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Preliminary Evaluation of Human Gingiva As an Extrapineal Site of Dentistry Section Melatonin Biosynthesis in States of Periodontal Health and Disease

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Preliminary Evaluation of Human Gingiva As an Extrapineal Site of Dentistry Section Melatonin Biosynthesis in States of Periodontal Health and Disease DOI: 10.7860/JCDR/2018/32451.11078 Experimental Research Preliminary Evaluation of Human Gingiva as an Extrapineal Site of Dentistry Section Melatonin Biosynthesis in States of Periodontal Health and Disease BALAJI THODUR MADAPUSI1, SURESH RANGA RAO2 ABSTRACT Melatonin Receptor 2 (MT2). Further flow cytometry experiments Introduction: Melatonin is a pineal gland hormone that plays were carried out in freshly obtained gingival tissue samples to an important role in periodontal homeostasis. Extra pineal quantify melatonin receptors. Immunohistochemistry experiment melatonin production has been found to occur in tissues like was performed on paraffin embedded tissue sections to study the ovaries, retina and gastrointestinal tract. It is not known the qualitative distribution of melatonin receptors. The results if the human gingiva could synthesise melatonin and whether were documented and analysed using SPSS software version melatonin receptors are present in the gingival tissues. 23.0. Aim: To investigate if human gingiva is an extrapineal site of Results: The current study showed that the human gingival melatonin biosynthesis in non smokers and current smokers tissues are an extrapineal site of melatonin biosynthesis. RT- with and without chronic generalised periodontitis. PCR, flow cytometry and immunohistochemistry techniques revealed that MT1 receptor was present in the human gingiva. Materials and Methods: The present study has a case control Furthermore statistical analysis revealed that the expression of design with a total of 60 participants divided into four groups AANAT, HIOMT and MT1 genes and the MT1 receptor protein namely, Periodontally Healthy Non smokers (PHN)-Group 1, Non levels were significantly lower in current smokers with and smokers with Chronic generalised Periodontitis (NCP)-Group without chronic generalised periodontitis compared to non 2, Periodontally Healthy current Smokers (PHS)-Group 3, and smokers. current Smokers with Chronic generalised Periodontits (SCP)- Group 4. Gingival tissue samples were obtained from the study Conclusion: It can be concluded that the gingiva could participants after obtaining informed consent, using an aseptic synthesise melatonin as a defense mechanism to protect protocol. Reverse Transcriptase Polymerase Chain Reaction against oxidative stress and inflammation. Habits like smoking (RT-PCR) was carried out to assess the presence of the genes can reduce the levels of gingival melatonin and MT 1 receptor encoding N-Acetyl Transferase (AANAT), Hydroxyindolyl-O- expression which could reduce the cytoprotective benefits of Methyltransferase (HIOMT), Melatonin Receptor 1 (MT1) and melatonin. Keywords: Antioxidant, Chronic periodontitis, Flow cytometry, Immunohistochemistry INTRODUCTION [14]. With this background, the present study was performed to The human gingiva is an integral part of the oral mucosa and forms the analyse if the gingiva could synthesise melatonin and inturn function soft tissue casing for the periodontal attachment apparatus. The human as an extrapineal site of melatonin production. The current study was gingiva has been identified as target tissue for synthesis, biochemical performed in non smokers and current smokers with and without transformation and action of hormones like oestrogen, progesterone, chronic generalised periodontitis to assess if the genes coding the testosterone and cortisol [1-4]. In recent times, another hormone, enzymes AANAT and HIOMT, the melatonin receptors MT1 and MT2 melatonin has been projected to play a significant role in periodontal are present in the gingival tissues. Current smokers were chosen as homeostasis and pathology. Melatonin, chemically denoted as smoking is a condition associated with oxidative stress and depletion N-acetyl-5-methoxytryptamine is synthesised predominantly by the of antioxidants such as melatonin. It was hence, planned to assess pinealocytes, the resident cells of the pineal gland during the dark phase the status of melatonin biosynthesis in this state. It is mandatory that of the day. A complex biochemical cascade operates to synthesise a tissue must have receptor for a particular drug/hormone to consider melatonin from its precursor tryptophan. The enzymes involved are it as a target tissue. Hence, a quantification of melatonin receptors tryptophan-5-hydroxylase, 5-hydroxytryptophan decarboxylase, and immunolocalisation was also carried out in the gingival tissue AANAT and HIOMT [5]. Melatonin performs numerous functions in the samples to analyse if the gingiva is a target for receptor mediated human body including free radical scavenging, immunomodulation, actions of melatonin in the above mentioned states. inhibition of carcinogenesis and stimulation of bone formation [6-9]. Extra pineal production of melatonin has been documented in other MATERIALS AND METHODS tissues such as the ovaries, retina and gastrointestinal tract [10- 12]. In the oral cavity, the salivary glands are known to possess the Study Design and Patient Selection biochemical machinery for melatonin biosynthesis [13]. A previous This Case control study was approved by the Institutional Ethics study showed the presence of melatonin in gingival tissue samples Committee of Sri Ramachandra University, Chennai and performed obtained from healthy individuals and chronic periodontitis patients between June 2016 to June 2017. A total of 60 volunteers were Journal of Clinical and Diagnostic Research. 2018 Jan, Vol-12(1): ZF01-ZF07 1 Balaji Thodur Madapusi and Suresh Ranga Rao, The Human Gingiva is an Extrapineal Site of Melatonin Biosynthesis www.jcdr.net recruited after taking written informed consent and allocated into were quantified using the Eppendorf Bio Spectrophotometer four groups namely, PHN-Group 1, NCP-Group 2, PHS-Group 3, and stored at −80°C until Complimentary Deoxyribonucleic Acid and current SCP-Group 4. The sample size was calculated by the (cDNA) conversion. A total of 1 µg of RNA was taken for the first statistical expert with support of relevant literature [12]. Based on the strand cDNA synthesis with the verso cDNA synthesis kit (ABgene) true probability of exposure a sample size of 15 volunteers per group using manufacturers protocol. The resultant cDNA samples were was arrived at, to reject the null hypothesis with a type 1 error of 5% quantified using the Eppendorf Bio Spectrophotometer and stored and a power of 80%. The PHN group comprised of 15 non smokers in −20°C until PCR amplification. A total of 250-500 ng of cDNA was who had clinically healthy gingiva without the signs of inflammation and used for PCR amplification. A 2X red dye mastermix from ampliquon bleeding on probing. The NCP group comprised of 15 non smokers was used for the reaction. The PCR reaction was carried out with with Chronic generalised periodontitis with at least the presence of the following conditions-Denaturation of 94°C for 30 seconds, 10 natural teeth who manifested clinical signs of gingival inflammation primer annealing for 30 seconds at temperature indicated in [Table/ and periodontal pockets with attachment loss in greater than 30% of Fig-1]. Extension step of 72°C for 30 seconds. PCR was carried the intraoral sites examined with radiographic evidence of bone loss out with 35 cycles for all genes. A 2% agarose gel was cast in tris [15]. The PHS group comprised of 15 cigarette smokers who were acetate EDTA buffer and 5-10 µL of the PCR reaction product was currently smoking and had smoked at least 100 cigarettes in their run at 100 volts for 45 minutes. A 1000 basepair ladder was also lifetime with clinically healthy gingiva without the signs of inflammation run with every gel to verify if the amplified fragments were of the and Bleeding On Probing (BOP) [16]. The SCP group comprised of right size. GAPDH was used as the house keeping gene for RTPCR 15 cigarette smokers who were currently smoking and had at least analysis. The bands were visualised using safe dye stain in a UV smoked 100 cigarettes in their lifetime with the clinical signs of chronic generalised periodontitis as described above. The general exclusion An- Prod- criteria for all groups in the present study were the presence of any GENE Primers nealing uct Temp size systemic disease, consumption of antibiotics, anti-inflammatory GAPDH Forward 5’-ACCACAGTCCATGCCATC-3’ 58°C 452 bp drugs, melatonin and antioxidant supplements in the past six months Reverse 5’-TCCACCACCCTGTTGCTG-3’ before the study. Patients with previous history of periodontal therapy, ANAAT Forward 5’- TGCCAGTGAGTTTCGCTGCCTC-3’ 58.6°C 242 bp pregnant and lactating women were also excluded from the study. Reverse 5’ACCTGTGCAGCGTCAGTGACTC-3’ HIOMT Forward - 5’ CCTGAAGCTGCTGAAAGTGG 3’ 57°C 371 bp Clinical Parameters Reverse 5’ GGACCTGTAGATGGCCGTAA 3’ All subjects were examined clinically for the following periodontal MT1 Forward 5’ CATCCTGTCGGTGTATCGGA 3’ 60°C 295 bp parameters: Plaque Index (PI) calculated by measuring the presence Reverse 5’ CGATGCCGGTGATGTTGAAT 3’ or absence of supragingival plaque biofilm around the buccal, mesial, MT2 Forward - 5’ GTGATCCTCTCCGTGCTCAG 3’ 58°C 273 bp distal and lingual regions of all teeth [17], gingival bleeding (BOP) Reverse 5’ GCCGGTAGATTCGGTGGTAG 3’ calculated as percentage positive sites that bleed upon probing in six [Table/Fig-1]: Primer sequences, annealing temperature and PCR product size of the GAPDH, AANAT, HIOMT, MT1 and MT2 genes. sites per tooth [18], probing pocket depth and Clinical Attachment PCR-Polymerase Chain Reaction, GAPDH-Glyceraldehyde-3-Phosphate
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