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Expression of N-acetyltransferases in Periodontal Granulation Tissue

¹ Department of Pharmacology,
² Department of Anatomy, and
³ Department of Human Genetics, Ernst Moritz Arndt University Greifswald, F.-Loeffler-Str. 23d, D-17487 Greifswald; and
⁴ Dental Clinics, Department of Periodontology, Rotgerberstr. 8, D-17487 Greifswald, Germany;

*corresponding author, meiselp{at}uni-greifswald.de

	ABSTRACT

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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

	INTRODUCTION

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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.

	MATERIALS & METHODS

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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)
For RNA extraction, the specimens were treated with lysis buffer containing mercaptoethanol (RLT buffer, Qiagen, Hilden, Germany), frozen in liquid nitrogen, and stored at -80°C. Isolation of RNA was performed by means of the RNeasy-Kit (Qiagen) following the protocol supplied by the manufacturer. Additionally, the samples were treated with proteinase K and DNase to improve the yield and to avoid contamination by genomic DNA. RT-PCR was carried out with the Omniscript-Kit (Qiagen), with oligo-dT as primers. Since NAT1 and NAT2 show high DNA sequence homologies (87%), specific primers (Table) were selected (Ilett et al., 1994; Macé et al., 1998). Amplification of GAPDH was performed for internal standardization. To exclude the co-amplification of genomic DNA, we performed a control PCR using a primer positioned outside the reading frame. With prevailing sequence homology, the possibility was to exclude NAT1 primer binding at NAT2 cDNA and vice versa. Therefore, to verify the identity of either NAT1 or NAT2 products, we purified the cDNA amplification products (QIAquick PCR-Purification Kit, [Qiagen]) and cloned them using the pGEM^®-T Easy vector (Promega, Madison, WI, USA). After transformation in competent cells (Epicurian Coli^® XL 10 Gold cells^®, Stratagene, La Jolla, CA, USA), DNA fragments were cut from the isolated plasmids with use of the restriction endonuclease EcoRI and sequenced by dideoxy methodology with the ALF DNA Sequencer.

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).

	RESULTS

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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.

View larger version (35K): [in this window] [in a new window]   Figure 1. NAT expression in granulation tissue. (A) RT-PCR products representing mRNA: N1-NAT1, N2-NAT2, G-GAPDH, each individual in three lanes from left to right. M, length standards; L, liver; arrows indicate approx. molecular weight in kDa. No. 64 – male, age 37, non-smoker, moderate periodontitis. No. 69 – recall. No. 65 – male, age 34, non-smoker, advanced periodontitis. No. 70 – recall. No. 76 – female, age 36, non-smoker, advanced periodontitis. No. 77 – male, age 45, 20 cigarettes/day, moderate periodontitis. (B) Western blots of NAT2 in gingival granulation tissues (lanes): No. 1 – female, age 36, non-smoker, advanced periodontitis; No. 2 – female, age 56, non-smoker, moderate periodontitis; No. 95 – female, age 17, non-smoker, local early-onset periodontitis (EOP); No. 97 – male, age 34, 20 cigarettes/day, advanced periodontitis.

Western Blotting
NAT2 proteins were identified in all the dissected tissue specimens by Western blotting (Fig. 1b). Positive control reactions were obtained with samples from human liver, which is well-known as an NAT2-expressing tissue. The NAT2-specific bands had an apparent molecular weight of approximately 31 kDa. Non-specific bands were observed in both granulation tissue and liver. For NAT1, only faint bands were detected. Thus, for NAT1 in this test, the reaction remained uncertain.

Immunohistochemistry
Infiltration of the gingiva by inflammatory cells was shown by immunohistochemistry with the use of antibodies recognizing cell-specific markers. In all patients, the connective tissue was infiltrated by B- and T-lymphocytes, as shown by staining with antibodies against CD79a or CD3. Macrophages (CD68) were localized in the connective tissue as well as in epithelial layers. Neutrophils were distributed throughout the tissue—in some cases, in epithelial structures.

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). Though less perceptible, NAT1 was detected likewise within the connective tissue (fibroblasts), and in epithelial cells. In normal gingiva, epithelial calls were stained, and NAT1 was detectable also in endothelial cells. No reaction could be observed in sections incubated with the pre-immune serum of the rabbits from which the antibodies were raised.

View larger version (89K): [in this window] [in a new window]   Figure 2. Immunohistochemical detection of N-acetyltransferases NAT1 (left column, A-C-E) and NAT2 (right column, B-D-F) in inflamed (top, A + B) or normal (middle, B + C) gingival tissue, control with pre-immune serum (bottom, E + F). Arrows indicate endothelial cells; bars represent 50 µm.

	DISCUSSION

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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.

	ACKNOWLEDGMENTS

 
We thank Mrs. Ingrid Geissler and Mrs. Marlis Bansemir for their skillful technical assistance.

Received June 14, 2001; Last revision February 8, 2002; Accepted February 26, 2002

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