Jusciéle Brogin Moreli Lesões no DNA e capacidade de resposta celular de gestantes e recém-nascidos em regime de hiperglicemia de intensidade variada Tese apresentada à Faculdade de Medicina, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de Botucatu, para obtenção do título de Doutora em Ginecologia, Obstetrícia e Mastologia. Orientadora: Profa. Dra. Iracema de Mattos Paranhos Calderon. Coorientador: Prof. Dr. Carlos Frederico Martins Menck. Botucatu 2015 Dedicatória A Deus e Nossa Senhora, por me concederem o dom da vida. Aos meus pais Nelo Moreli e Marli Brogin Moreli, que dignamente me apresentaram à importância da família, ao caminho da honestidade e persistência e, sobretudo pela solida formação moral que alicerçam todas as minhas realizações. Ao meu irmão Leandro Brogin Moreli, que juntamente com Vaniele Marcia Vilela Moreli, acompanharam meu caminho e me possibilitaram ser tia e madrinha da Isabela Vilela Moreli, criança muito especial que escreveu o poema abaixo para o “livro” da madrinha. Dizer e Sentir Diga uma palavra e as flores se abrirão, Diga apenas o seu nome e os gestos sairão, Dê um sorriso e mil pessoas olharão, Dê um passo e você estará perto da multidão. Um caminho cheio de obstáculos, Mas todos eles são um grande passo para a reconciliação. Isabela Vilela Moreli (10 anos) Ao meu amor Flávio Henrique Fernandes Volpon, que com muita paciência, aceitou minhas frequentes ausências motivadas pela busca de conhecimentos durante a pós graduação. Obrigada por tudo e, principalmente, pela parceria na constituição da nossa família. A Dra. Iracema Calderon, o meu reconhecimento pela oportunidade de realizar este trabalho ao lado de alguém como você; meu respeito e admiração pela sua capacidade e pelo seu dom de incentivar novos desafios. Agradecimentos “Cada pessoa que passa em nossa vida, passa sozinha, é porque cada pessoa é única e nenhuma substitui a outra! Cada pessoa que passa em nossa vida passa sozinha e não nos deixa só porque deixa um pouco de si e leva um pouquinho de nós. Essa é a mais bela responsabilidade da vida e a prova de que as pessoas não se encontram por acaso." Charles Chaplin Os obstáculos encontrados durante os quatro anos de desenvolvimento do doutorado me possibilitaram uma convivência privilegiada com grandes pesquisadores e, principalmente, amigos. Assim, gostaria de agradecer, com muito carinho: Às pacientes participantes desse estudo Gestantes que colaboraram e permitiram o desenvolvimento desse estudo. À Dra. Débora Cristina Damasceno e aos alunos do Laboratório de Pesquisa Experimental de Ginecologia e Obstetrícia da Faculdade de Medicina de Botucatu: Aline Bueno, Aline Netto, Bruna Dallaqua, Fernanda Piculo, Gabriela Marine, Glilciane Morceli, Isabela Iessi, Joice Vernine, Mariana Arantes, Rafael Botaro Gelaleti, Silvana Barroso Corvino, Talísia e Yuri Sinzato, agradeço pelo apoio científico e pela possibilidade de obtenção e processamento das amostras. À Dra. Estela Bevilacqua e aos colaboradores do Laboratório de Estudos da Interação Materno Fetal e da Biologia do Trofoblasto da Universidade de São Paulo: Aline Rodrigues Lorenzon-Ojea, Caroline Borgato Guedes e Simone Corrêa-Silva. Agradeço o conhecimento científico que compartilhamos nos dois anos de convivência; pela oportunidade de participar e utilizar livremente o laboratório e, principalmente, pela amizade. Ao Dr. Carlos Menck e aos colaboradores do Laboratório de Reparo de DNA da Universidade de São Paulo: Clarissa Rocha e Rodrigo Fortunato. Pelo incentivo e preciosas opiniões, bem como pela disponibilização do laboratório para realização de parte deste trabalho. À Dra. Janine H Santos e Dr. Ronald P. Mason do Toxicology & Pharmacology Laboratory/Free Radical Metabolism Group do National Institute of Environmental Health Sciences (NIEHS) – NIH, agradeço pelos três meses de convivência e todo conhecimento compartilhado. Meu agradecimento especial pela oportunidade de conviver com Janine e seus preciosos filhos, Valentina e Lorenzo. Obrigada por estarem sempre ao meu lado nesse desafio e tornar tudo mais fácil e feliz. À Dra. Inés Quintela e Dr. Angel Carracedo do Centro Nacional de Genotipado (CeGen) de Santiago de Compostela, Espanha, pelos dois meses de estágio para aprendizado das novas tecnologias aplicadas a biologia molecular. À Ms. Valéria Romero e Dra. Magaly Sales Monteiro Professoras da graduação, incentivadoras e iniciadoras deste sonho À Dra. Marilza Vieira Cunha Rudge Por todo conhecimento compartilhado e incentivo ao desenvolvimento dos projetos. Aos Amigos e Familiares Aline Carvalho, Lygia Merini, Leticia Lima, Patrícia Soares, Rodrigo Barbano Weingrill, Sara Gomes, Carla Bandeira e todos amigos e familiares, agradeço pelos conhecimentos compartilhados, por me proporcionarem grandes momentos de alegria e por estarem comigo durante a realização do doutorado. À Maria Carvalho Pelo carinho incondicional durante 20 anos ao meu lado. Ao Escritório de Apoio à Pesquisa (EAP), da Faculdade de Medicina de Botucatu Pela ajuda no delineamento do projeto e análise estatística dos dados. Aos Funcionários da Seção de Pós-Gradução e Departamento de Ginecologia e Obstetrícia Pelo apoio e serviços prestados. À Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP Pela concessão do auxílio pesquisa (processo número 2011/18240-2), bolsa regular de doutorado (processo número 2011/13562-1) e bolsa BEPE (processo número 2012/23296-0) que possibilitaram o desenvolvimento dessa tese. À Fundación Carolina – Espanha Pela bolsa de estudo que possibilitou o estágio no Centro Nacional de Genotipado (CeGen). Ao Serviço de Divisão Técnica de Biblioteca e Documentação no campus da Unesp – Botucatu, pelo auxílio na pesquisa bibliográfica e elaboração da ficha catalográfica. Ao Laboratório Clínico da Faculdade de Medicina de Botucatu pela colaboração na realização de dosagens. E a todos aqueles que contribuíram de alguma forma para a realização deste trabalho... Sumário Apresentação 01 Artigo de Revisão 08 DNA damage and its cellular response in mother and fetus exposed to hyperglycemic environment Artigo Original 17 Hyperglycemia differentially affects maternal and fetal DNA integrity and DNA damage response Anexo 59 Apresentação Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 1 O Grupo de Pesquisa Diabete e Gravidez  Investigação Clínica e Experimental vem desenvolvendo pesquisas clínicas e experimentais nesta linha há mais de 30 anos. Os primeiros resultados, associando o teste de tolerância à glicose (TTG) e o perfil glicêmico no diagnóstico do diabete melito gestacional (DMG), caracterizaram um grupo de gestantes que, apesar do TTG normal, apresentam picos de hiperglicemia ao longo do dia evidenciados por alterações no perfil glicêmico. Essas gestantes foram consideradas como portadoras de hiperglicemia gestacional leve (HGL) e classificadas no grupo IB de Rudge. Além de outros resultados perinatais adversos (RPA), característicos dos filhos de mães diabéticas, as gestações complicadas por HGL tem risco atribuível de morte perinatal comparável ao observado no grupo de gestantes diabéticas e 10 vezes maior que aquelas com resposta normal aos dois testes diagnósticos. A partir desses resultados, as gestantes com HGL são tratadas com o mesmo protocolo das diabéticas acompanhadas no Serviço Especializado de Diabete e Gravidez da Faculdade de Medicina de Botucatu/Unesp [1-3]. A literatura mais recente reconhece que a hiperglicemia materna, de qualquer intensidade e independente do diagnóstico de DMG, deve ser controlada pelo risco de RPA [4-5]. Tal constatação validou a identificação e o controle da hiperglicemia das gestantes do grupo IB de Rudge, protocolo que vem sendo praticado há mais de 30 anos em nosso serviço. Na busca pelos fatores envolvidos no desfecho adverso dessas gestações complicadas por hiperglicemia, os resultados do nosso grupo de pesquisa associam a hipóxia intrauterina e a hiperglicemia materna de intensidade variada. Entre outras, esta associação leva a alterações morfológicas e funcionais da placenta, Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 2 caracterizadas por comprometimento da vascularização da superfície de trocas materno-fetal, relacionadas a aumento e/ou diminuição dos marcadores da proliferação vascular [6-8], incremento da apoptose [9] e alterações no perfil de citocinas placentárias [10-12]. Paralelamente, nossos estudos translacionais, utilizando modelo experimental em ratas wistar prenhes, com diabete induzido por streptozotocin, relacionaram a intensidade da hiperglicemia materna e o nível de estresse oxidativo, e consequente aumento de danos do DNA, com a ocorrência de RPA nessas gestações [13-17]. Outros estudos da literatura apontam aumento na incidência de câncer em pacientes diabéticos, decorrente da associação estresse oxidativo e hiperglicemia. Nestas condições, mecanismos de reparo de DNA são ativados, na tentativa de garantir a sobrevivência e manter a integridade do genoma. Entretanto, a falha desses mecanismos de reparo pode levar a acúmulo de danos no DNA e favorecer tanto a apoptose das células como o desenvolvimento de câncer [18-20]. Em dezembro de 2009, os resultados do consenso entre a Associação Americana de Diabete e a Sociedade Americana de Câncer destacaram que, em ambos os sexos, o diabete melito tipo 2 (DM2) está associado com risco aumentado de câncer no fígado, pâncreas, cólon e bexiga. Individualizando os sexos, mulheres diabéticas apresentam aumento no risco de câncer de mama e de endométrio e, os homens, redução no risco de câncer de próstata [21]. Alguns estudos destacam, também, a associação entre DMG e câncer. Uma coorte de 37926 mulheres com histórico de DMG identificou risco relativo de 7,1 para desenvolver câncer pancreático [22]. Na Nova Zelândia, a população de mulheres Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 3 com DMG tem maior risco de câncer de mama, o que não foi confirmado na população americana [23,24]. Recentemente, o DMG também foi associado a linfoma não-Hodgkin e leucemia mieloide aguda [25]. Apesar do ponto comum, associação entre hiperglicemia e risco de câncer, os resultados da literatura ainda são controversos e os fatores e vias envolvidos ainda não estão totalmente esclarecidos. Nesse contexto, o interesse em contribuir para minimizar essa lacuna definiu o presente projeto, objeto dessa tese de doutorado. Inicialmente, a análise crítica da literatura relacionada ao tema resultou em um artigo de revisão, “DNA damage and its cellular response in mother and fetus exposed to hyperglycemic environment”, já publicado [26]. Posteriormente, o desenvolvimento do projeto, em si, resultou em um segundo artigo, agora original, “Hyperglycemia differentially affects maternal and fetal DNA integrity and DNA damage response”. A elaboração de dois artigos definiu a forma de apresentação dessa tese, intitulada “Lesões no DNA e capacidade de resposta celular de gestantes e recém-nascidos em regime de hiperglicemia de intensidade variada”. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 4 Referências 01. Rudge MVC, Peraçoli JC, Berezowski AT, Calderon IMP, Brasil MAM. The oral glucose tolerance test is a poor predictor of hyperglycemia during the pregnancy. Braz J Med Biol Res. 1990; 23:1079 - 89. 02. Rudge MV, Calderon IM, Ramos MD, Abbade JF, Rugolo LM. Perinatal outcome of pregnancies complicated by diabetes and by maternal daily hyperglycemia not related to diabetes. A retrospective 10-year analysis. Gynecol Obstet Invest. 2000; 50:108-12. 03. Rudge MVC, Calderon IMP, Ramos MD, Brasil MAM, Rugolo LMSS, Bossolan G, et al. Hiperglicemia materna diária diagnosticada pelo perfil glicêmico: um problema de saúde pública materno e perinatal. RBGO. 2005; 27(11):691-7. 04. Metzger BE, Lowe LP, Dyer AR, Trimble ER, Chaovarindr U, Coustan DR, et al. Hyperglycemia and adverse pregnancy outcomes. N Engl J Med. 2008; 358:1991- 2002. 05. Weinert LS. International Association of Diabetes and Pregnancy Study Groups recommendations on the diagnosis and classification of hyperglycemia in pregnancy: comment to the International Association of Diabetes and Pregnancy Study Groups Consensus Panel. Diabetes Care. 2010; 33:675-82. 06. Calderon IMP, Damasceno DC, Amorin RL, Costa RAA, Brasil MAM, Rudge MVC. Morphometric study of placental villous and vessels in maternal hyperglycemia, gestational and overt diabetic pregnancies. Diabetes Res Clin Pract. 2007; 78:65-71. 07. Carvalho-Silva, SAL. Dopplervelocimetria da artéria umbilical e controle Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 5 glicêmico materno como marcadores de alterações vasculares e apoptóticas placentárias [tese]. Botucatu: Faculdade de medicina, Universidade Estadual Paulista; 2010. 08. Pietro L, Daher S, Rudge MV, Calderon IM, Damasceno DC, Sinzato YK, et al. Vascular endothelial growth factor (VEGF) and VEGF-receptor expression in placenta of hyperglycemic pregnant women. Placenta. 2010; 31:770-80 09. Sgarbosa F, Barbisan LF, Brasil MA, Costa E, Calderon IM, Goncalves CR, et al. Changes in apoptosis and Bcl-2 expression in human hyperglycemic, term placental trophoblast. Diabetes Res Clin Pract. 2006; 73:143-9. 10. Brogin Moreli J, Cirino Ruocco AM, Vernini JM, Rudge MV, Calderon IM. Interleukin 10 and tumor necrosis factor-alpha in pregnancy: aspects of interest in clinical obstetrics. ISRN Obstet Gynecol. 2012;230742. 11. Moreli JB, Morceli G, De Luca AK, Magalhães CG, Costa RA, Damasceno DC, et al. Influence of maternal hyperglycemia on IL-10 and TNF-α production: the relationship with perinatal outcomes. J Clin Immunol. 2012; 32:604-10. 12. Moreli JB, Corrêa-Silva S, Damasceno DC, Sinzato YK, Lorenzon-Ojea AR, Borbely AU, et al. Dynamics changes in the TNF-alpha/IL-10 ratio in hyperglycemic- associated pregnancies. 2015: In press. 13. Damasceno DC, Volpato GT, de Mattos Paranhos Calderon I, Cunha Rudge MV. Oxidative stress and diabetes in pregnant rats. Anim Reprod Sci. 2002; 72:235-44. 14. Spada AP, Damasceno DC, Sinzato YK, Campos KE, Faria PA, Dallaqua B, et al. Oxidative stress in maternal blood and placenta from mild diabetic rats. Reprod Sci. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 6 2014; 21:973-77. 15. Lima PH, Damasceno DC, Sinzato YK, de Souza Mda S, Salvadori DM, Calderon Ide M, et al. Levels of DNA damage in blood leukocyte samples from non-diabetic and diabetic female rats and their fetuses exposed to air or cigarette smoke. Mutat Res. 2008; 653:44-9. 16. Lima PH, Sinzato YK, de Souza Mda S, Braz MG, Rudge MV, Damasceno DC. Evaluation of level of DNA damage in blood leukocytes of non-diabetic and diabetic rat exposed to cigarette smoke. Mutat Res. 2007; 628:117-22. 17. Lima PH, Sinzato YK, Gelaleti RB, Calderon IMP, Rudge MVC, Damasceno DC. Genotoxicity evaluation in severe or mild diabetic pregnancy in laboratory animals. Exp Clin Endocrinol Diabetes. 2012; 120:303–07. 18. Friedberg EC. DNA damage and repair. Nature. 2003; 421:436-40. 19. Berra CM, Menck CF, Di Mascio P. Oxidative stress, genome lesions and signaling pathways in cell cycle control. Quimica Nova. 2006; 29:1340-44. 20. Kryston TB, Georgiev AB, Pissis P, Georgakilas AG. Role of oxidative stress and DNA damage in human carcinogenesis. Mutat Res. 2011; 711:193-01. 21. Giovannucci E, Harlan DM, Archer MC, Bergenstal RM, Gapstur SM, Habel LA, et al. Diabetes and cancer: a consensus report. Diabetes Care. 2010; 33:1674-85. 22. Perrin MC, Terry MB, Kleinhaus K, Deutsch L, Yanetz R, Tiram E, et al. Gestational diabetes as a risk factor for pancreatic cancer: a prospective cohort study. BMC Med. 2007; 5:25. 23. Troise R, Weiss H, Hoover R. Pregnancy characteristics and maternal risk of Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 7 breast câncer. Epidemiology. 1998; 9:641-47. 24. Dawson SI. Long-term risk of malignant neoplasm associated with gestational glucose intolerance. Cancer. 2004; 100:149-55. 25. Sella T, Chodick G, Barchana M, Heymann AD, Porath A, Kokia E, et al. Gestational diabetes and risk of incident primary cancer: a large historical cohort study in Israel. Cancer Causes Control. 2011; 22:1513-20. 26. Moreli JB, Santos JH, Rocha CR, Damasceno DC, Morceli G, Rudge MV, et al. DNA damage and its cellular response in mother and fetus exposed to hyperglycemic environment. Biomed Res Int. 2014; doi: 10.1155/2014/676758. Artigo de Revisão Artigo Original Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 17 Hyperglycemia Differentially Affects Maternal and Fetal DNA Integrity and DNA Damage Response1 Jusciele B. Moreli1, Janine H. Santos2, Aline Rodrigues Lorenzon-Ojea3, Simone Corrêa-Silva1,3, Rodrigo S. Fortunato4, Clarissa Ribeiro Rocha5, Marilza V. C. Rudge1, Débora C. Damasceno1, Estela Bevilacqua3, Iracema M. P. Calderon1 1Graduate Program in Gynecology, Obstetrics and Mastology, Botucatu Medical School, São Paulo State University / UNESP, São Paulo, Brazil. 2Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences / NIEHS, North Carolina, USA. 3Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo / USP, São Paulo, Brazil. 4Laboratory of Molecular Radiobiology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro / UFRJ, Rio de Janeiro, Brazil. 5DNA Repair Laboratory, Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo / USP, São Paulo, Brazil. Corresponding author: Iracema Mattos Paranhos Calderon Departamento de Ginecologia e Obstetrícia, Faculdade de Medicina de Botucatu - UNESP Distrito de Rubião Jr. s/n; CEP − 18618-000 / Botucatu − SP / Brasil E-mail: calderon@fmb.unesp.br and juscielemoreli@gmail.com Phone: +55 14 3880-1383 1Artigo formatado nas normas editoriais da revista International Journal of Biological Sciences mailto:calderon@fmb.unesp.br mailto:juscielemoreli@gmail.com Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 18 Abstract Objective: The purpose of this study was to assess markers of oxidative stress, DNA damage and the cellular response in maternal and umbilical cord blood of pregnancies complicated by hyperglycemia. Methods: One hundred forty-four pregnant women, classified in normoglycemics (ND), mild gestational hyperglycemia (MGH), gestational diabetes mellitus (GDM) and type 2 diabetes mellitus (DM2), were included. The nuclear and mitochondrial DNA damage were evaluated by gene-specific quantitative PCR and the expression of genes and proteins involved in base excision repair (BER) pathway were assessed by real time PCR and western blot, respectively. The apoptosis was evaluated in vitro experiment. These analyses were performed in samples from maternal and umbilical cord blood. Results: The mothers with GDM and DM2 were characterized by oxidative stress, increase of nuclear and mitochondrial DNA damage and decrease expression of genes and proteins involved in BER. In addition, the levels of hyperglycemia were associated to in vitro cellular apoptosis. The newborns of diabetic mothers presented increase of BER genes and proteins expression, and the hyperglycemia environment in vitro was not able to induce apoptosis. Blood levels of DNA damage in umbilical cord were similar among groups. Conclusions: In this study, maternal hyperglycemia observed in GDM and DM2 groups was associated to oxidative stress and, consequently with nuclear and mitochondrial DNA damage. However, integrity of DNA from umbilical cord blood cells was preserved, suggesting the better involvement of DNA repair mechanisms in these fetuses. Key words: Diabetes, Pregnancy, DNA damage, DNA repair Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 19 Introduction Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia resulting from a defect in insulin action and/or production [1]. In pregnancy, hyperglycemia poses both short-term and long-term risks to the health of women and their offspring [2]. Newborns (NB) of hyperglycemic mothers exhibit an increased risk for malformations, macrosomia, hypoxia, and perinatal death, which are associated with hypoglycemia, hyperbilirubinemia, hyperinsulinemia and hyperleptinemia [3-5]. During adult life, these metabolic alterations increased risk of metabolic, cardiovascular and malignant diseases [2,5]. Oxidative stress has been widely studied as an important link between hyperglycemia and its complications, including alterations in embryonic and fetal development during pregnancy [6-8]. The basal explanation is that hyperglycemia leads to mitochondrial reactive oxygen species (ROS) overproduction and thus can induce protein oxidation, lipid peroxidation and DNA damage in both mitochondrial (mtDNA) and nuclear DNA (nDNA) [6,9- 11]. Different degradation processes can remove oxidized lipids and proteins; however, DNA has to be repaired or even be removed in the case of mtDNA damage. The mtDNA is more vulnerable than nDNA to ROS-mediated lesions [10-12]. One of several reasons that underlie this observation is that only base Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 20 excision repair (BER) removes mtDNA lesions [13]. BER is the main mechanism involved in the removal ROS-induced lesions i.e., oxidized bases, AP sites, and single strand breaks. BER is initiated by DNA glycosylases (i.e., human 8-oxoguanine DNA glycosylase - hOGG1), which recognize and remove specific damaged or inappropriate bases, forming AP sites. These are then cleaved by an AP endonuclease, such as human AP Endonuclease (APE-1), forming a single strand break. The resulting break can then be processed by either short-patch or long-patch BER. In the short-patch, APE- 1 interacts with DNA polymerase beta (POLβ) leading to single-nucleotide gap synthesis. In the long-patch, APE-1 similarly interacts with Flap endonuclease 1 (FEN-1) for synthesis of 2-10 new nucleotides [14]. When the rate of DNA damage exceeds the cellular capacity to repair it, the accumulation of errors can overwhelm the cell and result in cell death or in the fixation of mutations that may be transmitted to future generations if they occur in germ cells. Mutations that occur in somatic cells can lead to changes in cellular functions, which can contribute to cancer development [15,16]. The literature acknowledges that the repair capacity of DNA damage plays a critical role in the maintenance of genome stability [17]. Specifically, in DM type 2, it has been shown that patients have increased levels of nuclear DNA damage, high rate of lymphocyte apoptosis, and decreased repair capacity Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 21 of hydrogen peroxide- and doxorubicin-induced DNA damage [18,19]. In human diabetic pregnancies, data related to genome stability are still limited. A pilot study in health pregnant women associated DNA damage and risk of GDM [20] but no studies involving diabetic pregnant women and DNA damage and/or repair are to our knowledge available. Data from animal models support the correlation between diabetes and DNA damage with two studies in rats with streptozotocin-induced diabetes reporting high levels of nuclear DNA damage in maternal and NB leukocytes [21-23]. The objective of this study was to assess DNA damage and its cellular response in maternal and umbilical cord blood of pregnancies complicated by hyperglycemia. Our hypothesis was that, in these pregnancies, hyperglycemia and oxidative stress may affect both maternal and fetal DNA integrity and cellular response. Materials and Methods Ethics statement This study was conducted in the Diabetes and Pregnancy Service of the Botucatu Medical School/UNESP, Brazil, and was approved by the local Research Ethics Committee (protocol #507/2012). Written informed consent was obtained from all subjects according to the principles of the Declaration of Helsinki. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 22 Study design and subjects selection One hundred forty-four pregnant women with several degrees of hyperglycemia were enrolled to this cross-sectional study. To calculate the sample size, it was considered a completely randomized experiment with four groups. Supposing that 20 degrees of liberty would be sufficient to reduce the residual variance and to reveal the effect of groups, the minimum subject for each group of mothers and newborns would be six. Pregnant women with Diabetes mellitus type 2 (DM2; n = 23) were referred to our service with a confirmed diagnosis. The research subjects without pre- gestational diabetes underwent 75 g-GTT test, recommended by ADA [1], and the glucose profile (GP) test, recommended by Rudge [24], between 24th and 28th gestational weeks. According to the 75 g-GTT and GP results, pregnant women were classified into the following study groups: non- diabetic (ND; normal 75-g GTT and GP; n = 46), mild gestational hyperglycemia (MGH; normal 75-g GTT and abnormal GP; n = 30) or gestational diabetes mellitus (GDM; abnormal 75-g GTT first reported during the pregnancy; n = 45) (Figure 1). The inclusion criteria were as follows: (a) hyperglycemia defined at the maximum gestational age of 28-30 weeks for women with MGH and GDM, and 20 weeks for DM2 pregnant women; (b) prenatal and delivery care at the Service; (c) absence of clinically diagnosed infections and negative Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 23 serology for HIV and syphilis, multiple pregnancies, DM1, fetal malformations, fetal death, alcohol or illicit drugs habits; (d) deliveries before the 36th week of gestation. In MGH or GDM pregnant women the hyperglycemia was controlled through a combination of lifestyle changes, individualized diet and exercise; insulin therapy was used when necessary. Patients with DM2 followed the same protocol but received insulin therapy since the start of treatment to replace oral hypoglycemic agents. The goals of maternal glycemic control were fasting glucose ≤ 95 mg/dL, 1 hour postmeal ≤ 140 mg/dL, and 2 hours postmeal ≤ 120 mg/dL, resulting in a daily glycemic mean (GM) ≤ 120 mg/dL [1]. Sample collection Maternal blood samples were collected from 36th week of pregnancy and just prior to the beginning of labor. The umbilical cord blood was collected intradelivery, shortly after clamping. The samples of maternal or umbilical cord blood were collected in Vacutainer tubes (Becton Dickinson, USA) treated with EDTA, serum or heparin tubes. Characterization of the population The population of pregnant women was characterized by age, body mass index (BMI) in pre-pregnancy and third trimester of pregnancy, weight gain during pregnancy, gestational age at delivery, presence of hypertension Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 24 (gestational or chronic hypertension), glycemic mean (GM) and glycated hemoglobin (HbA1c) levels. The GM was calculated by arithmetic mean of plasma glucose levels evaluated in all GPs during treatment. Plasma glucose levels were evaluated by glucose oxidase method (Glucose Analyzer II Beckman, Fullerton, CA, USA). The HbA1c levels were evaluated by chromatography (high-performance liquid chromatography—D10™ Hemoglobin Testing System, Bio-Rad Laboratories, Hercules, CA, USA). The perinatal results were evaluated by the glucose, insulin, leptin (predictor of NB weight), hematocrit, hemoglobin, pH and total bilirubin in umbilical cord blood. The corporal weight, ponderal index (the weight/length3 X 100 ratio), NB classification in small (SGA), adequate (AGA), or large (LGA) for the gestational age (the weight/gestational age ratio) and 1st and 5th min Apgar scores were evaluated at delivery. Hematological parameters (i.e., hematocrit and hemoglobin) were determined in total blood samples with a Sysmex KX-21N (Roche). After evaluation of the hematological parameters, total blood aliquots were centrifuged (4°C for 15 min at 1.000×g) for plasma collection and remaining analyses. Bilirubin concentrations were evaluated by a colorimetric method using BuBc slides (VITROS Chemistry products, Johnson & Johnson), insulin levels were measured by a chemiluminescent immunoassay using microparticles (ARCHITECT insulin, Abbott Laboratories, São Paulo, SP, Brazil) Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 25 and leptin by ELISA kit (R&D system, MN, USA). Evaluation of oxidative stress Since imbalance between antioxidant and oxidants generates the condition of oxidative stress, estimation of total protein reduced thiols, 8-iso-PGF2α and the antioxidant capacity are useful in the prediction of this condition. - Total protein reduced thiols – indirect marker of protein oxidation Total reduced thiols were determined in a spectrophotometer (Spectra Max PLUS 384, Molecular Devices) using 5,5-dithionitrobenzoic acid (DTNB- Sigma Aldrich, St Louis, MO, USA). Thiol residues react with DTNB, cleaving the disulfide bond to give 2-nitro-5-thiobenzoate (NTB2), which ionizes to the NTB22 di-anion in water at neutral and alkaline pH. The NTB22 was quantified in a spectrophotometer by measuring the absorbance at 412 nm, and was expressed as mM of reduced thiols /ml of serum. - 8-Isoprostaglandin F2𝛼 levels (8-iso-PGF2α) – lipid peroxidation marker The 8-iso-PGF2α was detected using a commercially available Direct 8-iso- PGF2α EIA kit (Enzo Life Sciences, Farmingdale, USA). All procedures were carried out following the manufacturer recommendations. - Antioxidant capacity Antioxidant capacity in serum samples was measured using Amplex Red/horseradish peroxidase fluorescence assay (Invitrogen, Paisley, UK). Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 26 Serum samples were incubated with 20μM hydrogen peroxide for 30 minutes at 37 C. After this period, samples were incubated with Amplex Red (50 mM) and horseradish peroxidase type II (0.1 U/ml) in order to quantify the remaining hydrogen peroxide in each sample. Fluorescence readings were determined in GloMax®-Multi+ Microplate Multimode Reader (Promega) with Ex/Em = 530/580 nm. DNA damage analysis Gene-specific quantitative PCR (QPCR) was used to assay nDNA and mtDNA damage [10,11]. Briefly, total genomic DNA was isolated using QIAGEN Genomic Tip and Genomic DNA Buffer Set Kit (QIAGEN). The quantitation of the purified genomic DNA was performed fluorimetrically using PicoGreen dsDNA quantitation reagent (Molecular Probes). Lambda()/HindDIII DNA (Gibco) was used to generated a standard curve and to adjust the final DNA concentration to 3 ng/L. The “hot start” PCR were performed using the Gene Amp XL PCR Kit (Applied Biosystems) with 15 ng de DNA, 1X Buffer, 100ng/L of BSA, 200M of dNTPs, 20 pmol of each primer (Table 1), 1.3 mM final concentration of Mg++ and water to a total volume of 45L. The reaction was brought to 75C prior to addition of 1U/reaction of enzyme (0.5L of the polymerase in 4.5 L of sterile water). We quantitatively amplify an 8.9-kb and 221-bp fragment of the mitochondrial genome and 13.5-Kb of nuclear genome. Amplification of hyperglycemic samples (MGH, GDM and DM2 Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 27 groups) was compared to non hyperglycemic samples (ND group) and relative amplifications were calculated. These measurements were used to estimate the lesion frequency present in the DNA based on a Poisson distribution. Isolation of Peripheral Blood Mononuclear Cells (PBMC) Blood samples were collected in tubes containing heparin as anticoagulant. The samples were diluted in phosphate-buffered saline (PBS), layered onto Ficoll-Pacque (Pharmacia Biotech, Uppsala, Sweden), and centrifuged at 40 minutes at 1.800 rpm. The intermediate phase with the PBMC was collected and washed two times in Dulbecco’s modified eagle medium DMEM low glucose (12320-032-Gibco Invitrogen, Paisley, UK) supplemented with 10% of fetal bovine serum (FBS, Gibco Invitrogen, Paisley, UK). The 2.0x106 cells aliquots were frozen (liquid nitrogen) in FBS with 10% of dimethyl sulfoxide (Sigma Aldrich, St Louis, MO, USA). The samples were thawed in the same medium with 10% FBS. In order to verify the viability of PBMC after isolation and thawing, 1% Trypan blue (Gibco, Invitrogen, Paisley, UK) solution was added at 1:1 volume ratio. The number of dead PBMC in a sample of 100 cells was counted using a light microscope. Only samples with viability > 95% were used in the experiments. RNA extraction and cDNA synthesis RNA extraction and cDNA synthesis was performed in PBMC using power Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 28 SYBR® Green Cells-to-CT™ Kit (Ambion, Carlsbad, CA, USA) as recommended by the manufacturer. Real-time PCR Real-time PCR was used to evaluate the expression of genes involved in different steps of BER. This technique was performed using Power SYBR® Green PCR Master Mix in an Applied Biosystems 7500 Fast Real-Time PCR System (both from Applied Biosystems, Foster City, CA, USA). Real-time PCR was carried out using specific primers for hOGG1, APE-1, FEN-1, POLβ and GAPDH (Table 2). For negative controls, we used a complete DNA amplification mix in which the target cDNA template was replaced with water. The 2^ΔΔCT method of analysis was used with the GAPDH gene for normalization. All samples were run in triplicate. Amplifications were performed using the default cycling conditions: enzyme activation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, and annealing/extension at 60°C for 60 s. GeneAmp software (Applied Biosystems, Foster City, CA, USA) was used to quantify the expression levels (Quantitative PCR). Western blot Western blot was performed to quantify the expression of proteins involved in BER. Initially, PBMC from maternal and umbilical cord blood were lysed with a syringe in ice-cold RIPA buffer (1% NP-40, 0.25% Na-deoxycholate, 150 Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 29 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 50 mM Tris-HCl, pH 7.4), supplemented with a cocktail of protease inhibitors (Sigma Chemical Co, St Louis, MO, USA). Proteins were separated electrophoretically using 15% SDS- PAGE, and proteins on the gel were transferred to a 0.45 µm to nitrocellulose membrane (Millipore, Bedford, MA, USA). The transfer of proteins was confirmed by staining the membranes with a 10% Ponceau S solution (Sigma Aldrich, St Louis, MO, USA). The blotted membranes were blocked with TBS- T-milk 3% (140 mM NaCl, 20 mM Tris–HCl pH 7.4, 0.1% Tween-20, 3% powdered milk) for 1 h and washed three times with TBS buffer. Subsequently, membranes were incubated at 4°C with anti-hOGG1 (at 1:1000, Novus Biologicals, Littleton, CO, USA), anti-APE-1 (at 1:1000, Novus Biologicals, Littleton, CO, USA), anti- FEN-1 (at 1:1000, Novus Biologicals, Littleton, CO, USA) or anti-Polβ (at 1:500, Santa Cruz, CA, USA) in TBS-T-milk 3% overnight and washed three times with TBS buffer. The membranes were then exposed to horseradish peroxidase-conjugated antibody (1:1000, Jackson Immuno Research, USA) in TBS-T-milk 3% for 1 h and washed three times with TBS buffer. Immunoreactive bands (peroxidase activity) were detected by the Enhanced chemiluminescence method (ECL). The quantitative analysis of all DNA repair protein expression levels was performed by densitometry using Image J software (v. 1.43 u, NIH, Bethesda, MD, USA). β-actin levels estimated in the same membrane was used as loading control. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 30 PBMC cultures Considering that prolonged hyperglycemia with consequent oxidative stress and DNA damage are some of the triggers of apoptosis [9], the PBMC isolated from Non-diabetic mothers and their newborns were exposed to different glucose concentrations in vitro. Initially, PBMC were suspended in Dulbecco’s modified eagle medium DMEM low glucose (12320-032; Gibco Invitrogen, Paisley, UK), with 2% FBS, and 1% of gentamicin (Gibco Invitrogen, Paisley, UK). The cells were placed in 96 wells plate in the presence of 5 mmol/L (90 mg/dL, control sample – physiological concentration of glucose – equivalent of normoglycemia in the case of patients), 17.5 mmol/L (315 mg/dL, moderated concentration of glucose) and 30 mmol/L (540 mg/dL high glucose concentration – equivalent of severe hyperglycemia in the case of patients) of D-glucose (Sigma Aldrich, St Louis, MO, USA). Glucose concentrations in incubating medium followed values observed in patients and literature [9,25]. Cells were incubated in 5% CO2 incubator at 37°C for 48 h. Caspase 3/7 activity (Caspase-Glo® 3/7 Assay, Promega) and ATP levels (CellTiter-Glo® Luminescent Cell Viability Assay, Promega) were determined by luminescence in GloMax®-Multi+ Microplate Multimode Reader (Promega). Staurosporine 1mM (abcam, Cambridge, UK) was used to positive control of caspase and negative control of ATP analysis. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 31 Statistical Analysis Analysis of variance and Tukey’s multiple comparison test were used for quantitative variables with normal distribution. For quantitative variables with an abnormal distribution, the generalized linear model with gamma distribution and the log-link function and the LSMeans test were utilized for multiple comparisons. Analyses were performed using SAS software, version 9.1 and Prism taking into account a 95% level of statistical significance (p < 0.05). Results Maternal clinical background and perinatal outcomes evaluation GDM and DM2 patients were older (p < 0.0001) and had the highest BMI in the third trimester of pregnancy (p = 0.0003). In addition, the diabetics groups and MGH group had the highest pre-gestational BMI (p < 0.0001). The GM was progressively higher in relation to the severity of clinical conditions of the mother (p < 0.0001) and the HbA1c levels in the third trimester (p < 0.0001) confirmed the GM levels; therefore, the values of GM did not exceed the glycemic goals recommended by ADA [1] (Table 3). Newborns from MGH, GDM and DM2 groups had the highest leptin (p = 0.0162) and hematocrit levels (p = 0.0026). Glycemia and insulin in cord blood were not different among groups; the NB weight (p < 0.0001) and percentage of LGA (p < 0.0001) were increased only in MGH groups Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 32 (Table 04). Oxidative stress Levels of reduced thiols were significantly decreased in serum of mothers with GDM (p = 0.0043) and DM2 when compared with ND group (p = 0.027) (Figure 2-A1), and were not significantly different in the umbilical cord blood serum (Figure 2-B1). The 8-iso-PGF2α serum levels were higher in DM2 mothers than other groups (p = 0.0061) (Figure 2-A2), whereas the GDM group showed relevant augmentation in serum of umbilical cord blood when compared with ND group (p = 0.0217) (Figure 2-B2). No significant differences on the antioxidant capacity were observed in maternal serum (Figure 2-A3) or in NB (Figure 2-B3). Nuclear and mitochondrial DNA damage The levels of nDNA and mtDNA damage in maternal leukocytes (Figure 3-A1; Figure 3-A2, respectively) were higher in both GDM (p = 0.02) and DM2 groups (p = 0.0007) when compared to ND group. In contrast, no significant differences were found in nDNA or mtDNA damage in umbilical cord blood leukocytes (Figure 3-B1 and Figure 3-B2). mRNA expression of APE-1, FEN-1 and POLβ The mRNA hOGG1 expression was not detectable with the methodologies Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 33 employed in maternal or NB PBMC. The mRNA of APE-1, FEN-1 and POLβ were expressed in all study groups; in hyperglycemic groups were observed opposite results between mothers and NB (Figure 4). mRNA APE-1 expression was lower in mothers of DM2 group than others groups (p = 0.0008) (Figure 4-A1), and it was higher in NB of GDM group than others groups (p = 0.0032) (Figure 4-B1). mRNA FEN-1 expression was lower in GDM mothers than ND and MGH groups, and it was lower in mothers with DM2 compared to MGH group (p = 0.0006) (Figure 4-A2). In addition, the highest mRNA FEN-1 expression was found in NB of GDM and DM2 groups compared to ND group (p = 0.0016) (Figure 4-B2). mRNA POLβ expression was lower in mothers of GDM and DM2 groups compared to ND group (p = 0.0044) (Figure 4-A3), and it was higher in NB of DM2 group compared to ND group (p = 0.0036) (Figure 4-B3). The summary of these results are in Table 1S (supplementary archive). Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 34 Protein expression levels of hOGG1, APE-1, FEN-1 and POLβ The BER proteins evaluated were identified in all groups. The results are similar to observed in mRNA analysis and confirm the opposite response observed between mothers and NB of diabetic groups (Figure 5; Figure 6). The levels of hOGG1 expression were lower in mothers with GDM only when compared to DM2 group (p = 0.0105) (Figure 5-A1; Figure 5-a1), and it was higher in NB of DM2 group than ND and MGH groups (p = 0.0039) (Figure 5-B1; Figure 5-b1). APE-1 expression was lower in mothers of GDM and DM2 groups when compared to ND group (p = 0.0145) (Figure 5-A2; Figure 5-a2), and it was higher in NB of GDM group than others groups (p = 0.0164) (Figure 5-B2; Figure 5-b2). Maternal FEN-1 levels were similar in all groups (Figure 6-A1; Figure 6-a1); and increased in NB from GDM group when compared to ND and MGH groups (p = 0.0099) (Figure 6-B1; Figure 6- b1). POLβ expression was lower in mothers of MGH compared to ND group (p = 0.0363) (Figure 6-A2; Figure 6-a2), and it was elevated in NB of DM2 group when compared to others groups (p = 0.0077) (Figure 6-B2; Figure 6- b2). The summary of these results are in Table 1S (supplementary archive). Apoptosis and ATP production analysis The activity of effector caspases-3 and caspase-7 was elevated in PBMC isolated of mothers without diabetes and exposed to 30 mmol/L (540 mg/dL) of glucose compared with 95 mmol/L (90 mg/dL) concentrations (p < 0.001) Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 35 (Figure 7-A1). To confirm these results, we evaluated ATP levels in the same samples and observed ATP reduction in 30 mmol/L glucose concentration (p = 0.0480) (Figure 7-A2). Activation of caspases-3 our caspase-7 was not observed in PBMC of NB (Figure 7-B1); despite this, the ATP levels were reduced in 30 mmol/dL (540 mg/dL) glucose concentration (p = 0.0439) (Figure 7-B2). Discussion Almost all of the complications associated with diabetes in pregnancy are linked to maternal hyperglycemia [1]. In this study, pregnant women of MGH, GDM and DM2 groups had different hyperglycemia levels, which became progressively higher in relation to the severity of clinical conditions, and that impacted the results obtained solely for the mothers. The groups of mothers with GDM and DM2 were characterized by oxidative stress, increase of nuclear and mitochondrial DNA damage as well as decrease expression of genes and proteins involved in BER. In addition, the levels of hyperglycemia were associated to in vitro cellular apoptosis in maternal PBMC. It is widely accepted that hyperglycemia leads to oxidative stress. Important studies showed increased biomarkers of oxygen radical damage and abnormalities in the antioxidant defenses of diabetic patients [26]. Plasma and urinary concentrations of 8-iso-PGF2α were associated to DM2 in non- pregnant women [27,28], and to GDM in pregnant mothers [29]. Our Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 36 maternal results reinforce these data. In the present study, thiols in their reduced form were lower in GDM and DM2 groups, suggesting the presence of oxidized proteins. Levels of8-iso- PGF2α, a lipid peroxidation marker, was high only in the DM2 group. The differences among hyperglicemic levels overweight and obesity, with correspondent insulin resistance, may have influenced ROS production [30,31] and contributed to our results. Overweight, insulin resistance and hyperglycemia have been associated to differences in vascular disorders, cytokines production and apoptosis in placentas of diabetic mothers [32-36]. No difference was found in maternal antioxidant capacity after H2O2 treatment. Probably, antioxidant defenses were not enough to control the elevated oxidative stress in GDM and DM2 groups, as demonstrated by increased serum protein oxidation or lipid peroxidation in these diabetic mothers. This observation is consistent with previous studies in rats, which demonstrated higher levels of lipid peroxidation as gauged by malondialdehyde levels, even in presence of increased antioxidants enzymes in maternal blood of streptozotocin-induced diabetic pregnant rats [7]. Human studies report increased production of ROS and decrease in antioxidant defenses of diabetic pregnant women [37,38]. Thus, the results of our study reinforce that antioxidant defense in pregnancy complicated by diabetes or hyperglycemia is not enough to defending against the Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 37 exacerbated oxidative stress condition. Besides hyperglycemia and oxidative stress, our results showed that pregnant women with GDM or DM2 exhibited a significant increase in nDNA and mtDNA damage. Some studies have suggested that mtDNA is more susceptible than nDNA to genotoxic agents, most notably ROS [11,12]. Damage in DNA, if not repaired, could lead to mutations, which are associated to many different diseases including cancers, both in mother [39] and NB [40]. MtDNA damage can also lead to mitochondrial dysfunction, promote and maintain increased ROS production, which could leak out the mitochondria, affecting the nuclear genome. The maternal glucose can readily cross the placenta and it has always been associated with adverse perinatal outcomes [41-43]. In this study, NB of hyperglycemic mothers were characterized by elevated levels of leptin, increase in body weight, higher hematocrit levels and high rate of LGA. Furthermore, the NB of diabetic mothers presented increase of BER genes and proteins expression, and the hyperglycemia environment in vitro was not able to induce apoptosis in NB cells. Interesting, only NB from GDM mothers show increase in lipid peroxidation without association with DNA damage. Irrespective of the groups, blood levels of DNA damage in umbilical cord were similar. According to some studies, there is a compensatory response in umbilical Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 38 cord blood cells of NB exposed to hyperglycemia, relative to the higher telomerase activity, which is able to maintain telomere structure in nDNA [44]. Another explanation is related to the increase ratio of mitochondrial/nuclei hTERT, suggesting a protective effect on fetal mtDNA [29]. In contrast, our previous experimental studies showed higher levels of oxidative DNA damage in both, streptozotocin-induced diabetic pregnant rats and their NB, when glucose levels were ≥ 300mg/dL [21-23]. In this study, the maternal glucose was maintained at lower levels than those observed in diabetic rats, and this may explain the differences in offspring DNA damage between STZ-diabetic rats and diabetic mothers. The most intriguing result in the present study was that, in hyperglycemic conditions, the maternal and fetal compartment respond differently to DNA insults. The GDM and DM2 mothers exhibited nDNA and mtDNA damage, which were not observed in their newborns. In addition, these mothers had lowest expression of BER genes (APE-1, FEN-1, POLβ) and protein (hOGG1, APE-1), associated to apoptosis in high glucose concentrations (in vitro experiment). Conversely their NB showed increase of these genes and proteins expression and no in vitro induction of apoptosis in hyperglycemic concentrations. Overall, these results indicate that while damage in the mother genome was evident, the fetal genome was well protected. The umbilical cord contains at least three populations of stem cells, each Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 39 with unique features and properties. These stem cells possess highly efficient DNA repair network that becomes less efficient upon differentiation, and also have an anaerobic metabolism, which reduces mitochondrial number and oxidative stress [45,46]. The higher telomerase activity [44] and the increased mitochondrial/nuclei ratio of human translocation telomerase reverse transcriptase (hTERT) [29] have been also considered as a possible protective factor for DNA umbilical blood cells in hyperglycemic pregnancies. These data suggest that umbilical cord blood cells have potentially several mechanisms at play to protect the fetal DNA against genomic insults in hyperglycemic and oxidative stress conditions. The results of our study open a wide window for future researches. However, it is important to recognize its limitations, especially in respect to the difference between the number of mothers and NB evaluated. This was due to problems of inadequacy of some samples and difficulty to get and to process samples at night and weekend births. Although limited, our results highlight that protective mechanism for fetal DNA-damage may be dependent of glycemic levels, reinforcing yet again the importance of adequate maternal glycemic control in pregnancies complicated by diabetes. In conclusion, this study has demonstrated that maternal hyperglycemia observed in GDM and DM2 groups was associated to oxidative stress and, consequently with nDNA and mtDNA damage. However, integrity of DNA Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 40 from umbilical cord blood cells was preserved, suggesting the better involvement of DNA repair mechanisms in these fetuses. Supplementary Material Table 1S. Summary of results of gene and protein expression of BER Acknowledgments The authors would like to thank Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP (grant number 2011/18240-2; 2011/13562-1 and 2012/23296-0) for financial support and for the fellowships of JB Moreli, Dr. Carlos Frederico Martins Menck for scientific support and Dr. José Eduardo Corrente for statistics support. Competing Interests The authors have declared that no competing interest exists. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 41 References 1. American Diabetes Association (ADA). Standards of medical care in Diabetes - 2014. Diabetes Care. 2014;37:S14-S80. 2. Ornoy A. Prenatal origin of obesity and their complications: Gestational diabetes, maternal overweight and the paradoxical effects of fetal growth restriction and macrosomia. Reprod Toxicol. 2011; 32:205-12. 3. Calderon IMP, Kerche LTRL, Damasceno DC, Rudge MVC. Diabetes and pregnancy: an update of the problem. ARBS. 2007; 9:1–11. 4. Yang J, Cummings EA, O'connell C, Jangaard K. Fetal and neonatal outcomes of diabetic pregnancies. Obstet Gynecol. 2006; 10:644-50. 5. Lehnen H, Zechner U, Haaf T. Epigenetics of gestational diabetes mellitus and offspring health: the time for action is in early stages of life. Mol Hum Reprod. 2013; 19:415-22. 6. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001; 414:813-20. 7. Spada AP, Damasceno DC, Sinzato YK, Campos KE, Faria PA, Dallaqua B, et al. Oxidative stress in maternal blood and placenta from mild diabetic rats. Reprod Sci. 2014; 21:973-77. 8. Damasceno DC, Netto AO, Iessi IL, Gallego FQ, Corvino SB, Dallaqua B, et al. Streptozotocin-induced diabetes models: pathophysiological mechanisms Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 42 and fetal outcomes. Biomed Res Int. doi: 10.1155/2014/819065. 9. Arya AK, Pokharia D, Tripathi K. Relationship between oxidative stress and apoptotic markers in lymphocytes of diabetic patients with chronic non healing wound. Diabetes Res Clin Pract. 2011; 94:377-84. 10. Kovalenko OA, Santos JH. Analysis of oxidative damage by gene-specific quantitative PCR. Curr Protoc Hum Genet. 2009; Chapter 19:Unit 19.1. 11. Santos JH, Meyer JN, Mandavilli BS, Van Houten B. Quantitative PCR- based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods Mol Biol. 2006; 314:183-99. 12. Yakes FM, Van Houten B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc Natl Acad Sci U S A. 1997; 94:514-9. 13. Mandavilli BS, Santos JH, Van Houten B. Mitochondrial DNA repair and aging. Mutat Res. 2002; 509:127-51. 14. Mitra S, Boldogh I, Izumi T, Hazra TK. Complexities of the DNA base excision repair pathway for repair of oxidative DNA damage. Environ Mol Mutagen. 2001; 38:180-90. 15. Pácal L, Varvařovská J, Rušavý Z, Lacigová S, Stětina R, Racek J, et al. Parameters of oxidative stress, DNA damage and DNA repair in type 1 and type 2 diabetes mellitus. Arch Physiol Biochem. 2011; 117:222-30. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 43 16. Agnez-Lima LF, Melo JT, Silva AE, Oliveira AH, Timoteo AR, Lima-Bessa KM, et al. DNA damage by singlet oxygen and cellular protective mechanisms. Mutat Res. 2012; 15-28. 17. Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature. 2009; 461:1071-8. 18. Blasiak J, Arabski M, Krupa R, Wozniak K, Zadrozny M, Kasznicki J, et al. DNA damage and repair in type 2 diabetes mellitus. Mutat Res. 2004; 554:297-304. 19. Tatsch E, Bochi GV, Piva SJ, De Carvalho JA, Kober H, Torbitz VD, et al. Association between DNA strand breakage and oxidative, inflammatory and endothelial biomarkers in type 2 diabetes. Mutat Res. 2012; 732:16-20. 20. Qiu C, Hevner K, Abetew D, Enquobahrie DA, Williams MA. Oxidative DNA damage in early pregnancy and risk of gestational diabetes mellitus: A pilot study. Clin Biochem. 2011; 44:804-8. 21. Lima PH, Sinzato YK, de Souza Mda S, Braz MG, Rudge MV, Damasceno DC. Evaluation of level of DNA damage in blood leukocytes of non-diabetic and diabetic rat exposed to cigarette smoke. Mutat Res. 2007; 628:117-22. 22. Lima PH, Damasceno DC, Sinzato YK, de Souza Mda S, Salvadori DM, Calderon I de M, et al. Levels of DNA damage in blood leukocyte samples from non-diabetic and diabetic female rats and their fetuses exposed to air or cigarette smoke. Mutat Res. 2008; 653:44-9. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 44 23. Lima PH, Sinzato YK, Gelaleti RB, Calderon IM, Rudge MV, Damasceno DC. Genotoxicity evaluation in severe or mild diabetic pregnancy in laboratory animals. Exp Clin Endocrinol Diabetes. 2012; 120:303-7. 24. Rudge MVC, Peraçoli JC, Berezowski AT, Calderon IMP, Brasil MAM. The oral glucose tolerance test is a poor predictor of hyperglycemia during the pregnancy. Braz J Med Biol Res. 1990; 23:1079-89. 25. Oleszczak B, Szablewski L, Pliszka M. The effect of hyperglycemia and hypoglycemia on glucose transport and expression of glucose transporters in human lymphocytes B and T: an in vitro study. Diabetes Res Clin Pract. 2012; 96:170-8. 26. West IC. Radicals and oxidative stress in diabetes. Diabet Med. 2000; 17:171-80. 27. Davì G, Ciabattoni G, Consoli A, Mezzetti A, Falco A, Santarone S, et al. In vivo formation of 8-iso-prostaglandin f2alpha and platelet activation in diabetes mellitus: effects of improved metabolic control and vitamin E supplementation. Circulation. 1999; 99:224-9. 28. Devaraj S, Hirany SV, Burk RF, Jialal I. Divergence between LDL oxidative susceptibility and urinary F(2)-isoprostanes as measures of oxidative stress in type 2 diabetes. Clin Chem. 2001; 47:1974-9. 29. Li P, Tong Y, Yang H, Zhou S, Xiong F, Huo T, et al. Mitochondrial translocation of human telomerase reverse transcriptase in cord blood Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 45 mononuclear cells of newborns with gestational diabetes mellitus mothers. Diabetes Res Clin Pract. 2014; 103:310-8. 30. Collins AR, Raslová K, Somorovská M, Petrovská H, Ondrusová A, Vohnout B, et al. DNA damage in diabetes: correlation with a clinical marker. Free Radic Biol Med. 1998; 25:373-7. 31. Cerdá C, Sánchez C, Climent B, Vázquez A, Iradi A, El Amrani, et al. Oxidative stress and DNA damage in obesity-related tumorigenesis. Adv Exp Med Biol. 2014; 824:5-17. 32. Sgarbosa F, Barbisan LF, Brasil MAM, Costa E, Calderon IMP, Gonçalves CR, et al. Changes in apoptosis and Bcl-2 expression in human hyperglycemic, term placental trophoblast. Diabetes Res Clin Pract. 2006; 73:143-9. 33. Calderon IM, Damasceno DC, Amorin RL, Costa RA, Brasil MA, Rudge MV. Morphometric study of placental villi and vessels in women with mild hyperglycemia or gestational or overt diabetes. Diabetes Res Clin Pract. 2007; 78:65-71. 34. Pietro L, Daher S, RudgeMV, Calderon IM, Damasceno DC, Sinzato YK, et al. Vascular endothelial growth factor (VEGF) and VEGFreceptor expression in placenta of hyperglycemic pregnant women. Placenta. 2010; 3:7707-80. 35. Moreli JB, Morceli G, De Luca AK, Magalhães CG, Costa RA, Damasceno DC, et al. Influence of maternal hyperglycemia on IL-10 and TNF-α production: the relationship with perinatal outcomes. J Clin Immunol. 2012; Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 46 32:604-10. 36. Moreli JB, Corrêa-Silva S, Damasceno DC, Sinzato YK, Lorenzon-Ojea AR, Borbely AU, et al. Dynamics changes in the TNF-alpha/IL-10 ratio in hyperglycemic-associated pregnancies. 2015: In press: Diabetes Research and Clinical Practice. doi:10.1016/j.diabres.2015.01.005 37. Djordjevic A, Spasic S, Jovanovic-Galovic A, Djordjevic R, Grubor-Lajsic G. Oxidative stress in diabetic pregnancy: SOD, CAT and GSH-Px activity and lipid peroxidation products. J Matern Fetal Neonatal Med. 2004; 16:367-72. 38. Grissa O, Atègbo JM, Yessoufou A, Tabka Z, Miled A, Jerbi M, et al. Antioxidant status and circulating lipids are altered in human gestational diabetes and macrosomia. Transl Res. 2007; 150:164-71. 39. Polyak K, Li Y, Zhu H, Lengauer C, Willson JK, Markowitz SD, et al. Somatic mutations of the mitochondrial genome in human colorectal tumours. Nat Genet. 1998; 20:291-3. 40. Wu CS, Nohr EA, Bech BH, Vestergaard M, Olsen J. Long-term health outcomes in children born to mothers with diabetes: a population-based cohort study. PLoS One. 2012; 7:e36727. 41. Pedersen J, Bojsen-Moller B, Paulsen H. Blood sugar in newborn infants of diabetic mothers. Acta Endocrinol (Copenh). 1954; 15:33-52. 42. Jansson T, Powell TL. Role of the placenta in fetal programming: Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 47 underlying mechanisms and potential interventional approaches. Clin Sci (Lond). 2007; 113:1-13. 43. Fraser A, Lawlor DA. Long-term health outcomes in offspring born to women with diabetes in pregnancy. Curr Diab Rep. 2014; 14:489. 44. Cross JA, Temple RC, Hughes JC, Dozio NC, Brennan C, Stanley K, et al. Cord blood telomere length, telomerase activity and inflammatory markers in pregnancies in women with diabetes or gestational diabetes. Diabet Med. 2010; 27:1264-70. 45. Ali H, Mulla FA. Defining umbilical cord blood stem cells. Stem Cell Discovery. 2012; 2:15-23. 46. Rocha CR, Lerner LK, Okamoto OK, Marchetto MC, Menck CF. The role of DNA repair in the pluripotency and differentiation of human stem cells. Mutat Res. 2013; 752:25-35. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 48 Table 1. Gene targets, primers pairs and cycles for QPCR F: foward; R: reverse. Table 2. Primers used for real time PCR In hOGG1 analysis was tested two pairs of primers (A and B) and HS01114116_G1 OGG1 Taqman Assay.(Applied Biosystems, Foster City, CA, USA). In all experimentes hOGG1 expresion was not detectable. Targets Genes Fragme nt length Primers Pairs Cycles Nuclear fragment Region near the β Globin gene 13.5 kb F: 5′-CGA GTA AGA GAC CAT TGT GGC AG-3′ (GI 48510) 75C – 2 min 94C - 1 min 94C – 15 seg 64C – 12 min 72C – 10 min 21 cycles R: 5′-GCA CTG GCT TAG GAG TTG GAC T-3′ (GI 62007) Mitochodrial fragment 8.9 Kb F: 5′-TCT AAG CCT CCT TAT TCG AGC CGA-3′ (GI5999) 75C – 2 min 94C - 1 min 94C – 15 seg 64C – 12 min 72C – 10 min 17 cycles R: 5′-TTT CAT CAT GCG GAG ATG TTG GAT GG-3’ (GI14841) Normalize Mitochodrial small fragment 221bp F: 5′-CCC CAC AAA CCC CAT TAC TAA ACC CA-3′ (GI14620) 75C – 2 min 94C - 1 min 60C – 45 seg 72C – 45 seg 72C – 10 min 17 cycles R: 5′-TTT CAT CAT GCG GAG ATG TTG GAT GG-3′ (GI14841) Primer Forward 5’-3’ Reverse 5′-3′ TM (°C) hOGG1 (A) GTGGACTCCCACTTCCAAGA CGATGTTGTTGTTGGAGGAA 55 hOGG1 (B) GTTCTGCCTTCTGGACAATCT CCATACTTGATCCGCTAGTACAC 55 APE-1 CTGCCTGGACTCTCTCATCAATAC CCTCATCGCCTATGCCGTAAG 57 FEN-1 CGGGCTGTGGACCTCATC CAAGTCGCCGCACGAT 58 POLB GTGCAGAGTCCAGTGGTGACA CAGTTTTGGCTGTTTGGTTGATT 57 GAPDH CAAGAGCACAAGAGGAAGAGAG CTACATGGCAACTGTGAGGAG 55 Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 49 Table 3. Maternal clinical background Clinical data are presented as means ± standard deviation or n (%). 1: Evaluated in pre-pregnancy; 2: Evaluated in the third trimester of pregnancy; BMI: body mass index; GA: Gestational age at delivery; GM: Glycemic mean; HbA1c: Glycated hemoglobin. Values followed by different letters and same index significantly differ (p < 0.05). ns: not significant (p > 0.05). ND (n = 46) MGH (n = 30) GDM (n = 45) DM2 (n = 23) p Age (years) 26.1±7.9a0 28.4±6.4a0 30.9±4.9b0 33.2±7.1b0 <0.0001 BMI 1 (kg/m2) 26.4±5.0a1 31.6±7.9b1 33.3±7.1b1 34.8±5.9b1 < 0.0001 BMI 2 (kg/m2) 31.1±5.5a2 35.0±7.3a2 37.4±6.5b2 37.2±9.9b2 0.0003 Weight gain (kg) 12.3±5.1 9.9±6.1 10.3±8.0 10.2±6.5 ns GA (weeks) 39.5±1.7a3 39.3±1.1a3 38.9±1.3a3 37.5±0.8c3 <0.0001 Hypertension 03 (6.52) 12 (40.00) 07 (15.55) 11 (47.82) ns GM (mg/dL) 83.1±7.8a5 91.3±11.7b5 101.9±14.2c5 109.6±13.8d5 < 0.0001 HbA1c (%) 5.3±0.4a6 5.5±0.5b6 5.6±0.5b6 6.5±1.1c6 < 0.0001 Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 50 Table 4. Perinatal results Data are presented as means ± standard deviation or n (%). PI: ponderal index (weight/length3 x100); weight/gestational age classification: small (SGA), adequate (AGA), and large (LGA) for gestational age; Ht: hematocrit; Hb: hemoglobin. Values followed by different letters and same index significantly differ (p < 0.05). ns: not significant (p > 0.05). ND (n = 27) MGH (n = 20) GDM (n = 21) DM2 (n = 19) p Glycemia (mg/dL) 65.5±19.3 61.50±15.9 73.5±30.9 68.9±22.6 ns Insulin (μU/mL) 5.3±5.4 9.3±13.7 8.9±9.1 8.0±5.5 ns Leptin (pg/mL) 52.1±63.5a0 219.4±311.9b0 147.3±107.2b0 253.8±361.5b0 0.0162 Weight (g) 3198.8±421.2a1 3637.2±579.2b1 3317.2±559.7a1 3070.2±488.5a 1 <0.0001 PI (g/cm3) 0.028±0.002 0.028±0.003 0.029±0.003 0.030±0.003 ns SGA 03 (11.1) 03 (15.0) 0.0 (0.0) 0.0 (0.0) <0.0001 AGA 22 (81.5) 13 (65.0) 20 (95.2) 16 (84.2) LGA 02 (7.4) 04 (20.0) 01 (4.8) 03 (15.8) Ht (%) 48.0±5.5a2 49.4±4.7b2 51.8±5.5b2 53.4±7.5c2 0.0026 Hb (g/dL) 15.9±2.2 16.1±1.5 16.6±1.8 17.0±2.3 ns pH 7.26±0.12 7.28±0.10 7.27±0.09 7.30±0.07 ns Bilirubin (mg/dL) 2.02±0.57 2.10±0.73 2.03±0.74 2.22±0.63 ns Apgar 1 < 7 05 (18.52) 05 (25.00) 03 (14.29) 02 (10.53) ns Apgar 5 < 7 01 (3.70) 02 (10.00) 0 (0) 0 (0) ns Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 51 Figure 01. Definition of study groups and sample size.The MGH and GDM diagnosis was established between 24th and 28th gestational weeks according the 75 g-GTT and glucose profile results. The DM2 were referred to the Diabetes and Pregnancy Service with a confirmed diagnosis. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 52 Figure 02. Evaluation of oxidative stress markers in serum from mothers [A] and umbilical cord blood [B]. A1 and B1: Total reduced thiols determined by DTNB method. A2 and B2: 8-iso-PGF2α evaluated by EIA method. A3 and A4: Antioxidant capacity after incubation with 20μM hydrogen peroxide for 30 minutes at 37 °C. Hydrogen peroxide not degraded by samples was determined by Amplex Red/ horseradish peroxidase fluorescence assay. Values as mean ± SEM, *p < 0.05 vs others groups; ** p < 0.05 vs appointed study group. n=15/group. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 53 Figure 03. Nuclear [1] and mitochondrial [2] DNA damage from maternal [A] and umbilical cord blood [B]. DNA damage determined by Gene-specific quantitative PCR (QPCR). Values as mean ± SEM, ** p < 0.05 vs appointed study group. n=15/group. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 54 Figure 04. Gene expression of APE-1 [1], FEN-1 [2] and POLβ [3] in PBMC isolated from maternal [A] and umbilical coord blood [B]. Each reaction run in triplicates. All expression was normalized by GAPDH. hOGG1 expression was not detected with the methodologies employed. Values as mean ± SEM; *p < 0.05 vs others groups; ** p < 0.05 vs appointed study group. n=15/group. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 55 Figure 05. Protein expression of hOGG1 [1] and APE-1 [2] in PBMC isolated from maternal [A,a] and umbilical coord blood [B,b]. The relative band intensities from western blot experiments were normalized to the level of β-actin and analyzed with Image J software [A1, A2, B1, B2]. Values as mean ± SEM; *p < 0.05 vs others groups; ** p < 0.05 vs appointed study group. n = 08/group. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 56 Figure 06. Protein expression of FEN-1 [1] and POLβ [2] in PBMC isolated from maternal [A,a] and umbilical coord blood [B,b]. The relative band intensities from western blot experiments were normalized to the level of β-actin and analyzed with Image J software [A1, A2, B1, B2]. Values as mean ± SEM; *p < 0.05 vs others groups; ** p < 0.05 vs appointed study group. n = 08/group. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 57 Figure 07. Caspase 3/7 activity [1] and ATP levels [2] in PBMC isolated from maternal [A] and umbilical cord blood [B] of non-diabetic group. PBMC were exposet to 5 mmol/L (90 mg/dL), 17.5 mmol/L (315 mg/dL) and 30 mmol/L (540 mg/dL) of glucose during 48h. Control ST: Staurosporine 1mM. Values as mean ± SEM, ** p < 0.05 vs appointed study group. n=06/group. Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 58 Table 1S. Summary of results of gene and protein expression of BER ND MGH GDM DM2 Gene Protein Gene Protein Gene Protein Gene Protein Mothers hOGG1 nd APE-1 nd * FEN-1 nd   **  POLβ nd   *  Newborns hOGG1 nd APE-1 nd FEN-1 nd     POLβ nd     nd: not detected; ns: not significant;  increase of expression.;  decrease of expression.; * compared only with MGH group; ** compared only with DM2 group. Anexo Tese de Doutorado  Jusciéle Brogin Moreli, 2015 ___________________ 59