CARLOS AUGUSTO NASSAR Avaliação morfométrica e estereométrica dos tecidos periodontais de ratos imunossuprimidos por Tacrolimus (FK506) ARARAQUARA 2006 UNIVERSIDADE ESTADUAL PAULISTA FACULDADE DE ODONTOLOGIA DE ARARAQUARA CARLOS AUGUSTO NASSAR Avaliação morfométrica e estereométrica dos tecidos periodontais de ratos imunossuprimidos por Tacrolimus (FK506) ARARAQUARA 2006 UNIVERSIDADE ESTADUAL PAULISTA FACULDADE DE ODONTOLOGIA DE ARARAQUARA Tese apresentada ao Programa de Pós-Graduação da Faculdade de Odontologia de Araraquara, Universidade Estadual Paulista para a obtenção do título de Doutor em Periodontia. Orientador: Prof. Dr. Luis Carlos Spolidorio Nassar, Carlos Augusto Avaliação morfométrica e estereométrica dos tecidos periodontais de ratos imunossuprimidos por Tacrolimus (FK506) Carlos Augusto Nassar. – Araraquara : [s.n.], 2006. 126 f. ; 30 cm. Tese (Doutorado) – Universidade Estadual Paulista, Faculdade de Odontologia. Orientador : Prof. Dr. Luis Carlos Spolidorio 1. Tacrolimo 2. Doenças periodontais 3. Cemento dentário 4. Gengiva 5. Osso alveolar I. Título. Ficha catalográfica elaborada pela Bibliotecária Ceres Maria Carvalho Galvão de Freitas CRB 8/4612 Serviço Técnico de Biblioteca e Documentação da Faculdade de Odontologia de Araraquara / UNESP DADOS CURRICULARES CARLOS AUGUSTO NASSAR Nascimento 21 de maio de 1973 Ribeirão Preto – SP Filiação Jorge Nassar Emília Josephina Gallo Nassar 1992 – 1995 Curso de Odontologia – Faculdade de Odontologia de Araraquara – UNESP – São Paulo 1998 – 2000 Curso de Pós-Graduação em Periodontia, nível de Mestrado – Faculdade de Odontologia de Araraquara – UNESP. 2003 – 2006 Curso de Pós-Graduação em Periodontia, nível de Doutorado – Faculdade de Odontologia de Araraquara – UNESP. ...A DEUS “pela certeza de que para todos os acontecimentos da vida, bons ou ruins, existe uma razão norteada pela justiça divina.” ...À MINHA ESPOSA PATRICIA, pela dedicação, luta, determinação, amor e paciência para que todos os meus sonhos se realizassem. Acima de tudo, eterna companheira, incansável colaboradora e meu verdadeiro amor. A grande responsável por esta conquista e por todas da minha vida. ...AOS MEUS PAIS JORGE E EMÍLIA, exemplos de união, coragem, luta, renúncia, fé, carinho e dedicação, incondicionais a seus filhos... Os verdadeiros exemplos de vida. ...AOS MEUS IRMÃOS E CUNHADA: VALÉRIA, PATRICIA, SUMÁIRA, JORGE E ESTELA, meus melhores amigos, verdadeiros em tudo o que fazem. Pela força, atenção, compreensão e apoio dados ao irmão caçula, principalmente pelo amor e carinho em todos os momentos. ...AOS MEUS SOBRINHOS: LEONARDO, GIOVANNA, VINÍCIUS E HENRIQUE, Pelas brincadeiras e momentos de alegria que mantiveram meu espírito criança capaz de esquecer os problemas e enxergar as dificuldades com simplicidade. ...AOS MEUS SOGROS VALDEMIR E ROSEMARI E AOS MEUS CUNHADOS: RENATO E GABRIELA, RODRIGO E NATÁLIA, E JUNINHO, pelo constante apoio, carinho e amizade. Exemplos de vida e de seres humanos. ...AO PROF. DR. LUÍS CARLOS SPOLIDORIO não só o orientador e professor, mas acima de tudo um verdadeiro amigo e exemplo para ser seguido por toda a minha vida. Agradeço pela atenção a esta tese e principalmente pelo apoio em todos os momentos. Mesmo nos momentos difíceis estava pronto para nos receber. ...AO PROF. DR. JONI AUGUSTO CIRELLI, pela amizade, confiança e apoio. Espero poder retribuir-lhe tudo o que já fez e que Deus continue protegendo e iluminando você nesta brilhante caminhada... AGRADECIMENTOS Minha sincera gratidão aos que, direta ou indiretamente, contribuíram para a realização deste trabalho, em particular: À Faculdade de Odontologia de Araraquara, nas pessoas de sua Diretora, Profa. Dra. Rosemary Adriana Chiérici Marcantonio, e Vice-Diretor, Prof. Dr. José Cláudio Martins Segalla. Ao coordenador do Curso de Pós-Graduação – Área de Periodontia, Prof. Dr. Carlos Rossa Júnior, e a todos os docentes do Curso de Pós-Graduação, pela excelente formação, dedicação e exemplo. Aos amigos e Docentes da Disciplina de Periodontia, Prof. Dr. Benedicto Egbert Corrêa de Toledo, Prof. Dr. Ricardo Samih Georges Abi Rached, Prof. Dr. Elcio Marcantonio Junior, Prof. Dr. José Eduardo Cezar Sampaio, Profa. Dra. Rosemary Adriana Chiérici Marcantonio, Prof. Dr. Joni Augusto Cirelli, Prof. Dr. Carlos Rossa Junior e Profa. Dra. Silvana Regina Perez Orrico, pela formação e orientação. Aos amigos do Curso de Pós-Graduação em Periodontia, em especial Ana Emília, Carla e Joseane, pelos momentos de descontração, companheirismo e caráter; estejam certas de que tais atitudes jamais serão esquecidas. A Patrícia, Morgana e Denise pelo apoio, ajuda, cooperação, compreensão, amizade e dedicação na realização deste trabalho, podem contar sempre com a minha eterna gratidão e amizade. A todos os funcionários da Disciplina de Periodontia, D. Cidinha, Claudia, D. Maria do Rosário, D. Teresinha, Maria José, Thelma, Sueli e Toninho, cujo trabalho, dedicação e compreensão possibilitou a realização deste trabalho. À Regina Lúcia, pela cooperação e paciência e acima de tudo à competência no trabalho. Aos professores e funcionários do Departamento de Fisiologia e Patologia, em especial, Profa. Denise, Profa. Rita, Prof. Beto, Prof. Tato e Prof. Vanderlei; e em especial também ao José Antonio pela oportunidade, disponibilidade e ajuda. À Profa. Teresa Pepato e ao Prof. Iguatemi pela disponibilidade e ajuda no Laboratório de Bioquímica da Faculdade de Farmácia, e em especial aos funcionários Marcos e Valéria, do Laboratório de Bioquímica, pela ajuda, compreensão e disposição. Aos funcionários do Biotério, Donizeti e Betinha, pelo carinho, cuidado e atenção dedicados aos ratos do experimento. A CAPES e A FAPESP, que possibilitaram a ajuda financeira. Aos funcionários da Seção de Pós-Graduação, Mara, Rosângela, Vera, Silvia e Guilherme, pela paciência, competência e cooperação no decorrer de todo o Curso. A todos os funcionários da Faculdade, em especial aos funcionários da Biblioteca, Adriano, Ceres, Eliane, Eliane Scarso, Maria Aparecida, Maria Helena, Maria Inês, Maria José, Marley, Odete, Sandra e Silvia pela colaboração e paciência. A todos os meus amigos sempre presentes nesta minha caminhada: Ana Emília, Fernando, Maurício, Morgana, Luis Fernando, Henrique e Ju Moraes, pela convivência, festas, brincadeiras, apoio e sonhos compartilhados. Ao Curso de Odontologia de Cascavel da Universidade Estadual do Oeste do Paraná – UNIOESTE, nas pessoas do Digníssimo Reitor, Alcebíades Luiz Orlando e do Coordenador do Curso Prof. Júlio Katuhide Ueda, pelo apoio e compreensão dispensados para a realização deste Curso. Aos professores da Disciplina de Periodontia da UNIOESTE, Profa. Patricia e Profa. Adriane, pela cooperação, ajuda e compreensão. Aos professores e amigos de Cascavel, Adriano, Christian, Edo, Frank, Roberto e Juliana, Julio, Sandra e Julia e Felipe, Tanaka, Neto, Veridiana, Daniela e Ricardo, pela convivência, festas, apoio e amizade nesta longa e difícil caminhada... MUITO OBRIGADO!!!! HOMENAGEM “Aos animais que participaram desta pesquisa, que silenciosamente se deixaram levar pelas mãos do Homem. As mesmas mãos que os afagaram e os sacrificaram. Sabiam do destino desde o início, pois seus olhos não enganam, mas mesmo assim tiveram a coragem de servir à ciência. Ficam aqui os meus sinceros agradecimentos aos dóceis e amáveis ratos.........” SUMÁRIO INTRODUÇÃO ------------------------------------------------------------------------ 17 PROPOSIÇÃO ------------------------------------------------------------------------ 22 DISCUSSÃO -------------------------------------------------------------------------- 102 CONCLUSÃO ------------------------------------------------------------------------- 108 REFERÊNCIAS ----------------------------------------------------------------------- 110 RESUMO ------------------------------------------------------------------------------- 121 ABSTRACT ---------------------------------------------------------------------------- 124 INTRODUÇÃO 18 Os transplantes de órgãos, hoje corriqueiros, representam o coroamento de séculos de aperfeiçoamento da cirurgia, concomitante ao aprofundamento dos conhecimentos dos mecanismos de rejeição dos transplantes, que estão associados ao crescente desenvolvimento das técnicas e conceitos da imunologia e, do entendimento sobre os genes seus produtos e funções. Simultaneamente ao enriquecimento dos conhecimentos dos fenômenos biológicos fundamentados na biologia molecular, o desenvolvimento de drogas imunossupressoras tem propiciado sucesso dos transplantes e aumento na sobrevida dos transplantados. A necessidade de se evitar a rejeição levou ao desenvolvimento de novas drogas imuno-moduladoras e as pesquisas sobre as maneiras de se introduzir tolerância aos tecidos transplantados, métodos que na verdade têm uma aplicação mais geral no tratamento de várias doenças imunes, como o dano tecidual imuno-mediado na hipersensibilidade e na auto-imunidade (SPOLIDORIO et al., 2004). Na última década ocorreram avanços substanciais na compreensão de eventos que controlam o sistema imune e dos efeitos seletivos das drogas, capazes de alterar a função imune produzindo não apenas imunodeficiências, mas também uma imunopotencialização. Os agentes mais comumente utilizados na imunoterapia sistêmica são os esteróides, ciclofosfamida, azatioprina, mofetil micofenolato, metotrexato, sirolimus (rapamicina), anticorpos monoclonais, ciclosporina (CsA) e tacrolimus (FK506) (DUNN et al., 2001; van MOURIK e KELLY, 2001; DEBRAY et al., 2003; MENGARELLI et al., 2003). Há alguns anos junto com o aprimoramento de técnicas cirúrgicas, as drogas imunossupressoras, principalmente às de última geração, propiciaram de modo incontestável avanços significativos no sucesso na inibição da rejeição de transplantes. O grande sucesso dos transplantes culminou com a introdução da terapêutica imunossupressora com CsA, por Borel et al., em 1976, sendo atualmente substituída pelo FK506, que apresenta uma potência maior e com menores efeitos indesejáveis (OTT et al., 2003). A CsA é um polipeptídeo cíclico hidrofóbico e lipofílico, composto por 11 aminoácidos de fórmula C62 H111 O12 (peso molecular 1202.6 kDa) e isolado de 19 duas espécies de fungos Trichoderma polysporum rifai e Cylindrocarpo lucidum (DALEY e WYSOCKI, 1984). Quando descoberta, foi inicialmente utilizada como agente antifúngico, sendo produzida comercialmente a partir da cultura do fungo Tolyplocadium inflatum Gams (BOREL et al., 1976; SEYMOUR e JACOBS, 1992). Após aprovação pelo Food and Drugs Administration , em 1985, tem sido empregada como um dos principais agentes imunossupressores em indivíduos órgãos–transplantados (DUNN et al., 2001). Em 1984, um novo agente imunossupressor, FK506, foi descoberto no Japão e usado pela primeira vez em 1989. O FK506 é um macrolídeo produzido pela fermentação com Streptomyces tsukubaensis, e agiria nas células mediadoras de imunidade por inibição nas sínteses de citocinas nas concentrações de linfócitos CD4 (BADER et al., 1998). O FK506 possui ação de inibição de síntese de citocinas nos linfócitos CD4, sendo que age também na resposta imune através da inibição da formação de linfócitos-T citotóxicos responsáveis pela rejeição de enxertos, da supressão da ativação de células-T por inibir a produção de linfocinas como interleucina-2(IL-2), receptor de IL-2 e interferon gama, inibindo ainda, a ativação de células-B e bloqueando a transcrição de fator de necrose tumoral alfa (BADER et al., 1998; OETTINGER-BARAK et al., 2001). O FK506, entretanto, não inibe a proliferação secundária de células ativadas em resposta a IL-2, nem a droga interfere com a apresentação do antígeno ou modifica a função do fagócito mononuclear ou da célula Natural killer (PETERS et al., 1993; SPENCER et al., 1997; PLOSKER e FOSTER, 2000). Alguns dos efeitos do FK506 podem também ser mediados através da modulação de atividades celulares tais como a inibição da ativação de óxido nítrico sintase (KAIBORI et al., 1999; HAMALAINEN et al., 2002) (uma enzima envolvida na geração dos radicais livre de oxigênio que causam subseqüentemente danos aos tecidos) e indução de apoptoses (MIGITA et al.,1995, 1997, 1999). O seu uso foi estabelecido como uma alternativa de droga imunossupressora em substituição a CsA, sendo que possui uma atividade imunossupressora até 100 vezes maior que a própria CsA (ZICCARDI et al.,, 1991). Entretanto, o FK506 produz efeitos secundários indesejáveis incluindo 20 nefrotoxicidade, neurotoxicidade e indução de estados de diabetes (JAMES et al., 2000), principalmente quando associado ao uso de esteróides (FINNI et al., 2004). O FK506 é rapidamente absorvido no trato gastrintestinal (PLOSKER e FOSTER, 2000; SCOTT et al., 2003), sendo a taxa e a extensão da absorção reduzida na presença do alimento (PETERS et al., 1993; SPENCER et al., 1997; PLOSKER e FOSTER, 2000), demonstrado nos estudos em animais, que indicaram que o FK506 está distribuído extensamente na maioria dos tecidos incluindo os pulmões, o baço, o coração, o rim, o pâncreas, o cérebro, o músculo e o fígado (PETERS et al., 1993; VENKATARAMANAN et al., 1995; SPENCER et al., 1997; PLOSKER e FOSTER, 2000; WALLEMACQ e VERBEECK, 2001; CHRISTIANS et al., 2002). O FK506 submete-se ao metabolismo extensivo no fígado, com menos de 1% de droga inalterada excretada na urina e metabolizada também na mucosa intestinal (VENKATARAMANAN et al., 1995; PLOSKER e FOSTER, 2000). Os dados animais indicam que a excreção biliar é a rota principal para a eliminação dos metabólitos, com eliminação fecal em 90% da dose administrada (PLOSKER e FOSTER, 2000; SCOTT et al., 2003). Aventa-se a hipótese que fatores como dosagem, biofilme dentário, idade e gênero do paciente, assim como, fatores genéticos, tratamento concomitante com outras drogas, possam ser fatores de risco, isto é, estar associados com aumento da prevalência, da extensão ou da severidade do aumento gengival induzido pela CsA (SEYMOUR et al., 2000). Recentemente Montebugnoli et al. (2000) e Spolidorio et al. (2004), verificaram em humanos e modelos experimentais, respectivamente, que o tempo de tratamento com CsA poderia influenciar positivamente na involução do aumento gengival. Essa informação abre outro cenário de investigação pouco explorado em humanos e em animais de laboratório. Entretanto, há relatos na literatura que os ossos de maneira geral assim como o processo alveolar (EPSTEIN, 1996; HOFBAUER et al., 1999; HOFBAUER e HEUFELDER, 2000; 21 FU et al., 2001; SHEN et al., 2001) e o cemento (AYANOGLOU e LESTY, 1997; AYANOGLOU, 1998, 1999) são acometidos pela CsA desequilibrando as suas homeostases. Ainda são escassos os relatos da ação do FK506 sobre os tecidos gengivais e no processo alveolar, sendo que em diversos tratamentos, principalmente em transplantes de fígado, ocorre a substituição da CsA por essa droga imunossupressora, para que se possam reduzir os efeitos indesejáveis (JAMES et al., 2000). Atualmente existem poucos relatos de crescimento gengival induzidos pelo uso do FK506 (ADAMS e FAMILI, 1991; OETTINGER-BARAK et al., 2001), mas na realidade, essas alterações são atribuídas a outras drogas, como a prednisolona e nifedipina, que são administrados concomitantemente com o FK506. Sugere-se que a substituição da CsA pelo FK506 possa reduzir significativamente esse efeito indesejável (KOHNLE et al., 1998; JAMES et al., 2000, 2001; HERNANDEZ et al., 2000; THORP et al., 2000). Os dados apresentados na literatura da ação do FK506 sobre o tecido ósseo, são incertos e conflitantes. Alguns trabalhos experimentais mostram perda óssea (CVETKOVIC et al., 1994; ROMERO et al., 1995; PARK et al., 1996; CAYCO et al., 2000; STEMPFLE et al., 2002), enquanto que em outros trabalhos, o FK506 exerceu efeitos favoráveis no metabolismo ósseo, promovendo diferenciação osteoblástica, mantendo, assim, a densidade mineral óssea normal em modelos experimentais (INOUE et al., 2000; GOFFIN et al., 2002). 22 PROPOSIÇÃO 23 Baseado nas informações, anteriormente citadas, pode-se aventar alguns questionamentos: a administração do FK506 em ratos induz aumento gengival? Como se comporta a lesão gengival com o decorrer do tempo de tratamento? Partindo da premissa que o FK506 induz perda óssea, pode-se questionar se esse processo evolui proporcionalmente com o tempo de tratamento. E a eventual alteração do metabolismo ósseo está na dependência do aumento do número de osteoclastos ou nas disfunções de Ca2+ e fosfatase alcalina? A partir dos dados da literatura e desses questionamentos, acima citados, o presente trabalho terá como objetivos: - Determinar através de morfometria as características do epitélio e tecido conjuntivo da gengiva de ratos tratados com FK506 por vários períodos. - Determinar a concentração sérica de Ca2 + e fosfatase alcalina em todos os períodos de tratamento, dos ratos tratados com FK506. - Determinar através da estereometria a densidade volumétrica do osso de ratos tratados com FK506. - Determinar as dimensões morfométricas do cemento da região de primeiros molares tratados por vários períodos com FK506. - Determinar a concentração sérica de glicemia em todos os períodos de tratamento, dos ratos tratados com FK506. 24 TACROLIMUS: A OVERVIEW WITH RELATION AT PERIODONTAL DISEASE Submetido à publicação no periódico Oral Diseases 25 Tacrolimus: An overview with relation at periodontal tissue Carlos Augusto Nassar, Patricia Oehlmeyer Nassar, Denise Carleto Andia, Morgana Rodrigues Guimarães, Luis Carlos Spolidorio. Abstract Introduction: In the last years, the improvement of surgical techniques and the use of the immunosuppressive drugs, mainly those of the last generation, have propitiated significant advance and success in preventing rejection of transplants. The molecular mechanisms of the inhibition of T cell activation by tacrolimus are well understood. However, a frequent complication was reported in patients following organs transplant that were treated with tacrolimus. It was observed the development of side effects. Objective: This paper reports an overview of the action of tacrolimus considering the relation and effects in the periodontal tissue. Discussion: Some works have evaluated the skeletal effects of tacrolimus in humans or in animals, and the results obtained are uncertain and conflicting. Both cardiac and liver transplant recipients and animals have sustained rapid bone loss with tacrolimus-based immunosuppression. Other works have noted that bone loss decreases with tacrolimus and this fact has a favorable effect in bone metabolism, because it induces the differentiation of the osteoblastic, maintaining the normal bone mineral density in experimental models. Conclusion: The results indicate that the use of tacrolimus may be beneficial for suitable patients with marked gingival overgrowth, but the action of the drug on bone tissues are uncertain and conflicting. Keywords: tacrolimus; gingival tissue; alveolar bone; side effects 26 Introduction In the practical clinic, the organ transplantation aims to improve a functional deficit, unless the donor and the recipient be genetically identical, the antigens of graft may provoke a reply of immunologic rejection. A transplant can stimulate all the mechanisms (specific and non-specific) of the humoral and cellular immunity (Dunn et al., 2001). During the last decade, substantial advances were obtained to understand the events that control the immune system and the selective effects of drugs that may alter the immune function, producing immunosuppression. The agents more commonly used in the systemic immunotherapy are steroidal drugs like cyclofosfamid, azathioprine, mycophenolate mofetil, metotrexate, sirolimus (rapamycin), monoclines antibodies, cyclosporine-A (CsA) and tacrolimus (FK506) (Dunn et al., 2001; van Mourik and Kelly, 2001; Debray et al., 2003; Mengarelli et al., 2003). In the last years, the improvement of surgical techniques and the use of immunosuppressive drugs, principally of the last generation, provided significant advance and success in the inhibition of the rejections of the transplants (Mengarelli et al., 2003). The molecular mechanisms of the inhibition of T cell activation by tacrolimus have been understood (Shibasaki et al., 2002). The cytokine synthesis may be inhibited by Tacrolimus and this drug influences the formation of cytotoxic T-lymphocytes responsible for graft rejection. The engagement of T cell receptor with MHC/peptide normally provokes a calcium-dependent intracellular signal that results in an activation of the calcium/calmodulin-dependent phosphatase calcineurin. This leads to the dephosphorylation of NF-AT, allowing the translocation into the nucleus, where it enhances the binding of a transcription factor of the gene encoding for pro-inflammatory cytokines such as IL-2, IL-3, IL-4, IFN-γ and TNF-α. Tacrolimus enters in the cytoplasm and forms complexes with their immunophilin, called tacrolimus binding protein (FKBP-12). The tacrolimus-immunophilin complexes inhibit calcineurin activity and hence prevent nuclear translocation of NF-AT and cytokines gene transcription (Bader 27 et al., 1998; Oettinger-Barak et al., 2001). The use of tacrolimus was established as an alternative in substitution to CsA, due to the biggest immunosuppressive activity, 500 times more efficient than CsA (Ziccardi et al., 1991). However, a frequent complication was reported in patients after organs transplant and treated with tacrolimus. These patients showed the development of nephrotoxicity, neurotoxicity and induction of diabetes state (James et al., 2000). According our knowledge, there are few studies about the effect of tacrolimus on gingival tissues and cementum (Adams and Famili, 1991; James et al., 2000; Oettinger-Barak et al., 2001). Some works have evaluated the skeletal effects of tacrolimus in humans or in animals. Data presented in the literature considering the action of tacrolimus on bone tissues are uncertain and conflicting. Both cardiac (Stempfle et al., 1998) and liver (Park et al., 1996; Cayco et al., 2000; Stempfle et al., 2002) transplant recipients or animals (Cvetkovic et al., 1994; Romero et al., 1995) have sustained rapid bone loss with tacrolimus-based immunosuppresion. However, other works suggesting a decreasing in the bone loss with tacrolimus treatment and this fact has a favorable effect in the bone metabolism, promoting differentiation of the osteoblastic, maintaining the normal bone mineral density in experimental models (Inoue et al., 2000; Goffin et al., 2002). Pharmacodynamic Properties Tacrolimus (FK506), a macrolide immunosuppressant, inhibits cellular and humoral immune responses via several mechanisms of action. The principal effect involves the inhibition of calcineurin, a serine-threonine phosphatase (Fig. 1). Inactivation of calcineurin via complex formation with the immunophilin FK506 binding protein 12 (FKBP12) prevents the translocation of the transcription factor of activated T cells that promotes interleukin-2 (IL-2) mediated proliferation of helper T cells (Plosker et al., 2000; Scott et al., 2003). Tacrolimus may suppress T-cell activation inhibiting the production of lymphokines, such as IL-3, IL-4, IL-6, alpha tumor factor necrosis (TNFα), beta tumor factor necrosis (TNFβ) and 28 gamma interferon (INFγ). Tacrolimus inhibits B-cell activation through its action of T-cells and by blocking the transcription of the alpha-TNF gene (Bader et al., 1998; James et al., 2000; Scott et al., 2003) (Fig. 2). However, tacrolimus does not inhibit the secondary proliferation of activated cells in response to IL-2, and does not interfere with antigen presentation or modify mononuclear phagocyte or natural killer cell function (Peters et al., 1993; Spencer et al., 1997; Plosker, et al., 2000). Some effects of tacrolimus may also be mediated by modulation of cellular activities, such as inhibition of nitric oxide synthetase activation (Kaibori et al., 1999; Hämäläinem et al., 2002) (an enzyme involved in the production of oxygen-free radicals that cause tissue damage) and apoptosis induction (Migita et al., 1995; Migita et al., 1997; Migita et al., 1999). Absorption and Distribution Tacrolimus may be absorbed rapidly, but incompletely, in the gastrointestinal tract. The peak of tacrolimus concentration in whole-blood (Cmax) by oral administration is obtained after 1-2 hours, approximately. (Plosker et al., 2000; Scott et al., 2003). The oral bioavailability of tacrolimus is poor, with average bioavailability of 25% (range 4-93%), and is similar between adult (25%) and pediatric (31%) transplant recipients (Venkataramanan et al., 1995; Plosker et al., 2000; Wallemacq et al., 2001; Christians et al., 2002; Scott et al., 2003). The rate and the extent of tacrolimus absorption are reduced in the presence of food (Peters et al., 1993; Spencer et al., 1997; Plosker et al., 2000). A study with 15 healthy volunteers was realized with a single oral dose of tacrolimus, 5mg, and showed that food had a clinically significant effect in reducing the relative bioavailability, as well as slowing the absorption, but did not influence the half-life. (Bekersky et al., 2001a; Staatz & Tett, 2004). Other studies investigated the effect of meal timing. Bekersky et al. (2001b) analyzed four treatments: I- fasting for 10 hours; II- ingestion 1 hour before breakfast; III- ingestion after breakfast; IV- ingestion 1.5 hours after 29 breakfast. Tacrolimus absorption in the fasting state (I) had a greater relative bioavailability than other treatments. The AUC averaged 312, 276, 205 and 203 µg/ h/L were employed for treatments I, II, III and IV, respectively. Absorption was significantly prolonged after meal. Animal studies indicated that tacrolimus is widely distributed in most tissues, including lungs, spleen, heart, kidney, pancreas, brain, muscle and liver (Peters et al., 1993; Venkataramanan et al., 1995; Spencer et al., 1997; Plosker et al., 2000; Wallemacq et al., 2001; Christians et al., 2002). Tacrolimus may cross the placenta with umbilical cord plasma concentrations that are approximately one-third of those in maternal plasma. In addition, levels in breast milk were reported to be similar to those observed in the plasma (Venkataramanan et al., 1995; Wallemacq et al., 2001). Metabolism and elimination Tacrolimus is metabolized, extensively, by CYP3A isoenzymes (mainly) in the liver and intestinal wall, with < 0.5% of the parent drug appearing unchanged in the urine or excrements. Expression of these enzymes varies widely. The CYP3A subfamily consists of four isoforms: CYP3A4, CYP3A5, CYP3A7 and CYP3A43. These isoforms overlap substrate specificity, then, it is difficult to separate the relative contributions to the metabolism of the drug (Staatz & Tett, 2004). Tacrolimus undergoes extensive metabolism in the liver, with less than 1% of unchanged drug excreted in the urine, and it is also metabolized in the intestinal mucosa, in a minor extension (Venkataramanan et al., 1995; Plosker et al., 2000). More than 95% of tacrolimus metabolites are eliminated by the biliary route. Urinary excretion accounts for elimination of 2.4% of the drug, on average. Biliary obstruction may increase the concentration of tacrolimus metabolites in the blood (Staatz & Tett, 2004). Animal data indicate that biliary excretion is the main route for elimination of metabolites, 90% (or more) of the administered dose can be eliminated through the excrements (Plosker et al., 2000; Scott et al., 2003). 30 Side effects Many side effects are dose dependent and are related to the sites where tacrolimus concentration is high (Shibasaki et al., 2002). The side effects associated with tacrolimus-based immunosuppression are given in Table 1. An overview of the action of tacrolimus on gingiva, bone and cementum are demonstrated. Tacrolimus and Disturbances in Glucose Metabolism One of the more serious side effects associated with tacrolimus treatment is the development of a pos-transplantation diabetes mellitus (PTDM), predisposing the patient to complications of diabetes mellitus and to the risk of decreased patient and graft survival (Maes et al., 2001; Scott et al., 2003). The infections are associated with the rapid deterioration of graft function in renal allograft, considering that hyperglycemia and hyperinsulinemia promote atherosclerosis and then, the risk of cardiovascular complications increases. (Drachenberg et al., 1999; Scott et al., 2003). The influence of diabetes in periodontal tissue is being investigated. It is difficult to get definitive conclusions about the specific effect of diabetes in periodontal tissue, but many alterations are described, including trend to the generalized gingival overgrowth, dental abscesses, periodontitis and dental loss. Perhaps, the principal alteration of non-controlled diabetes was the reduction of the defense mechanism and the increase of the susceptibility to the infections, leading to the destructive periodontal disease (Caldeira et al., 2005) or other oral complications, as showed in Table 2. The introduction of calcineurin inhibitors associated with the current use of lower doses of steroids reduced the incidence of PTDM (3-14% of patients). However, PTDM remains as an important complication after organ 31 transplantation (Drachenberg et al., 1999) and seems to be associated to the immunosuppression therapy (Maes et al., 2001). Direct comparisons can be made between tacrolimus and diabetogenesis. Wakugami et al. (1993) realized histological examination and showed that tacrolimus and CsA prevent the reduction in the average size of islets and in the area of β cells in rats, and suggested that the administration of tacrolimus might be a more useful tool to prevent the development of insulin-dependent diabetes mellitus. Drachenberg et al. (1999) showed persistent damages in glucose metabolism after pancreas transplantation, and these effects have been attributed to various causes including damages in the insulin secretion and in the systemic drainage of the pancreas. According to the current immunosuppressive regimens employed for pancreas transplants, there are morphological abnormalities of islet cells correlated with drug levels and with damage of glucose metabolism. Tacrolimus showed an increasing in abnormalities in glucose metabolism, observed in patients. The use of pulse steroids with high levels, comparing to tacrolimus, are likely associated with hyperglycemia. The cell damage appears to be reversible and dose dependent. On the other hand, another field that needs research relates the glucose metabolism disorders as the primary endpoint after the solid organ transplantation. Although all comparisons between calcineurin inhibitors concerning PTDM showed three to five times higher incidence with tacrolimus, many questions remain without answers due to the different criteria used for the diagnosis of diabetes mellitus (based on insulin requirement) or due to the high trough levels of tacrolimus targeted in the initial studies. Data about the mechanism of glucose metabolism disorder in presence of tacrolimus are contradictories (Maes et al., 2001). Functional abnormalities associated with tacrolimus include decrease in insulin secretion, and more specifically, inhibition of the synthesis of insulin, due to the mRNA transcriptional defect or to the induced hyperinsulinemia that promotes insulin resistance, although in concordance to the animal studies 32 morphological evidence of islet cell toxicity in pancreas biopsies from patients that received tacrolimus (Van Hoff et al., 1999; Maes et al., 2001). The deleterious effects of calcineurin inhibitors in β cells are morphologically characterized by degranulation, vacuolization, swelling of rough endoplasmatic reticulum, Golgi apparatus, and mitochondria. The association between this class of agents and lipid metabolism abnormalities has been studied and well- documented, but the mechanism remains unclear (Mora, 2005). Hyperglycemia is a commom metabolic disturb associated with calcineurin inhibitor treatment (Scott et al., 2003). 51% of tacrolimus recipients required a treatment for hyperglycemia at 3 months, reducing to 19% of patients at 1 year follow-up. Notably, with tacrolimus treatment, whereas 47% of these liver transplant recipients had received insulin therapy for 3 months, only 13% of these patients remained in the insulin therapy at 1 year (Scott et al., 2003). Recently in our laboratory, a gradual time-related progress was observed at longer periods of treatment (180 and 240 days). In those periods, the values of glycemia levels were similar to the control rats. These results are in agreement with Hricik et al. (1991) that showed that the toxic damage with tacrolimus may be reversible, and suggested that the abnormalities of glucose metabolism may be slower to normalize after prolonged treatment with this drug. Maes et al. (2001) demonstrated the relevant role of the time in the reduction of the glycemia level. Those authors suggested that the abnormalities of glucose metabolism may be normalized after prolonged therapy with tacrolimus. After long-term administration of Tacrolimus or by an interruption of the drug administration, the disturbance in glucose metabolism would be reversible, but remains unclear by other factors (Maes et al., 2001; Scott et al., 2003; Mora, 2005). 33 Tacrolimus and Gingival Overgrowth Gingival overgrowth is characterized by an increase in gingival volume, generally appearing at the interdental papillae and not extending beyond the mucogingival junction. Drug-induced gingival overgrowth is associated with chronic usage of CsA, phenytoin and calcium channel blockers (Ellis et al., 2004). Where gingival overgrowth is associated with important functional or esthetic effects, surgical treatments are possible, but can not prevent recurrence, which often arises because it is impossible to suspend the immunosuppressive, anti-epileptic or anti-arrhythmia treatments (Bader et al., 1998). Tacrolimus shares many unwanted effects common to other immunosuppressive agents. Many cases reported suggest that the severity of gingival overgrowth observed in patients using tacrolimus is less than that with CsA. The first report detailed a 59-year-old male hepatic transplant recipient changed from CsA to tacrolimus 3 years post-transplant. After 2 months on the new drug regime, the extent of gingival overgrowth was reported to have decreased by 50% to an “acceptable level” (Bader et al., 1998). Further cases support this reduction, and in some cases complete resolution, of gingival overgrowth when changed from CsA to tacrolimus (Hernandez et al., 2000; Thorp et al., 2000; James et al., 2000). Prevalence studies are limited and the methods used for assessing overgrowth and severity vary from study to study (James et al., 2001; Oettinger- Barak et al., 2001; Wondimu et al., 2001). Few authors do not define a cut-off point at which overgrowth is said to be present, and they do not regard the overgrowth to be clinically significant. Nevertheless, the consensus of the research would suggest that tacrolimus causes less overgrowth than CsA, and when the overgrowth occurs, it is less severe (Ellis et al., 2004). Table 3 showed these studies. Tacrolimus and CsA have been associated with similar side effects, but contradictory effects despite of the gingival overgrowth. Although it has been reported one case linking FK506 with gingival enlargement (Adams & Famili, 34 1991), most clinical trials that compare the effectiveness of tacrolimus and CsA after cadaveric organ transplants tend to suggest that patients treated with tacrolimus infrequently complain of gingival problems (James et al., 2001). Three patients have been observed for 12 months (Adams & Famili, 1991), but one of these exhibited no hyperplasia for the first 9 months posttransplant, but hyperplastic changes were evident when intraoral records were made at 12 months. This patient began with nifedipine 1 month posttransplant and Procardia XL at month 8 posttransplant. The etiology of this hyperplasia is unclear. In other study, Oettinger-Barak et al. (2001) showed that patients treated with tacrolimus manifested greater gingival overgrowth than compared to the controls, although significantly lower than CsA-treated patients. The authors compared this overgrowth with the experimental group and they observed that it was not significantly different. This fact suggests that this overgrowth could be primarily attributed to the past liver disease and to the concomitant edema associated with it, with little additional effect of tacrolimus. Other studies demonstrate that tacrolimus has no adverse effects on the gingival tissues, thus it has potential like an alternative immunosuppressant for individuals susceptible to develop CsA-induced gingival overgrowth (Bader et al., 1998; James et al., 2001), probably because tacrolimus is a potent anti- inflammatory and its action causes a decrease in the exudates formation and decreases polimorfonucleaters and monocytes (Singh et al., 2003) or may suppress TNFα, IL-1β and IL-6 cytokines inflammatory (Miyata et al., 2005). In a preliminary study, James et al. (2000) presented conclusive results and showed that the conversion to tacrolimus may be beneficial for suitable patients with marked gingival overgrowth related to immunosuppressive therapy with CsA. However, in the short-term at least, while the use of tacrolimus may be associated with substantial reduction in the severity of gingival overgrowth, it may only be in a minority of cases that there will be almost complete regression, according to Hernandez et al. (2000), Thorp et al. (2000) and Spolidorio et al. (2005), but contradictory results of Ellis et al. (2004) when patients who have had 35 alteration of their immunosuppressant from CsA to tacrolimus may persist demonstrated gingival overgrowth, which may be attributed to their ongoing calcium channel blocker therapy. The effective plaque dental control must be done and it does not depend of the maintenance of the gingival overgrowth and conversion from CsA to tacrolimus. Tacrolimus and Bone Disease Bone loss following transplantation is usually rapid in the early phase with stabilization after 1 year, although this general statement conceals the reality of wide variability between apparently similar patients and also between the different categories of organ recipients. Fracture rates are variable and often extremely high. Compared with expected rates in the normal population, fracture incidence was five times higher in male kidney recipients aged 25 to 64 years. In women, the incidences were 18 and 34-fold higher in kidney recipients aged 25- 44 years and 45-64 years, respectively. The high fracture rate saw in dialysis patients increases even after successful kidney transplantation, especially in diabetics. Bone loss rate following cardiac, liver and lung transplantation is strikingly high during the first 6 months with fracture rates match, ranging from 22% to 36%, 24% to 65% the age bands that received cardiac transplants and experimented fracture incidence 13-fold higher than expected (Cunningham, 2005) As indicated above, the impact of the posttransplantation environment is conditioned to a substantial degree by the type and severity of pretransplant morbidity. They experiment an exceptionally high fracture rate after transplantation – not surprising given that following transplantation an already poor quality skeleton is bombarded with negative influences, many of them iatrogenic (Fig. 3) (Cunningham, 2005). The calcineurin inhibitors, CsA and tacrolimus, have complex and incompletely understood actions on bone. On the plus side, both have reduced dramatically the requirement for prolonged high dose of glucocorticoids following 36 transplantation, although in most cases have not eliminated this requirement completely, albeit the principal impact of CsA, but not necessarily tacrolimus, at least experimentally and also clinically possibly, is to accelerate bone resorption (Fig. 4) (Cunningham, 2005). Tacrolimus therapy has been shown in some clinical reports to cause bone mineral loss as severe as that caused by CsA (Cvetkovic et al., 1994; Park et al., 1997; Cayco et al., 2000; Stempfle et al., 2002). In other studies tacrolimus does not appear to induce severe osteopenia by high-turnover bone metabolism in rat (Inoue et al., 2000) or in patients (Goffin et al., 2002; Goffin et al., 2003). In most cases, however, tacrolimus is being used in combination with steroids including prednisolone, and according to the effect of tacrolimus alone on bone mineral metabolisms it could not be accurately elucidate (Cunningham, 2005). Cvetkovic et al. (1994) showed, with histomorphometry indices, that tacrolimus causes accelerated bone remodeling, with resorption far in excess of formation, leading to a loss of trabecular bone volume, albeit with elevated doses at tacrolimus. Although the molecular mechanisms for the immunosuppression caused by CsA and tacrolimus have been extensively investigated, the precise mechanisms of the immunosuppressant-induced high-turnover osteopenia are still unclear, and there are several possibilities. First, the agents may have a direct toxic effect on bone cells (osteoclasts). Second, the agents may act directly on bone cells and affect their ability to secrete local autocrine factors or respond to systemic hormones. Finally, the agents may have an indirect action on bone via their effects on the immune system, decreasing cytokine/lymphokine production. In all probabilities, there is a combination of the latter two explanations that underlies the effects of the immunosuppressant on bone (Cvetkovic et al., 1994; Stempfle et al., 2002). Factors other than corticosteroids must influence osteopenia. Both CsA and tacrolimus have been shown to cause osteopenia in experimental animals. The contribution of CsA and tacrolimus is difficult to estimate here due to the 37 differences in steroid dose between the two drugs. However, no benefit of tacrolimus immunosuppression was seen over that of CsA. It may be that both or neither of these drugs have a major etiologic role in the production of osteopenia, considering that their effects are comparable. Tacrolimus may be more deleterious to bone (an effect which counteracts the effect of reduced steroids), or may increase renal dysfunction, and this fact may exacerbate the osteopenia (Park et al., 1996). The role of tacrolimus on the skeleton is not well understood. In one ex0perimental rat model, tacrolimus caused histologically an increase in bone formation and especially bone resorption, leading to a significant loss of trabecular bone volume that was even more pronounced than that observed with CsA. In contrast to CsA, effects of tacrolimus on biochemical markers include a decrease of ionized calcium, accompanied by an increase in parathyroid hormone (PTH) and no increase in 1,25(OH)2D (calcitriol) and osteocalcin after administration (Stempfle et al., 2002). However, high dose tacrolimus-based immunosuppressive regimen is associated with a rapid bone loss early after cardiac transplantation. Beyond the first 6 months after transplantation, calcium vitamin D and hormone supplementation in hypogonadism level sufficiently lead to bone mineral recovery, and low dose calcitriol should be substituted for at least 2 years as additional antiresorptive therapy (Stempfle et al., 2002). In contract with others studies, tacrolimus did not affect the excretion of urinary dexypyridinoline, a marker of bone resorption, whereas CsA increased it. The results of the study of Inoue et al. (2000) clearly demonstrate that, in comparison with CsA, tacrolimus did not cause severe bone loss. Thus, considering that tacrolimus causes less reduction in bone mineral density compared with CsA in rats, it can be expected that the tacrolimus-treated patients might show a lower incidence of fracturing compared with CsA (Inoue et al., 2000). Another recent study has shown that tacrolimus has the potential to elevate plasma and hepatic levels of insulin-like growth factor (IGF)-I in rats, but 38 it was not observed for CsA (Epstein et al., 1995). IGF-I is the most potent of IGFs and has potent osteogenetic properties on bone metabolism, evoking the idea that tacrolimus, unlike CsA, might have beneficial effects on bone metabolism (Epstein et al., 1995; Inoue et al., 2000). There are no previous data comparing the effect on alveolar bone height of CsA and tacrolimus, and there are no data regarding the effect of tacrolimus itself on alveolar bone height. In the study of Oettinger-Barak et al. (2002) with patients and radiographic analysis, there were no statistically significant differences in alveolar bone height between CsA and tacrolimus. Tacrolimus might exert a direct trigger effect on bone cells, affecting the osteoblasts more than the osteoclasts (thus, counteracting the effects of glucocorticoid), or an indirect effect through cytokines that affects bone cells. In this context, Inoue et al. (2000) recently showed that rats treated with tacrolimus at a dose used to prevent allograft rejection had no significant reduction in their bone mass. In addition, Yoshikawa et al. (2000) have reported that allogeneic cultured bone constructs implanted in rats treated with tacrolimus exhibited better bone formation than those implanted in control rats (Goffin et al., 2002). The potential advantage of tacrolimus over CsA on bone mineral density could be explained either by a better induction of IGF-1, a powerful activator of osteoblastic function, a more “bone-friendly” expression of the different T lymphocyte subclasses (Voggenreiter et al., 2000), or by an indirect effect through cytokines that affects bone cells (Goffin et al., 2003). Conclusion It may be concluded from the results that the conversion to tacrolimus may be beneficial for suitable patients with marked gingival overgrowth, comparing to the immunosuppressive therapy with CsA. Data presented in the literature about the action of tacrolimus on the bone tissues are uncertain and conflicting, because there are not data regarding the 39 effect of tacrolimus itself on alveolar bone height. But tacrolimus might exert a direct trigger effect on bone cells, affecting the osteoblasts more than the osteoclasts, or this drug may have an indirect effect through cytokines and affects bone cells. 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Wakugami T, Islam MR, Higa S, Murakami K, Mimura G (1993). Effect of FK506 on the development of diabetes in BB rats in a comparison with that of cyclosporine. Tokohu J Exp Med; 169(1): 21-29. Wallemacq PE, Verbeeck RK (2001). Comparative clinical pharmacokinetics or Tacrolimus in paediatric and adult patients. Clin Pharmacokinet; 40: 283-95. Wermers RA, Fatourechi V, Wynne AG, Kvols LK, Lloyd RV (1996). The glucagonoma syndrome. Clinical and pathologic features in 21 patients. Medicine (Baltimore). 75(2):53-63 Wondimu B, Nemeth A, Modeer T (2001). Oral health in liver transplant children administered cyclosporine A or tacrolimus. Int. J. Paediat. Dent.; 11: 424-429. 49 Yoshikawa T et al. (2000). In vivo osteogeneic capability of cultured allogeneic bone in porous hydroxyapatite: Immunosuppressive and osteogeneic potential of FK-506 in vivo. J. Bone Min Res; 15: 1147-1157. Ziccardi VB et al. (1991). Maxillofacial considerations in orthotopic liver transplantation. Oral Surg. Oral Med. Oral Pathol., St Louis; 71: 21-26. 50 Table 1: The side effects associated with tacrolimus based immunosuppression. Side effects Author(s) Gingival overgrowth Adams and Famili , 1991 Oettinger-Barak et al., 2001 Ellis et al.,2004 Hirsutism Kari and Trompeter, 2004 Pruritus Becker et al., 2006 Neurotoxicity Corruble et al., 2005 Umapathi and Chaudhri, 2005 Nephrotoxicity Corruble et al., 2005 Tamada et al., 2006 Abnormalities bone metabolism Scott et al., 2003 Staatz & Tett, 2004 Hypertension Crespo-Leiro, 2005 Alopecia Tricot et al., 2005 Risk of infections (viral, bacterial and fungal) Spencer et al., 1997 Plosker et al., 2000 Diarrhea Vitko et al., 2005 Diabetogenesis (disturbances in glucose metabolism) Marchetti & Navalesi, 2000 Baltar et al., 2005 Malignancies (particularly lymphoma and malignancy of skin) Scott et al., 2003 Gastrintestinal disturbances Staatz & Tett, 2004 Hyperkalaemia Staatz & Tett, 2004 Hypomagnesaemia Staatz & Tett, 2004 Serum lipids and lipoprotein changes Staatz & Tett, 2004 51 Table 2: The side effects associated with diabetes mellitus and oral manifestations. Side effects Author(s) Cheilosis Wermers et al., 1996 Burning mucosa and língua Costa et al., 2004 Dental caries Siudikiene et al., 2005 Oral candidiasis Kumar et al., 2005 Costa et al., 2004 Xerostomia Costa et al., 2004 Gingivitis Chapper et al., 2005 Salvi et al., 2005 Periodontitis Torrungruang et al., 2005 Khader et al., 2006 Clinical attachment loss Torrungruang et al., 2005 Khader et al., 2006 Dental abscesses Caldeira et al., 2005 52 Table 3: The studies of prevalence of gingival overgrowth or no associated with tacrolimus based immunosuppression. Drugs Prevalence of gingival overgrowth Author(s) Tacrolimus Positive Adams and Famili, 1991 Basile et al., 1998 Oettinger-Barak et al., 2001 Ellis et al., 2004 Tacrolimus Negative Hernandez et al., 2000 Thorp et al., 2000 James et al., 2001 Conversion from CsA to tacrolimus Negative Bader et al., 1998 Kohnle et al., 1998 James et al., 2000 Spolidorio et al., 2005 53 Fig. 1: The Chemical Structure of Tacrolimus (FK506) - The Pharmaceutical Society of Japan, 2004 54 Fig. 2: Immunophilin-mediated inhibition of T cell activation. Stimulation of the T cell receptor complex results in an inositol 1,4,5-triphosphate-mediated increase in intracellular calcium level resulting in IL-2 gene transcription. FK506 inhibit this process by binding their respective immunophilin, FKBP12, to inhibit the calcium/calmodulin- dependent protein phosphatase, calcineurin. FKBP-12 = FK506-binding protein; NFAT nuclear factor of activated T cells. NFAT IL-2 promotor IL-2 gene T cell Membrane FK506 FKBP-12 Ca2+ T cell receptor Calmodulin Calcineurin Activated Calcineurin Active NFAT Dephosphorylation Nucleus 55 Fig. 3: Impact of the posttransplant environment on the skeleton. The scenario during the early posttransplant phase is depicted. The adverse influences are dominated by glucocorticoids and calcineurin inhibitors, and in the case of kidney transplantation, reduced glomerular filtrate rate (GFR). PI, inorganic phosphate; PTH, parathyroid hormone; RANKL, receptor activator of NFKbeta ligand; OPG, osteoprotegerin (extracted of Cunningham, 2005). Transplantation Glucocorticoids GFR cyclosporin glucocorticoids tacrolimus cyclosporin/tacrolimus sex steroids PI calcitriol PTH (adynamic) via RANKL and OPG Bone formation Bone resorption uncoupling OSTEOPENIA (Cunningham, 2005) Transplantation Glucocorticoids GFR cyclosporin glucocorticoids tacrolimus cyclosporin/tacrolimus sex steroids PI calcitriol PTH (adynamic) via RANKL and OPG Bone formation Bone resorption uncoupling OSTEOPENIA (Cunningham, 2005) 56 Fig. 4: Proposed role of RANKL (receptor activator of NFKbeta ligand) and osteoprotegerin in the mediation of glucocorticoid and calcineurin inhibitor toxicity (extracted of Cunningham, 2005). Glucocorticoids Glucocorticoids CsA/tacrolimus CsA/tacrolimus RANKL osteoprotegerin osteoclastogenesis Bone resorption (Cunningham, 2005) Glucocorticoids Glucocorticoids CsA/tacrolimus CsA/tacrolimus RANKL osteoprotegerin osteoclastogenesis Bone resorption (Cunningham, 2005) 57 EFFECTS OF LONG-TERM FK506 THERAPY ON THE GINGIVAL TISSUES OF RATS – A MORPHOLOGICAL EVALUATION. Submetido à publicação no periódico Journal of Periodontal Research. 58 Effects of long-term FK506 therapy on the gingival tissues of rats - A morphological evaluation. Carlos Augusto Nassar, Patricia Oehlmeyer Nassar, Denise Carleto Andia, Morgana Rodrigues Guimarães, Luis Carlos Spolidorio Abstract Background: Tacrolimus (FK506) is an immunosuppressive drug in organ transplantation. It is suggested that FK506 is not induced gingival overgrowth. Nevertheless there are not studies exploring the effects of long-term FK506 therapy on gingival tissues. Objective: The purpose of this study was to evaluate the effects of long-term therapy with FK506 on the gingival tissue. Material and methods: Rats were treated for 60, 120, 180 and 240 days with daily subcutaneous injection of 1mg/Kg body weight of FK506. After histological processing, the oral and connective tissue, as well as, volume densities of fibroblasts (Vf), collagen fibers (Vcf) and others structures (Vo) were assessed at the region of the lower first molar. Results: After 60 and 120 days of treatment with FK506 not observed gingival alterations. After 180 and 240 days of treatment with FK506, evident gingival overgrowth associated a significant increase of epithelium and connective tissue was observed. There was an increase of Vf, Vcf and Vo. Conclusions: Within the limits of this experimental study, it can be concluded that FK506 induced increase of fibroblast and collagen tissue in parallel with the severity of the overgrowth after long-term of treatment. Key words: tacrolimus; gingival overgrowth; immunosuppression drugs; animal study 59 Introduction Tacrolimus (formerly known as FK506) was introduced as an immunosuppressive agent for use in organ transplants in 1987 and has gradually been gaining popularity. The pharmacodynamics of FK506 is very similar to cyclosporin A (CsA) and is a potent immunosuppressive agent used as an alternative to CsA to prevent graft rejection and to treatment autoimmune diseases (1, 2). FK506, a macrolide immunosuppressant, inhibits cellular and humoral immune responses via several mechanisms of action, with the central effect involving inhibition of calcineurin. Inactivation of calcineurin via complex formation with the immunophilin FK506 binding protein 12 (FKBP12) prevents the translocation of the transcription factor nuclear of activated T cells that promotes interleukin-2 (IL-2) mediated proliferation of helper T cells (1, 2). It has been used successfully to prevent renal, liver and cardiac allograft rejection, although it can cause side effects such as nephrotoxicity, neurotoxicity and glucose metabolism disorders. On the other hand, hyperlipidaemia, hypertension and hirsutism are less likely with FK506 than CsA (2). Unlike CsA, however, FK506 does not appear to induce gingival overgrowth (3, 4), albeit the some authors suggest, in a number of cases reports, that the severity of gingival overgrowth seen in patients taking FK506 is less than that with CsA (5, 6, 7). Prevalence studies are limited and the methods used for assessing overgrowth and severity varies from study to study (8, 9). Few authors define a cut-off point at which overgrowth is said to be present, but nor do they regard the overgrowth to be clinically significant. Nevertheless, the consensus of the research thus far would suggest that FK506 causes less overgrowth than CsA, and that when overgrowth does occur it is less severe (10), Up to now, clinical trials have only shown that FK506 treatment is not commonly associated with the development of gingival overgrowth. However, further observations are necessary in human and experimental models. The purpose of this study was to describe the histometry and densities of fibroblasts, collagen fibers and the others structures in the gingival tissue of rats that treated with FK506 for long-term. 60 Materials and methods Eighty male Holtzman rats (Rattus novergicus albinus) weighing 50g were housed under similar conditions in cages with access to food and water ad libitum. The animals were randomly distributed in eight groups of 10 animals each. All protocols described below were approved by the Institutional Experimentation Committee of the School of Dentistry of Araraquara, Araraquara, São Paulo, Brazil. Four groups were treated with FK-506 (Prograf® - Janssen Cilag, Brazil) injected subcutaneously in a daily dose of 1mg/Kg body weight (11, 12). Four groups were used as controls and received subcutaneous injection of saline solution during all periods. The experimental periods were 60, 120, 180 and 240 days. According to others authors, this dosage provides plasma peak and trough levels of FK506 of 11.2 ng/mL approximately (13, 14, 15). Control rats from the other from groups were daily injected subcutaneously with saline (NaCl 0.9%). All rats were weighed weekly. HISTOLOGY TECHNIQUES The rats were killed by an overdose of anesthesia (Ketamine - Francotar®, Virbac do Brazil Ind. e Com. Ltda, São Paulo, São Paulo, Brazil) at the end of experimental periods and the mandibles were carefully removed, and soaked in 10% formalin. Decalcification was carried out in solution of Morse (50 mL of 50% formic acid and 50 mL of 20% sodium citrate). Five micrometers (5µm) serial paraffin sections were made on the bucco-lingual aspects of the whole 1st left and right lower molar and stained with hematoxylin and eosin. Each 1st lower molar has a mesial-distal diameter of approximately 1mm, producing sections of 5 µm each. Histometric and stereological studies were made on the buccal gingiva. HISTOMETRY Gingival epithelium and connective tissue area measurements were made with the help of a Zeiss microscope at a magnification of 125x using a Sigma computer program (Mocha, Jandel Scientific, CA, San Rafael, USA). From each tooth, were made 10 measurements in sections of 50-µm intervals each. For 61 statistical analysis the mean from each animal was used, calculated from the 20 measurements obtained from the 1st right and left molars (Fig. 1). STEREOLOGY Volume densities of fibroblasts (Vf), collagen fibers (Vcf) and others structures (Vo), i.e. blood vessels, nerves and unidentified structures, were estimated according to the principles established by Dellesse (16), which were applied to histology by Weibel (17). The count was performed with the help of a Zeiss microscope, using oil immersion at a magnification of 1000X. A square lattice of 25 test points was projected into the microscope ocular, with the use of microvid system that connected the microscope to a computer. For each animal, 16 sections were selected (eight from the left molar and eight from the right), and 25 points were counted in each section. Vf, Vcf and Vo were expressed as percentages of the total points counted. STATISTICAL ANALYSIS Measurements were expressed as mean and standard deviation. Statistical analyses were made by 1-way analysis of variance (ANOVA) and Tuckey-test. Results HISTOLOGIC The gingival of the control rats as well as of the rats treated with FK506 for 60 and 120 days, showed normal morphology in all analyzed periods, showed keratinized stratified squamous epithelium. The interface between epithelium and connective tissue was strongly interdigitated: many tall, narrow connective tissue papillae project into the epithelium. Alternating with these is usually as taller, thin epithelial extension, projecting the underlying connective tissue. The connective tissue was dense, and showed fine collagen fibers that were interspersed with delicate vessels and fibroblasts. After 180 and 240 days, gingival overgrowth was observed in all gingival areas, but it was more evident on the buccal gingival tissue of the lower molar teeth. The gingival epithelium was hyperplastic, with 62 deep papilla interdigitations. The connective tissue was dense, and showed thick collagen fibers that were interspersed with delicate vessels and fibroblasts. It was observed little inflammatory cells. HISTOMETRIC FINDINGS Table 1 show the linear measurements (µm+SD) of the epithelium and connective tissue of the buccal gingiva for the 1st lower first molars of control and treated rats. The gingival of the control rats as well as of than FK506-treated rats for 60 and 120 days were similar (p>0.05). On the other hand, the linear values of the FK506-treated rats were significantly increased at 180 and 240 days of treatment compared with control as well as at 60 and 120 days (p<0.05). STEREOMETRIC FINDINGS The stereometric findings of the control group and of the groups treated with FK506 are demonstrated in Table 2. In the control groups, the volumetric densities of fibroblasts, collagen fibers and other structures were 11.87%, 66.66% and 21.47% in the buccal gingiva and stayed constant in all the studied periods. After 60 and 120 days of the treatment the values of Vf, Vcf and Vo were similar to the control group (p>0.05). There was a significant increase of Vf and Vcf at 180 and 240 days (p<0.05), while the volumetric densities of other structures decreased when compared of the control groups (p<0.05). Discussion The present study evaluated the gingival overgrowth following long-term administration of FK-506 in rats. Some studies have shown that FK506 not induced gingival overgrowth (3, 4) or suggested that the severity of gingival overgrowth seen in patients taking FK506 is less than that with cyclosporine (6, 18). For our knowledge, there are not available data evaluating the effects of long-term therapy with FK506 on the gingival tissue in rats, however, we investigated the effect of a systemic treatment of rats with the 63 immunosuppressant FK506 for long-term on histological, histometric and stereometric analysis. In this study, the use of a rat model allowed the strict control of some variables, such a genetic predisposition, gender and dose. In addition, the chosen dose 1 mg/Kg body weight was sufficient to achieve therapeutic FK506 serum levels (11, 12, 19), recently in our laboratory verified that serum levels of FK506 (data not publishing). This dose is clinically relevant and within the range of doses used in studies on organ and limb transplantation that usually are between 0.6 and 1.0 mg/Kg body weight (13, 14, 15, 20, 21), resulting in consistent responses. In agreement with previous studies (5, 6), this work showed that FK506 administration for brief periods of treatment (60 and 120 days) not induced gingival overgrowth. Interestingly, in the present study, gingival overgrowth was observed at longer periods of treatment (180 and 240 days) in all rats. The gingival overgrowth was more evident on the buccal gingival tissue of the lower molar teeth. The gingival epithelium was hyperplastic, with deep papilla interdigitations, with the connective tissue increased and dense, when compared with respective control groups. It was recently verified that after briefer periods of treatment with CsA, there was evident gingival overgrowth associated with a significant increase of epithelium and connective tissue. After 180 and 240 days of the treatment, there was a reduction of the gingival overgrowth with significant decreases of epithelium and connective tissue (22). These results are in agreement with a prospective longitudinal study (23) that showed the relevant role of time in reducing gingival overgrowth in heart transplanted patients undergoing CsA therapy from 6 to 18 months after transplantation. A time-dependent pattern of gingival overgrowth in nifedipine-treated animals was also demonstrated by Fu et al. (24). Therefore FK506 not present the same effect on the gingival tissue. The exact pathogenesis of FK506-induced gingival overgrowth after long period of treatment is not known. We have been to speculate that the gingival 64 overgrowth could be the result of a gradual sensibilization of the gingival fibroblasts as well as of gingival epithelium. After long-term of FK506 therapy can have a action direct or indirect on determined population of gingival fibroblasts and collagen fibers via cytokine and growth factors. Fibroblasts heterogeneity remains one of the key factors used to explain the variable response of the gingival tissue to the various drugs (25). Several in vivo and in vitro studies have investigated the changes in tissue composition and cellular function that accompany drug-induced gingival overgrowth, mainly those induced by CsA or calcium channel blockers, most of the attention to date has focused on cytokine expression alterations (26, 27, 28). In line with the present work, Frizell et al. (29) verified that FK506 increases hepatic collagen and increase RNA levels of transforming growth factor beta 1 and collagens I, III and IV, reducing hepatic fibrosis after 240 days of treatment. Some cytokine is a multifunctional peptide that regulates diverse biologic activities including cell growth, cell death or apoptosis, cell differentiation, and extracellular matrix synthesis (30). Gagliano et al. (31) found that FK506 strongly induced matrix metalloproteinases (MMP)-1 gene expression, and the pattern was similar for, MMP-1 collagenolytic levels in fibroblasts after FK506. By contrast, MMP- 2mRNA levels increased 48 and 72 h after FK506 treatment, while gelatinase protein levels seemed unaffected by FK506. In the same fibroblast samples they evaluated the effect of CsA on MMP levels, and found that CsA lowered MMP-1 in the supernatants of CsA treated fibroblasts (32). This confirms that MMP-1 has a key role in the mechanisms of gingival overgrowth development. In the present work suggest that special attention should be given to the clarification of the mechanisms of action of FK506 on the index fibroblasts proliferation and collagen fibers in vitro and in vivo as well as in the protein synthesis and colagenolytic activity, mainly with longer periods of treatment with FK506. 65 Nevertheless, detailed studies are still needed to clarify the possible cellular and molecular mechanisms involved in the effect of the FK506 on the gingiva after long-term administration. Within the limits of this experimental study, it can be concluded that FK506 induced increase of fibroblast and collagen tissue in parallel with the severity of the overgrowth after long-term of treatment. Acknowledgements We are especially grateful to José Antônio Sampaio Zuanon for the carefully histological processing and FAPESP for the financial support. References 1-Faulds D, Goa KL, Benfield P. Cyclosporin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in immunoregulatory disorders. Drugs 1993; 45(6): 953-1040. 2-Spencer CM, Goa KL, Gillis JC. Tacrolimus: an update of its pharmacology and clinical efficacy in the management of organ transplantation. Drugs 1997; 54:925-975. 3-McKaig SJ, Kelly D, Shaw L. Investigation of the effect of FK506 (tacrolimus) and cyclosporin on gingival overgrowth following paediatric liver transplantation. Int J Paediatr Dent. 2002; 12(6): 398-403. 4-James JA et al. Tacrolimus (FK506) is not associated with gingival overgrowth in renal transplant patients. J. Clin. Periodontol 2001; 28:848-852. 5-Hernandez G et al. Reduction of severe gingival overgrowth in a kidney transplant patient by replacing cyclosporin A with tacrolimus (FK506). J. Periodontol. 2000; 71:1630-1636. 6-James JA et al. Reduction in gingival overgrowth associated with conversion from cyclosporin A to tacrolimus (FK506). J. Clin. Periodontol 2000; 27:144-148. 66 7-Thorp M et al. The effect of conversion from cyclosporine to tacrolimus (FK506) on gingival hyperplasia, hirsutism and cholesterol. Transplantation 2000; 69(6): 1218-1224. 8-Oettinger-Barak O et al. Periodontal changes in liver cirrhosis and post- transplantation patients. I: Clinical findings. J. Periodontol. 2001; 72:1236-1240. 9-Wondimu B, Nemeth A, Modeer T. Oral health in liver transplant children administered cyclosporine A or tacrolimus. Int. J. Paediat. Dent. 2001; 11:424- 429. 10-Ellis JS, Seymour RA, Taylor JJ, Thomason JM. Prevalence of gingival overgrowth in transplants patients immunosuppressed with tacrolimus. J Clin Periodontol.2004; 31:126-131. 11-Jiang H et al. Effect of FK 506 on heart allograft survival in highly sensitized recipient rat in comparison with cyclosporine. Transplant. Proc. 1991; 23:540. 12-Jiang H et al. Immunosuppressive effects of FK 506 on rat renal allograft survival, in comparison with cyclosporine. Transplant. Proc. 1995; 27:367. 13-Li S, Louis LB 4th, Kawaharada N, Yousem SA, Pham SM. Intrathymic inoculation of donor bone marrow induces long-term acceptance of lung allografts Ann Thorac Surg. 2003 Jan; 75(1): 257-263. 14-Muramatsu K, Kurokawa Y, Kuriyama R, Taguchi T, Bishop AT. Gradual graft-cell repopulation with recipient cells following vasularized bone and limb allotransplantation. Microsurgery. 2005 Nov 9; 25(8): 599-605 15-Voggenreiter G, Siozos P, Hunkemoller E, Heute S, Schwarz M, Obertacke U. Immunosuppression with FK506 has no influence on fracture healing in the rat. Bone. 2005 Aug; 37(2): 227-233. 16-Delesse MA. Procedé mécanique pour determiner la composition des roches. CR. Acad. Sce.1848; 25:544. 17-Weibel ER. Selection of the best method in stereology. J. Microsc 1974; 100:261-269. 67 18-Bader G, Lejeune S, Messner M. Reduction of cyclosporine-induced gingival overgrowth following a change to tacrolimus (FK506). A case history involving a liver transplant patient. J. Periodontol 1998; 69:729-732. 19-Akahane M et al. Osteogenic phenotype expression of allogeneic rat marrow cells in porous hydroxyapatite ceramics. J. Bone Miner. Res 1999 Apr; 14(4): 561-568. 20-Kaihara S, Bessho K, Okubo Y et al. Effect of FK-506 on osteoinduction by recombinant human bone morphogenetic protein-2. Life Sciences 2002; 72:247- 256. 21-Fukunaga J, Yamaai T, Yamachika E et al. Expression of osteoclast differentiation factor and osteoclastogenesis inhibitory factor in rat osteoporosis induced by immunosuppressant FK506. Bone 2004 Mar; 34(3): 425-431. 22-Spolidorio LC, Spolidorio DM, Holzhausen M, Nassar PO, Nassar CA. Effects of long-term cyclosporin therapy on gingiva of rats--analysis by stereological and biochemical estimation. Pesqui Odontol Bras. 2005 Apr-Jun; 19(2):112-118. 23-Montebugnoli L, Servidio D, Bernardi F. The role of time in a reducting overgrowth in heart-transplanted patients following cyclosporin therapy. J. Clin. Periodontol, 2000; 27(8): 611-614. 24-Fu E et al. Nifedipine-induced gingival overgrowth in rats: brief review and experimental study. J Periodontol 1998; 69: 765-771. 25-Cotrim P, Martellli Junior H, Graner E, Sank JJ, Colleta RD. Cyclosporin A induces proliferation in human gingival fibroblasts via induction of transforming growth factor beta-1. J Periodontol 2003; 74:1625-1633. 26-Coletta RD, Reynolds MA, Martelli-Junior H, Graner E, Almeida OP, Sauk JJ. Testosterone stimulates proliferation and inhibits interleukin-6 production of normal and hereditary gingival fibromatosis fibroblasts. Oral Microbiol Immunol. 2002 Jun;17(3):186-192. 27-Araujo CS, Graner E, Almeida OP, Sauk JJ, Coletta RD. Histomorphometric characteristics and expression of epidermal growth factor and its receptor by epithelial cells of normal gingiva and hereditary gingival fibromatosis. J Periodontal Res. 2003 Jun; 38(3): 237-241. 68 28-Ruhl S, Hamberger S, Betz R et al. Salivary proteins and cytokines in drug- induced gingival overgrowth. J Dent Res. 2004 Apr;83(4):322-326. 29-Frizell E, Abraham A, Doolittle M et al. FK506 enhances fibrogenesis in vitro and in vivo models of liver fibrosis in rats. Gastroenterology 1994; 107:492-498. 30-Bauer M, Schuppan D. TGF-beta 1 in liver fibrosis: time to change paradigms? FEBS Lett 2001; 502:1-3. 31-Gagliano N, Moscheni C, Dellavia C, Stabellini G, Ferrario VF, Gioia M. Immunosuppression and gingival overgrowth: gene and protein expression profiles of collagen turnover in FK506-treated human gingival fibroblasts. J Clin Periodontol. 2005; 32(2): 167-173. 32-Gagliano N, Moscheni C, Dellavia C et al. Effect of cyclosporin A on human gingival fibroblast collagen turnover in relation to the development of gingival overgrowth: an in vitro study. Biomed Pharmacother 2004; 58(4): 231-238. 69 Fig. 1- Schematic illustration showing the regions where linear measurements were made. E, epithelium thickness; C, epithelium crest; H, gingival connective tissue height; L, connective tissue width 70 Table 1 – Linear measurements (µm+ SD) of epithelium thickness (E), epithelium crest (C), gingival connective tissue height (H) and connective tissue width (L) of the buccal gingival of first lower molars of normal rats and treated with FK506 in various periods of treatment. PERIODS TREATMENT 60d 120d 180d 240d E Control 45.8+0.6 43.3+ 0.4 48.5+1.4 44.6+0.8 FK506 45.7+1.9 a 43.4+2.0 a 67.5+0.2 b* 87.0+0.3 c* C Control 46.9+0.4 41.3+ 0.7 45.7+1.8 47.7+0.7 FK506 46.4+2.1 a* 41.2+1.5b 69.8+0.2 c* 69.0+0.2 c* H Control 415.9+1.1 414.9+ 1.2 418.8+0.7 409.7+1.8 FK506 415.7+0.9 a 413.8+0.5 b* 493.8+ 0.7c* 570.8+0.8 d* L Control 141.9+ 1.5 147.4+ 0.9 166.1 + 2.3 139.0+0.5 FK506 139.7+0.5 a* 145.5 + 0.7 b* 218.5+ 0.5 c* 260.5+0.6 d* Different letters represent statistically significant difference among means in the same group. *p<0.05, statistical significance vs. control rats in the same period. 71 Table 2 – Volumetric densities of fibroblasts (Vf), collagen fibers (Vcf) and others structures (Vo) from the buccal gingival region of the first lower molar in control and FK506 rats. Values present means + SEM. The results are expressed as percentages. PERIODS TREATMENT 60d 120d 180d 240d Vf Control 11.87+0.3 11.01+0.2 10.93+0.4 10.81+0.2 FK506 11.80+ 1.0 a 11.40+ 1.1 b* 12.40+1.1c* 12.80+1.2d* Vcf Control 66.66+0.2 66.70+0.3 65.90+0.2 68.68+0.3 FK506 66.76+1.8 a 66.18+2.3 a 69.00+1.8b* 73.2+1.9 c* Vo Control 21.47+0.6 22.29+0.3 20.17+0.3 20.51+0.2 FK506 21.44+1.5 a 21.80+1.9 a 18.60+1.4b* 14.00+1.5 c* Different letters represent statistically significant difference among means in the same group. *p<0.05, statistical significance vs. control rats in the same period 72 THE EFFECTS OF LONG-TERM FK506 THERAPY ON THE ALVEOLAR BONE AND CEMENTUM OF RATS. Submetido à publicação no periódico Journal of Periodontal Research. 73 The effects of long-term FK506 therapy on the alveolar bone and cementum of rats. Carlos Augusto Nassar, Patricia Oehlmeyer Nassar, Denise Carleto Andia, Morgana Rodrigues Guimarães, Luis Carlos Spolidorio Abstract Background: The calcineurin inhibitors, cyclosporine A (CsA) and tacrolimus (FK506) have complex and incompletely understood actions on bone. FK506 is an alternative therapy used for transplant patients with refractory graft rejection or those intolerant to CsA, without the adverse effects frequently attributed to CsA therapy; however bone loss following transplantation is usually rapid in the early phase of the transplant. Objectives: The objectives of the present study were to evaluate the effects of long-term therapy with tacrolimus on the alveolar bone metabolism and cementum. Material and methods: Rats were treated for 60, 120, 180 and 240 days with daily subcutaneous injection of 1mg/Kg body weight of FK506. After period experimental, blood collection was obtained and serum levels calcium and alkaline phosphatase (ALP) were measured. After histological processing, the alveolar bone and cementum, as well as, volume densities of bone (Vb) and osteoclasts (Vo) were assessed at the region of the lower first molar. Results: There was a tendency towards a statistically significant decrease in ALP levels with FK506; however serum calcium levels increased during long periods when compared with the control group. After 60, 180 and 240 days of treatment with FK506 not observed Vb and Vo alterations. After 120 days of treatment, evident decrease Vb associated a significant Vo was observed, but it was not showed alveolar bone loss. Not observed any alterations of cementum in rats treated with FK506. 74 Conclusion: So, it may be concluded that FK506 administration has not induced side effects on the periodontium. Key-words: alkaline phosphatase; calcium; FK506; alveolar bone; cementum Introduction Progressive bone loss after transplantation is one of the side effects after transplantation. Several lines of evidence have suggested that preexisting bone diseases immunosuppressive drugs (1). Cyclosporin A (CsA) and tacrolimus (FK506) are the most commonly used immunosuppressant to reduce the rejection of allogeneic organ transplant. Although structurally unrelated CsA and FK506 have similar cellular effects on T-cell activation and the lymphokine- monokine cascade (2, 3). FK506 is an alternative therapy used for transplant patients with refractory graft rejection or those intolerant to CsA, without the adverse effects frequently attributed to CsA therapy; however bone loss following transplantation is usually rapid in the early phase of the transplant (4). Experimentally have suggested that CsA accelerates bone resorption and leads quickly to a severe high turnover osteopenic state, albeit some studies demonstrated that CsA decrease bone resorption and increased bone formation in rats (5), and in alveolar bone also decrease bone resorption with imbalance in the metabolism bone (6). However, a severe osteopenia responsible for osteoporotic fractures in transplantation recipients has been described by some authors (7,8). Furthermore, an increased osteoclasia and decreased bone formation at periodontal sites have been observed in the alveolar bone of rats treated with CsA (9). On the other hand suggested that systemic CsA administration induced the formation of new cementum in rat molar, and they are not reversible after cessation of therapy (10). The action of FK506 on cementum was not still accounted and in the bone, in vitro, is still uncertain and experimental data obtained from animal models suggests that FK506 is an osteopenic agent (8). However, more recent 75 studies suggest that FK506 is less osteotoxic than CsA (11,12). In contrast to CsA, effects of FK506 on biochemical markers include a decrease in ionized calcium, accompanied by an increase in parathyroid hormone (PTH) and no increase in 1,25(OH)2D (calcitriol) and osteocalcin after administration (8,13). In general, long-term administration of immunosuppressant at high doses induces high-turnover osteopenia. In a previous study, however, Yoshikawa et al (14) reported that low-dose and short-term application of FK506 promoted bone formation in cultured allogeneic and isogeneic rat bone grafts. Indeed, they showed that FK506 had an excellent effect on bone formation; however, they used osteoconductive porous hydroxyapatite with the bone grafts, thus, the effect of FK506 on osteoinduction in the absence of osteoconduction remains to be clarified (15). The process of bone formation consists of differentiation of the mesenchymal cells into osteoblasts, osteoblasts proliferation, bone matrix deposition and mineralization. Enhanced expression of alkaline phosphatases (ALP) occurs at the maturation stage of osteoblastic differentiation, and an increase in osteocalcin expression occurs at the mineralization stage (16), although serum calcium showed correlation with bone formation, however, this may decrease with kidney disease for long-term (1). Therefore, the objectives of the present study were to evaluate the effects of long-term therapy with tacrolimus on the alveolar bone metabolism and cementum. Material and methods Animals Eighty male Holtzman rats (Rattus novergicus Albinus) weighing 50g were housed under similar conditions in cages with access to food and water ad libitum. The animals were randomly distributed in eight groups of 10 animals each. All protocols described below were approved by the Institutional Experimentation Committee of the School of Dentistry of Araraquara, Araraquara, São Paulo, Brazil. Four groups were treated with FK506 (Prograf® - Janssen Cilag, Brazil) injected subcutaneously in a daily dose of 1mg/Kg body 76 weight (17,18). Four groups were used as controls and received subcutaneous injection of saline solution during all periods. The experimental periods were 60, 120, 180 and 240 days. The rats were weighed weekly and monitored for abnormal appearance of coat and abnormal activity levels. Calcium and alkaline phosphatase analysis At the end of experimental periods, the rats were anesthetized with 0.08mg/100g body weight Ketamine (Francotar®, Virbac do Brazil Ind. e Com. Ltda, São Paulo, São Paulo, Brazil ) and 4-5ml of blood was obtained by direct cardiac puncture in heparinized capillary tubes for immediate calcium measurements, using an ICA-1 ionized calcium analyzer (Radiometer Company, Copenhagen, Denmark); other blood samples were centrifuged and the serum stored at -70°C until assay. Total serum alkaline phosphatase activity was measured colorimetrically ( ALP Kit-Sera Pak, Bayer AG, Elitech, France) using paranitrophenyl phosphate as the substrate. Alkaline phosphatase activity was measured by the absorbance at 405 nm, using Technicon SMA-24 (Technicon, Domont, France). The units (U/dL) of enzyme activity in the ex