0 UNESP – Universidade Estadual Paulista Faculdade de Odontologia de Araraquara JOÃO PAULO STEFFENS EVIDÊNCIAS DA ASSOCIAÇÃO ENTRE TESTOSTERONA E DOENÇA PERIODONTAL NO SEXO MASCULINO Araraquara 2013 1 UNESP – Universidade Estadual Paulista Faculdade de Odontologia de Araraquara JOÃO PAULO STEFFENS EVIDÊNCIAS DA ASSOCIAÇÃO ENTRE TESTOSTERONA E DOENÇA PERIODONTAL NO SEXO MASCULINO Tese apresentada ao programa de Pós- Graduação em Odontologia - Área de Periodontia, da Faculdade de Odontologia de Araraquara, da Universidade Estadual Paulista para título de doutor em Odontologia. Orientador: Prof. Dr. Luis Carlos Spolidorio Coorientador: Prof. Dr. Carlos Rossa Jr. Araraquara 2013 2 JOÃO PAULO STEFFENS EVIDÊNCIAS DA ASSOCIAÇÃO ENTRE TESTOSTERONA E DOENÇA PERIODONTAL NO SEXO MASCULINO COMISSÃO JULGADORA TESE PARA OBTENÇÃO DO GRAU DE DOUTOR Presidente e Orientador: Prof. Dr. Luis Carlos Spolidorio 2o examinador: Profa. Dra. Estela Sasso Cerri 3o examinador: Prof. Dr. Hernandes Faustino de Carvalho 4o examinador: Prof. Dr. Francisco Humberto Nociti Junior 5o examinador: Prof. Dr. Giuseppe Alexandre Romito Araraquara, 20 de setembro de 2013. 3 DADOS CURRICULARES JOAO PAULO STEFFENS NASCIMENTO 21 de março de 1985 – Blumenau – Santa Catarina FILIAÇÃO Vilson Davi Steffens Miracélia Bernardina Duarte Steffens 2002 - 2005 Graduação em Odontologia Universidade Federal do Paraná 2006 - 2007 Especialização em Periodontia Universidade Positivo 2008 - 2009 Pós-Graduação em Odontologia – Nível de Mestrado Universidade Estadual de Ponta Grossa 2010 - 2013 Pós-Graduação em Odontologia – Nível de Doutorado Faculdade de Odontologia de Araraquara Universidade Estadual Paulista – UNESP 2010 - 2011 Especialização em Saúde da Família Universidade Federal de Santa Catarina 2012 - 2013 Pós-Graduação – Postdoctoral Fellow Department of Appied Oral Sciences The Forsyth Institute, Cambridge, MA, EUA 4 Dedico este trabalho, fruto de meu empenho e de um sonho iniciado há mais de 10 anos, a todas as pessoas que me incentivaram, apoiaram, e acreditaram em mim durante todo meu processo educacional. Não chegaria a lugar algum caminhando sozinho... 5 AGRADECIMENTOS Primeiramente, a DEUS, por infinitos motivos, mas principalmente por permitir tão frequentemente que a Sua vontade e os meus sonhos sejam coincidentes. Ao meu orientador, Prof. Dr. Luis Carlos Spolidorio , que não se contentou em ser somente um brilhante mentor acadêmico, mas também me ofereceu sua amizade e empenhou esforços na captação de recursos e realização deste trabalho. Ao meu coorientador, Prof. Dr. Carlos Rossa Jr , pela atenção e auxílios frequentes, indispensáveis desde a concepção do projeto até a redação dos manuscritos. Aos Pesquisadores Dr. Thomas E. Van Dyke e Dr. Alpdogan Kantarci , pela amizade, pela orientação, e por proporcionarem a experiência no exterior que tanto me engrandeceu pessoal e profissionalmente. A Jackeline Starr e Xiaoshan Wang pela colaboração nas análises estatísticas. À minha família , de origem e estendida, não tenho palavras para agradecer. Ao meu pai , minha mãe , minha irmã ... Aos meus tios e primos sempre presentes na minha vida... E aos amigos que elegi família , em especial aos da Família Simioni . A todos vocês, muito obrigado por acreditarem em mim e entenderem o sacrifício da distância e da ausência. 6 Aos Professores e funcionários envolvidos no Programa de Pós- Graduação em Odontologia , em especial a Mara Amaral, e aos Professores que me deram aulas , muito obrigado pela oportunidade e por compartilharem seu conhecimento! Aos Profs. Drs. Denise Spolidorio, Élcio Marcantonio Junior, Joni Cirelli, José Eduardo Cezar Sampaio, Raquel Scarel Caminaga, Adriana Marcantonio e Silvana Orrico , muito obrigado pelo aprendizado proporcionado! Aos Professores e funcionários das Disciplinas de Periodontia e Patologia , em especial a Silvana, da Secretaria do Departamento de Fisiologia e Patologia, onde realizei estágios em docência, agradeço a oportunidade e o auxílio constante. Aos alunos e colegas do Programa de Pós-Graduação em Odontologia , agradeço a convivência. Aos amigos que tive oportunidade de fazer nesta jornada, em especial aos que mais convivi, como Pablo Dallari Ramalho Lucas, Rubens Moreno e Telma Bedran, Rafael Molon e Érica Dorigatti D’Ávila, Shelon Souza e Matheus Bandeca, Leila Coimbra, Lívia Finoti, Livia Perussi, Jônatas Esteves, Marcell Medeiros e Giovana Anovazzi, Fernanda Rocha, Fausto Frizzera, João Antônio Chaves, entre outros, muito obrigado pela amizade! À Universidade Estadual Paulista “Júlio de Mesquita Filho”- UNESP , e à Faculdade de Odontologia de Araraquara , na pessoa de sua Diretora Profa. Dra. Andréia Affonso Barreto Montandon, como Instituições e também por todas as pessoas que nela trabalham, estudam ou frequentam, agradeço imensamente a oportunidade. 7 Ao Forsyth Institute , na pessoa do seu presidente, Dr. Philip Stashenko , e aos funcionários e colegas do Departament of Applied Oral Sciences , em especial a Ricardo e Flávia Teles, Hatice Hasturk, Bruno Herrera, Luciano e Tina Andrada, Francisco e Mônica Groppo, Çigdem Pasali, Bahar Eren Kuru, Maha Bahaman, Daniel Nguyen, Olivia Nguyen, Danielle Stephens, Ahmed Zarrough, Aggasit, Carla Cugini, Josephine Hirschfeld, Geisla Soares, Rafael De Oliveira Dias, Sabrina Zani, Panpan Wang, por compartilharem seu conhecimento, pela recepção, amizade e convivência na minha experiência no exterior. Aos estagiários que conviveram comigo e colaboraram na execução deste projeto: Yagmur Denis, Ali Murat Gali, Dehan Elcin, George Aoude, Shivani Shrivastava. Agradeço pela paciência e pela oportunidade de prática em orientação. Aos colegas do Grupo de Pesquisadores e Universitários Brasileiros em Boston (PUBBoston) agradeço pelo convívio e conhecimento compartilhado. A todos os meus antigos, mas sempre admirados, mestres em Periodontia com quem tive a oportunidade de conviver e aprender muito, em especial Profa. Dra. Marilia Compagnoni Martins, Profa. Dra. Tatiana Miranda Deliberador, Prof. Dr. Fábio André dos Santos e Prof. Dr. Gibson Luiz Pilatti , muito obrigado pelo incentivo e amizade. À Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP , pela concessão de bolsa de Doutorado (Processo FAPESP 2010/09658-0) e auxílio à pesquisa (Processo FAPESP 2010/12021-4). 8 Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), pela concessão de auxílio financeiro referente ao Edital Universal (Processo 470870/2011-7). À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), pela concessão de bolsa PDSE (Processo 5258-11-1). A todos que contribuíram , direta ou indiretamente, para a realização deste trabalho... MUITO OBRIGADO!!! 9 “Talvez não tenha conseguido fazer o meu melhor, mas lutei para que o melhor fosse feito. Não sou o que deveria ser; não sou o que irei ser. Mas, graças a Deus, não sou o que era antes.” (Martin Luther King) 10 Steffens JP. Evidências da associação entre testosterona e doença periodontal no sexo masculino [tese de Doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2013. RESUMO A hipótese deste trabalho é que hormônios sexuais participam da etiopatogenia da doença periodontal (DP). Diferentes níveis de evidência científica testaram esta hipótese, avaliando: i. associação entre hormônios sexuais e DP em homens; ii. a influência de níveis sub- e suprafisiológicos de testosterona (T) sobre a DP em ratos; iii. se este mecanismo de ação envolve respostas de osteoblastos e osteoclastos in vitro. Dados do III NHANES relacionados com diagnóstico de DP e mensuração hormonal em homens com 30+ anos foram analisados para correlacionar estas duas variáveis. Em ratos, níveis subfisiológicos foram alcançados através da orquiectomia e níveis suprafisiológicos pelo tratamento com T. Metade dos animais em cada grupo foi submetida à DP utilizando–se modelo de ligadura. In vitro, células RAW264.7 foram diferenciadas em osteoclastos na presença de T (1nM-1µM) e identificados por TRAP. Cultura primária de osteoblastos murinos foi utilizada para avaliar a expressão de osteocalcina, RANKL e OPG na presença de T. Em homens, altos níveis de T biodisponível e baixa razão estradiol:T se correlacionaram significativamente com DP. Em idosos, baixos níveis de AAG, metabólito da dihidrotestosterona, também apresentaram correlação significativa. Em ratos, níveis sub- e suprafisiológicos de T aumentaram significativamente a perda óssea e modularam a expressão de citocinas inflamatórias. In vitro, doses fisiológicas de testosterona preveniram a osteoclastogênese e diminuíram a expressão de osteocalcina, RANKL e razão RANKL:OPG por osteoblastos. Concluiu-se que a T modula a resposta do hospedeiro à DP no sexo masculino, regulando a diferenciação de osteoclastos direta e indiretamente (via osteoblastos). Palavra – chaves: Hormônios Esteroides Gonadais, Periodontite, Inflamação 11 Steffens JP. Evidence of the association between testosterone and periodontal disease in males [tese de Doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2013. ABSTRACT The hypothesis of this work is that sex hormones participate in the etiopathogenesis of periodontal disease (PD). Different levels of scientific evidence tested that hypothesis, evaluating: i. The association between sex hormones and PD in men; ii. The influence of sub- and supraphysiologic testosterone (T) levels on PD in rats; iii. If that mechanism of action involves osteoblast and osteoclast responses in vitro. Data from NHANES III related to diagnosis of PD and hormones measurement in 30+- year-old men were assessed to correlate those two variables. In rats, subphysiologic levels were obtained by orchiectomy and supraphysiolgic levels by T treatment. Half of the animals in each group received PD using a ligature model. In vitro, RAW264.7 cells were differentiated into osteoclastos in the presence of T (1nM-1µM) and identified by TRAP-staining. Murine osteoblast primary culture was used to evaluate the expression of osteocalcin, RANKL and OPG in the presence of T. In men, high levels of bioavailable T and low estradiol:T ratio significantly correlated with PD. In older men, low levels of AAG, a metabolite of dihydrotestosterone, also presented a significant correlation. In rats, low and high T levels significantly increased bone loss and modulated the expression of inflammatory cytokines. In vitro, physiologic T doses prevented osteoclastogenesis and decreased the expression of osteocalcin, RANKL and RANKL:OPG ratio produced by osteoblasts. We concluded that T modulates host response to PD in males, regulating osteoclast differentiation direct and indirectly (through osteoblasts). Keywords: Gonadal Steroid Hormones; Periodontitis; Inflammation 12 SUMÁRIO 1 INTRODUÇÃO...........................................................................................12 2 CAPÍTULOS...............................................................................................15 2.1 Capítulo 1: Sex hormones correlate with periodontitis in men: Results from NHANES III ....................................................................18 2.2 Capítulo 2: The effect of supra- and subphysiologic testosterone levels on ligature-induced bone loss in rats - A radiographic and histologic pilot study ……………………..42 2.3 Capítulo 3: The impact of testosterone on periodontal bone loss…. .. 51 2.4 Capítulo 4: Resolvin D2 ameliorates testosterone-derived downregulation of osteocalcin and osteoprotegerin on primary murine osteoblastos................................... .............79 2.5 Capítulo 5: Telomere length and its relationship with chronic diseases - New perspectives for periodontal research ……………101 3 CONCLUSÃO..........................................................................................109 REFERÊNCIAS........................................................................................111 APÊNDICE A – MATERIAL E MÉTODOS..............................................113 ANEXO A – APROVAÇÃO DO COMITÊ DE ÉTICA...............................120 ANEXO B – PERMISSÃO DAS REVISTAS CIENTÍFICAS....................121 12 INTRODUÇÃOINTRODUÇÃOINTRODUÇÃOINTRODUÇÃO 13 1 Introdução O envelhecimento populacional é observado como uma tendência em muitos países do mundo. No Brasil, o Instituto Brasileiro de Geografia e Estatística (IBGE) estimou que, para a próxima década, a taxa de crescimento da população de 0 a 24 anos seja negativa, enquanto a população com 75 anos ou mais aumentará em 3,81%, chegando a um crescimento de 7,43% entre 2030 e 20504. A transição demográfica, e consequente envelhecimento populacional, levam a uma transição epidemiológica, o que significa que a prevalência e incidência de doenças infecto- contagiosas decresce enquanto doenças crônicas se tornam mais frequentes. Dentre os vários fatores tempo-dependentes que podem desencadear diferentes doenças crônicas, a diminuição/supressão da produção de hormônios sexuais deverá constituir um importante problema, sendo que a mesma está intimamente relacionada com a resposta do hospedeiro às condições do organismo e externas14. A testosterona, principal hormônio sexual no homem, é sintetizada pelas células de Leydig e pelo córtex adrenal,9 apresentando efeitos biológicos morfogênicos irreversíveis e efeitos reversíveis excitatórios/de manutenção13. A manutenção de níveis fisiológicos de testosterona é crítica para a saúde masculina. Níveis baixos de testosterona são extensamente correlacionados com uma série de alterações da normalidade clínica, incluindo perda de libido e função sexual, perda de força muscular, fadiga, alterações cognitivas e de humor,2 bem como aumento de marcadores para doença cardiovascular,3 mortalidade,7 diabetes mellitus,15,16 síndrome metabólica6 e risco aumentado para fratura óssea8,11,12,17-19. Por outro lado, doses suprafisiológicas de testosterona, como aquelas apresentadas por usuários de anabolizantes esteroidais androgênicos que buscam estética e/ou melhora na performance de atividades físicas, também têm sido 14 associadas com consequências médicas graves, incluindo complicações cardiovasculares, endócrinas e psiquiátricas1,5. Recentemente, o termo “Endocrinologia Reprodutiva Periodontal” foi proposto para a representar a área de estudo que avalia a possível participação dos hormônios esteroides sexuais na patogênese e progressão da doença periodontal10. Nossa hipótese principal foi que a testosterona exerce importante influência sobre a resposta imunoinflamatória e metabolismo ósseo, não apenas em situações homeostáticas e fisiológicas, mas também em condições associadas à inflamação crônica, como a doença periodontal. O objetivo deste trabalho de tese foi avaliar a relação entre testosterona e doença periodontal em diferentes níveis de evidência científica: • Associação entre níveis hormonais e presença e severidade de periodontite em homens; • Relação causal entre modulação dos níveis séricos de testosterona e alterações no periodonto e na resposta do hospedeiro à periodontite experimental em ratos; • Mecanismo de ação das observações in vivo utilizando cultura de osteoblastos e precursores de osteoclastos. 15 CAPÍTULOSCAPÍTULOSCAPÍTULOSCAPÍTULOS 16 2 Capítulos A relação entre testosterona e a doença periodontal no sexo masculino foi avaliada em diferentes níveis de evidência científica: - Em humanos, através de um estudo observacional transversal utilizando dados de uma amostra representativa da população norte-americana: Capítulo 1: Steffens JP, Wang X, Spolidorio LC, Van Dyke TE, Starr J, Kantarci A. Sex hormones correlate with periodontitis in men: Results from NHANES III. Artigo a ser submetido ao Journal of Dental Research. - Para se avaliar a relação causal entre andrógenos e severidade da doença periodontal, foi realizado um estudo piloto em ratos para validação de metodologia: Capítulo 2: Steffens JP, Coimbra LS, Ramalho-Lucas PD, Rossa Jr. C, Spolidorio LC. The effect of supra- and subphysiologic testosterone levels on ligature-induced bone loss in rats - A radiographic and histologic pilot study. J Periodontol 2012; 83: 1432-9. - Para aprofundar a compreensão desta relação, e também para se identificar possíveis mecanismos de ação, foram realizados ensaios in vivo (ratos) e in vitro: Capítulo 3: Steffens JP, Coimbra LS, Rossa Jr C, Kantarci A, Van Dyke TE, Spolidorio LC. The impact of testosterone on inflammation-induced periodontal bone loss in rats. Artigo a ser submetido ao Journal of Bone and Mineral Research. Capítulo 4: Steffens JP, Herrera BS, Stephens D, Spolidorio LC, Kantarci A, Van Dyke TE. Resolvin D2 ameliorates testosterone-derived downregulation of 17 osteocalcin and osteoprotegerin on primary murine osteoblasts. Artigo submetido ao Hormones and Metabolic Research. - Para sugerir novas perspectivas sobre a relação entre envelhecimento e doença periodontal, que não pelo mecanismo hormonal, realizou-se a revisão da literatura: Capítulo 5: Steffens JP, Masi S, D’Aiuto F, Spolidorio LC. Telomere length and its relationship with chronic diseases - New perspectives for periodontal research. Arch Oral Biol 2013; 58: 111-7. Uma descrição detalhada dos materiais e métodos utilizados pode ser encontrada no Apêndice A. Todos os procedimentos experimentais foram aprovados pela Comissão de Ética no Uso de Animais (CEUA) da Faculdade de Odontologia de Araraquara – Unesp (Anexo A). As permissões das revistas científicas para reprodução dos artigos publicados estão dispostas no Anexo B. 18 CAPÍTULO 1CAPÍTULO 1CAPÍTULO 1CAPÍTULO 1 19 Sex Hormones Correlate with Periodontitis in Men: Results from NHANES III * Joao Paulo Steffens, DDS, MSc, PhD student.1,2 Xiaoshan Wang, PhD.2 Luis Carlos Spolidorio, DDS, PhD.1 Thomas E. Van Dyke, DDS, PhD.2 Jacqueline Starr, PhD.2,3,4 Alpdogan Kantarci, DDS, PhD.2 1 Department of Physiology and Pathology, School of Dentistry at Araraquara, UNESP- Universidade Estadual Paulista, SP, Brazil. 2 Department of Applied Oral Sciences, The Forsyth Institute, Cambridge, MA, USA. 3 Department of Oral Health Policy and Epidemiology, Harvard School of Dental Medicine, Boston, MA, USA. 4 Department of Epidemiology, University of Washington, Seattle, WA, USA. Corresponding Author: Dr. Joao Paulo Steffens. Rua Humaita, 1680. Araraquara, SP, Brazil. Phone: +55 (16) 3301-6479. E-mail: joaopaulosteffens@hotmail.com. * De acordo com as instruções do periódico Journal of Dental Research. Disponível em: http://mc.manuscriptcentral.com/societyimages/jdr/JDR%20Instructions%20for%20A uthors%202.19.13%20edits.pdf. 20 ABSTRACT Sex hormones have the ability to influence inflammation and bone turnover. The goal of this study was to explore the potential association between sex hormones and periodontitis using data from the Third National Health and Nutrition Examination Survey (NHANES). The dataset from 772 individuals aged 30+ years who had blood tests for assessment of serum testosterone, estradiol, sex hormone binding globulin (SHBG) and androstenediol glucuronide (AAG) levels were included in the analysis. Bioavailable testosterone (CBT) and estradiol over testosterone ratio (ETR) were also calculated. Periodontitis was defined as a combination of clinical attachment loss (CAL)≥3mm and probing pocket depth (PPD)≥4mm. The presence and severity of periodontitis were correlated with categories of the sex hormones. The prevalence of persons presenting PPD≤3mm or CAL≤2mm was decreased when low or high testosterone levels were present, as well as in men with low estradiol. When adjusted for confounding factors, high CBT and low ETR displayed a significant odds ratio for both presence and severity of the disease. The strength of association between sex hormones (or their interactions) and periodontitis was different according to the age intervals studied. Our findings suggest that sex hormones present an age-dependent association with both presence and severity of periodontitis in men. Keywords: gonadal steroid hormones; periodontitis; inflammation; androgens. 21 INTRODUCTION Sex steroid hormones are known to regulate a variety of functions, such as growth, reproduction and differentiation (Nava-Castro et al., 2012). In men, the main sex steroid hormone is testosterone, which is primarily produced by the Leydig cells in the testicles, but can also be derived from the adrenal precursor dehydroepiandrosterone. The production of testosterone by the testes is regulated through a hypothalamus-pituitary axis feedback mechanism (Mawhinney and Mariotti, 2013). Testosterone can act directly, by binding to the nuclear androgen receptor, or indirectly. The indirect action is mediated by the enzyme 5α-reductase, expressed in tissues like the liver, kidneys, skin and prostate, that converts testosterone to dihydrotestosterone (DHT), which is metabolically more active than testosterone and binds the androgen receptor (AR) with greater affinity. Also, testosterone can be converted to estradiol by the enzyme aromatase, produced by macrophages and fibroblasts in the liver, kidneys, brain and adipose tissue, which exerts its action by binding to estrogen receptors (ER) α, β and G protein-coupled receptor 30 (GPR30) (Ohlsson and Vandenput, 2009; Vandenput and Ohlsson, 2010). Physiologic testosterone levels vary greatly between individuals and also throughout a man’s life. Peaks of testosterone are achieved three times in life: in the middle of embryo development, during the 2nd-5th month of age, and in puberty (Harvey and Berry, 2009). However, after the 40 years of age, total testosterone levels start to decline gradually and continuously at a rate of 1-2% a year (Gray, 2005). Also, the globulin that binds the hormone with great affinity, SHBG, increases, further reducing the amount of bioavailable testosterone (free and albumin-bound testosterone) (Yeap, 2009). 22 Low testosterone levels have been extensively correlated with medical disorders, such as low libido, sexual dysfunction and muscle strength loss, fatigue, cognitive and mood alterations (Bain, 2010). Low testosterone has also been reported to be associated with increased low grade systemic inflammation biomarkers (tumor necrosis factor-alpha, macrophage inflammatory protein 1-alpha and 1-beta) (Bobjer et al., 2013), markers for cardiovascular disease (Del Fabbro et al., 2010) and mortality (Laughlin et al., 2008), diabetes mellitus (Selvin et al., 2007; Stellato et al., 2000), metabolic syndrome (Laaksonen et al., 2004) and increased risk for bone fracture (Meier et al., 2008; Mellstrom et al., 2006). In rats, we have described that both low and high serum testosterone levels modulate periodontal bone loss (Steffens et al., 2012). However, it is not clear whether the impact of testosterone in men’s health, especially in bone metabolism, is derived from the activation of ARs, by testosterone and DHT, or through its aromatization to estradiol (Clarke and Khosla, 2009). Recently, the term ‘Periodontal Reproductive Endocrinology’ was proposed to represent the field of Periodontology dedicated to studying the interactions between sex steroid hormones and the periodontium (Mariotti, 2013). Along those same lines, the objective of this study was to explore the potential impact of abnormal serum levels of sex steroid hormones on the prevalence and severity of periodontitis in men using data from the Third National Health and Nutritional Examination Survey (NHANES III). MATERIALS AND METHODS Study Design 23 Third National Health and Nutrition Examination Survey (NHANES III) was a cross-sectional study conducted from 1988 to 1994 by the National Center for Health Statistics (NCHS), which is part of the Centers for Disease Control and Prevention (CDC). This program of studies was designed to assess the health and nutritional status of adults and children in the United States. It was designed as a multistage, stratified, clustered probability sample of the US civilian non-institutionalized population at least 2 months old. The protocols for the conduct of NHANES III were approved by the institutional review board of the NCHS, CDC. Informed consent was obtained from all participants (Plan and operation of the Third National Health and Nutrition Examination Survey, 1988-94. Series 1: programs and collection procedures, 1994). A subset of male participants in the first phase of NHANES III (1988-1991) had concentrations of testosterone, SHBG, androstenediol glucuronide (AAG, a metabolite of DHT) and estradiol recorded. These measurements were made on stored serum specimens from 1,637 men aged 12 or more who were examined in the morning sample of the first phase of NHANES III (1988-1991). Blood was drawn after an overnight fast for participants during either an examination at the medical examination center or during an abbreviated examination at home (Selvin et al., 2007). Competitive electrochemiluminescence immunoassays on the 2010 Elecsys autoanalyser (Roche Diagnostics, Indianapolis, IN, USA) were used to quantify testosterone, estradiol and SHBG concentrations, while AAG was detected by an enzyme immunoassay (Diagnostic Systems Laboratories, Webster, TX, USA). The lowest detection limits [and coefficients of variation] of the assays were: testosterone 0.02ng/mL [5.9%(2.5ng/mL), 5.8% (5.5ng/mL)]; estradiol 5pg/mL [6.5%(102.7pg/mL), 6.7%(474.1pg/mL)]; AAG 0.33ng/mL [9.5%(2.9ng/mL), 5%(10.1ng/mL)]; SHBG 3nM 24 [5.3%(5.3nM), 5.9%(16.6nM)]. The assay of stored serum specimens was conducted at the Children’s Hospital, Boston, MA, and the protocol was approved by the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health and the NCHS, CDC (Rohrmann et al., 2011). Among individuals who had their serum specimens assessed, we identified participants who were 30 years old or older, had at least 6 teeth present and had received periodontal examination (n=772). Thirty years of age was chosen because of the rarity of periodontitis in younger individuals. According to NHANES criteria, each individual had 2 randomly selected quadrants evaluated: one upper and one lower quadrant. Only fully erupted teeth were analyzed (excluding third molars), and a maximum of 14 teeth per individual was examined. Periodontal measurements used in this study included clinical attachment loss (CAL) and probing pocket depth (PPD). These measurements were performed in the mesio-buccal and mid-buccal sites of each tooth. PPD was described as the distance between the free gingival margin and the bottom of the pocket/sulcus, while CAL was defined as the distance between the cement-enamel junction and the bottom of the pocket/sulcus. All measurements were performed by trained dentists using NIDR periodontal probes (Albandar et al., 1999). Classification of Sex Hormone Levels Total testosterone levels in serum were classified as very low (≤2.3 ng/mL), low (2.3-3.5 ng/mL), and reference (>3.5-7.67 ng/mL) (Wang et al., 2008), whilst the 90th percentile was used as cutoff for ‘high’ values (>7.67 ng/mL) (Rohrmann et al., 2011). Calculated bioavailable testosterone (CBT) was performed based on previously described equations (Vermeulen et al., 1999) and the albumin value was standardized as 4.5 g/dL. Low CBT (<1 ng/mL) and high CBT (>4.2 ng/mL) cutoffs 25 were based on generally accepted levels. Estradiol over testosterone ratios (ETR) were calculated by dividing estradiol levels by total testosterone values. The 10th and 90th percentiles were used as lower and higher limit cutoffs, respectively, for all measurements and interactions (CBT, estradiol, ETR, SHBG and AAG) (Rohrmann et al., 2011). Classification According to Extent and Severity of Periodontitis We used the classification of extent and severity of periodontitis proposed in the latest NHANES periodontal analysis by Eke et al., 2012: - Severe periodontitis: presence of 2 or more interproximal sites with CAL≥6 mm (not on the same tooth) and 1 or more interproximal site(s) with PPD≥5 mm. - Moderate periodontitis: 2 or more interproximal sites with 4 mm ≤CAL<6 mm (not on the same tooth), or 2 or more interproximal sites with PPD≥5 mm, also not on the same tooth. - Mild periodontitis: 2 or more interproximal sites with 3 mm ≤CAL<4 mm and 2 or more interproximal sites with PPD≥4 mm (not on the same tooth) or 1 site with PPD≥5 mm. Total periodontitis was the sum of severe, moderate and mild cases of periodontitis (Eke et al., 2012). Statistical Analysis All data analyses were performed with STATA version 12 with SVY package, which uses weights to account for the multi-stage stratified, clustered sampling method of NHANES III. Possible confounding factors considered in this analysis were defined a priori as follows: age (continuous), current smoking (yes/no), alcohol drinking frequency (drinks per week, continuous), waist-to-hip ratio, race/ethnicity (non- 26 Hispanic white / non-Hispanic black / others), diabetes mellitus (self-reported, yes/no). We fit logistic regression models to estimate the association between presence or absence of periodontal disease and the various hormones. Ordinal logistic models were used to further evaluate the association of covariates with the severity of periodontal diseases. Unless otherwise stated, all values are expressed as mean ± SEM. RESULTS After assessing and screening the NHANES III dataset, 772 men (age 45.5±0.5 years) were included in this study. The baseline characteristics of the study population are shown in table 1. Among the people presenting with the reference total testosterone levels, 61.1 ± 3.2% had PPD ≤3 mm and 46.8 ± 2.5% had AL ≤2 mm, indicating periodontal health. The prevalence of periodontal health was decreased in men presenting with testosterone levels lower or higher than the reference group. Table 2 contains the raw non-adjusted data for prevalence of periodontitis by category (PPD and CAL) for various sex hormone concentrations. After classification of presence and severity of periodontitis and statistical adjustments, high CBT levels and low ETR were the only variables to correlate with periodontitis. The adjusted odds ratios for presence and severity of periodontitis based on sex steroid hormone concentrations can be found in table 3. Table 4 demonstrates the prevalence and adjusted odds ratio for presence and severity of periodontitis by sex steroid hormone concentrations grouped according to age. Low ETR correlates with the presence and severity of periodontitis in young adult men (30-45 years). In middle-aged adults (46-60 years), we observed 27 a correlation with the severity, but not presence of periodontitis. We noted a similar trend of association of age with high total testosterone levels. In addition, low estradiol and high ETR were correlated with the presence of periodontitis. In older men (>60 years) only low AAG correlated with presence and severity of periodontitis. DISCUSSION Sex hormones are important for a variety of characteristics that individuals develop throughout life. Several factors may contribute to hormone variations in males, which can be physiologic (e.g. puberty), pathologic (e.g. Klinefelter syndrome and hypergonadotropic hypogonadism), pharmacologic (e.g. steroid abuse and testosterone replacement therapy), genetic (e.g. race and ethnicity) or behavioral (e.g. smoking and alcohol consumption). Physiological testosterone levels are believed to regulate inflammation, since low testosterone levels have been linked to the presence of several inflammatory medical disorders (Maggio and Basaria, 2009; Traish et al., 2009a; Traish et al., 2009b; Traish et al., 2009c). Similarly, anabolic-androgenic steroid abuse that results in high levels of testosterone has adverse actions, such as alterations in the cardiovascular, central nervous and endocrine systems (Basaria, 2010). We have previously demonstrated bimodal actions of testosterone; both low and high testosterone levels increase bone resorption in an inflammatory dental model in rats (Steffens et al., 2012). Many epidemiological studies have shown men at higher risk for presence and severity of attachment loss and destructive periodontal disease than women (Albandar, 2002; Corbet et al., 2001; Mack et al., 2004; Susin et al., 2005). Although it is plausible that this difference could be hormonally mediated, gender-based differences in behavior could also explain, at least in part, the greater prevalence. 28 Men tend to have poorer oral hygiene, increased consumption of alcohol and tobacco, and less utilization of oral health care services than women (Haytac et al., 2013). In the elderly population, men were reported to have higher prevalence and greater severity of periodontitis, as shown in cross-sectional (Holtfreter et al., 2010; Mack et al., 2004) and longitudinal studies (Hirotomi et al., 2002; Ogawa et al., 2002). Several mechanisms have been proposed to explain the regulation of periodontal disease by sex steroid hormones, including: (i) hormones increase growth of pathogenic microflora; (ii) promote an alteration in vascular characteristics; (iii) periodontal tissue responses are exacerbated by immune-endocrine interactions; (iv) specific populations of fibroblasts and epithelial cells are modulated by sex steroid hormones (Mariotti and Mawhinney, 2013). However, the host response in a multifactorial disease, such as periodontal disease, most probably cannot be simplified and represented by one mechanism of action only; a combination of factors is likely involved. Previous observational studies conducted to evaluate the possible role of sex hormones in the development of destructive periodontal disease reported no significant correlations between hormone levels and the prevalence of periodontitis (Daltaban et al., 2006; Unsal et al., 2008). However, design features of some of these studies limit conclusions and preclude direct comparisons with our data (for instance, small sample size and restriction to hypogonadic men). In a prospective study of a cohort of community-dwelling ambulatory men aged 65 years or older, no correlation was observed between sex hormones and the progression of periodontitis (Orwoll et al., 2009). The criteria for defining periodontitis were different (proximal CAL≥5mm in at least 30% of teeth examined), but the outcome was similar showing 29 no significant correlation between total testosterone or estradiol and periodontitis in our oldest age group. We observed a linear relationship between total testosterone levels and presence or severity of periodontitis, though the 95% confidence intervals were very wide. If the clinically generally accepted level of 10 ng/mL is used as a high cutoff value, the correlation is still significant, but the small sample size could bias the result (n=10). To our knowledge, we are the first group to report that high CBT and low ETR are correlated with periodontitis. This observation is consistent with high testosterone and low estradiol levels being harmful to the periodontium. The sex hormone estradiol alone, which is generally accepted as the main regulator of bone loss in men, did not associate with periodontitis in any age group. However, we assessed the levels of the hormone only in serum, whereas estradiol can be generated locally in the tissues (such as bone) using testosterone as a precursor. As a matter of fact, some studies have shown that testosterone treatment has a suppressive effect on leukocyte count on orchiectomized mice and young male rats (Kamis and Ibrahim, 1989; Yao et al., 2003). Those results also showed that testosterone treatment decreases monocyte count, CD4+/CD8+ ratio, and also inhibits proliferative responses of lymphocytes (Yao et al., 2003). Due to the immunosuppressive properties of testosterone, it can be suggested that high endogenous levels of that hormone increases susceptibility to infectious diseases, such as periodontitis. Even though a smaller prevalence of healthy subjects (as inferred from CAL≤2 mm and PPD≤3 mm) can be observed in the reference levels of total testosterone when compared to the other groups (Table 2), we could not see this same trend after adjusting for confounding factors. After adjustments, very low and low testosterone 30 levels were associated with a non-significant decrease in the odds ratios for prevalence and severity of periodontitis (Table 3), a direction opposite to what we had proposed. This difference with other medical disorders and our animal findings can be attributed to the fact that periodontitis is a multifactorial disease and highly depends on patient-based biofilm control. That was also the rationale for investigating presence and severity of periodontitis, as to test whether sex hormones could interfere in the initiation or progression of periodontitis. The sex hormones (and their interactions) exhibit age-dependent associations with periodontitis. ETR correlates with the presence and severity of periodontitis in young and middle-aged adults. In middle-aged adults, low estradiol correlates with the presence of periodontitis and high total testosterone correlates with severity of periodontitis. In older men only low AAG correlates with presence and severity of periodontitis, reinforcing the importance of androgens on this disease. Among the various limitations of a reliance on NHANES III data is the difficulty in developing accurate measures of disease and disease severity (Albandar, 2011; Eke et al., 2010). The primary disadvantage is that data collection was from only two sites per tooth in two randomly assigned quadrants, an approach that underestimates the prevalence of periodontitis. However, our objective was not to give the absolute prevalence of periodontitis in the studied population, but rather compare the prevalence in groups that were evaluated with the same criteria. Our findings suggest that high CBT and low ETR correlate with periodontitis prevalence and severity in men, and these correlations are age-dependent. In older men (>60 years) low AAG, a metabolite of DHT, significantly correlates with higher prevalence and severity of periodontitis. Causality remains to be determined. 31 ACKNOWLEDGEMENTS JPS is a recipient of a scholarship from the São Paulo State Research Foundation (FAPESP), São Paulo, SP, Brazil (#2010/09658-0) and from the Coordination for the Improvement of Higher Education Personnel (CAPES), Brasília, DF, Brazil (#5258- 11-1). JPS and LCS hold research support grants from FAPESP (#2010/12021-4) and from the National Council for Scientific and Technological Development (CNPq), Brasília, DF, Brazil (#470870/2011-7). LCS holds a Productivity Scholarship from CNPq. Supported in part by grant R01 DE15566 from the National Institute of Dental and Craniofacial Research (NIDCR), Bethesda, MD, United States. REFERENCES Albandar JM, Brunelle JA, Kingman A (1999). Destructive periodontal disease in adults 30 years of age and older in the United States, 1988-1994. J Periodontol 70(1):13-29. Albandar JM (2002). Global risk factors and risk indicators for periodontal diseases. Periodontol 2000 29(177-206. Albandar JM (2011). Underestimation of periodontitis in NHANES surveys. J Periodontol 82(3):337-41. Bain J (2010). Testosterone and the aging male: to treat or not to treat? Maturitas 66(1):16-22. Basaria S (2010). Androgen abuse in athletes: detection and consequences. J Clin Endocrinol Metab 95(4):1533-43. 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Yao G, Liang J, Han X, Hou Y (2003). In vivo modulation of the circulating lymphocyte subsets and monocytes by androgen. Int Immunopharmacol 3(13- 14):1853-60. Yeap BB (2009). Testosterone and ill-health in aging men. Nat Clin Pract Endocrinol Metab 5(2):113-21. 37 Table 1: Characteristics (SEM) of the study population. Overall Unweighted sample number (n) 772 Age (years) 45.5 (0.5) Race/Ethnicity (prevalence, %) Non-Hispanic White 79 (3.0) Non-Hispanic Black 8 (1.0) Other 13 (2.0) Diabetes (prevalence, %) 5 (1.0) Current smoking (prevalence, %) 30 (2.0) Education: high school and above (prevalence, %) 54 (4.0) Body Mass Index (BMI) 26.91 (0.31) Waist-to-hip ratio (WHR) 0.96 (0.005) Alcohol drinking frequency (times/month) 14.00 (1.07) Total Testosterone* (ng/mL) 4.85 (0.10) Estradiol* (pg/mL) 34.70 (0.77) SHBG* (nM) 34.96 (0.65) AAG* (ng/mL) 11.10 (0.36) PPD, mean (mm) 1.66 (0.04) CAL, mean (mm) 1.40 (0.06) Periodontitis (prevalence, %) 30 (2.0) *geometric mean. SHBG= sex hormone binding globulin; AAG= androstenediol glucuronide; PPD= probing pocket depth; CAL= clinical attachment loss 38 Table 2: Prevalence (SEM) of persons presenting categories of probing pocket depth (PPD in mm) and clinical attachment loss (CAL in mm) according to sex hormone concentrations. PPD≤3 33.5-7.67 ng/mL) 61.1(3.2) 22.5(2) 16.4(2.3) 46.8(2.5) 28.5(2.3) 24.7(2.8) 90th percentile (>7.67 ng/mL) 46.5(9.7) 38.6(8.9) 14.9(3.9) 38.4(6.6) 35.7(6.2) 25.9(5) CBT Low (<1 ng/mL) 49.5(9.6) 32.9(9.2) 17.6(6.3) 23.7(9.7) 43.4(9) 32.9(9.3) Reference (1-4.2 ng/mL) 57.9(2.8) 24.4(1.7) 17.7(2.2) 46(1.9) 30.2(2.2) 23.8(2.3) High (>4.2 ng/mL) 57.8(11.7) 31.4(7.6) 10.8(8.3) 40(10.5) 41.2(7.5) 18.8(9.3) Estradiol 10th percentile (<24.88 pg/mL) 70.8(6.1) 15.2(5.2) 14.1(6.9) 52.7(8.8) 25.8(6.7) 21.5(4.6) Reference 58.2(2.9) 25(1.9) 16.8(2) 45.6(2) 32.6(2.4) 21.8(2.2) 90th percentile (>49.2 pg/mL) 38.9(7) 35.1(4.4) 25.9(7.5) 31.1(6) 24.8(6) 44.1(6.1) ETR (*1,000) 10th percentile (<0.0047) 65.1(7.8) 26.7(7) 8.2(3.4) 62.4(7.4) 19.9(5.3) 17.7(5.5) Reference 57.7(2.9) 24.1(1.8) 18.1(2.6) 43.8(2.2) 31.8(2.4) 24.4(2.5) 90th percentile (>0.0109) 49.3(7.1) 31(5.1) 19.7(6) 36.1(5.5) 38(5.8) 25.9(6.1) SHBG 10th percentile (<20.7 nM) 56.8(8.3) 25.3(8) 18(5.9) 51.6(8.1) 29.7(6.5) 18.7(5.4) Reference 58.3(3.1) 24(2.2) 17.7(2.1) 45.7(2) 31(2.4) 23.2(2.2) 90th percentile (>59.2 nM) 52.5(6.1) 33.3(5) 14.3(4.3) 30.7(7) 33.4(6.2) 35.9(6) AAG 10th percentile (<5.26 ng/mL) 51.9(7.9) 35.6(7.4) 12.4(4.4) 21.8(5.3) 35.7(6.6) 42.6(7.3) to be continued... 39 Reference 57(3.4) 23.7(1.8) 19.3(2.7) 48.4(2.4) 28.8(2.5) 22.8(2.4) 90th percentile (>22.28 ng/mL) 68.3(5) 25(5.9) 6.7(3.1) 41.9(6.2) 47.2(6.7) 10.9(3.9) TT= total testosterone; CBT= calculated bioavailable testosterone; ETR= estradiol over testosterone ratio; SHBG= sex hormone binding globulin; AAG= androstenediol glucuronide ... continued. 40 Table 3: Adjusted* OR (95% CI) for presence and severity of periodontitis according to sex steroid hormone concentrations. Presence Severity OR (95% CI) OR (95% CI) TT** Very Low (≤2.3 ng/mL) 0.31(0.06-1.63) 0.3(0.07-1.34) Low (2.3-3.5 ng/mL) 0.45(0.19-1.04) 0.49(0.2-1.24) Reference (>3.5-7.67 ng/mL) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 90th percentile (>7.67 ng/mL) 2.21(0.95-5.11) 2.07(0.96-4.43) CBT Low (<1 ng/mL) 0.44(0.12-1.53) 0.36(0.11-1.11) Reference (1-4.2 ng/mL) 1.00 (1.00-1.00) 1.00 (1.00-1.00) High (>4.2 ng/mL) 4.67(1.04-20.88)† 3.62(1.14-11.46) † Estradiol 10th percentile (<24.88 pg/mL) 0.88(0.41-1.86) 0.8(0.43-1.49) Reference 1.00 (1.00-1.00) 1.00 (1.00-1.00) 90th percentile (>49.2 pg/mL) 1.12(0.45-2.77) 0.83(0.4-1.72) ETR 10th percentile (<0.0047) 3.28(1.11-9.72)† 4.43(1.62-12.11)† Reference 1.00 (1.00-1.00) 1.00 (1.00-1.00) 90th percentile (>0.0109) 0.6(0.23-1.59) 0.61(0.23-1.6) SHBG 10th percentile (<20.7 nM) 0.8(0.27-2.36) 0.76(0.28-2.06) Reference 1.00 (1.00-1.00) 1.00 (1.00-1.00) 90th percentile (>59.2 nM) 1.91(0.81-4.55) 1.72(0.68-4.35) AAG 10th percentile (<5.26 ng/mL) 1.81(0.78-4.18) 1.82(0.89-3.72) Reference 1.00 (1.00-1.00) 1.00 (1.00-1.00) 90th percentile (>22.28 ng/mL) 0.92(0.37-2.27) 1.11(0.42-2.91) * adjusted for age, race/ethnicity, smoking, education, waist-to-hip ratio, diabetes mellitus, and alcohol drinking. ** further adjusted for estradiol. † (bold) statistically significant. TT= total testosterone; CBT= calculated bioavailable testosterone; ETR= estradiol over testosterone ratio; SHBG= sex hormone binding globulin; AAG= androstenediol glucuronide 41 Table 4: Prevalence [%(SEM)] and adjusted* OR (95% CI) for presence and severity of periodontitis by sex steroid hormone concentrations grouped according to age intervals. 30-45 years 46-60 years 61+ years %(SEM) Presence Severity %(SEM) Presence Severity %(SEM) Presence Severity TT Very low 4(2.7) 1.57(0.15-16.19) 1.47(0.16-13.3) 2.3(1.7) 0.08(0-2.93) 0.07(0-5.31) 6.4(2.6) 0.35(0.04-2.72) 0.34(0.04-2.7) Low 6(3.2) 0.93(0.12-7.27) 0.91(0.15-5.62) 7.8(2.6) 0.28(0.07-1.09) 0.26(0.07-1.02) 18.9(6.1) 0.29(0.04-2.24) 0.48(0.04-5.13) Ref. 70.9(7.5) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 69.6(6.9) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 72(7.7) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 90th perc. 19.1(7.7) 1.59(0.4-6.29) 1.52(0.41-5.6) 20.3(5.8) 4.31(0.84-22.2) 3.7(1.02-13.49)† 2.7(2.3) 2.4(0.15-39.22) 1.97(0.18-21.29) CBT Low - - - 3.4(2.1) 2.55(0.53-12.22) 8.58(0.28-266.52) 11.8(3.8) 0.71(0.16-3.08) 0.57(0.2-1.64) Ref. 82.2(8.2) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 92.8(3.3) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 88.1(3.8) 1.00 (1.00-1.00) 1.00 (1.00-1.00) High 11.9(8) 3.28(0.5-21.64) 3(0.62-14.67) - - - - - - Estradiol 10th perc. 16.3(6.3) 0.7(0.16-3.06) 0.63(0.15-2.63) 18.4(5.7) 8.27(1.69-40.38) 2.15(0.63-7.29) 8.3(3.1) 1.22(0.14-10.89) 0.8(0.18-3.54) Ref. 77.4(6.4) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 75.8(5.9) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 75.7(5.4) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 90th perc. 6.4(4) 0.97(0.17-5.6) 0.95(0.18-5.12) 5.8(2.7) 0.87(0.31-2.47) 0.78(0.3-2.07) 16(5.6) 1.11(0.13-9.41) 0.74(0.13-4.06) ETR 10th perc. 8.3(5) 5.24(1.26-21.76)† 8.88(1-78.77)† 13.3(5) 2.43(0.45-13.14) 5.26(1.31-21.07)† 3.4(2.4) 1.34(0.22-7.99) 0.95(0.3-3.05) Ref. 82.1(6.6) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 76.3(4.9) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 78.6(5.5) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 90th perc. 9.6(3.1) 1.55(0.19-12.37) 1.41(0.23-8.61) 10.5(2.6) 0.3(0.14-0.67)† 0.26(0.11-0.64)† 18(4.3) 0.46(0.09-2.45) 0.56(0.08-4.19) SHBG 10th perc. 8.4(3.3) 0.54(0.19-1.57) 0.53(0.19-1.52) 6.7(1.8) 0.79(0.19-3.34) 0.75(0.19-2.99) 4.8(4.1) 0.3(0.01-10.18) 0.19(0.01-5.83) Ref. 84(3.9) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 76.8(4.3) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 73.8(5.7) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 90th perc. 7.6(3.2) 0.96(0.16-5.82) 0.89(0.16-4.99) 16.5(3.8) 2.51(0.61-10.25) 2.97(0.79-11.19) 21.3(4.9) 0.8(0.21-3.04) 0.56(0.16-1.91) AAG 10th perc. 11.3(6) 1.62(0.32-8.33) 1.35(0.28-6.55) 18.4(6.6) 0.8(0.2-3.21) 1.09(0.3-3.96) 23.9(6.6) 7.41(1.51-36.34)† 5.15(2.01-13.21)† Ref. 79.5(5.4) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 76.5(7.2) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 63.5(8.7) 1.00 (1.00-1.00) 1.00 (1.00-1.00) 90th perc. 9.2(4.3) 1.19(0.24-5.86) 1.17(0.26-5.22) 5.1(3.1) 0.32(0.08-1.23) 0.41(0.05-3.33) 12.6(4.7) 2(0.52-7.67) 3.13(0.7-14.01) *adjusted for age, race/ethnicity, smoking, education, waist-to-hip ratio, diabetes mellitus and alcohol drinking. † (bold) statistically significant. TT= total testosterone; CBT= calculated bioavailable testosterone; ETR= estradiol over testosterone ratio; SHBG= sex hormone binding globulin; AAG= androstenediol glucuronide; Ref.= reference; Perc.= percentile. 42 CAPÍTULO 2CAPÍTULO 2CAPÍTULO 2CAPÍTULO 2 The Effect of Supra- and Subphysiologic Testosterone Levels on Ligature-Induced Bone Loss in Rats — A Radiographic and Histologic Pilot Study Joao P. Steffens,* Leila S. Coimbra,* Pablo D. Ramalho-Lucas,* Carlos Rossa Jr.,† and Luis C. Spolidorio* Background: Testosterone is the primary male sexual hor- mone, and varying concentrations of the hormone mediated by physiologic, pathologic, or pharmacologic mechanisms may induce large variations in the body. Data regarding the general role of testosterone in mediating inflammation are still inconclusive. Therefore, the purpose of this study is to as- sess the consequences of supra- and subphysiologic levels of testosterone on ligature-induced bone loss in rats. Methods: Three male adult Holtzman rats were used to ob- serve the course of serum testosterone concentration follow- ing orchiectomy (Ocx) and testosterone injections. Another 60 rats were randomly assigned to the following groups: 1) sham-operation controls (n = 10); 2) sham-operation and ligature-induced bone loss (n = 10); 3) orchiectomy without ligature (Ocx; n = 10); 4) Ocx and ligature (n = 10); 5) Ocx plus 250 mg/kg body weight intramuscular testosterone es- ters injection without ligature (Ocx+T; n = 10); and 6) Ocx, T, and ligature (n = 10). The ligatures were placed 30 days post- orchiectomy (or sham-operation) and maintained for 15 days. Thereafter, the rats were sacrificed, and their hemimandibles were used for radiographic evaluation of bone loss along with histologic and histometric analyses of gingival tissue. Results: The results indicated a significant increase in bone loss in the Ocx and Ocx+T groups in the presence and absence of inflammation, respectively. In addition, the Ocx and Ocx+T groups presented increased gingival area accompanying ligature-induced bone loss. Conclusions: Both sub- and supraphysiologic testoster- one levels may influence bone metabolism, but only subphy- siologic levels significantly increase ligature-induced bone loss. Moreover, testosterone has a regulatory effect on the gingival area. J Periodontol 2012;83:1432-1439. KEY WORDS Bone remodeling; gingival overgrowth; inflammation; testosterone. A ndrogens are hormones respon- sible for regulating primary and secondary sexual characteristics in men.1 Testosterone, the primary male sex hormone, is related to the develop- ment and maintenance of muscle mass, erythropoiesis stimulus, increased brain perfusion, influence of mood and cog- nition, and bone health.2 Testosterone rises to comparable adult male levels during three life phases: 1) the midpoint of embryonic development, 2) the first 2 to 5 months of life, and 3) throughout puberty.1,3 Approximately 95% of the testoster- one in a healthy man is produced by the testes, whereas the other 5% is con- verted into testosterone from adrenal- produced precursors.4,5 Testosterone may act directly by binding to the intra- cytoplasmic androgen receptor or can be converted to dihydrotestosterone (DHT) or estrogen. DHT is responsible for an even greater activation of the androgen receptor, whereas estrogen, the primary regulator of bone homeostasis in men, acts by binding to estrogen receptors.4,5 After 40 years of age, testosterone levels in men may decrease gradually and continuously at a rate of 1% to 2% per year. It is estimated that 50% of men aged 60 years or older may have considerably decreased testosterone levels.3,6 Clinically, low testosterone * Department of Physiology and Pathology, School of Dentistry at Araraquara, São Paulo State University, Araraquara, São Paulo, Brazil † Department of Diagnosis and Surgery, São Paulo State University. doi: 10.1902/jop.2012.110658 Volume 83 • Number 11 1432 levels may influence sexual function and libido, muscle strength, bone density, fatigue, cognition, mood, adipose tissue, or even affect periodontal disease.6-8 In contrast, supraphysiologic levels of testoster- one may be induced by synthetic drugs used by body builders and other athletes to increase performance. The effects of these drugs include an increase in body weight, fat-free mass production, and enlarge- ment of muscle size and mass. The reported side effects are sexual dysfunction; liver toxicity; and alterations of the psyche, behavior, and cardiovas- cular system.9 Additionally, a case-control study reported that the prolonged use of anabolic andro- genic steroids is associated with significant levels of gingival enlargement.10 However, the effect of testosterone on inflamma- tion remains unclear. Androgens have been shown to be protective in certain conditions, and harmful in others.11 They contribute to inflammation by influencing the activity of leukocytes and inflam- matory cells such as neutrophils,12 monocytes,13 macrophages,14 mast cells,15 and platelets.16,17 Al- though androgens protect males from inflammatory disorders such as atherosclerosis and rheumatoid arthritis,18-20 they may also exacerbate wound in- flammation.21,22 Polymerase chain reaction mRNA analysis sug- gests that androgen receptors are also expressed in human periodontal and gingival tissue. In addition, testosterone may modulate its own receptors, which indicates that supra- and subphysiologic levels of the hormone could modify tissue response.23 Many in vitro experiments using periodontal cells and tissues have been performed to identify the role of testosterone on inflammatory markers such as prostaglandins and interleukins, as well as on the proliferative capacity of bone cells.24-27 However, in vivo experiments are still needed to understand the influence and consequences of the variations of testosterone levels in the periodontium. There- fore, the primary purpose of this pilot study is to evaluate the consequences of supra- and subphy- siologic testosterone levels on ligature-induced bone loss in rats. MATERIALS AND METHODS Animals A total of 63 male adult Holtzman rats (Rattus norvegicus albinus) weighing 300 to 400 g were housed under similar conditions in cages with ac- cess to food and water ad libitum. During the entire experimental protocol, the rats were kept in a quiet room with controlled temperature (23 – 2�C), humidity (65% to 75%), and a 12-hour light–dark cycle. All experimental protocols were approved by the local Ethics Committee for Animal Experimenta- tion and conducted in accordance with the guidelines of the Brazilian College of Animal Experimentation. Orchiectomy and the Dynamics of Testosterone Levels After Exogenous Testosterone Administration Three rats were used to evaluate the dynamics of testosterone levels after orchiectomy and testoster- one injections. Surgical orchiectomy was performed under anesthesia using ketamine (1 mL/kg/body weight [bw]) and xylazine (0.4 mL/kg/bw). In brief, after a scrotal incision, both testicles were removed and the incision sutured under sterile conditions. The rats were given acetaminophen (300 mg/kg/bw; orally) for postoperative pain relief and an intramus- cular dose of penicillin and streptomycin (1 mL/kg/ bw). After the procedure, the animals were kept in separate cages for 7 recovery days. Three days postorchiectomy, the animals were given a single intramuscular injection of a long-lasting mixture of testosterone esters (30 mg testosterone propionate, Figure 1. Diagram illustrating the points used to evaluate the connective tissue and epithelial height and width measurements. Connective tissue (1) and buccal epithelium (2) areas were obtained by multiplying each tissue’s height by its width. Modified from Corrêa et al. (2005).32 J Periodontol • November 2012 Steffens, Coimbra, Ramalho-Lucas, Rossa, Spolidorio 1433 60 mg testosterone phenylpropionate, 60 mg testos- terone isocaproate, and 100 mg testosterone dec- anoate),‡ 250 mg/kg/bw, diluted to 0.1 mL in corn oil.28 Venous blood (400 mL) was collected from the tail at baseline; 1, 2, and 3 days postorchiectomy; and 8 hours, 1, 2, 3, and 7 days post-testosterone injection. A blood sample was centrifuged for 10 minutes at 3,000 rpm to obtain approximately 150 mL of serum. Each serum sample was analyzed for total testosterone levels using a chemiluminescence- based immunoassay.§ Experimental Protocol Sixty rats were randomly assigned to the following groups: 1) sham-operation controls (Sham; n = 10); 2) sham-operation and ligature-induced bone loss (Sham+L; n = 10); 3) orchiectomy without lig- ature (Ocx; n = 10); 4) Ocx and ligature (Ocx+L; n = 10); 5) Ocx plus testosterone injection without lig- ature (Ocx+T; n = 10); and 6) Ocx, T, and ligature (Ocx+T+L; n = 10). Thirty days postorchiectomy (or sham operation), the ligatures were placed bilaterally on the mandibular first molars as pre- viously described and maintained for 15 days.29 The Ocx+T group received injections of testos- terone esters (250 mg/kg/bw, intramuscularly; di- luted to 0.1 mL in corn oil)28 every 7 days until the rats were sacrificed, starting 3 days postorch- iectomy. At the end of the experimental period, the rats were sacrificed via an overdose of anesthesia. Radiographic Evaluation After the rats were sacrificed, the mandibles were removed, and the right hemimandible was evaluated radiographically as previously described.30 In brief, digital x-ray images were used to estimate alveolar bone loss by examining the distance between the cemento-enamel junction (CEJ) and the height of alveolar bone in the mesial root surfaces of the mandibular right first molars. The analysis was conducted with the aid of software.i Histologic and Histometric Evaluation The left hemimandibles were soaked in formalin (10%) for 48 hours. A quick decalcifying agent containing EDTA, so- dium and potassium tartrate, chloric acid, and deionized water¶ was used for a 10-hour decalcification. Serial paraffin 5-mm-thick sections were made on the mesial and distal aspects of the whole mandibular right first molars and stained with hematoxylin and eosin. The gingival epithelium and connective tissue areas were measured in a modification of a previously de- scribed method.31,32 In brief, histologic sections of each rat were photographed at 100· magnification, and the connective tissue and epithelial height and width were measured with the aid of software.# The connective tissue and buccal epithelium areas were obtained by multiplying each tissue’s height by width (Fig. 1). Statistical Analyses One-way ANOVA, Tukey post hoc tests, and pair- wise comparisons (Student t test) were used to as- sess the differences among the quantitative data. Whenever the groups did not reach homogeneity of variances or did not present a normal distribution, the Kruskal-Wallis (Dunn post hoc) or Mann-Whit- ney U tests were used. A paired t test was used to assess baseline and final weight differences. The data were expressed as mean – SEM. All tests were Figure 2. Mean (– SEM) testosterone serum levels (ng/mL) at baseline, postorchiectomy, and post-testosterone injection (n = 3). OCX = orchiectomy; T= testosterone injection. ‡ Durateston, MSD Animal Health, Milton Keynes, UK. § Immulite 2000, Diagnostic Products Corporation, Gwynedd, UK. i ImageTool for Windows 3.0, The University of Texas Health Science Center at San Antonio, San Antonio, TX. ¶ Allkimia, Campinas, São Paulo, Brazil. # BioEstat 5.0, Manuel Ayres, Belém, Porto Alegre, Brazil. Effects of Testosterone in the Periodontium Volume 83 • Number 11 1434 performed using software,** and the significance level was set at P = 0.05. RESULTS All animals were alive at the end of the experimental period. The animals in the group that received a tes- tosterone injection demonstrated aggressive be- havior and were placed in isolated cages. The three animals that were used to evaluate the dynamics of testosterone levels after orchiectomy and testoster- one injection presented mean baseline testosterone levels of 2.01 – 0.07 ng/mL, which decreased to non-detectable levels 24 hours postorchiectomy. The testosterone injections resulted in a mean 11- fold maximum increase compared with the base- line serum levels 8 hours after drug administration. Seven days postinjection, testosterone levels de- creased to values comparable to the baseline. The effect of orchiectomy and testosterone injec- tion on the serum levels of testosterone is shown in Figure 2. The mean baseline weight for each group was: 1) Sham = 388 – 4 g; 2) Sham+L = 381 – 2 g; 3) Ocx = 386 – 5 g; 4) Ocx+L = 384 – 7 g; 5) Ocx+T = 398 – 4 g; and 6) Ocx+T+L = 379 – 6 g (P = 0.1). The mean final weight for each group was: 1) Sham = 445 – 8 g; 2) Sham+L = 445 – 5 g; 3) Ocx = 442 – 9 g; 4) Ocx+L = 418 – 8 g; 5) Ocx+T = 404 – 4 g; and 6) Ocx+T+L = 379 – 8 g. Paired statistical testing indicated that the baseline and final weight mea- surements significantly differed for all groups (P <0.0001), except for the Ocx+T and Ocx+T+L groups (P >0.05). Bone loss was successfully induced by ligature; the non-ligature groups significantly differed from the ligature-induced bone loss groups (P <0.05). When inflammation was absent (non-ligature groups), a tes- tosterone injection after orchiectomy significantly increased bone loss compared with either the orchiec- tomy alone or sham operation groups. In contrast, in the presence of inflammation (ligature groups), orchi- ectomized, but not testosterone-treated rats, pre- sented an increased bone loss compared with sham operation controls (P <0.05). The mean bone loss (– SEM) for each group is shown in Figure 3. Histometric analyses indicated that, in the pres- ence of inflammation, both supra- and subphysio- logic levels of testosterone significantly increased the gingival area, but there was no increase in gingi- val area when inflammation was absent. Ligature- induced inflammation resulted in a significantly increased buccal epithelium in Ocx and Ocx+T groups compared with sham operation controls (P <0.05), whereas connective tissue was signifi- cantly increased in only the Ocx+T group. The mean (– SEM) gingival area (mm2) for each group at each experimental period is listed in Table 1. Representative histologic images of each experi- mental group are shown in Figure 4. Figure 3. A) Mean (– SEM) distance from CEJ to alveolar bone height (ABH) (mm) measured in mesial root surfaces for each group (n = 10 animals/group). Same letters indicate no statistically significant difference (t test; P >0.05). B) Representative radiographs of each experimental group. ** BioEstat 5.0, Manuel Ayres. J Periodontol • November 2012 Steffens, Coimbra, Ramalho-Lucas, Rossa, Spolidorio 1435 DISCUSSION The effects of testosterone hormone on the peri- odontium and periodontal cells have been assessed both in humans8,10 and in vitro,33,34 but there is in- sufficient evidence to fully understand the exact effect of testosterone on periodontal tissues and inflammation in general. This issue is important, given the increasing age of most populations and the associated hormonal imbalances.1 Using an animal model enables evaluation of the effects of hormonal imbalances in vivo with the additional advantage of enabling ex vivo analyses. Sex steroid hormones, such as testosterone, carry out their function in adipose tissues by both geno- mic and non-genomic mechanisms, which leads to lipolysis.35 As testosterone regulates the amount and distribution of adipose tissues, it would be ex- pected that supraphysiologic levels would lead to fat burning compatible with the lack of statistically significant differences between final and baseline weights observed in the testosterone-treated rats. Ad- ditionally, the stress provided by weekly injections of a medication could have contributed to this finding. Bone loss was significantly induced by ligatures in every ligature group, thus validating the method used. The authors observed that orchiectomized rats presented increased bone loss when compared with sham operation controls, but this difference was not observed when the ligature was not placed around the teeth, even though measuring bone loss only in the mesial surface of the tooth could result in an underestimation of bone loss. Daltaban et al.8 demonstrated that in humans, clinical attachment levels were not statistically different in patients with hypergonadotropic hypogonadism compared with controls, although these authors observed a nega- tive correlation between gingival index and free tes- tosterone levels. In rats, bone marrow plasma and the bone marrow cell extract receptor activator of nuclear factor-kappa B ligand (RANKL), an essen- tial cytokine for bone resorption, are significantly in- creased 1 and 2 weeks postorchiectomy.36 It would, therefore, be expected that a decrease of testoster- one levels would also decrease peripheral estrogen levels (also a product from testosterone conver- sion), which are closely related to bone metabolism regulation. However, in rats, 30-day orchiectomy significantly decreased serum testosterone and DHT levels, but not estrogen, whereas supraphy- siologic testosterone treatment was related to sig- nificantly increased serum testosterone, DHT, and estrogen levels.37 Testosterone injections significantly increased both the connective tissue and epithelial areas in the presence of inflammation when compared with sham operation controls (P <0.05). These results are in accordance with findings in humans that revealed the association between prolonged use of steroids and gingival enlargement.10 The fact that sub- or supraphysiologic levels of testosterone did not influence the gingival area of non-ligature ani- mals could be at least partially attributed to the inflammation-induced higher conversion of testos- terone to DHT.33,34 Gingival fibroblasts present an- drogen receptors (but not estrogen receptors), and DHT upregulates their proliferation.25 However, our results clearly demonstrate that orchiectom- ized animals also present increased epithelial area when compared with sham operation controls. In fact, one study suggests that bone may contain an intraskeletal reservoir of sex steroids that are capable of producing biologic effects, and 30-day orchiectomy did not alter the testosterone or es- trogen reservoir but instead increased DHT levels by 39%.37 This suggests a role for the increased in- traskeletal DHT reservoir following orchiectomy or other testosterone-related mechanisms that may be involved in gingival tissue regulation. Supraphysiologic levels of testosterone were induced by the injection of testosterone esters, which resulted in a mean 11-fold maximum increase when compared with baseline serum levels 8 hours after drug administration. This represents a light hor- mone overdose (drug abusers usually take 10 to 100 times higher doses than those used for medical Table 1. Mean (– SEM) Gingival Area (mm2) for Each Group (n = 10 animals/group) Tissue Type Treatment Sham Ocx Ocx+T Buccal epithelium Control 6,662 – 947 3,438 – 383 4,875 – 1,260 Ligature 737 – 271* 7,245 – 1,826 14,995 – 8,209 Connective tissue Control 118,815 – 15,693 121,159 – 10,734 99,436 – 7,806 Ligature 62,264 – 24,305† 120,319 – 16,287 147,186 – 8,295 * Statistically significant difference compared with Ocx and Ocx+T (Mann-Whitney U; P =0.01). † Statistically significant difference compared with Ocx+T (Mann-Whitney U; P =0.02). Effects of Testosterone in the Periodontium Volume 83 • Number 11 1436 conditions).10 Our results clearly demonstrate that the medication dose used (250 mg/kg) provides 7-day supraphysiologic testosterone levels, thus indicating that the method used provided high levels of testosterone throughout the experiment. Supraphysiologic levels of testosterone groups were also subjected to orchi- ectomy to verify the effect of synthetic hormone on the tis- sues and to serve as controls for the orchiectomy group. We did not use a hormone repo- sition group (physiologic levels) because the non-operation an- imals served as normal tes- tosterone–level controls. As this was an initial study, only the consequences of ab- normal testosterone levels on rats’ periodontal tissues in the presence or absence of inflam- mation were assessed. However, the mechanisms by which tes- tosterone deficiency excessively influenced bone remodeling or epithelial and connective tissue areas of periodontal tissues in health and disease should be further investigated. CONCLUSIONS Both sub- and supraphysio- logic levels of testosterone may influence bone metabo- lism, but only subphysiologic levels significantly increase ligature-induced bone loss, thus suggesting a protective effect of normal testosterone levels on inflammation-induced al- veolar bone resorption. Last, testosterone has a regulatory effect on the gingival area. ACKNOWLEDGMENTS The authors thank the São Paulo Research Foundation (FAPESP), São Paulo, SP, Brazil, for their fi- nancial support (Grants #2010/ 09658-0 and #2010/12021-4). LCS is a fellowship recipient from theNationalCouncil forScientific and Technological Development (CNPq), Brası́lia, DF, Brazil. We also thank the Clinical Analysis Laboratory, Américo Brasiliense State Hospital, Américo Brasiliense, SP, Brazil, and the Foundation for the Development of Pharmaceutical Sciences (FUNDECIF), Araraquara, SP, Brazil, for the chemiluminescence-based immunoassay analysis. The authors report no conflicts of interest related to this study. Figure 4. Representative histologic images of each experimental group (H&E staining). J Periodontol • November 2012 Steffens, Coimbra, Ramalho-Lucas, Rossa, Spolidorio 1437 REFERENCES 1. Harvey J, Berry JA. Andropause in the aging male. J Nurse Pract 2009;5:207-212. 2. Bain J. Testosterone and the aging male: To treat or not to treat? Maturitas 2010;66:16-22. 3. Gray M. Andropause and the aging man: Separating evidence from speculation. Adv Nurse Pract 2005;13: 22-27, quiz 28. 4. Vandenput L, Ohlsson C. Sex steroid metabolism in the regulation of bone health in men. J Steroid Biochem Mol Biol 2010;121:582-588. 5. Ohlsson C, Vandenput L. The role of estrogens for male bone health. Eur J Endocrinol 2009;160:883- 889. 6. Gooren LJ. Androgens and male aging: Current evidence of safety and efficacy. Asian J Androl 2010; 12:136-151. 7. Bhasin S. Regulation of body composition by andro- gens. 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Methods to quantify sex steroid hormones in bone: Applications to the study of androgen ablation and administration. Am J Physiol Endocrinol Metab 2010;299:E841- E847. Correspondence: Professor Dr. Luis Carlos Spolidorio, Rua Humaitá, 1680, Araraquara, São Paulo, Brazil. Fax: 55-16- 33016488; e-mail: lcs@foar.unesp.br. Submitted November 6, 2011; accepted for publication January 10, 2012. J Periodontol • November 2012 Steffens, Coimbra, Ramalho-Lucas, Rossa, Spolidorio 1439 mailto:lcs@foar.unesp.br 51# ## CAPÍTULO 3 52# ## The Impact of Testosterone on Inflammation-Induced Periodontal Bone Loss in Rats* Joao Paulo Steffens, DDS, PhD student1 Leila Santana Coimbra, DDS, PhD1 Carlos Rossa Jr, DDS, PhD2 Alpdogan Kantarci, DDS, PhD3 Thomas E. Van Dyke, DDS, PhD3 Luis Carlos Spolidorio, DDS, PhD1 1 Department of Physiology and Pathology, Univ Estad Paulista – UNESP, School of Dentistry at Araraquara, SP, Brazil. 2 Department of Diagnosis and Surgery, Univ Estad Paulista – UNESP, School of Dentistry at Araraquara, SP, Brazil. 3 Department of Applied Oral Sciences, The Forsyth Institute, Cambridge, MA, USA. Reprint Requests / Corresponding Author Dr. Luis Carlos Spolidorio, Rua Humaita, 1680, Araraquara, SP, Brazil. Zip Code 14801-903. Phone: +55 16 3301-6479. E-mail: lcs@foar.unesp.br. No Supplemental Data Disclosure All authors state that they have no conflicts of interest. # ## # # # # ######################################################## *# De# acordo# com# as# instruções# do# periódico# Journal( of( Bone( and( Mineral( Research.# Disponível# em:# http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1523I 4681/homepage/ForAuthors.html# # # 53# Abstract Testosterone, the main androgen, can be present at different levels within an individual’s lifespan, which could alter host susceptibility to diseases. Periodontitis is an infectious disease that leads to inflammation and consequent bone loss around the teeth. The objective of this study was to evaluate the impact of different testosterone levels on ligature-induced periodontitis in vivo, assessing associated bone markers and cytokines expression, as well as to investigate the impact of testosterone on osteoclastogenesis in vitro. A total of 80 male adult rats were used in the study and subjected to treatments that resulted in subphysiologic (L), normal, or supraphysiologic (H) serum concentrations of testosterone. Forty rats were subjected to bilateral orchiectomy and 40 rats received testicular sham- operation. Twenty of the sham-operated animals received flutamide (F), an androgen receptor antagonist. Three days after orchiectomy, 20 of the rats started receiving testosterone injections. Four weeks after surgery, half of the rats received a subgingival cotton ligature around the lower first molars, which is an experimental model for periodontitis, and were killed two weeks later. In vitro, osteoclasts were generated from RAW264.7 precursors for 4 days. Test groups were treated with testosterone at doses of 1, 10, 100 nM or 1µM. F (100nM) was added to the 100nM testosterone group. In ligated animals, gingival levels of IL-1β were increased in L group, while F significantly reduced gingival IL-6 when compared to sham-operated animals. Linear analysis of bone loss using micro-computed tomography was increased for L, F and H groups when compared to ligated controls. Similarly, the number of osteoclasts was significantly reduced only when 10 and 100nM testosterone was added, which was successfully reversed by F. Testosterone also dose-dependently reduced TNF and RANTES. Testosterone modulates host response to periodontitis in rats, interfering at a molecular level in the progression of the disease. Keywords: testosterone; periodontitis; inflammation; androgens; gonadal steroid hormones. # # 54# Introduction Aging can be defined as a gradual and time-dependent decline of various physiological functions, leading to the end of lifespan [1]. Nine hallmarks of aging have been described: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication [2]. In 2000, the term “Inflammaging” was proposed to represent the low-grade pro-inflammatory status developed during the aging process, that predisposes the individual to age-related diseases [3]. Indeed, several experiments support this concept; aging mammals express an increase in genes related to the immune-inflammatory response; activation of nuclear factor kappa B (NF-κB) signaling (a key regulator of inflammation); and increased serum pro-inflammatory cytokines [4-8]. It was recently demonstrated that the aging process induces activation of inhibitor of nuclear factor kappa B kinase subunit beta (IKK-β) and NF-κB in the hypothalamus of mice, and mechanistic studies revealed that those proteins inhibited gonadotropin-releasing hormone (GnRH) [1]. In men, hypothalamic GnRH is responsible for regulating the release of interstitial cell-stimulating hormone by the pituitary gland, which in turn stimulates the Leydig cells to produce testosterone [9]. The decline of GnRH, and consequently testosterone, indicates the closing of the reproductive period of life – which is necessary for the quality of the species – but also contributes to systemic aging [1]. Declines in testosterone have broader consequences beyond reproduction. The maintenance of physiologic levels of testosterone is critical for overall men’s health. Cross- sectional and prospective studies show that low testosterone levels are related to increased serum soluble interleukin (IL)-6 receptor (sIL-6r) in older men, although results for IL-6 were controversial [10, 11]. In addition, testosterone replacement therapy in hypogonadal men significantly decreased serum pro-inflammatory cytokines IL-1β and tumor necrosis factor (TNF) [12]. A recent cross-sectional study demonstrated that macrophage inflammatory # # 55# protein 1-alpha (MIP-1α), 1-beta (MIP-1β) and TNF are negatively associated with total testosterone in young men, before any concurrent manifestation of age-related systemic diseases, suggesting that low testosterone levels also promotes an aging-independent pro- inflammatory status [13]. Other studies have shown that testosterone treatment of orchiectomized mice and young male rats suppresses leukocyte counts [14, 15]. The results also showed that testosterone treatment decreased monocyte counts, CD4+/CD8+ ratio, and inhibited proliferative responses of lymphocytes [15]. Due to the immunosuppressive properties of the doses of testosterone used, animals that were treated with testosterone were more susceptible to infection [14]. Periodontitis is a chronic inflammatory disease affecting the supporting structures of teeth that is characterized by an overproduction of innate immune cytokines, such as IL-1β, IL-6, and TNF, leading to tissue breakdown, and consequent loss of clinical attachment and alveolar bone [16]. The loss of bone in periodontitis is thought to be a consequence of altered coupling of bone resorption and formation. The disease etiology is infectious; it is initiated and maintained by specific microorganisms organized on the tooth surfaces as an oral biofilm. Since the pathogens associated with periodontitis are commensal, susceptibility and host response are key regulators of disease initiation and progression, and have been investigated at genetic, cellular and molecular levels [17]. Since periodontitis is an infection-derived inflammatory disease, we used a model of experimental periodontal disease in rats to test the hypothesis that sub- and supraphysiologic testosterone levels impact host susceptibility to periodontal bone loss. The objective of the present study is to evaluate the actions of testosterone in an experimental model of periodontitis in vivo, assessing associated bone markers and cytokine expression, as well as to investigate the impact of testosterone on osteoclastogenesis in vitro. # # 56# Materials and Methods Animals Eighty male adult Holtzman rats weighing 300-400g were kept in cages under similar conditions (controlled temperature 23±2ºC, humidity 65-75% and 12-hour light-dark cycles). Food and water were provided ad libitum. Randomization of animals was performed using a riffle method. All experimental protocols were approved by the Institutional Ethics Committee for Animal Experimentation (protocol #25/2010) and performed in accordance with the guidelines of the Brazilian Society of Science on Laboratory Animals (SBCAL). This study conforms to the ARRIVE guidelines. Induction of Sub- and Supraphysiologic Serum Testosterone Levels After 1 week of acclimatization, forty rats received orchiectomy to suppress testosterone production. Briefly, a scrotal incision was performed for bilateral testicular removal and the incision was sutured under anesthesia using ketamine [1 mL/kg/body weight (bw)] and xylazine (0.4 mL/kg/bw) under sterile conditions. The rats were given acetaminophen (300 mg/kg/bw; orally) for postoperative pain and a single intramuscular dose of penicillin and streptomycin (1 mL/kg/bw). After the procedure, the animals were kept in individual cages for recovery for 7 days. Starting three days after orchiectomy, twenty of the rats received 250 mg/kg of a long-acting mixture of testosterone esters – 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Durateston, MSD, Campinas, SP, Brazil). The medication was diluted to 0.1mL in corn oil and injected intramuscularly every 7 days until sacrifice. Twenty other rats received the same surgical procedure except for the testicular removal and were considered sham-surgery controls. Assessment of the Role of Androgen Receptors on Testosterone-Related Responses Flutamide (Sigma-Aldrich, Saint Louis, MO, USA; 50mg/kg), an androgen receptor antagonist, was administered intragastrically, every other day, to twenty sham-surgery # # 57# controls until sacrifice. Flutamide was diluted to 25mg/mL in distilled water and Tween-20. The treatment was started three days after sham-surgery [18]. Induction of Experimental Periodontal Disease Four weeks after orchiectomy (or sham-surgery), half of the rats in each group (n=10/group) were anesthetized as above. A 3.0 cotton ligature was placed in a subgingival position around the lower first molar tooth. Ligatures enable bacterial accumulation leading to inflammation and bone loss. The other 10 animals in each group served as controls. The ligatures were maintained for 2 weeks at which time all rats were sacrificed. A schematic representation of the in vivo study design is shown in figure 1. Blood Assessment A blood sample was collected from every animal at the end of the experiment. After clotting for 45 minutes at room temperature, the sample was centrifuged for 10 minutes at 3,000 rpm to obtain serum. Each serum sample was analyzed for total testosterone levels using a chemiluminescence-based immunoassay (Immulite 2000, Diagnostic Products Corporation, Gwynedd, UK); biochemical colorimetric tests for calcium (Ca2+), alkaline phosphatase (ALP) and phosphorus (P) (Bioclin, Quibasa, Belo Horizonte, MG, Brazil); and enzyme-linked immunosorbent assays (ELISA) for the detection of interleukin (IL)-1β and IL-6, using commercially available kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Micro Computed Tomography (µCT) Analyses For quantitative and qualitative three-dimensional (3D) analysis of the alveolar bone, mandibles of 5 animals per group were scanned using a µCT system (Skyscan, Aartselaar, Belgium). The specimens were scanned at a resolution of 18 µm in all three spatial dimensions. CTan/CTvol software (Skyscan) was used for imaging and analysis. The region of interest (ROI) was interpolated and drawn including all bone medial to the mesial roots of # # 58# the second molar to the mesial surface of the first molar, using a slice-based method. Bone volume fraction (BV/TV) was analyzed by the CT-scan software. Additionally, the linear distance between the cemento-enamel junction and alveolar bone was measured on the mesial surface of the first molars using Dataviewer 1.4.3, (Skyscan) software. The measurement was performed three times by a calibrated individual, who was blinded to the treatment groups, and under the same background conditions. The mean of all three measurements was considered one sample and used for statistical analysis. Local Expression of Cytokines in the Tissue The mucogingival tissues around the first molars of the remaining 5 animals per group were removed and processed for concentrations of IL-1β and IL-6 using commercially available ELISA kits (R&D Systems), according to the manufacturer’s instructions. Total protein content in each sample was determined using the Bradford method and the results were used for normalization. Increasing Testosterone Concentration and Osteoclast-like Cells The RAW264.7 murine monocyte/macrophage cell line was obtained from ATCC (Manassas, VA, USA) and cultured in T-75 flasks containing MEM-alpha supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin until approximately 90% cell confluence, with media replacement every three days. On day 4, cells were seeded at 2.5x104 cells/mL in 96-well plates (200µL/well) in the same media with the addition of 50ng/mL recombinant murine sRANKL (PeproTech, Rocky Hill, NJ, USA). Testosterone (Sigma-Aldrich) was diluted in DMSO to a stock concentration of 100 mM and further dilutions were performed using medium. The tested doses included 1nM, 10nM, 100nM and 1µM. 1% DMSO was added to each control well. Flutamide (Sigma- Aldrich), 1µM, was used alone or in addition t