Local Application of Melatonin Into Alveolar Sockets of Beagle Dogs Reduces Tooth Removal-Induced Oxidative Stress
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Volume 78 • Number 3
Local Application of Melatonin Into
Alveolar Sockets of Beagle Dogs
Reduces Tooth Removal–Induced
Oxidative Stress
Antonio Cutando,* Carlos Arana,† Gerardo Gómez-Moreno,* Germaine Escames,† Ana López,†
Marı́a J. Ferrera,* Russel J. Reiter,‡ and Darı́o Acuña-Castroviejo†§
Background: The antioxidant and anti-inflammatory hor-
mone melatonin is secreted by saliva into the oral cavity,
where it may protect the mucosal and gingival tissues from
radical damage. To date, no studies have addressed the po-
tential beneficial role of melatonin in the acute inflammatory
response that follows oral surgical interventions, especially
tooth extractions. The aim of this study was to determine
R
eactive oxygen species (ROS),
whether tooth extraction induces changes in plasma oxidative including superoxide anion radi-
stress levels, and whether melatonin treatment may counter- cal, hydrogen peroxide, and the
act these changes. hydroxyl radical, and reactive nitrogen
Methods: Maxillary and mandibular premolars and molars of species (RNS), including nitric oxide
16 adult Beagle dogs were extracted under general anesthesia. (NO) and the peroxynitrite anion, are
Eight dogs were treated with 2 mg melatonin placed into the al- common byproducts produced by nor-
veolar sockets, whereas the other eight dogs received only ve- mal aerobic metabolism of oral cavity
hicle. Lipid peroxidation (LPO) and nitrite plus nitrate (NOx) cells or by inhalation of oxidizing agents
levels were determined in plasma, whereas glutathione (GSH) in tobacco smoke and other air pollut-
and glutathione disulfide (GSSG) levels and glutathione perox- ants.1-4 Moreover, activation of the
idase (GPx) and reductase (GRd) activities were measured in immune system by inflammatory pro-
red blood cells before and 24 hours after tooth extraction. cesses, such as chronic periodontitis,
Results: Removal of the premolars and molars caused a increases ROS-RNS generation.3 Al-
significant rise in plasma LPO and NOx levels and in the eryth- though ROS are necessary for defense
rocyte GSSG/GSH ratio, whereas melatonin treatment restored of the host, they also expose oral tissues
the normal values of these parameters. Also, melatonin slightly to oxidative damage.2 The mucosal
increased erythrocyte GRd activity without changing GPx activity. barrier is the first line of defense against
Conclusion: For the first time to our knowledge, the results flora growing in the oral cavity. In mu-
show that during the immediate postoperative period following cosal cells, the production of NO by the
tooth extraction, there is a significant increase of oxidative expression of inducible NO synthase
stress, which is counteracted by the administration of melatonin (iNOS) serves as a chemical barrier to
into the alveolar sockets. J Periodontol 2007;78:576-583. limit bacterial plaque invasion. How-
ever, iNOS expression by oral epithelial
KEY WORDS
cells is associated with diminished cell
Antioxidant; free radicals; mouth; oral surgery; oxidative viability, which may depend on the peroxy-
stress. nitrite formation.4,5 These reactive spe-
cies are involved in the pathogenesis of
several oral processes, including recur-
* Department of Special Care in Dentistry, School of Dentistry, University of Granada,
Granada, Spain. rent aphthous ulceration,6 leukoplakia,7
† Department of Physiology, Institute of Biotechnology, University of Granada. lichen planus,8 and especially in oral
‡ Department of Cellular and Structural Biology, The University of Texas Health Science
Center at San Antonio, San Antonio, TX. cavity cancer and periodontal inflam-
§ Clinical Analysis Unit, San Cecilio’s University Hospital, Granada, Spain. matory disease.9-12
doi: 10.1902/jop.2007.060244
576J Periodontol • March 2007 Cutando, Arana, Gómez-Moreno, et al.
Periodontal disease in its destructive phase is ethylenediamine dihydrochloride, sulphanilamide, tri-
considered to be initiated and perpetuated by Gram- chloroacetic acid, and phosphoric acid were purchased,i
negative bacteria that colonize the subgingival area. and aMT was obtained.¶ All other reagents were of the
When stimulated by periodontal pathogens, host cells highest purity available.
release proinflammatory cytokines, whereas massive
Animals, Surgery, and Treatment
polymorphonuclear cell migration to the gingival
The study was performed in 16 male Beagle dogs ob-
crevicular fluid leads to abnormal spreading of ROS.13,14
tained from the Veterinary Faculty, University of
Additionally, macrophage infiltration to the periodon-
Córdoba, Córdoba, Spain. The animals were maintained
tal tissues increases iNOS and NO, with the latter being
in the University’s facility in individual kennels in a
related to the pathogenesis of periodontitis and subse-
12:12 light-dark cycle (lights on at 7:00 am) at 22C –
quent bone loss.4,15 Usually, an increase in free radical
2C with regular chow and tap water. Animals were
production coexists with a decrease in the antioxidant
14 months of age at the time of the study and weighed
defense system.6 The imbalance between the prooxi-
16 to 18 kg. All experiments were approved and per-
dant and antioxidant systems may lead to further
formed according to the Spanish Government Guide
oxidative damage of periodontal tissues.2,16-18
and the European Community Guide for animal care.
Previous studies reported that oral inflammatory
Both upper and lower maxillary and mandibular
processes, such as periodontitis, can trigger signals
premolars and molars of the 16 Beagle dogs were ex-
that increase not only plasma melatonin (aMT) levels
tracted under general anesthesia. The anterior group
but also aMT levels in the oral cavity, where the indol-
of teeth was conserved so that the dogs could main-
amine may exert an antioxidant role.19,20 The direct
tain an appropriate masticatory function. All inter-
action of aMT as a free radical scavenger of both
ventions were supervised by the veterinarian of the
ROS and RNS19,21,22 is complemented with an in-
Animal Experimentation Service of the University of
direct stimulatory effect of the antioxidant enzymes,
Granada. Fifteen minutes before general anesthesia,
including glutathione peroxidase (GPx) and reductase
the animals received an intramuscular injection of 0.5
(GRd), superoxide dismutase, and catalase.23-25 Due
to 1 mg/kg acepromazine maleate, an anxiolytic. Gen-
to its stimulatory effect on GRd, aMT favors the recy-
eral anesthesia included ketamine plus chlorbutol, 5 to
cling of glutathione (GSH) from glutathione disulfide
8 mg/kg intravenously; 0.5 to 1 mg/kg acepromazine
(GSSG), maintaining a high GSH/GSSG ratio.26
maleate as coadjuvant; and 0.05 mg/kg atropine.
aMT also promotes the de novo synthesis of GSH by
Dexamethasone isonicotinate (2 ml intramuscularly)
promoting the activity of g-glutamyl-cysteine synthe-
and amoxicillin (2 ml intramuscularly) were adminis-
tase.27 Additionally, aMT is also capable of reducing
tered at the end of surgery and every 2 days for a total
NO and peroxynitrite generation because of its ability
of 4 days.
to inhibit iNOS activity and expression, which increase
After the tooth extractions and before suturing,
the tissue damage that accompanies inflammation.28,29
eight dogs received aMT applied into the extraction
aMT also may exert an immunoenhancing role in the
wounds and gingival tissue surrounding the premolar
oral cavity because patients with severe periodontal
and molar area. The following groups of dogs were
status with bone damage and gingival involvement
included: 1) control group (Con), consisting of all 16
show concomitant high interleukin-2 and aMT levels,
dogs sampled 1 hour before tooth extractions; 2)
which may stimulate CD4 lymphocytes in response to
vehicle-treated group (Veh), consisting of eight dogs
periodontal disease.30,31
with tooth extractions but without postextraction
It is now well established that the acute inflamma-
treatment; and 3) aMT-treated group (aMT), consist-
tory response of gingival tissue during the first 24 to 48
ing of eight dogs receiving 2 mg powder aMT into the
hours postextraction causes an important polymor-
alveolar sockets and surrounding gingival tissue after
phonuclear leukocyte infiltration, which is respon-
surgical removal of the tooth.
sible, in part, for the increase in ROS and RNS
Blood samples were taken from the vena cephalica
generation.32 Thus, the aim of this study was to test
antebrachii 1 hour before tooth extraction (control
whether plasma changes reflect the acute inflamma-
samples) and 24 hours after the surgical procedure.
tory response caused by tooth extractions in Beagle
Blood was rapidly transferred to cold EDTA-K–con-
dogs, and whether aMT treatment could modify any
taining tubes and centrifuged at 3,000 · g for 10 min-
observed changes.
utes at 4C. Plasma aliquots were stored at -80C for
MATERIALS AND METHODS lipid peroxidation (LPO) and nitrite plus nitrate (NOx)
determination. GSH and GSSG levels and GPx and
Chemicals
GRd activities were determined in red blood cells.
GSH, GSSG, GRd, nicotinamide adenine dinucleotide
phosphate (NADPH), cumene hydroperoxide, ophtha- i Sigma-Aldrich, Madrid, Spain.
laldehyde, N-ethylmaleimide, methanol, N-(1-naphthyl) ¶ Helssin Chemicals, Biusca, Switzerland.
577Melatonin and Oral Oxidative Stress Volume 78 • Number 3
The cells were separated from the plasma and washed determined by the methemoglobin method.36 The
two times with 0.9% sodium chloride solution. Red concentration of GSH and GSSG was expressed in
blood cell aliquots were stored at -80C until assays micromoles per gram of Hb.
were performed.
Determination of GPx and GRd Activities
LPO Determination Aliquots of saline-washed red blood cells were thawed
Malonaldehyde and 4-hydroxyalkenals concentra- and hemolized (1:20) with 10 mM phosphate buffer,
tions provide a convenient index of lipid peroxidation. 1 mM EDTA-Na2, pH 6.5, at 4C for 5 minutes, and
These lipid peroxidation products were determined centrifuged at 20,000 · g for 15 minutes at 4C. For
with a special kit.# The kit takes advantage of a chro- GPx determination, 120 ml supernatant was incubated
mogenic reagent that reacts with malonaldehyde and in a final volume of 3 ml with 100 mM phosphate buffer
4-hydroxyalkenal (4HDA) at 45C yielding a stable containing 1 mM EDTA-Na2, pH 7.5, in the presence of
chromophore with maximal absorbance at the 586- 30 ml of 20 mM NADPH, 100 ml of 60 mM GSH, and 4 ml
nm wavelength.33 Plasma LPO levels were expressed (1 international unit [IU]) GRd for 5 minutes at room
in nanomoles per milliliter. temperature. A total of 100 ml of 36 mM cumene hydro-
peroxide solution was added, and GPx activity was
NOx Determination measured following the oxidation of NADPH for 3 min-
Levels of NOx were measured in plasma previously utes at 340 nm37 in a spectrophotometer.‡‡ GRd activ-
treated with nitrate reductase. Then, pretreated plasma ity was measured in 35 ml supernatant incubated in a
aliquots were incubated with 100 ml of Griess reagent final volume of 508.5 ml with 100 mM phosphate-
(0.1% N-[1-naphthyl] ethylenediamine dihydrochlor- EDTA-Na2 buffer, pH 7.5, containing 2.5 mM GSSG
ide; 1% sulfanilamide in 5% phosphoric acid; 1:1) at for 5 minutes at room temperature. A total of 8.5 ml
room temperature for 20 minutes.34 The absorbance NADPH 12 mM was added, and NADPH oxidation
at 550 nm was measured with a spectrophotome- was followed for 3 minutes at 340 nm37 in an ultravio-
ter.** NOx concentrations were calculated by com- let (UV) spectrophotometer.§§ In both cases, non-
parison to the absorbance of a standard solution of enzymatic NADPH oxidation was subtracted from
known sodium nitrite concentration and expressed the overall rate. The activity of both enzymes was ex-
in nanomoles per milliliter. pressed in micromoles per minute per gram Hb.
Measurement of GSH and GSSG Statistical Analysis
Both GSH and GSSG were measured by a fluorometric All data are expressed as the mean – SEM. One-way
method,35 which was slightly modified. Aliquots of analysis of variance followed by the Student t test
saline-washed red blood cells were thawed and he- was used to compare the differences between groups.
molized (1:20) with 10 mM phosphate buffer, 1 mM PJ Periodontol • March 2007 Cutando, Arana, Gómez-Moreno, et al.
traction levels (1.98 – 0.13 versus 1.328 – 0.13
mmol/minute/g Hb, respectively; PMelatonin and Oral Oxidative Stress Volume 78 • Number 3
relationships among aMT, the immune system, and treated dogs. Thus, the observed differences in the
oral status are not well defined. Reduced oral health cellular pool of GSH in the vehicle-treated animals re-
(with advanced periodontal processes with gingival flect a generalized oxidative stress. These alterations
tissue damage and bone loss) serves as a trigger for may also produce changes in the GSH redox cycling
increases in salivary aMT levels,29,30 which in turn enzymes. In fact, the increase in GPx activity after
stimulates the CD4 lymphocytes.30,31 However, there tooth extraction likely reflects the activation of the an-
are no published reports related to aMT oxidative tioxidant machinery. However, the measured rise in
stress interactions during surgical oral interventions, GSSG was not adequately metabolized to GSH be-
including after tooth extractions. For the first time to cause of only a slight increase in GRd activity.
our knowledge, the current results show the existence Recently, an inverse relationship between salivary
of significant oxidative stress during the immediate aMT levels and periodontal status was found.40 This
postoperative period following tooth extraction, with study40 supported a protective role of aMT against
the changes being counteracted by local aMT applica- free radicals produced by inflammatory periodontal
tion into the alveolar sockets after tooth removal. diseases. Herein, we found that the application of
One day after oral surgery, the dogs that did not re- aMT into the alveolar sockets after tooth removal re-
ceive aMT exhibited a significant increase in parame- duced significantly the oxidative stress parameters in
ters of plasma oxidative stress as a consequence of both the plasma and the erythrocytes. Increased
the damage and the inflammatory process that fol- levels of LPO caused by tooth removal were counter-
lows surgical intervention. After tooth removal, bacte- acted by aMT at 24 hours after surgery. The ability of
ria of the oral cavity colonize the surface of the blood aMT to efficiently reduce the oxidation of lipids under
clot that covers the alveolar socket, granulation tis- a variety of conditions where free radicals are gener-
sue, wound epithelium, and the adjacent gingival tis- ated is well established.20,21,29,41 It is likely that aMT
sue.15,38 These events cause an acute inflammatory achieves this high degree of lipid protection by neu-
response of the gingival mucosa, which surrounds tralizing the radicals (i.e., hydroxyl radical and peroxy-
the blood clot. Thus, during the first 24 to 48 hours nitrite) that initiate the process of lipid breakdown.
postextraction, edema and vasodilatation are ob- aMT positions itself among the membrane lipids in
served in the periphery of the alveolar socket with a such a way as to impede the oxidation of the polyun-
marked infiltration of polymorphonuclear leuko- saturated fatty acids.42-44
cytes.15,38 Gingival tissue infiltration by polymorpho- In the present study, aMT also counteracted NOx
nuclear leukocytes and monocytes, whose principal levels that were increased after oral surgery; in fact,
function after tooth extraction is phagocytosis of the indole reduced NOx concentrations below those
bacteria, is also responsible for the generation of measured in plasma before tooth extraction. The ef-
ROS.1,2,13,14 Besides the inflammatory process, other fect of aMT on NOx levels may depend, at least in part,
mechanisms, including breakdown of the gingival fi- on its ability to scavenge nitrite.41,45 Furthermore, in
bers, damage to periodontal vessels, and the mechan- vivo studies have documented that aMT inhibits iNOS
ical mutilation to oral tissues as a consequence of expression and activity in experimental models of
tooth extraction, also participate in the oral damage sepsis in rats and mice.28,29,40 Increased iNOS activ-
after tooth removal.15,38 Together, these events par- ity and expression are related to several oral mucosal
ticipate in promoting oxidative stress generated by the inflammatory diseases;4,5,7,11,12 thus, elevated iNOS
inflammatory process. In turn, the increased ROS stim- activity probably contributes to the overproduction of
ulate the production of proinflammatory cytokines, NO and peroxynitrite during the inflammatory pro-
transcription factors, such as nuclear factor-kappa cess following tooth removal. The inhibition of iNOS
B (NF-kB), and vascular cell adhesion molecules, by aMT likely reduces NOx levels, thereby diminish-
thereby increasing the progression of the inflamma- ing gingival damage and postextraction oxidative
tory process and the synthesis of RNS such as NO stress in the oral cavity. Additionally, aMT could also
and peroxynitrite.32,39 decrease nitrosative stress in gingival cells by directly
Both ROS and RNS locally generated in the oral neutralizing peroxynitrite.45
cavity after tooth removal can enter the circulation. Besides reductions in plasma markers of oxidative
In fact, plasma levels of LPO and NOx, which reflect (LPO) and nitrosative (NOx) damage, aMT also
the increased production of ROS and RNS, respec- reduced significantly the GSSG/GSH ratio, the best
tively, were significantly higher 24 hours after tooth index of intracellular oxidative damage in erythro-
extraction. Overproduction of lipid hydroperoxides cytes. In addition to the direct scavenging activity of
and aldehyde products causes depletion of GSH, dis- aMT, which reduces GSH consumption,26 aMT also
rupting mucosal turnover.1,9 Our results document increased GRd activity, which may account for the
these changes because GSSG levels and the GSSG/ reduction of GSSG and increase of GSH levels,
GSH ratio were significantly elevated in the vehicle- thereby providing the cell with additional GSH.23,26
580J Periodontol • March 2007 Cutando, Arana, Gómez-Moreno, et al.
Besides protecting GRd per se from oxidative de- system aids in wound healing and reduces the recov-
struction, the effect of aMT on GRd activity also may ery. Patients with compromised antioxidant defenses
depend on a genomic effect of the indolamine to in- in the oral cavity or with pathologies associated with
crease the expression of the enzyme.24,25 Regulation oxidative stress, such as diabetes, Parkinson’s dis-
of the GSH redox cycling is probably of great signifi- ease, autoimmune disorders, periodontal disease, or
cance for oral tissue homeostasis, because GSH is a aphthous ulceration, have elevated levels of ROS-
major endogenous antioxidant in the cell. GSH plays RNS, which aggravates the damage to gingival tissue,
an important role in cellular protection from oxidative delaying the regeneration processes. The current re-
damage of lipids, proteins, and nucleic acids.46 Addi- sults suggest that local application of aMT may be
tionally, GSH regulates the metabolism and activity useful in preventing inflammatory and infectious
of other proteins and it interacts synergistically with complications induced by oxidative stress after tooth
other components of the antioxidant defense system, extraction.
such as vitamins C and E and superoxide dismut-
ase.47,48 ACKNOWLEDGMENTS
Although these data support the ability of aMT to This work was partially supported by grants PI04-
reduce oral surgery–dependent oxidative stress, the 1610, PI03-0817, and G03-137 from the Institute of
two-faced character of ROS-RNS should be noted.49 Health Carlos III, Spain; PTR 1995-0885-OP from
Although overproduction of ROS-RNS should be con- the Ministry of Education and Science, Spain; and
sidered a protective response of the immune system CTS-263 and CTS-101 from the Gobern of Andalusia,
to prevent bacteria infection, it also results in oxidative Spain.
stress and cell damage. By contrast, beneficial effects
of these radicals occur at low to moderate concentra-
REFERENCES
tions, and involve physiologic roles in a number of cel-
1. Minczykowski A, Woszczyk M, Szczepanik A,
lular signaling pathways.49 Thus, the organism tends Lewandowski L, Wysocki H. Hydrogen peroxide and
to prevent an excess of ROS-RNS activating the anti- superoxide anion production by polymorphonuclear
oxidative response. In this regard, exogenous admin- neutrophils in patients with chronic periapical granu-
istration of aMT should not be considered a treatment loma, before and after surgical treatment. Clin Oral
to minimize ROS-RNS. Instead, aMT may help in pre- Investig 2001;5:6-10.
2. Waddington RJ, Moseley R, Embery G. Reactive oxy-
venting the overproduction of free radicals by main- gen species: A potential role in the pathogenesis of
taining their basal levels. Whether the effects of aMT periodontal diseases. Oral Dis 2000;6:138-151.
reported here favor healing reaction is yet unclear 3. Paquette DW, Williams RC. Modulation of host inflam-
and requires further research. matory mediators as a treatment strategy for peri-
odontal diseases. Periodontol 2000 2000;24:239-252.
CONCLUSIONS 4. Daghigh F, Borghaei RC, Thornton RD, Bee JH. Hu-
man gingival fibroblasts produce nitric oxide in re-
Our results document a significant increase in the sponse to proinflammatory cytokines. J Periodontol
levels of oxidative stress, as measured by blood 2002;73:392-400.
parameters, in the immediate postoperative period 5. Kimura S, Yonemura T, Kaya H. Increased oxidative
following tooth removal. These results suggest that product formation by peripheral blood PMN leuko-
cytes in human periodontal diseases. J Periodontal Res
the use of antioxidants, such as aMT, may be a bene- 1993;28:197-203.
ficial therapy after surgical procedures in the oral cav- 6. Sculley DV, Langley-Evans SC. Periodontal disease is
ity.50 Locally, administration of aMT into the alveolar associated with lower antioxidant capacity in whole
sockets successively counteracted oxidative and ni- saliva and evidence of increased protein oxidation.
trosative stress in blood, presumably also reflecting Clin Sci (Lond) 2003;105:167-172.
7. Beevi SS, Rasheed AM, Geetha A. Evaluation of oxida-
the reduction of damage in the oral cavity. Overpro- tive stress and nitric oxide levels in patients with oral
duction of ROS-RNS in gingival cells after tooth ex- cavity cancer. Jpn J Clin Oncol 2004;34:379-385.
traction contributes to postextraction inflammatory 8. Chaiyarit P, Ning MA, Hiraku Y, et al. Nitrative and
and infectious complications. The subsequent in- oxidative DNA damage in oral lichen planus in relation
crease in the damage to oral tissues may, in turn, de- to human oral carcinogenesis. Cancer Sci 2005;96:
553-559.
lay postextraction wound healing and regeneration of 9. Cerutti PA. Oxy-radicals and cancer. Lancet 1994;344:
gingival tissue surrounding the alveolar bone. Thus, 862-863.
increased oxidative stress after tooth removal would 10. Altman LC, Baker C, Fleckman P, Luchtel D, Oda D.
be related to a poor postextraction prognosis and Neutrophil-mediated damage to human gingival epi-
evolution, mainly when a concomitant inflammatory thelial cells. J Periodontal Res 1992;27:70-79.
11. Lohinai Z, Stachlewitz R, Virag L, Szekely AD, Hasko
disease is present in the oral cavity.40 Because the an- G, Szabo C. Evidence for reactive nitrogen species
tioxidant defense system in the oral cavity counter- formation in the gingivomucosal tissue. J Dent Res
acts this oxidative stress, stimulation of this defense 2001;80:470-475.
581Melatonin and Oral Oxidative Stress Volume 78 • Number 3
12. Page RC, Kornman KS. The pathogenesis of human ride-induced expression and activity of mitochon-
periodontitis: An introduction. Periodontol 2000 1997; drial nitric oxide synthase in rats. FASEB J 2003;17:
14:9-11. 932-934.
13. McMillan MD. Neutrophils in the molar tooth extraction 30. Gómez-Moreno G, Cutando-Soriano A, Arana C, et al.
wound in the rat: A transmission electron microscope Melatonin expression in periodontal disease. J peri-
(TEM) study. J Oral Pathol Med 1999;28:297-302. odont Res 2007; in press.
14. Shapira L, Borinski R, Sela MN, Soskolne A. Super- 31. Cutando A, Silvestre FJ. Melatonin: Implications at the
oxide formation and chemiluminescence of peripheral oral level. Bull Group Int Rech Sci Stomatol Odontol
polymorphonuclear leukocytes in rapidly progres- 1995;38:81-6.
sive periodontitis patients. J Clin Periodontol 1991;18: 32. Brennan PA, Thomas GJ, Langdon JD. The role of
44-48. nitric oxide in oral diseases. Arch Oral Biol 2003;48:
15. Deguchi S, Hori T, Creamer H, Gabler W. Neutrophil- 93-100.
mediated damage to human periodontal ligament- 33. Esterbauer H, Cheeseman KH. Determination of
derived fibroblast: Role of lipopolysaccharide. J aldehidic lipid peroxidation products: Malonaldehide
Periodontal Res 1990;25:293-299. and 4-hydroxynonenal. Methods Enzymol 1990;186:
16. Diab-Ladki R, Pellat B, Chahine R. Decrease in the 407-421.
total antioxidant activity of saliva in patients with peri- 34. Verdon CP, Burton BA, Prior RL. Sample pretreatment
odontal diseases. Clin Oral Investig 2003;7:103-107. with nitrate reductase and glucose-6-phosphate dehy-
17. Ohashi M, Iwase M, Nagumo M. Elevated production drogenase quantitatively reduces nitrate while avoid-
of salivary nitric oxide in oral mucosal diseases. J Oral ing interference by NADP+ when the Griess reaction
Pathol Med 1999;28:355-359. is used to assay for nitrite. Anal Biochem 1995;224:
18. Ma N, Tagawa T, Hiraku Y, Murata M, Ding X, 502-508.
Kawanishi S. 8-Nitroguanine formation in oral leuko- 35. Hissin PJ, Russel H. A fluorometric method for deter-
plakia, a premalignant lesion. Nitric Oxide 2006;14: mination of oxidized and reduced glutathione in tis-
137-143. sues. Anal Biochem 1976;74:214-226.
19. Acuña-Castroviejo D, Martı́n M, Macı́as M, et al. 36. Van Kampen EJ, Zijlstra WG. Determination of he-
Melatonin, mitochondria, and cellular bioenergetics. moglobin and its derivatives. Adv Clin Chem 1965;8:
J Pineal Res 2001;30:65-74. 141-187.
20. Tan DX, Reiter RJ, Manchester CL, et al. Chemical 37. Jaskot RH, Charlet EG, Grose EC, Grady MA, Roycroft
and physical properties and potential mechanisms: JH. An automatic analysis of glutathione peroxidase,
Melatonin as a broad spectrum antioxidant and free glutathione-S-transferase, and reductase activity in ani-
radical scavenger. Curr Top Med Chem 2002;2: mal tissue. J Anal Toxicol 1983;7:86-88.
181-197. 38. Altman LC, Baker C, Fleckman P, Luchtel D, Oda D.
21. Tan DX, Manchester LC, Reiter RJ, Qi WB, Karbownik Neutrophil-mediated damage to human gingival epi-
M, Calvo JR. Significance of melatonin in antioxidative thelial cells. J Periodontal Res 1992;27:70-79.
defense system: Reactions and products. Biol Signals 39. Xie Q, Kashiwabara Y, Nathan C. Role of transcription
Recept 2000;9:137-159. factor NF-kB/Rel in induction of nitric oxide synthase.
22. León J, Acuña-Castroviejo D, Escames G, Tan DX, J Biol Chem 1994;269:4705-4708.
Reiter RJ. Melatonin mitigates mitochondrial malfunc- 40. Cutando A, Galindo P, Gómez-Moreno G, et al. Rela-
tion. J Pineal Res 2005;38:1-9. tionship between salivary melatonin and severity of
23. Pablos MI, Agapito MT, Gutierrez I, et al. Melatonin periodontal disease. J Periodontol 2006;77:1533-1538.
stimulates the activity of the detoxifying enzyme glu- 41. Escames G, López LC, Tapias V, et al. Melatonin
tathione peroxidase in several tissues of chicks. counteracts inducible mitochondrial nitric oxide syn-
J Pineal Res 1995;19:111-115. thase-dependent mitochondrial dysfunction in skeletal
24. Antolı́n I, Rodrı́guez C, Sainz RM, et al. Neurohormone muscle of septic mice. J Pineal Res 2006;40:71-78.
melatonin prevents cell damage: Effect on gene ex- 42. Reiter RJ, Tan DX, Manchester LC, Qi W. Biochem-
pression of antioxidative enzymes. FASEB J 1996; ical reactivity of melatonin with reactive oxygen and
10:882-890. nitrogen species. Cell Biochem Biophys 2001;34:
25. Rodrı́guez C, Mayo JC, Sainz RM, et al. Regulation of 237-256.
antioxidant enzymes: A significant role for melatonin. 43. Garcı́a JJ, Reiter RJ, Guerrero JM, et al. Melatonin
J Pineal Res 2004;36:1-9. prevents changes in microsomal membrane fluidity
26. Martı́n M, Macı́as M, Escames G, León J, Acuña- during induced lipid peroxidation. FEBS Lett 1997;
Castroviejo D. Melatonin but not vitamins C and E 408:297-300.
maintains glutathione homeostasis in t-butyl hydro- 44. Bongiorno D, Ceraulo L, Ferrugia M, Fillizzola F,
peroxide-induced mitochondrial oxidative stress. FASEB Ruggierello A, Liveri VT. Localization and interactions
J 2000;14:1677-1679. of melatonin in dry cholesterol/lecithin mixed reversed
27. Urata Y, Honma S, Goto S, et al. Melatonin induces micelles used as cell membrane models. J Pineal Res
gamma-glutamylcysteine synthetase mediated by ac- 2005;38:292-298.
tivator protein-1 in human vascular endothelial cells. 45. Blanchard B, Pompon D, Ducrocq C. Nitrosation of
Free Radic Biol Med 1999;27:838-847. melatonin by nitric oxide and peroxynitrite. J Pineal
28. Crespo E, Macı́as M, Pozo D, et al. Melatonin inhibits Res 2000;29:184-192.
expression of the inducible NO synthase II in liver and 46. Reiter RJ. Melatonin: Lowering the high price of free
lung and prevents endotoxemia in lipopolysaccharide- radicals. News Physiol Sci 2000;15:246-250.
induced multiple organ dysfunction syndrome in rats. 47. Kir HM, Dillioglugil MO, Tugay M, Eraldemir C,
FASEB J 1999;13:1537-1546. Ozdogan HK. Effects of vitamins E, A and D on MDA,
29. Escames G, León J, Macı́as M, Khaldy H, Acuña- GSH, NO levels and SOD activities in 5/6 nephrec-
Castroviejo D. Melatonin counteracts lipopolysaccha- tomized rats. Am J Nephrol 2005;25:441-446.
582J Periodontol • March 2007 Cutando, Arana, Gómez-Moreno, et al.
48. Conner EM, Grisham MB. Inflammation, free radicals, oxygen species. Crit Rev Oral Biol Med 1999;10:
and antioxidants. Nutrition 1996;12:274-277. 458-476.
49. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M,
Telser J. Free radicals and antioxidants in normal phys- Correspondence: Prof. Dario Acuña-Castroviejo, Depart-
iological functions and human disease. Int J Biochem ment of Physiology, Faculty of Medicine, Avenida de
Cell Biol 2007;39:44-84. Epub 2006 Aug 4. Madrid 11, E-18012 Granada, Spain. Fax: 34-958-
50. Battino M, Bullón P, Wilson M, Newman H. Oxidative 246295; e-mail: dacuna@ugr.es.
injury and inflammatory periodontal diseases: The
challenge of anti-oxidants to free radicals and reactive Accepted for publication September 27, 2006.
583You can also read
