic aspects of the contralateral right side(unligated side) with no resorption of the alveolar bone,severe bone resorption with root exposure in theuntreated group (EPD) (Fig. 3B) and reduction in boneloss in animals subjected to experimental periodontitisand treated with aminoguanidine (5 mg/kg) (Fig. 3C) orL-NAME (20 mg/kg) (Fig. 3D). Figure 3E shows themacroscopic appearance of periodontium subjected toexperimental periodontitis and co-treated with L-NAMEand L-arginine, where severe bone loss with root expo-sure is observed. The histological analysis of the region

between the first and second molars of the control group,not subjected to experimental periodontitis, shows thestructure of the normal periodontium, where gingiva (G),

40171.qxd 6/7/05 12:46 PM Page 958

959

J Periodontol • June 2005 Leitão, Ribeiro, Chaves, Rocha, Lima, Brito

Figure 3.Macroscopic aspects from periodontium of rats subjected to EPD.A) macroscopic aspects of normal periodontium; B) periodontiumsubjected to EPD, showing severe bone resorption with root exposure orthose subjected to EPD and treated with AG (C) or L-NAME (D),showing reduction in bone loss.The inhibitory effect of l-NAME on boneloss was reversed by simultaneous administration of L-arginine (E).

Figure 4.Histopathology from periodontium of rats subjected to EPD. Photo-micrographs of region between the first and second molars. A) Normalmaxilla, showing cementum (C), alveolar bone (AB), gingiva (G),periodontal ligament (PDL), and dentin (D). B) Maxilla after 11 days ofEPD, showing severe inflammatory infiltrate, with extensive cementumdestruction and total resorption of the alveolar process. Periodontiumsubjected to EPD and treated with AG (C) or L-NAME (D), showingdiscrete cell influx and preservation of the alveolar process andcementum. E) Periodontium of rat subjected to experimental periodontitisand treated with L-NAME and simultaneous administration of L-arginine,showing inflammatory cell infiltration and destruction of alveolar bone lossand cementum (bars = 250 µm; H&E stain; original magnification ×40).

periodontal ligament (PDL), alveolar bone (AB), cemen-tum (C), and dentin (D) can be observed (Fig. 4A;Table 1). The histopathology of the periodontium of theanimals subjected to periodontitis that received no treat-ment (EPD) revealed inflammatory cell infiltration cou-pled with severe cementum destruction and completealveolar process resorption (Fig. 4B; Table 1), receivingmedian score 3 (range, 2 to 3), whereas a reduction ofinflammatory cell infiltration and a partial preservationof the cementum and of the alveolar process was foundin the periodontium of animals subjected to experimentalperiodontitis and treated with aminoguanidine (5 mg/kg)(Fig. 4C; Table 1) or L-NAME (20 mg/kg)(Fig. 4D;Table 1). Both groups received score 1 (range, 1 to 2)(Table 1). These values were statistically significant

40171.qxd 6/7/05 12:46 PM Page 959

960

Nitric Oxide Synthase Prevents Alveolar Bone Resorption Volume 76 • Number 6

Table 1.

Histological Analysis of Rat Maxillae WithExperimental Periodontitis

Experimental Group

Naive EPD L-NAME L-NAME + L-arg. AG

Scores 0(0-0) 3(2-3) 1(1-2)* 3(2-3) 1(1-2)*

Data are reported as medians with range in parentheses. *P <0.05 was considered significantly different compared to the EPD group.

Figure 5.Effect of L-NAME and L-NAME + L-arginine on leukocyte counts ofrats. Each point represents the mean ± SEM of total leukocytes (A),neutrophils (B), or mononuclear cells (C) ×106/ml of six animals.*P <0.05 indicates significant difference from the EPD group; †P <0.05indicates significant difference from the group treated with L-NAME(ANOVA; Bonferroni’s test).

(P <0.05), when compared to the EPD group. Co-admin-istration of L-arginine and L-NAME reversed L-NAMEeffects, scoring 3 (range, 2 to 3) (Figure 4E, Table 1).

The experimental periodontal disease caused a pro-nounced leukocytosis at 6 hours and 7 and 11 daysafter ligature placement when compared to the sameparameters measured at time zero, which was con-sidered as normal control (Figs. 5A and 6A). Theleukocytosis observed at 6 hours was due to a neu-trophilia (Figs. 5B and 6B), whereas lymphomono-cytosis predominated at day 11 (Figs. 5C and 6C).L-NAME (20 mg/kg) treatment inhibited (P <0.05) theleukocytosis observed on the seventh and eleventhdays (Fig. 5A), reducing the number of mononuclearcells, when compared to EPD group (Fig. 5C). Thislatter effect of L-NAME was also reversed by L-arginine (600 mg/kg) (Figs. 5A and 5C). AG (5 and10 mg/kg) treatment significantly reduced (P <0.05)the leukocytosis and the increase in mononuclear cellson the eleventh day (Fig. 6). Neither L-NAME noraminoguanidine reversed the weight loss, comparedto EPD group (data not shown).

DISCUSSIONIn the present study, we have shown that NO is involvedin the inflammatory events and bone loss associated toperiodontal disease. Either the non-selective NOS inhi-bitor L-NAME or the selective iNOS inhibitor amino-guanidine significantly and dose-dependently inhibitedthe alveolar bone loss. This effect was associated witha reduction in the local inflammatory cell influx. Theinhibitory effect of L-NAME was reversed by the co-administration of L-arginine, but not by D-arginine, theinactive enantiomer of L-arginine, suggesting that theeffect of L-NAME on alveolar bone loss was most likelydue to a specific inhibition of NO synthesis.

Previous data have shown that NO participates inthe inflammatory response in periodontal tissues.21,22

In agreement with these data, a study has detectedincreased L-arginine and L-citrulline levels in theinflamed gingiva of patients with periodontitis, whichare the precursor and byproduct of NO synthesis,respectively.23 Periodontopathogenic bacteria wereshown to upregulate NO synthesis in mouse macro-

phages.24-26 Furthermore, increased iNOS expressionwas demonstrated in periodontal disease tissue com-pared to healthy tissue.21

40171.qxd 6/7/05 12:46 PM Page 960

961

J Periodontol • June 2005 Leitão, Ribeiro, Chaves, Rocha, Lima, Brito

Figure 6.Effect of aminoguanidine on leukocyte counts of rats. Each pointrepresents the mean ± SEM of total leukocytes (A), neutrophils (B), ormononuclear cells (C) ×106/ml of six animals. *P <0.05 indicatessignificant difference from the EPD group (ANOVA; Bonferroni’s test).

The pathogenesis of periodontitis involves the releaseof inflammatory mediators leading ultimately to alveolarbone loss.27 Among these mediators, prostaglandins,

following cycloxygenase 2 activation, and cytokinessuch as IL-1 and TNF-α seem to be important media-tors in peridontitis.20,28,29 These proinflammatorycytokines induce the NO production,5,30-33 and thenNO may act as a stimulator of bone resorption.13,34

Thus, the inhibition of ABL and inflammatory param-eters in EPD after administration of NOS inhibitors addsupport for a role of NO in periodontitis.

It has been proposed that NO downregulates theexpression of adhesion molecules in the vascularendothelium, thereby decreasing neutrophil traffickinginto inflamed tissues.35,36 However, in the presentstudy, we clearly demonstrated that the NOS inhibitorsdecreased inflammatory cell infiltration in the perio-dontal tissue. Actually, there are data showing thatNOS inhibition may even promote increased neutrophilmigration in septic peritonitis.37 The mechanisms of thereduction of the inflammatory cell infiltration byL-NAME and AG in the present EPD model are notstraightforward. NO can exert its effects either directlyor via the formation of potent oxidants. For instance,during inflammatory reactions, where large amounts ofNO and superoxide are formed, the combination ofboth leads to the formation of reactive nitrogen species,such as the peroxynitrite anion, a toxic product of NOwhen combining with superoxide, which can nitrate thephenolic ring of tyrosine residues in proteins.38,39 Thetissue injury induced by peroxynitrite may lead to anexcessive local amplification of the immune response,resulting in migration of inflammatory cells. In accor-dance with our data, it was shown that L-NAME inhib-ited the edema formation induced by carragenin, aninflammatory agent that induce increase in vascularpermeability by inducing a massive leukocyte emi-gration, in rat skin.40

Coupled with the reduction of local inflammatoryparameters, we also found that both L-NAME and AGinhibited of the lymphomonocytosis observed after11 days. This latter effect of L-NAME could be reversedby the co-administration of L-arginine. In addition, theL-NAME-treated rats also exhibited inhibition of lym-phomonocytosis in the seventh day. The leukocytosisthat happens in this periodontitis model is probablysecondary to the intense local inflammatory reaction,with the possible contribution of bacterial growth.Therefore, by inhibiting the inflammation locally, NOSinhibitors would also decrease the systemic reactionassociated with periodontitis. In accordance with thesedata, a previous study also demonstrated a pronouncedleukocytosis at 6 hours and 11 days after the ligatureplacement in rats, due to neutrophilia and lympho-monocytosis, respectively.20 However, leukocytosis hasnot been detected in humans with periodontal disease.

We observed that in the groups treated with thelower AG doses, neither with 5 or 10 mg/Kg L-NAME(data not shown) had a significant alveolar bone loss

40171.qxd 6/7/05 12:46 PM Page 961

962

Nitric Oxide Synthase Prevents Alveolar Bone Resorption Volume 76 • Number 6

reduction, despite the use of the same experimentalprotocol. Additionally, treatment with 40 mg/kg L-NAMEand 100 mg/kg AG (data not shown), under the sameconditions, did not inhibit the alveolar bone resorptionor the local inflammatory changes. These apparentlycontradictory results raise the possibility that basallevel of NO, produced by the constitutive NOSenzymes (cNOS), may exert an inhibitory effect onbone resorption, since a previous study proposed thatbasal NO production may exert a tonic inhibitory effecton osteoclast activity.41 There is a report showing thatAG is a selective iNOS inhibitor.42 However, in thepresence of calcium, calmodulin, and other cofactors,AG can also inhibit constitutive NOS.36 Thus, the inhi-bition of cNOS by AG could be an explanation for thelack of effect of the highest AG doses in preventingbone loss in the present study.

Additionally, several lines of evidence also suggestthat nitric oxide overproduction is associated with perio-dontal disease. The presence of iNOS in inflamed gin-gival tissue in localized juvenile periodontitis patientshas been demonstrated.43 Several reports state thatnitric oxide generated by iNOS is required for antimi-crobial activity and immune modulation.44-46 Accord-ingly, a recent report showed that mice lackinginducible nitric oxide synthase demonstrate impairedkilling of Porphyromonas gingivalis, a primary agentof severe periodontitis.47 Hence, it might well be thathigher doses of the NOS inhibitors, due to a virtualabolition of local NO production, would interfere withthe periodontal homeostasis in host-microbial defensemechanisms.

In conclusion, our data demonstrate that NOS inhibi-tion decreases alveolar bone resorption in a periodon-titis model. This effect was associated with reductionof local inflammation. The possible contributions ofreactive nitrogen species to these events, generatedsecondary to NO production deserve additionalinvestigation.

ACKNOWLEDGMENTSThe authors thank Maria Silvandira França Pinheiro,Department of Physiology and Pharmacology, and JoséIvan Rodrigues de Sousa, Department of Morphology,Faculty of Medicine, Federal University of Ceara, Brazil,for technical assistance. This work was supported bythe Brazilian Agency for Scientific and TechnologicalDevelopment.

REFERENCES1. Moncada S, Palmer RMJ, Hibbs JR, Higgs AE. Nitric

oxide: Physiology, pathophysiology and pharmacology.Pharmacol Rev 1991;43:109-142.

2. Radomski MW, Palmer RM, Moncada S. Endogenousnitric oxide inhibits human platelet adhesion to vascu-lar endothelium. Lancet 1987;2:1057-1058.

3. Murad F. Cyclic guanosine monophosphate as a medi-

ator of vasodilation. J Clin Invest 1986;78:1-5.4. Beckman JS, Beckman TW, Chen J, Marshall PA,

Freeman BA. Apparent hydroxyl radical production byperoxynitrite: Implications for endothelial injury fromnitric oxide and superoxide. Proc Natl Acad Sci (USA)1990;87:1620-1624.

5. Liew FY. Regulation of nitric oxide synthesis in infec-tious and autoimmune diseases. Immunol Lett 1994;43:95-98.

6. Bogdan C, Vodovotz Y, Paik J, Xie Q, Nathan C. Mech-anism of supression of nitric oxide synthase expressionby interlukin-4 in primary mouse macrophages. J Leuko-cyte Biol 1994;55:227-233.

7. Stefanovic-Racic M, Stadler J, Evans CH. Nitric oxideand arrhrits. Arthrits Rheum 1993;36:1036-1044.

8. Evans DE, Ralston SH. Nitric oxide and bone. J BoneMiner Res 1996;11:300-305.

9. Griffiths MJD, Messent M, MacAllister RJ, Evans TW.Aminoguanidine selectively inhibits inducible nitric oxidesynthase. Br J Pharmacol 1993;110:963-968.

10. Bredt DS, Zinder SH. Nitric oxide: A physiologic mes-senger molecule. Annu Rev Biochem 1994;63:175-195.

11. Anggard E. Nitric oxide: Mediator, murder, and medi-cine. Lancet 1994;75:12-24.

12. Tepperman BL, Brown JF, Korolkiewicz R, Whittle BJR.Nitric oxide synthase activity, viability and cyclic GMPlevels in rat colonic epithelial cells: Effect of endotoxinchallenge. J Pharmacol Exp Ther 1994;271:1477-1482.

13. Ralston SH, Ho LP, Helfrich M, Grabowski PS, JohnstonPW, Benjamin N. Nitric oxide: A cytokine induced reg-ulator of bone resorption. J Bone Miner Res 1995;10:1040-1049.

14. Da Rocha FAC, Brum-Fernandes AJ. Evidence that per-oxynitrite affects human osteoblast proliferation anddiferentiation. J Bone Miner Res 2002;17:434-442.

15. Lohinai Z, Benedek P, Fehér E, et al. Protective effectsof mercaptoethylguanidine, a selective inhibitor ofinducible nitric oxide synthase, in ligature-induced perio-dontitis in the rat. Br J Pharmacol 1998;123:353-360.

16. Lohinai Z, Mabley JG, Feher E, Marton A, Komjati K,Szabo C. Role of the nuclear enzyme poly(ADP-ribose)polymerase in the pathogenesis of periodontitis. J DentRes 2003;82:987-992.

17. Di Paola R, Marzocco S, Mazzon E, et al. Effect of amino-guanidine in ligature-induced periodontitis in rats. J DentRes 2004;83:343-348.

18. Sallay K, Sanavi F, Ring I, Pham P, Behling UH, NowotnyA. Alveolar bone destruction in the immuno-suppressedrat. J Periodontal Res 1982;17:263-274.

19. Samejima Y, Ebisu S, Okada H. Effect of injection withEikenella corrodens on the progression of ligature-induced periodontitis in rats. J Periodontal Res 1990;25:308-315.

20. Lima V, Bezerra MM, Alencar VBM, et al. Effects of chlor-promazine on alveolar bone loss in experimental perio-dontal disease in rats. Eur J Oral Sci 2000;108:123-129.

21. Lappin DF, Kjeldsen M, Sander L, Kinane DF. Induciblenitric oxide synthase expression in periodontitis. J Perio-dontal Res 2000;25:369-373.

22. Lohinai Z, Stachlewitz R, Virág L, Székely AD, Haskó G,Szabó C. Evidence for reactive nitrogen species forma-tion in the gingivomucosal tissue. J Dent Res 2001;80:470-475.

23. Matejka M, Partyka L, Ulm C, Solar P, Sinzinger H. Nitricoxide synthesis is increased in periodontal disease. JPeriodontal Res 1998;33:517-518.

24. Blix IJ, Helgeland K. LPS from Actinobacillus actino-

40171.qxd 6/7/05 12:46 PM Page 962

963

J Periodontol • June 2005 Leitão, Ribeiro, Chaves, Rocha, Lima, Brito

mycetemcomitans and production of nitric oxide in murinemacrophages J774. Eur J Sci 1998;106:576-581.

25. Frolov I, Houri-Hadad Y, Soskolne A, Shapira L. In vivoexposure to Porphyromonas gingivalis up-regulates nitricoxide but supresses tumor necrosis factor-a productionby cultured macrophages. Immunology 1998;93:323-328.

26. Shapira L, Champagne C, Van Dyke TE, Amar S. Strain-dependent activation of monocytes and inflammatorymacrophages by lipopolysaccharide of Porphyromonasgingivalis. Infect Immun 1998;66:2736-2742.

27. Assuma R, Oates T, Cochran D, Amar S, Graves DT. IL-1and TNF antagonists inhibit the inflammatory responseand bone loss in experimental periodontitis. J Immunol1998;160:403-409.

28. Galbraith GMP, Hagan C, Steed RB, Sanders JJ, JavedT. Cytokine production by oral and peripheral blood neu-trophils in adult periodontitis. J Periodontol 1997;68:832-838.

29. Bezerra MM, Lima V, Alencar VBM, et al. Selectivecyclooxygenase-2 inhibition prevents alveolar bone lossin experimental periodontitis in rats. J Periodontol 2000;71:1009-1014.

30. Green SJ, Nacy CA, Schreiber RD, et al. Neutralizationof gamma interferon and tumor necrosis factor alphablocks in vivo synthesis of nitrogen oxides from L-arginineand protection against Francisella tularensis infection inMycobacterium bovis BCG-treated mice. Infect Immun1993;61:689-698.

31. Kawakami K, Tohyanma M, Qifeng X, Saito A. Expres-sion of cytokine and inducible nitric oxide synthasemRNA in the lungs of mice infected with Cryptococcusneoformans: Effects of interleukin-12. Infect Immun1997;65:1307-1312.

32. Ralston SH, Todd D, Helfrich MH, Benjamin N, GrabowskiP. Human osteoblast-like cells produce nitric oxide andexpress inducible nitric oxide synthase. Endocrinology1994;135:330-336.

33. Ralston SH, Grabowski PS. Mechanisms of cytokineinduced bone resorption: Role of nitric oxide, cyclicguanosine monophosphate, and prostaglandins. Bone1996;19:29-33.

34. Brandi ML, Hukkanen M, Umeda T, et al. Bidirectionalregulation of osteoclast function by nitric oxide synthaseisoforms. Proc Natl Acad Sci (USA) 1995;92:2954-2958.

35. Kubes P, Suzuki M, Granger DN. Nitric oxide: An endoge-nous modulator of leukocyte adhesion. Proc Natl AcadSci (USA) 1991;88:4651-4655.

36. Peng HB, Spiecker M, Liao JK. Inducible nitric oxide:An autoregulatory feedback inhibitor of vascular inflam-mation. J Immunol 1998;161:1970-1976.

37. Dal Secco D, Paron JA, Oliveira SHP, Ferreira SH, SilvaJS, Cunha FQ. Neutrophil migration in inflammation:Nitric oxide inhibits rolling, adhesion and induces apop-tosis. Nitric Oxide 2004;9:153-164.

38. Beckman JS, Koppenol WH. Nitric oxide, superoxide, andperoxynitrite: The good, the bad, and ugly. Am J Physiol1996;271:C1424-C1437.

39. Szabo C. The role of peroxynitrite in the pathophysiol-ogy of shock, inflammation and ischemia-reperfusioninjury. Shock 1996;6:79-88.

40. Martin SW, Stevens AJ, Brennan BS, Davies D, RowlandM, Houston JB. The six-day-old rat air pouch model ofinflammation: Characterization of the inflammatoryresponse to carrageenan. J Pharmacolog Toxicolog Meth1994;32:139-147.

41. Kasten TP, Collin-Osdoby P, Patel N, et al. Potentiationof osteoclast bone-resorption activity by inhibition ofnitric oxide synthase. Proc Natl Acad Sci (USA) 1994;91:3569-3573.

42. Laszlo F, Evans SM, Whittle BLR. Aminoguanidineinhibits both constitutive and inducible nitric oxide syn-thase isoforms in rat intestinal microvasculature in vivo.Eur J Pharmacol 1995;272:169-175.

43. Gaspirc B, Masera A, Skaleric U. Immunolocalization ofinducible nitric oxide synthase in localized juvenile perio-dontitis patients. Connect Tiss Res 2002;43:413-418.

44. Kroncke K, Fehsel K, Kolb-Bachofen V. Nitric oxide:Cytotoxicity versus cytoprotection – how, why, when,and where? Nitric Oxide: Biol Chem 1997;1:107-120.

45. Allaker RP, Mendez LSS, Hardie JM, Benjamin N. Antimi-crobial effect of acidified nitrite on periodontal bacteria.Oral Microbiol Immunol 2001;16:253-256.

46. Shibata K, Warbington ML, Gordon BJ, Kurihara H, VanDyke TE. Nitric oxide synthase activity in neutrophilsfom patients with localized aggressive periodontitis. JPeriodontol 2001;72:1052-1058.

47. Gyurko R, Boustany G, Huang PL, et al. Mice lackinginducible nitric oxide synthase demonstrated impairedkilling of Porphyromonas gingivalis. Infect Immun 2003;71:4917-4924.

Correspondence: Dr. Gerly Anne de Castro Brito, Departamentode Fisiologia e Farmacologia, Faculdade de Medicina, Uni-versidade Federal do Ceará, Rua Coronel Nunes de Melo,1127, Rodolfo Teófilo, 60.430-270 Fortaleza, Ceará, Brazil.Fax: 55-85-288-8333; e-mail: gerlybrito@hotmail.com.

Accepted for publication October 25, 2004.

40171.qxd 6/7/05 12:46 PM Page 963