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Expression of N-acetyltransferases in Periodontal Granulation Tissue
1 Department of Pharmacology, Correspondence: *corresponding author, meiselp{at}uni-greifswald.de
Smoking is a major risk of periodontal diseases. At the site of first contact, the gingiva is exposed to aromatic amines and polycyclic hydrocarbons. These are metabolized by the N-acetyltransferases (NAT), leading to local detoxification and/or activation reactions contributing to the risk of periodontal destruction in smokers. The purpose of this study was to detect the expression of N-acetyltransferase isoenzymes NAT1 and NAT2 in periodontal granulation tissue. In 24 specimens obtained from periodontitis patients or control subjects, mRNA encoding for NAT1 and NAT2 was detected by RT-PCR, and proteins were identified by immunohistochemistry. In periodontal granulation tissues, immunoreactivity for NAT1 and NAT2 was detected in infiltrating leukocytes and fibroblasts. In normal gingiva, both enzymes were found in epithelial cells, whereas NAT1 was also detected in endothelial cells. The results suggest that these enzymes may play a role in the defense against xenobiotics and the accelerated progression of periodontal disease in smokers.
Key Words: N-acetyltransferase • NAT1 • NAT2 • expression • periodontitis • gingiva
Periodontal disease is an infectious, chronic inflammatory disease characterized by loss of connective tissue attachment and alveolar bone in association with the formation of a pocket. It may eventually lead to tooth loss. It is well-known that smoking is one of the major environmental risk factors for a wide variety of diseases, including periodontal disease. The association between tobacco smoke and the prevalence and severity of periodontal diseases and subsequent tooth loss was shown in several epidemiological studies (for review, see Kinane and Chestnutt, 2000). Several toxic heterocyclic amines have been identified in cigarette smoke. Acetylation of these substances catalyzed by arylamine N-acetyltransferases (NAT) is a prerequisite for either the elimination of or for activation to carcinogenic compounds, depending on the tissue-specific expression of these enzymes. The role of N-acetyltransferases in environmentally related cancer risk has been widely studied (Hein et al., 2000). There are two active N-acetyltransferase isoenzymes, NAT1 and NAT2, characterized by different substrate specificities. Both enzymes are expressed polymorphically in humans, leading to different phenotypes generally designated as "slow" or "rapid" acetylators. The two human isoenzymes NAT1 and NAT2 show different tissue expression. While human NAT2 was found mainly in liver and intestine, human NAT1 is expressed in many tissues, including erythrocytes, bladder, lymphocytes, and neural tissue, as well as liver and intestine (Ward et al., 1995; Macé et al., 1998). The heterogeneity of NAT1 and NAT2 mRNA expression may reflect the organ-specific toxicity of various arylamine drugs and carcinogens (Debiec-Rychter et al., 1999; Windmill et al., 2000). Recently, we reported in a case-control study that individuals who smoked and expressed a slow acetylation phenotype had more periodontal breakdown than non-smokers or rapid acetylators, regardless of their smoking status (Meisel et al., 2000; Kocher et al., 2002). We hypothesized that smoking and slow-acetylating individuals could be more prone to periodontal disease due to their impaired capacity to eliminate smoke-derived xenobiotics such as toxic heterocyclic amines. The mechanisms by which NATs could influence the extent and severity of periodontal disease, however, are currently unknown. Since NAT2 is expressed mainly in the liver, it seems unlikely that liver metabolism of xenobiotics could affect the natural course of periodontitis, contributing to the inflammatory response to bacterial pathogens in the gingiva. Alternatively, localization of the enzymes at the site of first contact with cigarette smoke could explain the higher risk of smoking in slow-acetylating subjects suffering from periodontal diseases. In the present study, by RT-PCR, Western blotting, and immunohistochemistry, we demonstrate the expression of both NAT1 and NAT2 in the lymphocyte-infiltrated gingival tissue of patients suffering from severe periodontitis.
Seventy-three periodontally diseased patients, previously untreated (mean age, 49.6 yrs [34–65], 43 females, 30 males), were selected from subjects referred to the Unit of Periodontology, University of Greifswald. All subjects gave their written informed consent, and the study was approved by the local ethics committee. Subjects displayed periodontal disease with moderate to severe bone loss; otherwise they were healthy. In three initial treatment sessions, the patients were instructed in efficient supragingival plaque control, and subgingivial scaling was carried out while the subjects were under local anesthesia. If, after 3 mos, pockets were still present with probing depth  6 mm, flap surgery was carried out. Following an intrasulcular incision and reflection of a mucoperiosteal flap, the periodontal granulation tissue was removed and the tooth surface instrumented. Control specimens were obtained from subjects undergoing tooth extraction because of caries (probing depth < 3 mm, interdental tissue used). The excised specimens were then treated as required for subsequent studies. From the 73 patients, 103 samples were obtained, of which 24 provided material sufficient for final analyses.
mRNA Expression (RT-PCR)
Immunological Techniques Polyclonal antisera against NAT1 and NAT2 were raised in rabbits. Animals were immunized with antigens produced from specific peptide sequences: NAT1, LEAIFDQVVRR; and NAT2, PEGVYCLVGFILTY (Affina Immuntechnik, Berlin, Germany), providing antigenicity scores of 1.136 and 1.222, respectively. For cell differentiation, monoclonal antibodies were used: mouse anti-human neutrophil elastase (Chemicon International, Inc., Temecula, CA, USA), mouse anti-human CD68 macrophage (DAKO, Carpinteria, CA, USA), mouse anti-human B-cell CD79a (DAKO, Glostrup, Denmark), and rabbit anti-human T-cell CD3 (DAKO). Homogenates of the tissue specimens were separated by electrophoresis (SDS-PAGE). NAT1 and NAT2 proteins were detected by Western blotting with the antisera mentioned. Alkaline-phosphatase-conjugated anti-rabbit IgG was used as a secondary antibody. Binding was visualized with the BCIP/NBT system, BCIP being the substrate (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium). Tissue specimens were fixed in buffered formalin (3.5%) for 12 hrs, dehydrated, and embedded in paraffin according to standard protocols. Sections 6 µm thick were mounted on glass slides (SuperFrost, Menzel Braunschweig, Germany) and de-paraffinized by means of xylene and a series of ethanols. Endogenous peroxidase activity was blocked prior to immunohistochemistry by incubation in 0.3% H2O2/methanol. After being washed in PBS, slides were subjected to 10 mmol/L citrate buffer and incubated in a microwave oven (20 min, 700 W). Immunohistochemistry was done with the use of the Vecastain-PO-Kit (Vector Laboratories, Burlingame, CA, USA). In brief, blocking (20 min) was followed by incubation with primary antibodies (NAT1, NAT2, CD79a, 1:50; neutrophil elastase, CD68, CD3, 1:100) overnight at 4°C. After incubation with secondary antibody (30 min, 22°C) and being washed in PBS, slides were subjected to ABC reagent (45 min). Visualization was done after slides were washed with 0.1% diamino-benzidine in PBS/0.1% H2O2. Control reactions to demonstrate specificity of antibody binding were done by omitting the primary antibody or incubation with the rabbit pre-immune serum. Slides were counterstained with hematoxylin (HE) and mounted in glycerol-gelatin (Merck Darmstadt, Germany).
mRNA Expression (RT-PCR) After extraction of mRNA and amplification of cDNA, products of NAT1 and GAPDH were identified in all specimens tested. In contrast, gene products of NAT2 were identified in most (N = 15) but not all of the samples studied so far (Fig. 1A  ). This difference was independent of the subjects' smoking behavior. In cases of positively detected NAT2 amplificates, the intensity of the bands was weaker compared with those of NAT1 and GAPDH.
To exclude the possibility that NAT2 primers had bound non-specifically to NAT1 sequences due to high sequence homologies, we purified and sequenced PCR products of two tissue specimens. The resulting DNA fragment sequence shows complete concordance with the NAT2 sequences from gene databases (accession number D90040; Ohsako and Deguchi, 1990).
Western Blotting
Immunohistochemistry
With both the NAT1- and NAT2-specific antiserum, cells infiltrating the connective tissue were stained in the inflamed specimens. Immune reactions were seen around leukocytes, sometimes also within the epithelial plasma (Fig. 2
In this study, we report for the first time the expression of N-acetyltransferases (NAT1 and NAT2) in the periodontal tissues of patients suffering from periodontitis. N-acetyltransferases are important enzymes in phase II metabolism of xenobiotics (Trepanier et al., 1997). There are two isoforms of the enzyme, NAT1 and NAT2, which are very differently expressed in various tissues. NAT2 expression is mostly tissue-specific; however, in some tissues, both enzymes may be expressed together (Hein et al., 2000). In situ hybridization experiments demonstrated mRNAs of NAT1 as well of NAT2 together in the epithelial cells of the colon. In the kidney, NAT1 is expressed only in proximal tubulus cells, and NAT2 in both proximal and distal cells (Debiec-Rychter et al., 1999). In contrast to the liver, with its expression of high activities of NAT2, in most other tissues the ubiquitous NAT1 exhibit the highest expression levels (Hickman et al., 1998; Kawakubo et al., 2000). Expression of both NAT1 and NAT2 mRNA was observed in liver, gastrointestinal tract tissues (esophagus, stomach, small intestine, and colon), urinary tract, bladder, and lung. Acetylation of exogenous substances by the enzymes is considered to accomplish de-toxification, mainly by N-acetylation. However, O-acetylation reactions by NATs are possible, which may also lead to substance activation producing cytotoxic by-products. Bladder cancer in persons exposed to arylamines is one of the best-studied examples (Hein et al., 1992). In periodontal diseases, smoking is one of the most important risk factors leading to increased severity, early onset, and augmented tooth loss. There exists controversy about the role of nicotine in the modulation of blood flow to the gingiva (Palmer et al., 1999). Arylamines are products of cigarette smoke and, when de-toxified insufficiently by N-acetyltransferases, may have undesirable effects on the gingiva. Therefore, differences in the expression of the isoenzymes as well as in activity differences by NAT2 polymorphisms may be relevant for the periodontal risk imposed by tobacco products. To our knowledge, there are no reports on the presence of N-acetyltransferases in the gingiva. Expression of these enzymes in the granulation tissue suggests that metabolism of toxic xenobiotics having first contact within the oral cavity may contribute to the pathogenesis of the disease. So far, these results support studies showing an association between polymorphism of NAT2 and adult periodontal disease (Meisel et al., 2000; Kocher et al., 2002). To date, possible pathways leading from smoke products to periodontal destruction via xenobiotic metabolism remain speculative. Due to the heterogeneous character of inflammatory tissue, the question arises as to which cell types are relevant for tissue activity. Positive staining reactions were observed in all specimens, especially in the pocket epithelial cells of the retepegs. In non-inflamed gingiva, retepegs and connective tissue papillae are lacking between the junctional epithelium and its underlying connective tissue. In contrast, retepegs are visible in most of the connective tissue sections studied, indicating the process of inflammation. Furthermore, expression in immune-competent cells attracted by the inflammatory process is possible. NAT1 is expressed in mononuclear leukocytes, monocytes, and neutrophils (Cribb et al., 1991). Here also, NAT2 immunoreactivity was shown in those cells, independent of the variation in acetylation of arylamine substrates by NAT2 which is attributable to NAT1 activity. Numbers and types of inflammatory cells and mediators are dependent on the extent of inflammation and thus may be the expression of toxic reactions to environmental stimuli (Seguir et al., 2000; Lappin et al., 2001). Substrate-dependent regulation of NAT activity was also reported (Butcher et al., 2000). Moreover, proportions of infiltrating inflammatory cells (B- and T-cells, macrophages) in granulation tissue reflect differences in the etiology and immunopathology of the disease (Lappin et al., 1999). Well-known instances of drug metabolizing activity leading to unwanted drug effects are the gingival hyperplasias induced by phenytoin, nifedipine, or cyclosporine. It was suggested that metabolism of these drugs by cytochromes P450 expressed in the gingiva causes this condition (Zhou et al., 1996). In cancer pathogenesis, interactions are known between cytochrome-activated cigarette smoke products (mainly cytochromes CYP1A1 and 1A2) and N-acetyltransferases (Badawi et al., 1996). Activation of polycyclic aromatic hydrocarbons from cigarette smoke by cytochromes and subsequent local de-toxification by N-acetyltransferases are possible. This could explain the association of the slow-acetylating phenotype among patients with more severe periodontal disease. The many different types of heterocyclic amines from tobacco smoke and from other sources (grilled meat, bacterial products) undergo different reaction pathways in which the N-acetyltransferases NAT1 and NAT2 participate in changing proportions (King et al., 2000). Moreover, oxidative pathways competing for NAT substrates (e.g., myeloperoxidase) may predominate in cases of low or absent NAT activities.
We thank Mrs. Ingrid Geissler and Mrs. Marlis Bansemir for their skillful technical assistance. Received for publication June 14, 2001. Revision received February 8, 2002. Accepted for publication February 26, 2002.
Journal of Dental Research, Vol. 81, No. 5,
349-353 (2002) This article has been cited by other articles:
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