Resumen de Effect of Low-Dose Doxycycline on Serum Oxidative Status, Gingival Antioxidant Levels, and Alveolar Bone Loss in Experimental Periodontitis in Rats
Background: Subantibiotic doses of doxycycline (low-dose doxycycline [LDD]) have been widely used in periodontal treatment for enzymatic inhibition and related anti-inflammatory properties. The aim of the present study is to verify the possible effects of LDD on oxidative stress in relation to periodontal attachment loss associated with ligature-induced experimental periodontal disease in rats.
Methods: Thirty female Wistar albino rats were divided into three study groups as follows: 1) control (C) rats; 2) rats with experimental periodontitis (PED); and 3) rats with PED that were treated with doxycycline (PED + LDD). PED was induced by placing ligatures around the cervix of the maxillary second molars for 21 days. The PED + LDD group was treated orally with doxycycline (6 mg/kg) for 21 days after the ligature was placed. After 21 days, the rats were euthanized, and samples of the right maxilla were defleshed and used for histologic and morphometric analyses. The gingival tissue of the left maxilla was used for the analysis of lipid peroxidation (malondialdehyde [MDA]) and antioxidant enzymes (catalase, glutathione peroxidase, and superoxide dismutase). Levels of serum total antioxidant status (TAS)/total oxidant status (TOS) and oxidative stress index (OSI) were also analyzed.
Results: Alveolar bone loss was significantly higher in the PED group compared with the PED + LDD and C groups (P <0.05). Doxycycline exhibited the most prominent inhibition on gingival tissue levels of MDA and antioxidant enzymes (P <0.05). Doxycycline also significantly reduced TOS and OSI levels (P <0.05) but increased the TAS level.
Conclusion: Doxycycline helps to prevent periodontal tissue breakdown by inhibiting local and systemic oxidative stress.
Periodontitis, a chronic inflammatory disorder of the periodontium in adults, is characterized by loss of connective tissue (CT) and bone around the teeth.1 Oral bacteria and their products (e.g., lipopolysaccharides [LPSs] and proteases) are responsible for the initiation of periodontitis. Conversely, the progression of periodontitis depends on the host responses to bacterial pathogens.2 It has been observed that the invading bacteria trigger the release of cytokines such as interleukin-1 and tumor necrosis factor-a, leading to elevated numbers and activity of polymorphonuclear cells (PMNs). In response to periodontal pathogens, PMNs release destructive reactive oxygen species (ROS),3,4 proteinases, and other factors.5,6 ROS generation in periodontal disease causes bone resorption and degradation of the CT and increases the activity of matrix metalloproteinases (MMPs), resulting in an imbalance.7 ROS is a collective term that includes oxygen-derived free radicals, such as the superoxide radical (O2-), hydroxyl radical (-OH), and nitric oxide radical species, and the non-radical derivatives of oxygen, such as hydrogen peroxide (H2O2) and hypochlorous acid, and they are produced within the cell in all aerobic organisms.6 In healthy organisms, protection against the harmful effects of ROS on cells is obtained by maintaining a balance between oxidants and antioxidants. In a balanced cell state, ROS are produced as a normal product of cellular metabolism, and the level of ROS can be stabilized by an antioxidant defense system, including enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). In a state of cellular imbalance, the levels of oxidants outweigh the levels of antioxidants. If a cell is exposed to more reactive oxygen compounds than it can instantly degrade, it is under oxidative stress.8 The presence of oxidative stress may be tested in one of three ways: 1) direct measurement of the ROS; 2) measurement of the resulting damage to biomolecules; and 3) detection of antioxidant levels.9,10 Directly measuring ROS might seem the preferred method, but many ROS are extremely unstable and difficult to measure directly. The measurement of the damage on proteins, DNA, RNA, lipids, or other biomolecules is preferred by many scientists. These assays include various measures of DNA oxidation, lipid peroxidation, and protein oxidation (protein carbonyls).11 The most commonly used criterion of oxidative stress is that based on the determination of the peroxidation products of lipids, particularly polyunsaturated fatty acid residues of phospholipids, which are susceptible to attack by ROS.12 Malondialdehyde (MDA) is one of the final products of the peroxidation of polyunsaturated fatty acids in cells. An increase in free radicals causes overproduction of MDA. Therefore, the MDA level is commonly known as a marker of oxidative stress and peroxidative tissue injury.13 Another assay is based on the measurement of the levels of specific antioxidant enzymes, which are intracellular ROS preventative enzymes: SOD, CAT, and GSH-Px.9,10 SOD reduces the highly reactive O2- to the less reactive H2O2, which is detoxified to water by CAT and GSH-Px. GSH-Px has an important role in the cellular defense against cytotoxic lipid peroxidation products.14 Miller et al.15 created a new test to measure the total antioxidant status, which is referred to as total antioxidant capacity. This is also named as total antioxidant activity,16 total antioxidant power,17 or total antioxidant status (TAS).18 The major advantage of this test is the possibility to measure the antioxidant capacity of all antioxidants in a biologic sample and not just the antioxidant capacity of a single compound or other synonyms. In recent years, for the evaluation of individual oxidative stress, tests of TAS and total oxidant status (TOS) have been developed by Erel,19,20 because the measurement of individual antioxidant molecules is not feasible and because the antioxidant effects of these molecules are additive.
Antibiotics have been shown to be helpful in periodontal treatment.21 Tetracyclines, including doxycycline and minocycline, have long been recognized as adjunctive antibiotics in periodontal treatment because of their effectiveness on Gram-negative anaerobic periodontopathogens, such as Aggregatibacter actinomycetemcomitans, which is commonly found in the subgingival plaque. Although initially attributed to their antimicrobial properties,22 the clinical efficacy of tetracyclines in periodontitis has been suggested recently to be attributable to their intrinsic anti-inflammatory activity.23 Thereupon, novel �low doses� or non-antibacterial formulations of tetracyclines, such as subantimicrobial-dose doxycycline (SDD) or low-dose doxycycline (LDD) as an adjunctive treatment of periodontal disease, have been approved by the US Food and Drug Administration and other national regulatory agencies in Canada and Europe. Several studies have shown that SDD suppresses host-derived MMPs in the periodontal lesion and thereby inhibits the pathologic degradation of various collagens, including types I, III, and IV, while preserving other constituents of the periodontal tissues (fibronectin, proteoglycan ground substance, elastin, and basement membranes) and inhibiting bone resorption.24-27 Furthermore, it has been found that SDD prevents complications of �regular-dose� doxycycline administration, such as the emergence of antibiotic-resistant microorganisms in the subgingival plaque.23,28 Besides inhibiting MMPs, doxycycline has antioxidant effects that may contribute to the beneficial effects shown in recent studies.29-31 Akamatsu et al.32 reported that doxycycline significantly reduced the levels of O2-, H2O2, and -OH in human neutrophils.
Recent medical and dental research has focused on the role of ROS, lipid peroxidation products, and antioxidant systems in the pathology of periodontitis. These studies have demonstrated that the level of ROS molecules increases in patients with periodontitis and that ROS molecules induce additional oxidative damage to the gingival tissue and periodontal ligaments and elicit osteoclastic bone resorption.6,33-46 However, to the best of the authors� knowledge, there is no published investigation that evaluated the effect of low doses of doxycycline on the oxidative/antioxidant status and the defense enzymes of oxidative stress together with alveolar bone loss in experimental periodontitis (PED) in rats.
In light of these literature findings, the present study aims to verify the possible effects of LDD on oxidative stress and the efficacy of LDD in reducing progressive alveolar bone loss and clinical attachment loss (AL) in ligature-induced experimental periodontal disease in Wistar rats.
