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Role of free radicals and antioxidant defences in oral

cavity-related pathologies

T. Iannitti1, V. Rottigni2,3, B. Palmieri2,3

1Department of Physiology, School of Medicine, University of Kentucky Medical Center, Lexington, KY, USA; 2Poliambulatorio delSecondo Parere, Modena, Italy; 3Department of General Surgery and Surgical Specialties, University of Modena and Reggio EmiliaMedical School, Surgical Clinic, Modena, Italy

Free radicals play a key role in the development of sev-

eral pathological conditions. Therefore, several methodshave been developed to measure oxidative stress from

bodily fluids including blood, urine and, more recently,

saliva. Free radical and antioxidant defences within the

oral cavity may play a key role in odontostomatological

pathologies. This review provides an update of the liter-

ature concerning the association of oxidative stress with

pathological conditions associated with the oral cavity. It

focuses on the diagnostic and therapeutic importance

of the tests based on saliva specimens in a preventive

perspective.

J Oral Pathol Med (2012)

Keywords: antioxidant; dentistry; free radical; oral cavity;

oxidative stress; periodontal; saliva

Introduction

Oxidative stress has been described as a process derivedfrom the inability of the bodys endogenous antioxidantdefences to scavenge free radical species and has beenrelated to many pathologies such as ageing, cardiovas-cular disease, neurodegenerative disorders, cancer, com-plex regional pain syndrome and many others (1). Thisevidence suggests that free radicals play a key role in thedevelopment of several pathological conditions. Free

radicals are highly reactive chemical species, character-ized by very short half-life. They are made up of a singleatom or several atoms that form a molecule with a freeelectron. This electron is responsible for the highreactivity of free radicals, which can bind other freeradicals or subtract an electron from the surroundingmolecules (2). Living cells are constantly exposed tooxidants deriving from a variety of sources that can be

exogenous, i.e. pollution, ozone, radiations, chemical

substances and pathogenic micro-organisms, and endog-enous, such as the mitochondrial electron transportchain, inflammatory cells and enzymes (3). When freeradical-derived oxidative damage to nucleic acids, pro-teins and lipids of the cellular and extracellular matrix isobserved, it produces damages of clinical importanceand severity. One of the most crucial concerns is themutation-induced carcinogenesis, the damage to cellularmembrane lipoproteins and lipid-mediated oxidativedamage leading to tegument ageing evolution. Oxidativedamage represents the main threat for the genomeintegrity in the greater part of living organisms. Reactiveoxygen species (ROS) can be generated as a product ofthe normal aerobic cell metabolism which, although is

limited to the mitochondria, can lead to the formationof oxidized bases, apurinicapyrimidinic sites (AP) andDNA breaks. 7,8-Dihydro-8-ossiguanina (8-oxoG) and2-hydroxy-adenine (2-OH-A) represent the two mainpre-mutagen bases (4, 5). The elevated mutageniccharacteristics of these oxidized bases are the conse-quence of their ability to create unusual pairings. Inparticular, the 8-oxoG can pair with the cytosine andadenine nucleotides with the same efficiency, causing thetransversions GC fi TA e A fi CG. The 2-OH-A canpair in an unconventional way leading to the transver-sion GC fi TA.

The 8-oxoG is a particularly frequent lesion, and its

danger to the cell is evident from the presence of severalcontrol systems, which are able to remove the oxidizedbase. The presence of ROS inside a cell can cause notonly DNA direct oxidation, but also purine andorpyrimidine oxidation of the nucleotide pool (dNTPs).The oxidized dNTPs can be incorporated in the DNAby the polymerases involved in the replication during thesynthesis phase.

Low-density lipoproteins (LDL) that contain hun-dreds of phospholipids, cholesteryl esters and triglyce-rides are particularly targeted by the attack of freeradicals and therefore subject to lipid peroxidation. TheLDL oxidative modifications alter their biologicalproperties increasing the atherogenic power. Oxidized

Correspondence: Dr. Tommaso Iannitti, Department of Physiology,School of Medicine, University of Kentucky Medical Center, Lexing-ton, KY 40536-0298, USA. Tel: +393282813314, E-mail: [email protected] for publication February 14, 2012

doi: 10.1111/j.1600-0714.2012.01143.x

J Oral Pathol Med

2012 John Wiley & Sons A/S All rights reserved

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LDL stimulate the endothelial cells to produce inflam-matory markers leading to potential innate and adaptiveimmune responses; they are also involved in the forma-tion of foam cells that inhibit macrophage motility andNO-induced vasodilatation (6). Among lipids, fattyacids are the most commonly subject to oxidationbecause the double bonds, which compose their chem-ical structure, are easily attacked by free radicals,leading to lipidic oxidation. When free radicals interactwith unsaturated fatty acids, which are found in cellmembranes or inside lipoproteins, the lipid peroxidationmechanism is automatically triggered off. They areconverted into the primary product, commonly calledlipid peroxide, and consequently a chain reaction istriggered off, leading to the amplification of the lipop-eroxide number with the formation of dienes, lipidhydroperoxide, cyclic endoperoxide and malondialde-hyde (MDA) (7).

The difficulty in measuring oxidative stress withhistochemical, spectrophotometric or proteomic andgenomic assays and its aetiopathogenic link with path-

ological conditions are among the reasons that have holdback research and development in this field so far.Therefore recently, new practical and reliable instru-ments have been set up. They only require a blood dropor a sample of urine or saliva and are able to provide ageneric value of the patients oxidative stress quantifyinghis amount of free radicals. Furthermore, several othertests, which are not found in the literature, are availableon the market. An example is given by the BioxTestOraxx (Bioxvita Diagnostics, Getvital HealthcareGmbH, Klosterweg, BiliesKastel, Germany), a commer-cially available colorimetric test able to measure theincrease in free radicals through the analysis of urinespecimens. This test is based on the ability of pararos-

aniline, a triphenylmethanol derivative, to bind thealdehyde compounds normally found in the urines,leaving a coloration that can vary between pink andviolet, but its reliability has not been proven yet.Recently, a new non-invasive instrument (FRAS 4EVOLVO Free radical system; H&D, Parma, Italy;Fig. 1) has been introduced in the clinical setting. Itanalyses a sample of saliva giving the grade of oxidativestress in the oral cavity, and it can be useful for the studyof particular pathological conditions affecting this dis-trict. Everything has been achieved thanks to an instru-ment that allows the measurement of the antioxidantconcentration starting from a simple saliva specimen.

The patient receives a cotton swab that has to be rolledfor a minute in his mouth to stimulate salivation. Theswab is then weighed inside a glass and subsequentlysqueezed out to allow the collection of saliva. In a cuvettecontaining R1 reagent, 40 ll of the reactive reagent R2 isadded, and after shaking the cuvette for 10 s, the blankreading is obtained. After that, 10 ll of the previouslycollected saliva is withdrawn and introduced into thecuvette containing the two reagents, and after agitation,it is inserted in the instrument to beread. At the end ofthereading, the instrument prints the result directly, andafter that, it is confronted with the reference values. Withthis simple procedure, it is possible to give a comparative

diagnosis of the current pathological condition in which

the role of free radicals has been identified. It involves thepathogenesis with pre- and post-medical or surgicaltherapy references and protects the oral cavity healthand, in a more general sense, the patient.

This article provides an update of the literatureconcerning the association of oxidative stress withpathological conditions associated with the oral cavity.It includes the pathologies associated with otolaryngol-ogy and the ones related to the orocervicofacialdistrict, focusing on the diagnostic and therapeuticimportance of the tests based on saliva specimens in apreventive perspective.

Searching criteriaA PubMed search was performed by using the followingkey words (separately or combined): antioxidant,oxidative stress, free radical, dentistry, periodontal,saliva and oral cavity. Selected papers, includingreviews, were chosen on the basis of their content(quality and novelty). The main focus was on freeradical species and antioxidants in relation to the oralcavity.

Salivary physiology in the redox balance

A consistent number of recent studies have showed how

oxidative stress can cause pathologies affecting theperiodontium, although the periodontitis triggeringevent is due to the presence of bacteria forming abiofilm in the subgingival plaque. However, the diseaseprogression would be due to the immune system adverseresponse to these organisms that, through the polymor-phonucleated leucocytes, causes an acute inflammationreleasing ROS (8). The resulting disequilibrium betweenthe ROS generated in this process and the existingantioxidants leads to a situation of oxidative stresscausing pathological conditions at the level of theperiodontium, but also other diseases, such as rheuma-toid arthritis (9), acute respiratory distress syndrome

Figure 1 FRAS 4 EVOLVO, H&D, Parma, Italy Free RadicalAnalytical System used for antioxidant identification in saliva speci-mens.

Role of free radical and antioxidant defences in oral cavity

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(10), AIDS (11), Alzheimers disease (12) and Parkin-sons disease (13).

Several studies show how free radical levels increase insaliva and in crevicular gingival fluid (CGF) resulting in afailure to keep their protective function within the oralcavity.Saliva is the biological liquidof the oral cavity thatis generated for the most part, i.e. 65%, by thesubmandibular glands, for the 20% by parotid, for the78% by the sublingual glands and in a low percentage byminor salivary glands localized in various mucosal sites ofthe oral cavity. Saliva production is around 11.5 l daily(14). Water is the most abundant component of saliva(99%), followed by a variety of electrolytes includingsodium, potassium, calcium, magnesium, bicarbonateand phosphate. In saliva, there are several inorganiccompounds, that is, urea, ammonia, uric acid, glucose,cholesterol, fatty acids, mono-, di- and triglycerides,glycolipids,amino acids,hormones, steroids and proteins,and they all exert a key role in the maintenance of the oralcavity protection or health. They include mucins, amy-lases, agglutinins, glycoproteins, lysozyme, peroxidase,

lactoferrin and secretory IgAs (15). All these substancesplay an important role in the function of saliva:

1 Bicarbonates, phosphates and urea modulate pH;2 Proteins and mucins act as disinfectant and play

an important preventive role against micro-organ-isms;

3 Calcium, phosphate and proteins are all involved inthe modulation of tooth mineral integrity;

4 Immunoglobulins and enzymes contribute to thebactericidal action, in particular lysozyme, which isable to lyse the Gram-positive bacterial wallcatalysing the beta 1, four bond hydrolysis betweenN-acetylmuramic acid (NAM) and the N-acetyl-

glucosamine (NAG), which are the maincomponents of peptidoglycan.

Changes in the composition of these compounds maybe indicative of the patients health. Therefore, salivacan be used as a diagnostic tool, and its componentsmay be used as pathological markers (see Fig. 2).

Antioxidants, free radicals and odontostoma-tology-related diseases

Antioxidant defensive systems also play an importantrole in the oral cavity. Among them, salivary peroxidase(SPO) and myeloperoxidase (MPO) represent the two

most important ones (16). SPO catalyses thiocyanate ion(SCN)) peroxidation to generate oxidation products,such as O2SCN), O3SCN), (SCN)2, HOSCN andOSCN) that inhibit the growth and the metabolism ofmany micro-organisms. MPO is able to catalyse CL)

with a consequent formation of hypochlorite (OCL)),a ROS that induces the cleavage of peptide bonds witha consequent formation of a low molecular weightpotential bactericide compound, that is, chloramine.According to several studies, the myeloperoxidase couldaccumulate while sleeping, when the salivary flux is low,i.e. between meals, removing the original products fromthe polymorphonuclear neutrophils.

Crevicular gingival fluid, an exudate of the periodon-tal tissue, which pours in the gingival sulcus passingthrough the sulcular epithelium, is similar to thecomposition of saliva and, in part, its possible expres-sion. Passing through the deepest tissues, it catches thesubproducts of the local metabolism. It is largelydemonstrated that variations in the type and quantityof several components of CGF, as its total antioxidantcapacity (TAOC), is an indicator of the periodontiumhealth status. This fact is supported by Chapple et al.

(8), who measured the CGF TAOC in 17 patients withslight chronic periodontitis and 18 patients with seriouschronic periodontitis, comparing them to 32 controls.This study showed that at baseline, the total antioxidantlevels were lower in patients affected by the disease ifcompared to healthy controls (680 371 lMteq in illpatients compared to 1129 722 lMteq observed inhealthy subjects), pointing out that this parameter iscompromised during the periodontium inflammation.TAOC levels can increase until they reach the onesfound in controls as a consequence of non-surgicaltreatment at the level of the periodontium. Anotherresearch group (17) also analysed free radical produc-

tion in the CGF level, stating that free radical produc-tion is involved in the pathophysiological progression ofperiodontal diseases because it might contribute tooxidative stress generation. The authors investigated therole of glutathione peroxidase (GPx), an antioxidantenzyme of lactoferrin and myeloperoxidase, i.e. twoglycoproteins that reflect the amount of oxidative stressand the role of interleukin 1 beta (IL-1b), a pro-inflammatory cytokine that plays a key role in theregulation of the inflammatory and immunitary reactionin the periodontium-associated diseases in humans.They collected CGF from 27 patients, aged 2462 years.Nineteen patients were affected by periodontitis, while

Cystic

Fibrosis

Sjgrens

Syndrome

Viral

Infections

Cardio

vascular

Diseases

Cancer

Bacterial

Infections

Alzheimers

Disease

Saliva

Figure 2 Pathologies in which saliva can be used for medicaldiagnosis.


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eight subjects were healthy and were used as controls.ELISA test was used for the purposes of this study.A significant increase in GPx, lactoferrin, myeloper-oxidase and IL-1b was observed in subjects affected bythe disease, if compared to healthy controls. Moreover,they were positively correlated to plaque and gingivalindices. The presence of these markers of oxidative stressis strictly correlated to the severity of the disease, and inparticular, the disequilibrium in the myeloperoxi-daseIL-1b levels and GPxlactoferrin could be theresult of the tissue damage previously generated by ROSand fuelled by the pathogens that were present in theoral cavity. Superoxide anion has also been studied inrelation to the disease affecting the oral cavity. A studyperformed by Krol (18) in patients affected by peri-odontitis aimed at determining the production ofsuperoxide anion in blood, measuring the reduction ofcytochrome c, identifying the effects of oxidative stressin patients with periodontal disease and analysing thelipid peroxidation and the degradation of DNA bases inperipheral and gingival blood. Moreover, this study

wanted to prove whether there was a correlationbetween superoxide anion production, lipid peroxida-tion, 8-hydroxy-deoxyguanosine (8-OHdG) concentra-tion and clinical parameters. The total antioxidantstatus in peripheral and gingival serum was also studiedand correlated with the periodontal clinical status;several periodontitis risk factors, related to oxidativestress, were evaluated too. Fifty-six patients, affected byuntreated periodontitis, and 25 healthy volunteers wereenrolled in this study. The results evidenced a negativecorrelation between cytochrome c reduction and theindex of periodontal disease, a positive correlationbetween oxidised anti-LDL autoantibodies in gingivalblood and the levels of 8-OHdG in venous blood.

Moreover, a negative correlation between the 8-OHdGconcentration in venous blood and the total antioxidantstatus in patients gingival blood and a positive corre-lation in healthy controls were observed. Increasedlevels of 8-OhdG in gingival blood were also observed,while significantly lower levels of total antioxidant statusin venous blood, from patients affected by periodontitisif compared to healthy controls, were reported. Therewas also a negative correlation between gingival blood8-OhdG concentration and the total antioxidant statusin venous blood serum from patients affected byperiodontitis, if compared to healthy controls. Thisstudy points out that oxidative stress in patients affected

by periodontitis is expressed by elevated concentrationsof ROS and associated with a suppression of gingivalblood antioxidant activity and may contribute to a rapidformation of lesions in periodontal tissues. In 2002, forthe first time, Takane et al. (19) evaluated the levels of8-OHdG in saliva of patients affected by periodontitis,before and after periodontal treatment, to demonstratethat there is a strong connection between the presence ofthis molecule in their saliva and periodontal healthstatus. Seventy-eight patients with periodontitis and 17healthy controls participated in this study and 8-OhdGsaliva levels were detected by immunoenzymatic essay.The mean value of 8-OhdG levels in subjects affected by

periodontitis increased significantly if compared to thevalue of healthy subjects (4.28 0.10 ngml and1.56 0.10 ngml, respectively), and a decrease in thisparameter was observed after a therapy, concluding that8-OHdG levels in saliva are proportional to theperiodontium health status. Another study (20) alsoevaluated pro-inflammatory and oxidative stress mark-ers in gingival tissues from patients affected by chronicperiodontitis. Eighteen subjects were divided into twogroups: an experimental one (age: 52.9 5.0) and acontrol one (age: 51.1 9.6). For both groups, cata-lase, GPx, glutathione S-transferase, glutathione reduc-tase (GR), total glutathione, redox glutathione,glutathione oxidase, thiobarbituric acid-reactive sub-stances and myeloperoxidase were evaluated. This studyshowed that, in subjects affected by periodontitis, therewas a significant increase in gingival tissues concerningmyeloperoxidase, GPx, glutathione S-transferase, oxi-dized glutathione and thiobarbituric acid-reactive sub-stances if compared to the control group, showing acorrelation between oxidative stress and periodontitis.

Therefore, research on antioxidant total power in salivaand serum of patients affected by pathologies of theperiodontium is a useful approach to determine aconnection between disease and oxidative stress, high-lighting that the disequilibrium between ROS andantioxidants, in favour of ROS, leads to the productionof free radicals and a defective antioxidant activity ofsaliva. This evidence was also supported by a studyperformed by Diab-Ladki et al. (21) who compared theantioxidant activity of saliva from 17 patients affectedby periodontal disease, requiring a dental treatment, tothe one from 20 healthy controls. The results showedthat healthy subjects saliva was more efficient (4050%)in the removal of in vitro-generated free radicals. In

patients affected by periodontitis, saliva antioxidantactivity decreased significantly, although this fact didnot interfere with the levels of the three main tracedantioxidants, namely uric acid, albumin and ascorbicacid (AA). Another study (3) focused on the evaluationof TOC in serum samples from 30 subjects withclinically diagnosed periodontitis and 30 healthy con-trols using spectrophotometric quantification. A meanantioxidant value of 28.5 lgdl 4.6 was observed inpatients affected by periodontitis, and this value almostdoubled in control subjects (50.6 lgdl 3.1), showingthat the antioxidant ability decreases with periodontitisand suggesting a negative correlation with the peri-

odontal parameters.During the past few years, in the odontostomatolog-ical and oropharyngeal fields, several studies haveaddressed the issue of peculiar oral diseases related toa direct damage caused by an excess in free radicalproduction. The literature shows how MDA is one ofthe most studied products of polyunsaturated fatty acidperoxidation, which increases in an oxidative stressdependant manner. In a recent study (22), MDA levelsand total oxidative stress (TOS) in serum, saliva andGCF have been measured from 36 patients (19 men and17 women, aged between 32 and 55, with mean age40.66 5.31) affected by chronic periodontitis and


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from 28 healthy controls (13 men and 15 women, agedbetween 29 and 51 with mean age of 38.5 6.10). Dataanalysis showed that MDA values in saliva and in CGFand TOS values in serum, saliva and CGF weresignificantly higher in the group of patients affected bythe disease if compared to the control group, suggestingthat lipid peroxidation plays an important role in theperiodontal pathology. However, no significant differ-ences in serum MDA levels between the two groups wereobserved. It is noteworthy that MDA and TOS concen-trations appeared to be lower in saliva and higher ingingival crevicular fluid.

Mitochondrial DNA also represents an excellentbiomarker of oxidative stress and its possible deletions,in gingival tissues of patients affected by periodontitis,were studied by Canakci et al. (23). In this study, bloodand gingival tissue samples were collected from 30patients with chronic periodontitis and 30 healthy

controls. Possible mitochondrial DNA deletions, corre-sponding to 5 and 7.4 kbp, were analysed through PCR.No deletions were observed in the two groups of bloodsamples and in the mitochondrial DNA, correspondingto 7.4 kbp. In the mitochondrial DNA, deletions,corresponding to 5 kbp, were found in 24 ill subjects[on a total of 30 patients (80%)], while no deletions werefound in healthy subjects, showing that the ROSoverproduction can cause oxidative damage at thislevel. The studies concerning antioxidants, free radicalsand odontostomatology-related disease are summarizedin Table 1.

Oxidative stress and oral cavity tumours

On a worldwide scale, the tumours of the oral cavity,together with the ones associated with the larynxand pharynx, represent nearly 10% of all malignant

Table 1 Antioxidants, free radicals and odontostomatology-related diseases

Author Patients Methods ResultsTakane et al. (19) 78 patients with

untreated periodontitisand 17 healthy controls

Saliva samples were collected to evaluate8-OHdG production and eventualchanges after periodontal treatment

8-OHdG levels in subjects affected byperiodontitis were significantly increased ifcompared to healthy subjects. A decrease in8-OHdG levels was observed after treatment

Diab-Ladki et al. (21) 17 patients affected byperiodontal diseaserequiring a dentaltreatment and 20healthy controls

Saliva TAOC in removing free radicalslike xanthinexanthine oxidase systemwas determined

Uric acid, albumin and ascorbic acid weremeasured in saliva specimens

In healthy subjects, saliva was more efficient inthe removal of free radicals. In patients affectedby periodontitis, saliva antioxidant activitydecreased significantly although it did notinterfere with the production of uric acid,albumin and ascorbic acid

Wei et al. (17) 19 patients affected byperiodontitis and 8healthy controls

CGF levels of glutathione peroxidase,lactoferrin, myeloperoxidase and IL-1bwere analysed by ELISA test

A significant increase in glutathione peroxidase,lactoferrin, myeloperoxidase and IL-1beta wasobserved in subjects affected by periodontitis ifcompared to healthy controls

Krol (18) 56 patients affected by

periodontitis and 25healthy controls

The production of superoxide anion was

determined in peripheral and gingivalblood

TAOC status in peripheral and gingivalserum was also studied and correlatedwith periodontal clinical status

Increased levels of 8-OHdG in gingival blood

were observed in patients affected byperiodontitis. On the other hand, significantlower levels of TAOC status in venous bloodwere observed in these patients

Canakci et al. (23) 30 patients with chronicperiodontitis and 30healthy controls

Mitochondrial DNA was analyzed inblood and gingival tissue samplesto investigate its deletions

In mitochondrial DNA, deletions, correspondingto 5 kbp were found in 24 ill subjects (80%),while no deletions were found in healthysubjects

Chapple et al. (8) 17 patients with slightchronic periodontitis,18 patients with seriouschronic periodontitisand 32 controls

TAOC of CGF and plasma before andafter a non-surgical therapy wasevaluated

At baseline, CGF TAOC was lower in patientsaffected by disease

Periodontal therapy did not alter plasma TAOC,but CGF TAOC increased in patients withperiodontitis

Borges et al. (20) 18 subjects divided into2 groups: experimentalgroup including patients

with chronic periodontitisand control group

Catalase, glutathione peroxidase,glutathione S-transferase, glutathionereductase, total glutathione and reduced

glutathione, oxidized glutathione,thiobarbituric acid-reactive substancesand myeloperoxidase activity wereevaluated in gingival tissues

In subjects with periodontitis, there was asignificant increase in gingival tissues concerningmyeloperoxidase, glutathione peroxidase,

glutathione S-transferase, oxidized glutathioneand thiobarbituric acid-reactive substances

Akalin et al. (22) 36 patients affected bychronic periodontitisand 28 healthy controls

MDA levels and TOS in serum, saliva andCGF were measured

MDA values in saliva and in CGF and TOSvalues in serum, saliva and CGF were higher inpatients affected by periodontitis

No significant differences in serum MDA levelswere observed in both groups

Pendyala et al. (3) 30 subjects withclinically diagnosedperiodontitis and 30healthy controls

Serum TAOC was evaluated using aspectrophotometric quantification

In the control group, antioxidant values doubledif compared with patients affected by periodon-titis

TAOC, total antioxidant capacity.


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neoplasia in men and 4% in women. In Italy, every year,4500 cases of oral cavity tumours are diagnosed and3000 deaths are registered. Several studies have inves-tigated the presence of free radicals in this area todetermine a possible correlation between free radicalsand neoplasia incidence. As far as tongue carcinoma isconcerned (5% of incidence among all malignanttumours worldwide and 0.43% incidence in the worldspopulation), a potential correlation with a high index ofoxidative stress and a weak antioxidant ability has beenreported (24). This type of neoplasia, very frequent inIndia, has been selected in 60 patients affected bysquamous cell carcinoma (stadium IIIIV) comparedwith a group of 60 healthy controls to evaluate serumlipid peroxide, glutathione reductase (GSH), conjugateddienes, vitamin C and E, GPx and superoxide dismutase(SOD). Elevated levels of lipids, peroxides and dieneswere observed in serum of patients affected by this typeof carcinoma, while low levels of antioxidants weredetected. According to this study, we could suggest touse therapeutic and prophylactic supplementation with

vitamins and nutraceuticals, while the measurement offree radicals and the spontaneous antioxidant individualprofile might represent a predictive value of risk. Fiaschiet al. (25) compared the intratumoral and haematiclevels of GSH, AA and antioxidant enzymes in thetumour tissue affected by squamous cell carcinoma ofthe mouth to better understand the genotoxic andmutagen role of free radicals. Therefore, a study wasdeveloped to compare 18 patients affected by squamouscell carcinoma and 20 healthy controls comparing thelevels of GSH, AA, SOD, GPx and GR. The patients,affected by mouth cancer, had increased levels of GR,GPx, GSH and AA and decreased levels of SOD in thetumour tissue and in the surrounding healthy mucosa.

On the other hand, the same values of GSH, GPx, GR,AA and SOD had significantly decreased in blood frompatients with cancer if compared to healthy subjects.This interesting observation let us assume that theantioxidant defences are confined in the oral district totackle the neoplastic degeneration, although, in thisway, an antioxidant reserve is built in the tumour cellsand it could paradoxically protect them from necrosisand apoptosis because of oxidative stress.

The hypothesis of a programmed and therapeutic useof antioxidant compounds to reduce the frequency oforal tumour malignancy has still to be confirmed.Another study (26) evaluated plasma levels of thiobar-

bituric acid-reactive compounds (TBARS), vitamin E,GSH, SOD activity, catalase (CAT) and GPx. Thisstudy involved 48 patients, affected by different stages oforal cancer, who were divided into groups of 16 patientsat the II, III and IV stages and compared to 16 healthycontrols of the same age and sex. In this study, anincreased lipid peroxidation and a decrease in anti-oxidant enzymes were reported. Ma et al. (27) studiedthe formation of DNA damage in the oral epitheliumof 19 patients affected by oral leukoplakia usingdouble-labelled immunofluorescent immunohistochemistry.They reported that in the leucoplastic tissue, as well asin the dysplastic epithelium and with surrounding

phlogistic reaction, the accumulation of 8-nitroguanine,which represents DNA damage caused by nitric oxide,and of 8-oxo-7,8-dihydro-2-deoxyguanosine, which isanother marker of oxidative stress, induced DNAdamage.

None of these immunoreactions resulted positive innormal mucosa, while the nitric oxide synthetase, anenzyme that promotes the damage, was present at thesame time in leucoplastic tissue. Moreover, the histo-chemical examination showed the presence of nuclearproliferating antigen and p53 gene.

According to the authors, the increased nitrosylationlinked to the phlogistic state results in any case jointlyresponsible for the disproliferative event, and as such, itis subject to adjustment in a prophylactic manner. Inneoplastic diseases affecting other districts, the salivaryprofile also plays a key role allowing the achievement ofa better quality of life. Therefore, Avivi et al. (28)realized a study, on the damage induced by oralmucositis, in 25 patients affected by myeloma andtreated with melphalan at high doses to undergo

autologous bone marrow transplantation. Among thetests used to establish the oral mucositis level, after3 and 7 days from the transplantation, they measuredsecretory IgA and TAOC, while the damage to themucosa was measured by albumin and carbonyl assay.The iatrogenic mucositis, measured at day 3 and 7 post-transplantation, showed significant reductions in thesecretory IgA, and even more significant reductions inantioxidant levels, TAOC and uric acid levels. The sumof these investigations is without any doubt a usefulmeasure of iatrogenic damage, and it suggests the needto start an antioxidant therapy as a protective measure.The human studies related to oxidative stress and oralcavity tumours are summarized in Table 2.

Oxidative stress and various pathologies of theoral cavity

The pathology of the oral cavity also affects theWaldeyer ring corresponding to the adenotonsil tissue.On this subject, two studies described the relationshipbetween hypertrophy and pathology of the lymphatictissue and free radicals. Dogruer et al. (29) studied 29children affected by adenotonsillar hypertrophy com-pared to 51 age-matched controls. All the patientsshowed rhonchus and obstructive sleep apnoea andunderwent adenotonsillectomy under general anaesthe-

sia. Venous blood, withdrawn at baseline and 4 weekspost-surgery, was used to measure the levels of MDA,SOD, GSHPx and CAT. The results showed that thelevels of enzymes and MDA were very high pre-operatively, but they normalized post-operatively. Thelevels of catalase did not show any variations pre- andpost-operatively. This fact shows that there is animbalance between oxidation and antioxidation inyoung patients affected by adenotonsillar hypertrophyand surgery is able to restore the systemic balance.Kiroglu et al. (30) designed a study in 20 childrenaffected by chronic adenotonsillitis and 19 childrenwith adenotonsillar hypertrophy who underwent


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adenotonsillectomy. MDA pre-operative levels andserum and erythrocyte catalase of both compounds attonsillar and adenoidal levels were studied. All thechecked parameters were significantly higher thanexpected, showing that the two pathologies share thesame pathological mechanism and confirming the studypreviously described. Yilmaz et al. (31) also examined38 children with chronic tonsillitis and adenoid

hypertrophy, who were undergoing adenotonsillectomy,and compared them to 23 healthy controls. Levels ofcirculating retinol, b-carotene, alpha tocopherol,lycopene, AA, SOD, GSHx and peroxidation productand MDA, before and 1 month post-operatively, weremeasured. The same assay was used on the previouslycollected adenoid-tonsillar tissue. In the group ofpatients affected by pathology, an increased level ofantioxidants and a reduction in the oxidative damage,comparing pre- and post-operative values, wereobserved, although they never reached the healthysubjects balanced equilibrium. According to theauthors, the oxidative stress imbalance represents a

peculiar feature of the orolymphatic perioral pathologyin paediatric population, and it does not get back tonormal even after the elimination of pathological tissue.

Zhang et al. (32) conducted a study to detect andquantify DNA damage in human oral mucosa cells from25 smokers and 25 healthy controls. This study used amonoclonal antibody specific from MDA-DNA adductsand showed a significant difference in the raising of the

DNA damage. This method appears to be useful todetect MDA-DNA adducts, which could represent animportant biomarker of DNA damage. A study per-formed by Yamamoto et al. (33) showed how in rats, fedon a high-cholesterol diet, the topical application ofphlogogen compounds (lipopolysaccharides and prote-ases) in the gingival sulcus augmented the effect of dieton steatosis, inflammation and oxidative damage in theirliver. This phenomenon was measured quantifying thelevels of hexanol-lysine, a value that is a symptom ofsteatosis and inflammation and the concentration of8-hydroxydeoxyguanosine in the hepatic parenchyma.These values were significantly elevated and associated

Table 2 Oxidative stress and oral cavity tumours

Author Patients Methods Results

Fiaschi et al. (25) 18 patients affected bysquamous cell carcinoma ofthe mouth and 20 healthycontrols

Antioxidant levels were estimated intumour tissue and blood samples

Patients affected by squamous cellcarcinoma of the mouth showed anincrease in GR, GPx, GSH and AAvalues and a decrease in SOD activity intumour tissue and surrounding healthy

mucosa. The same values weresignificantly lower in the blood frompatients with cancer if compared tohealthy subjects

Manoharan et al. (26) 48 patients, affected by differentstages of oral cancer and 16healthy controls

Plasma levels of TBARS, vitamin E andGSH, and activity of SOD, CAT andGPx were assayed

Elevated lipid peroxidation and decline innon-enzymatic and enzymaticantioxidant status were noticed inpatients with oral cancer if compared tohealthy subjects. The TBARS levels hadgradually increased, whereasantioxidants were gradually reducedfrom stage II to stage IV of patients withoral cancer

Ma et al. (27) Biopsy specimens of 19 patientsaffected by leukoplakia andnormal oral mucosa (NM)obtained from 4 specimens

The formation of 8-nitroguanine and8-oxo-7,8-dihydro-2-deoxyguanosinewas investigated withimmunohistochemistry methods

Mutagenic 8-nitroguanine and8-oxo-7,8-dihydro-2-deoxyguanosineproduction was observed in oralepithelium of patients with leukoplakia.Little or no immunoreactivity wasobserved in normal oral mucosa

Avivi et al. (28) 25 patients affected bymyeloma treated withmelphalan followed byautologous SCT were enrolledto evaluate alterations in thenormal salivary composition

Salivary samples were collectedpost-transplantation to analyse secretoryIgA and antioxidant capacity

The degree of mucosal damage wasassessed by measuring the salivarycarbonyl and albumin levels

After transplantation, there was asignificant reduction in secretory IgA,TAOC and uric acid levels.

The increase in salivary Alb and carbonyllevels indicates mucosal and oxidativedamage

Sharma et al. (24) 60 patients affected byadvanced squamous cellcarcinoma of the tongueand 60 healthy controls

Single blood samples were taken to assesslipid peroxides, conjugated dienes, GSH,vitamin C and E, GPx and SODalterations

Elevated levels of lipids, peroxides anddienes and low levels of antioxidantswere detected in serum of patients withcancer

Rai et al. (43) Patients with oral leukoplakia,oral submucous fibrosis orlichen planus, and healthyindividuals

Salivary and serum oxidative markers,such as MDA, 8-OHdG and vitamins Cand E were measured just prior to theintake of curcumin, a pigment with

antioxidant, anti-inflammatory andpro-apoptotic activity

After a week from curcumin intake, serumand saliva vitamin C and E levelsincreased, while MDA and 8-OHdGdecreased in patients affected by

leukoplakia

GR, glutathione reductase; GPx, glutathione peroxidase; MDA, malondialdehyde; SOD, superoxide dismutase; TAOC, total antioxidant capacity.


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with the hepatic damage morphologically observed ifcompared to rats put on a normal diet, and alsodisplaying oxidative damage in the oral cavity andhepatic parenchyma with less serious morph-functionalconsequences. The steatosic phlogistic damage, conse-quently strengthened by an atherogenic diet at a highlevel of lipid oxidation, can lead to a real systemicdamage if this experimental observation will be con-firmed in clinical studies involving human subjects. Asto tooth motility and reabsorption of deciduous teeth,relevant from the orthodontic point of view, there areexperimental studies that show a correlation betweenosteoplasty or readsorption and manganese-dependentSOD activity and lipoperoxides (34). The orthodonticmotility of teeth (35) would be a function of the localactivity of nitric oxide promoted by the administrationofL-arginine in rodent studies. According to Poplawskiet al. (36), compounds, such as the urethane dimethac-rylate, used for orthodontic purposes, would determinethe cause of oxidative damage as they induce DNAdamage to the leucocytes in the oral mucosa that can be

counteracted by vitamin C and chitosan. In conclusion,toothpastes, according to Moore et al. (37), must betaken into consideration as a potential cause of lesionsto the soft tissues, as shown by experimental studies onkeratinocytes exposed to commonly available deter-gents. In particular, increasing concentrations wereevaluated in pluronic acid, showing that a partialoxidative damage exists in vivo and it is neutralized bysalivary flux. These investigations demonstrate theopportunity to regularly monitor the physiologicallevel of salivary antioxidants for homoeostasis espe-cially in the presence of exogenous insults, even if theyare introduced for hygienic sanitary purposes. Thehuman studies related to oxidative stress and various

pathologies affecting the oral cavity are summarized inTable 3.

Role of antioxidants in the oral cavity

Antioxidants, according to the definition given in 1989by Halliwell and Gutteridge, are substances which slowdown the rate at which something decays because ofoxidization. This definition includes both non-enzymatic and enzymatic compounds. The endogenousenzymatic antioxidants are represented by SOD,catalases and glutathione system. The SODs are a classof ubiquitous enzymes that efficaciously catalyse the

rupture of the superoxide anion in oxygen and hydrogenperoxide. In mammals, three different enzyme isoformswere identified. SOD1 was found in cytoplasm, innuclear compartments and in lysosomes, and it containsRa and Zn in its catalytic centre. SOD2, which exists asa homotetramer, presents Mn as cofactor, and it hasbeen localized at mitochondrial level and has beenobserved to play a role in the promotion of cellulardifferentiation and tumorigenesis and the protection ofpulmonary toxicity induced by hyperoxia. FinallySOD3, which is also a homotetramer, was found inextracellular fluids like human plasma, lymphatic fluidand cerebrospinal fluid (38). The catalase is an enzyme

that belongs to the class of oxidoreductase, and itcatalyses the following reaction:

2 H2O2 !O2 2 H2O

It is a tetramer that is constituted by four polypeptidicchains highly compacted which cause an unusualstability of this enzyme. Inside the tetramer, there are4 Fe groups that allow the reaction of the enzyme withhydrogen peroxide: it is therefore degraded to twomolecules of water and one of oxygen.

The glutathione system consists of GPx and glutathi-one S-transferase. This system is found in animals,plants and micro-organisms. The glutathione is atripeptide that is soluble in water and synthesizedstarting from glutamate amino acids, cysteine andglycine. Glutathione is one of the most importantantioxidants that are present at cellular level. It playsa key role in the detoxification of a variety of electro-philic compounds and peroxides through reactionscatalysed by GST and GST (39).

It has been observed that cells lacking in this enzymesuffer from oxidative stress with a consequent degener-ation at mitochondrial level. The tripeptide can existintracellularly in GSSG and GSH. The GPx is anenzyme that, as SOD and catalase, has the function toprotect the cell from the damage provoked by ROS. Itdetoxificates peroxides acting as an electron donor in thereduction reaction producing GSSG as a final product.The expression of this enzyme is induced by oxidativestress, and one of its aberrant expression has beenassociated with a variety of pathologies such as hepa-titis, HIV, skin, kidney, breast and intestine cancer. TheGST is a member of the family of enzymes that arecoevoluted with glutathione system. It catalyses the

conjugation of the glutathione to exogenous andendogenous electrophilic compounds including areneoxides, unsaturated carbonyls and organic acids (40).Many compounds that induce GST expression are oftensubstrates of the enzyme, suggesting that the inductionrepresents a mechanism of adaptive response. Instead,among the non-enzymatic antioxidants, we can findseveral compounds like vitamin C, vitamin E, vitaminA, carotenoids and polyphenols. L-AA or vitamin C is ahydrosoluble vitamin that plays numerous functions,including the antioxidant function, in the humanorganism. It is a compound that is very hydrosoluble,highly acidic and appears in the form of odourless and

tasteless crystals with a pH nearly 2.5. Vitamin C has astrong reducing action due to the presence of an enediolgroup; in particular, it is able to donate an electronforming semidehydroascorbic acid, which donates asecond electron generating dehydroascorbic acid. Theredox potential of these reactions is as follows: semiasc-orbicAA 0.28 V and dehydroascorbic acidsemi-hydroascorbic acid )0.17 V. This fact makes vitaminC an effective electron donor. Finally, dehydroascorbicacid is reduced by dehydroascorbate reductase regener-ating AA.

Vitamin E is made up of a group of eight naturalisoforms like alpha, beta, gamma and delta tocopherol


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and alpha, beta, gamma and delta tocotrienols, whichare known to display antitumoral properties because oftheir antioxidant features. Among these isoforms, alphatocopherol is a more potent form of vitamin E, and ithas a high biological and nutritional value. Tocopherolscannot be synthesized in humans and animals; they mustbe taken with the diet through vegetable oils such as soy,maize and sesame oil; they can also be found in cottonseeds and in hazelnuts. The tocotrienols are mostlypresent in palm oil (41).

Carotenoids are a class of organic pigments that canbe found in plants or photosynthetic organisms like

algae and some bacterial species. We know more than600 varieties of carotenoids, and they are normallydivided into two classes: carotenes, which are hydrocar-bons lacking in oxygen, and xanthophylls, which con-tain oxygen. They are accessorial pigments that duringthe photosynthetic process allow absorbtion of wave-lengths different from chlorophyll, protecting the latterfrom photo-oxidation. They protect against the effectsof unpaired oxygen and form ROS generated in thepresence of sunlight.

The typical colour of carotenoids, which goes frompale yellow to orange until bright red, is a direct

Table 3 Oxidative stress and various pathologies affecting the oral cavity

Author Patients Methods Results

Zhang et al. (32) 25 smokers (range 2030cigarettesday) and 25race-, sex- and age-matchednon-smokers

Oral mucosa cells were collected toevaluate DNA damage measuringMDA

DNA damage had increased in smokers ifcompared with non-smokers

Dogruer et al. (29) 29 children affected by

adenotonsillar hypertrophywho underwentadenotonsillectomy and 51age-matched controls

MDA, SOD, GSHPx and CAT levels

and activity were analysed in venousblood specimen

MDA levels and SOD-GSHPx activity

were significantly higher in thepre-tonsillectomy period than in thepost-tonsillectomy period, while CATactivity was not different pre- andpost-operation

Yilmaz et al. (31) 38 children with chronictonsillitis and adenoidhypertrophy who underwentadenotonsillectomy and 23healthy controls

Circulating retinol, beta-carotene,alpha-tocopherol, lycopene, ascorbicacid, SOD, GSH and MDA levelswere measured before and 1 monthafter operation

An increased level of antioxidants and areduction in oxidative damage,comparing pre- and post-operativevalues, were observed followingadenotonsillectomy

Kiroglu et al. (30) 20 children affected by chronicadenotonsillitis and 19children with adenotonsillarhypertrophy who underwentadenotonsillectomy

Pre-operative blood levels oferythrocyte and serum MDA andCAT, and adenoidal and tonsillartissue levels of MDA and CAT werestudied

All parameters were significantly higherthan expected, showing that the twopathologies share the same pathologicalmechanism

Ergun et al. (46) 21 patients affected by orallichen planus and 20 healthycontrols

TAOC and MDA were measured inserum and saliva

Salivary MDA was significantly higher,while serum TAOC was significantlylower in patients affected by oral lichenplanus if compared to healthy controls.A significant correlation between serumand saliva TAOC values was observed inpatients with oral lichen planus andcontrol subjects. A significant correlationbetween serum and saliva MDA values incontrol group was reported. A significantinverse correlation between salivaryMDA and TAOC values was observed inthe control group

Agha-Hosseini et al. (47) 30 patients affected by orallichen planus and 30 healthycontrols

Salivary MDA and TAOC levels wereassayed by thiobarbituric acid andferric-reducing antioxidant potential(FRAP) respectively in both groups

Higher levels of salivary MDA wereobserved in patients with oral lichenplanus

Upadhyay et al. (48) 22 patients affected by oral

lichen planus, 10 patients withoral lichenoid reaction and 15healthy controls

Protein thiol oxidation, MDA and

TAOC were estimated in both thegroups and compared with that ofnormal subjects

Serum MDA levels were significantly

higher if compared to healthy controls.Serum thiol levels were significantlylower in patients with oral lichen planusand oral lichenoid reaction if comparedto controls. Therefore, these studiespoint out that oxidative stress may play akey role in pathologies affecting the oralmucosa such as oral lichen planus andoral lichenoid reaction. Both salivary andserum MDA have increased in patientswith oral lichen planus confirmingits importance as an oxidative stressbiomarker

MDA, malondialdehyde; SOD, superoxide dismutase; TAOC, total antioxidant capacity.


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consequence of the molecular structure of these com-pounds. In fact, the polymeric chains that compoundthem are characterized by the presence of double bondsthat interact among them, allowing the electrons of theatoms involved to move more freely. As soon as thedouble bonds in the chain increase, there is also anincrease in the freedom of electron movement. This factallows the light spectrum, absorbed from these mole-cules, to decrease and, as a consequence, the reflectedlight wavelength increases appearing of a colour vergingon red (42).

Polyphenols are a family of around 5000 organicmolecules that are largely present in the vegetablekingdom. As meant by their name, they are character-ized by the presence of several phenolic groupsorganized in more or less complex structures, generallycharacterized by a high molecular weight. These com-pounds are the product of plant secondary metabolism.Polyphenols are natural antioxidants present in plants,and they can prevent oxidation of lipoproteins and exertpositive biomedical effects at cardiovascular level and in

diseases related to ageing and tumoral growth.The problem associated with the polyphenol intake

with diet is their low bioavailability. In particular, theyare found in bodily fluids in a non-native form, but asmetabolites (sulphates, methylates and glucoronates)after an extensive metabolization in the intestine andliver.

A recent study, performed in Belgium (43) in 2010,has examined the potential antioxidant, anti-inflamma-tory and pro-apoptotic effects of curcumin, a yellowpigment extracted from its plant, in subjects affected bytumours of the oral cavity like leukoplakia and submu-cous fibrosis, and by oral lichen planus. Twenty-fivepatients with oral leukoplakia (13 men, 12 women), 25

patients with oral submucous fibrosis (11 men, 14women), 25 patients with oral lichen planus (12 menand 13 women) and 25 healthy subjects, aged between 17and 50 years, were selected. Consequently, oxidativestress markers, present in saliva and serum, weremeasured. MDA was measured using TBARS accordingto the methods described by Buege and Aust (44),8-OhdG according to the method described byToyokuni (45) and vitamin C and E with liquidchromatography. The measures were executed beforecurcumin intake after a week from curcumin treatmentafter clinical cure for pre-cancerous lesions and after209 days. The values of vitamin C and E, measured in

serum and saliva, increased, while MDA and 8-OHdGdecreased after a week from curcumin intake ifcompared to the values before treatment in all the illpatients. The values were particularly significant afterthe cure of the disease, and in particular, the lesionentity improved as for the lesion size. Therefore, theseresults suggest that curcumin significantly increases theantioxidant local and systemic status and vitamin C andE levels, while lipid peroxidation and DNA damagedecrease in patients with pre-cancerous lesions, showingthat the anti-pre-cancerous effects of curcumin aremediated by pro-oxidative and antioxidative pathways.A study by Ergun et al. (46), involving 21 patients

affected by oral lichen planus (11 men, 10 women),showed significantly higher salivary MDA (z = 2.24,P = 0.03), while serum TAOC was significantly lower(z = 2.48, P = 0.01) if compared to healthy controls(n = 20; 11 men, nine women). This study also showeda significant correlation between serum and salivaTAOC values in patients with oral lichen planus(r = 0.714 and P = 0.0001) and control subjects(r = 0.69 and P = 0.001) and between serum andsaliva MDA values in control group (r = 0.464 andP = 0.04). A significant inverse correlation (r = )0.598and P = 0.005) between salivary MDA and TAOCvalues was observed in control group. The higher levelsof salivary MDA in patients affected by oral lichenplanus were also confirmed by another study involving30 patients affected by the same disease (15 men and 15women (47). Another study (48), involving 22 patientsaffected by oral lichen planus and 10 patients with orallichenoid reaction, also showed that serum MDA levelswere significantly higher in patients affected by thesepathologies if compared to healthy controls (n = 15;

P < 0.001). Serum thiol levels were also significantlylower in patients affected by oral lichen planus and orallichenoid reaction if compared to controls(P < 0.0047). These studies point out that oxidativestress may play a key role in pathologies affecting theoral mucosa such as oral lichen planus and orallichenoid reaction. Both salivary and serum MDA haveincreased in patients affected by oral lichen planusconfirming their importance as oxidative stress biomar-kers. The finding that serum thiol levels have decreasedin these patients is also of interest because plasma thiolsare considered key oxidative targets (49), furtherstrengthening the importance of oxidative stress in thesepathologies.

Conclusions

This literary excursus is meant to draw the clinical-scientific readerships attention to the role that thedental surgeon, the otolaryngologist and generally thephysician can play in an early biochemical and clinicaldiagnosis intended to characterize the diseases that arethe object of a specialized study.

In summary, this review points out the key role playedby oxidative stress in pathologies concerning the oralcavity. For instance, salivary 8-OHdG levels signifi-cantly increase in periodontitis, while they decrease after

treatment, strongly underlying the key role played bythis oxidized nucleoside of DNA in this condition (19).This finding is supported by another study showingincreased levels of 8-OHdG in periodontitis, although adifferent specimen was analysed, that is, gingival blood(18). Mitochondrial DNA deletions have also beendetected in subjects affected by chronic periodontitis(23), a condition that is also characterized by a decreaseat baseline TAOC values which, on the other hand,increase after therapy (8). Furthermore, in subjectsaffected by periodontitis, a significant increase in mye-loperoxidase, GPx, glutathione S-transferase, oxidizedglutathione and thiobarbituric acid-reactive substances


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has been reported in gingival tissue (20). Saliva, CGFand serum MDA levels, and saliva and CGF TOS valueswere also higher in patients affected by this condition(22). Another interesting feature of this condition is thesignificant increase in GPx, lactoferrin and myeloperox-idase, as reported by Wei et al. (17). In the setting ofperiodontal disease, saliva antioxidant activity decreasessignificantly, if compared to healthy peers (21). Anotherstudy showed how serum TAOC levels double inpatients affected by periodontitis, if compared tocontrols (3).

As to cancer affecting the oral cavity, in patients withsquamous cell carcinoma, GR, GPx, GSH and AAvalues had increased, while a decrease in SOD activitywas observed in tumour tissue and surrounding healthymucosa (25). An increase in oxidative stress in thiscondition was also confirmed by another research groupwho reported elevated levels of lipids, peroxides anddienes and low levels of antioxidants in serum frompatients with squamous cell carcinoma (24). In patientsaffected by oral cancer, lipid peroxidation increased, and

a decrease in non-enzymatic and enzymatic antioxidantstatus was reported, further supporting a key role ofoxidative stress in this condition (26). Patients affectedby myeloma present an increase in salivary Alb andcarbonyl levels, which are an indicator of mucosal andoxidative damage. Following transplantation, a signif-icant reduction in salivary secretory IgA, TAOC anduric acid levels was observed (28). Two studies alsoinvestigated the role of oxidative stress in leukoplakia.Mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2-de-oxyguanosine production was observed in oral epithe-lium of patients affected by this pathology (27).Curcumin intake promoted serum and saliva vitaminC and E increase, while MDA and 8-OHdG decreased in

patients with oral leukoplakia, oral submucous fibrosisor lichen planus, suggesting that an antioxidant treat-ment may be useful to face these conditions or as asupportive therapy (43).

It is also interesting that adenotonsillectomy results ina decrease in MDA levels and SOD-GSHPx activity inpatients affected by chronic tonsillitis and adenoidhypertrophy (31). Adenotonsillitis and adenotonsillarhypertrophy are pathologies sharing an increase inblood levels of erythrocyte and serum MDA andCAT, and adenoidal and tonsillar tissue levels ofMDA and CAT that decrease following adenotonsillec-tomy (30).

Oxidative stress has also been investigated in thecontext of oral lichen planus. For instance, salivaryMDA was significantly higher, while serum TAOCvalues were significantly lower in patients affected bythis condition. Furthermore, the same study showed asignificant correlation between serum and saliva TAOCvalues in patients with oral lichen planus and controlsubjects. A significant correlation between serum andsaliva MDA values in control group was reported (46).Another study also reported higher levels of salivaryMDA in patients affected by oral lichen planus (47).This evidence was also confirmed by Upadhyay et al.(48), who also reported the same finding in patients

affected by oral lichenoid reaction. Furthermore, thesame study showed that serum thiol levels were signif-icantly lower in patients with oral lichen planus and orallichenoid reaction if compared to controls. Buildingupon these foundations, these findings strongly under-line how oxidative stress may be involved in both orallichen planus and oral lichenoid reaction.

The syndromes or the diseases, induced or aggravatedby oxidative stress, can be evaluated at the right time,using corrective, instrumental, pharmaceutical or nutra-ceutical treatments able to support the primary treat-ment, namely antibiotics, anti-inflammatories, steroidsor being themselves the primary corrective substratumfor a particular pathology.

In our experience, the values of free radicals andantioxidants in saliva do not always match with thevalues found in peripheral blood, showing a diagnosticand selective specificity of these diagnostic parameters inrelation to pathologies affecting the oral cavity. Thismakes the method even more important in the dentalsurgeons hands. With the ex juvantibus experience of

antioxidant products already existing, and the onescoming shortly, there is a sound hope to get long-lastingrecoveries and more efficacy in technical-odontoiatricresults and prevent the development of leukoplakias andtumours that critically afflict each specialists clinicalexperience.

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Acknowledgements

The authors contributed equally to this work. This article was not

supported by grants.

Authors contribution

The authors hereby certify that all work contained in this review is original.

The authors claim full responsibility for the contents of the article.

Conflict of interest

The authors certify that there is no conflict of interest with any financial

organization regarding the material discussed in the manuscript.


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