Toxicity of the antitumor drug mitoxantrone in pediatric and adult mice

Toxicity of the antitumor drug mitoxantrone in pediatric and adult mice José Luís das Dores Sousa Mestrado em Bioquímica Departamento de Química e Bioquímica 2013 Orientador Doutora Vera Marisa Costa, Investigadora, REQUIMTE/FFUP Co-orientador Prof. Doutor Félix Carvalho, Professor Catedrático, FFUP Todas as correcções determinadas pelo júri, e só essas, foram efectuadas. O Presidente do Júri, Porto, ______/______/_________ INSTITUTO DE CIÊNCIAS BIOMÉDICAS ABEL SALAZAR UNIVERSIDADE DO PORTO FACULDADE DE CIÊNCIAS UNIVERSIDADE DO PORTO FACULDADE DE FARMÁCIA UNIVERSIDADE DO PORTO Toxicity of the antitumor drug mitoxantrone in pediatric and adult mice Dissertação de Candidatura ao grau de Mestre em Bioquímica da Universidade do Porto Orientadora: Doutora Vera Marisa Costa Investigadora do REQUIMTE, Faculdade de Farmácia da Universidade do Porto Co-orientador: Professor Doutor Félix Carvalho Professor Catedrático da Faculdade de Farmácia da Universidade do Porto Afiliação: REQUIMTE – Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto 2013 ACKNOWLEDGMENTS É impossível agradecer o suficiente a quem contribuiu, directa ou indirectamente, para a elaboração desta dissertação e para o meu crescimento a nível científico. À Doutora Vera Marisa Costa não há palavras suficientes para mostrar o quanto estou agradecido. Obrigado por me ter aberto a janela de cardiooncologia no presente ano curricular e por ter sempre acreditado nas minhas capacidades. A sua boa vontade, simpatia e paciência com que me acolheu, o excelente profissionalismo e o rigor mostrado, e a confiança, o suporte e as sugestões ao longo deste projecto foram decisivos e fundamentais. Mais uma vez, um MUITO obrigado! Ao Professor Doutor Félix Carvalho agradeço a sua partilha de conhecimentos e sabedoria, assim como os preciosos conselhos, enriquecendo-me a nível crítico e profissional. À Professora Doutora Maria Lourdes Bastos, agradeço a oportunidade de poder ter estado neste fantástico laboratório. Ao Professor Doutor José Duarte, obrigado por me ter dado a oportunidade de fazer a parte histológica da minha dissertação na FADEUP, por me ter dado conselhos e críticas úteis acerca do meu trabalho, e pela disponibilidade e simpatia que sempre mostrou comigo. Ao Professor Doutor Vítor Seabra, devo-lhe um enorme agradecimento por ter tornado possível a utilização do Biotério da CESPU e que foi fulcral para o desenrolar deste projecto. Obrigado pelas sugestões, disponibilidade e simpatia. Agradeço de uma forma muito especial a todos os outros professores e membros de investigação do Laboratório de Toxicologia da FFUP, pelas suas ajudas e pela tamanha amabilidade diária. Um agradecimento enorme à Cátia Faria, que considero o motor do laboratório: tudo foi mais fácil com a sua ajuda e prontidão para resolução de problemas, assim como por se ter sempre mostrado preocupada e atenciosa com o meu trabalho. Obrigado pela amizade! À D. Celeste, obrigado pela sua ajuda preciosa com os procedimentos histológicos e boa disposição logo pela manhã. À Rita Azevedo, um grande obrigado por todo o companheirismo desde o início, a ajudar integrar-me e a conhecer melhor o laboratório, e pela paciência infinita! À minha Joana, obrigado por todo o amor e suporte, por me teres sempre ouvido e ajudado quando mais precisei. Foste um pilar essencial nesta etapa da minha vida! Tudo era mais difícil sem ti! Por último, as pessoas mais importantes para mim: a minha família. Aos meus pais e irmã dedico este trabalho, pois sem eles eu não era nada do que sou hoje. Obrigado por me terem apoiado a 200% em todos os meus objectivos, e por terem sempre acreditado em mim. Tudo vos devo! Deixo aqui os agradecimentos à Fundação da Ciência e Tecnologia (FCT) pelo financiamento do projecto EXPL/DTP-FTO/0290/2012, co-financiado pelo Eixo I do Programa Operacional Fatores de Competitividade (POFC) do QREN, (COMPETE: FCOMP01-0124-FEDER-027749), sem o qual esta tese não seria possível. Abstract ix ABSTRACT Anticancer therapy has evolved greatly with the introduction of new drugs that increase life expectancy. However, the increased efficacy leads to several side effects and damage, especially for non-cancer tissues, thus compromising the survival or the quality of life of the treated-patients. Mitoxantrone (MTX) is an anticancer agent widely used and one of its major adverse effects is cardiac toxicity that can affect up to 18% of MTX-treated patients. One of the most concerning MTX-cardiac side effects is heart failure that may be detected only years after cancer therapy cessation. Although MTX has a toxic clinical profile similar to doxorubicin, their mechanisms of cardiotoxicity differ and MTX mechanisms of cardiotoxicity remain largely unknown. MTX is also used in pediatric cancer and in multiple sclerosis in children. Children that survive cancer potentially have a long life-expectancy after treatment, but little is known about the overall risks of this population to develop heart toxicity after MTX. Therefore, the present study aimed to evaluate the parameters and risk factors associated to MTX-cardiac toxicity as well as the toxicity towards the liver and kidneys, in pediatric (3 weeks) and adult (8-10 weeks) male CD-1 mice. Total cumulative doses of 4.5 mg/kg and 9.0 mg/kg of MTX were given as a result of 6 intraperitoneal injections (2 per week) in experiment 1. Cumulative doses of 7.0 mg/kg and 9.0 mg/kg MTX were used in experiment 2. This multiple administration procedure was used in order to mimic the human MTX therapy that comprises multiple MTX-administration in different cycles. After MTX administration, the animals were maintained, before sacrifice, in a drug-free period to allow the development of cumulative toxicity. In experiment 1, the surviving animals were sacrificed at day 20; in experiment 2, the 9.0 mg/kg MTX-treated animals were sacrificed one day after the last administration, whereas the 7.0 mg/kg MTX-treated animals were sacrificed 14 days after the last administration. The consumption of food and water, and animal weight were recorded along the experiments. After sacrifice, the following parameters were evaluated: total glutathione (GSHt), oxidized glutathione (GSSG), lipid peroxidation and adenosine triphosphate (ATP) on heart, liver, and kidney; cardiac apoptosis and histopathological examination; and finally aspartate aminotransferase (AST), alanine aminotransferase (ALT), total-creatine kinase (total-CK) and creatine-kinase MB (CK-MB) levels in the plasma. In the studies developed in the present dissertation, pediatric population revealed itself as more resistant to MTX-induced toxicity as, in experiment 1, some of these animals survived with the 9.0 mg/kg cumulative MTX dose. In both experiments, the average body weight of pediatric mice almost did not vary with time, oppositely to what happened with adults. Elevation of plasma AST in the survivors of 9.0 mg/kg MTX-treated animals in experiment 1 may indicate heart damage since no changes were seen in ALT. The decreases in AST and ALT in 4.5 mg/kg MTX-treated adults may be related to their xvi Table of Contents 3.4. Blood and tissue collection ............................................................................. 30 3.5. Measurement of hematological parameters, aminotransferases, total-CK and CK-MB ...................................................................................................................... 31 3.6. Determination of total glutathione (GSHt) and GSSG ..................................... 32 3.7. Assessment of lipid peroxidation .................................................................... 32 3.7.1. HPLC-DAD determination ........................................................................... 33 3.7.2. Fluorescence microplate determination ....................................................... 33 3.8. Determination of cellular ATP levels ............................................................... 33 3.9. Determination of caspase-3, -8 and -9 activities ............................................. 34 3.10. Processing of tissues for optic microscopy .................................................. 34 3.11. Protein determination .................................................................................. 35 3.12. Statistical analysis ....................................................................................... 36 4. RESULTS .......................................................................................................... 37 4.1. Experiment 1 ................................................................................................... 37 4.1.1. Animal survival, body weight and daily food / water consumption ............... 37 4.1.2. Plasma AST, ALT and CK-MB levels, and heart weight / body weight and liver weight / body weight ratios ................................................................................ 39 4.1.3. GSHt and GSSG cellular levels in heart, liver, and kidneys ........................ 41 4.1.4. Lipid peroxidation levels .............................................................................. 43 4.2. Experiment 2 ................................................................................................... 44 4.2.1. Body weight and daily food / water consumption ......................................... 44 4.2.2. Ratios of heart weight / body weight and liver weight / body weight ............ 47 4.2.3. Lymphocytes determination ......................................................................... 48 4.2.4. Plasma AST and ALT levels and AST/ALT ratio ......................................... 49 4.2.5. Total-CK and CK-MB levels ......................................................................... 49 4.2.6. GSHt and GSSG cellular levels in heart, liver and kidneys ......................... 51 4.2.7. Lipid peroxidation levels .............................................................................. 53 4.2.8. ATP levels ................................................................................................... 55 4.2.9. Caspase-3, -8 and -9 activities .................................................................... 57 4.2.10. Structural examination of heart ................................................................ 57 5. DISCUSSION ..................................................................................................... 61 5.1. General welfare of the MTX-treated animal model ......................................... 61 5.2. Biochemical blood analysis and organ damage .............................................. 63 Table of contents xvii 5.3. Oxidative stress mechanisms related to MTX ................................................. 65 5.4. Energetic (im)balance caused by MTX ........................................................... 69 5.5. Lymphocytes and MTX ................................................................................... 71 5.6. Histopathological examination of cardiac damage .......................................... 71 5.7. Apoptosis in the heart ..................................................................................... 72 6. CONCLUSIONS ................................................................................................ 75 7. REFERENCES .................................................................................................. 77 List of figures xix LIST OF FIGURES Figure 1 - (A) HER2 pathways and their protective effect in the heart. Heterodimerization of HER2-HER4 due to HER2 binding of neuregulin activates life-oriented signals and survival factors that minimize cardiotoxicity when the heart is exposed to anthracyclines; (B) Anthracyclines induce cardiotoxicity via oxidative stress mechanisms. Trastuzumab burdens cardiotoxicity by blocking the heterodimerization of HER2-HER4 and therefore the downstream signaling that is responsible for compensatory mechanisms. ............................ 4 Figure 2 - Mathematical formula to calculate body-surface area (BSA). ................................ 6 Figure 3 - Congestive heart failure (CHF) versus cumulative dose for doxorubicin, adapted from Ewer & Suter, 2010. ...................................................................................................... 6 Figure 4 - Chemical structure of doxorubicin (DOX) and daunorubicin (DNR). .....................10 Figure 5 - Progression of the myocardial injury to congestive heart failure (CHF) caused by anthracyclines. Adapted from Mann & Bristow (2005) and Ewer & Suter (2010)...................11 Figure 6 - Anthracyclines, such as doxorubicin (DOX), enter easily in cardiomyocytes through passive diffusion and become cardiotoxic after oneor two-electron reductive activation. The cardiotoxicity of anthracyclines is multifactorial. The cardiac damage caused by oxidative stress has probably two phases: early cardiotoxicity characterized by formation of semiquinones (one electron reduction) or aglycones that trigger an initial high oxidative stress burst and the late/chronic cardiotoxicity characterized by the less redox active but highly toxic secondary alcohols (two electron reduction), forming doxorubicinol (DOXOL) which forms a long-lived toxic reservoir due to its high hydrophilicity. See text for details. ....13 Figure 7 - Signaling pathways involved in anthracycline-induced toxicity. The principal mechanism of anthracycline damage is via the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), leading to lipid peroxidation and membrane damage. In mitochondria, ROS and calcium overload lead to the release of cytochrome c, which activates caspases and leads to apoptosis. Other mechanisms include damage to nuclear DNA, disturbance of energetic metabolism, disruption of sarcomere, and suppression of transcription factors, namely GATA-4 that regulates cell survival. ........................................15 Figure 8 - Chemical structure of mitoxantrone (MTX)...........................................................17 Figure 9 - Number of animals per cage according to EU recommendations and Revision of Appendix A (ETS 123) (Tecniplast, 2009).............................................................................28 Figure 10 - Schematic representation of the distribution of the animals by age group and doses (control, cumulative doses of 4.5 mg/kg or 9.0 mg/kg of MTX), as well as the timeline of the administration of MTX and time to sacrifice. ...............................................................29 xx List of figures Figure 11 - Schematic representation of the distribution of the animals by age group and cumulative concentrations (cumulative doses of 7.0 mg/kg or 9.0 mg/kg of MTX and respective controls), as well as the timeline of the administration of MTX. Oppositely to the cumulative dose of 7.0 mg/kg of MTX, no time was given to the dose of 9.0 mg/kg of MTXtreated animals for development of late cardiotoxicity and sacrifice was done 24h after the last administration. ...............................................................................................................30 Figure 12 - Survival curves after MTX intraperitoneal (i.p.) cumulative administration in adult (A) and pediatric (B) CD-1 mice. Results are expressed in percent survival. The initial number of the animals is six in each group. Black line represents saline-control treatment, light blue line represents the cumulative dose of 4.5 mg/kg MTX treatment and dashed dark blue line represents cumulative dose of 9.0 mg/kg MTX treatment. The vertical dashed red line represents the last MTX i.p. administration. ...................................................................37 Figure 13 - Average body weight in MTX-treated (exposed to cumulative dose of 4.5 mg/kg MTX and 9.0 mg/kg MTX) and control mice, in adult (A) and pediatric (B). Results in grams (g) are presented as mean ± standard deviation (SD), from six animals in each group, excepting after the 21 st day after which the percentage of survival was different from 100%. Light blue open squares (□) represent cumulative dose of 4.5 mg/kg MTX, dark blue triangles (∆) represent cumulative dose of 9.0 mg/kg MTX and black solid circles (●) represent salinecontrol. Statistical comparisons were made using two-way ANOVA followed by the Bonferroni post hoc test (*p < 0.05, **p < 0.01 and ***p < 0.001, treatment vs. control). In 9.0 mg/kg MTX-treated animals, it was not possible to make any statistical comparisons after day 21, since the number of the animals is different from controls. .......................................38 Figure 14 - Food consumption in MTX-treated (exposed to 4.5 mg/kg and 9.0 mg/kg cumulative dose) and control mice, in adult (A) and pediatric (B). Results in g/day/animal are presented as means ± standard deviation (SD), from six animals in each group up to day 21. Light blue open squares (□) represent cumulative dose of 4.5 mg/kg MTX, dark blue triangles (∆) represent cumulative dose of 9.0 mg/kg MTX and black solid circles (●) represent salinecontrol. Statistical comparisons were made using two-way ANOVA followed by the Bonferroni post hoc test (*p < 0.05, **p < 0.01 and ***p < 0.001, treatment vs. control). .......39 Figure 15 - Water consumption in MTX-treated (exposed to 4.5 mg/kg and 9.0 mg/kg cumulative dose) and control mice, in adult (A) and pediatric (B). Results in mL/day/animal are presented as means ± standard deviation (SD), from six animals in each group up to day 21. Light blue open squares (□) represent cumulative dose of 4.5 mg/kg MTX, dark blue triangles (∆) represent cumulative dose of 9.0 mg/kg MTX and black solid circles (●) represent saline-control. Statistical comparisons were made using two-way ANOVA followed by the Bonferroni post hoc test (*p < 0.05, **p < 0.01 and ***p < 0.001, treatment vs. control). .............................................................................................................................................39 List of figures xxi Figure 16 - Plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and its ratio in mice exposed to cumulative dose of 4.5 mg/kg MTX and 9.0 mg/kg MTX. Results, in units per liter (U/L), are presented as means ± standard deviation (SD). The number of animals varied between 2 to 6. (A and B) Plasma AST levels after MTX administration in adult and pediatric mice, respectively. Statistical comparisons were made using the t-test when two groups were considered and One-Way ANOVA, followed by the Student-Newman-Keuls post hoc test, when three groups were considered (*p < 0.05 and **p < 0.01, treatment vs. control). (C and D) Plasma ALT levels after MTX administration in adult and pediatric mice, respectively. Statistical comparisons were made using the t-test when two groups were considered and Kruskal-Wallis ANOVA on Ranks when three groups were considered. (E and F) AST/ALT ratio in adult and pediatric mice, respectively. Statistical comparisons were made using the t-test when two groups were considered and One-Way ANOVA, followed by the Student-Newman-Keuls post hoc test, when three groups were considered (*p < 0.05 treatment vs. control). .................................40 Figure 17 - (A and B) GSH / GSSG ratio in heart after MTX administration in adult and pediatric mice, respectively. Results are presented as means ± standard deviation (SD), and were obtained from 2-6 animals from each treatment group. Statistical comparisons were made using the t-test when two groups were considered and One-Way ANOVA, followed by the Student-Newman-Keuls post hoc test, when three groups were considered (*p < 0.05, treatment vs. control). ...........................................................................................................42 Figure 18 - (A and B) GSH/GSSG ratio in the liver after MTX administration in adult and pediatric mice, respectively. Results are presented as means ± standard deviation (SD), and were obtained from 2-6 animals from each treatment group. Statistical comparisons were made using the t-test when two groups were considered and Kruskal-Wallis ANOVA on Ranks, followed by the Dunn’s post hoc test, when three groups were considered (*p < 0.05, treatment vs. control). ...........................................................................................................43 Figure 19 - (A and B) Average body weight in 7.0 mg/kg MTX-treated adult and pediatric mice, respectively. (C and D) Average body weight in 9.0 mg/kg MTX-treated adult and pediatric mice, respectively. Results in grams (g) are presented as mean ± standard deviation (SD), from eight animals in each group, excepting in 7.0 mg/kg MTX-treated pediatrics (n = 7). Light blue open squares (□) represent cumulative dose of 7.0 mg/kg MTX treatment, dark blue triangles (∆) represent cumulative dose of 9.0 mg/kg MTX treatment and black solid circles (●) represent saline-control treatment. Statistical comparisons were made using two-way ANOVA followed by the Bonferroni post hoc test (*p < 0.05, **p < 0.01 and ***p < 0.001, treatment vs. control). ......................................................................................45 Figure 20 - (A and B) Food consumption in 7.0 mg/kg MTX-treated adult and pediatric mice, respectively. (C and D) Food consumption in 9.0 mg/kg MTX-treated adults and pediatrics, xxii List of figures respectively. Results in grams (g) are presented as mean ± standard deviation (SD), from eight animals in each group, excepting 7.0 mg/kg MTX-treated pediatrics (n = 7). Light blue open squares (□) represent cumulative dose of 7.0 mg/kg MTX treatment, dark blue triangles (∆) represent cumulative dose of 9.0 mg/kg MTX treatment and black solid circles (●) represent saline-control treatment. Small vertical line in 17 th day (x axis) indicates the last MTX administration. Statistical comparisons were made using two-way ANOVA followed by the Bonferroni post hoc test (*p < 0.05, **p < 0.01 and ***p < 0.001, treatment vs. control). .46 Figure 21 - (A and B) Water consumption in 7.0 mg/kg MTX-treated adult and pediatric mice, respectively. (C and D). Water consumption in 9.0 mg/kg MTX-treated adults and pediatrics, respectively. Results in grams mL/day/animal are presented as mean ± standard deviation (SD), from eight animals in each group, excepting 7.0 mg/kg MTX-treated pediatrics (n = 7). Light blue open squares (□) represent cumulative dose of 7.0 mg/kg MTX treatment, dark blue triangles (∆) represent cumulative dose of 9.0 mg/kg MTX treatment and black solid circles (●) represent saline-control treatment. Small vertical line in 17 th day (x axis) indicates the last MTX administration. Statistical comparisons were made using two-way ANOVA followed by the Bonferroni post hoc test (*p < 0.05, **p < 0.01 and ***p < 0.001, treatment vs. control). ................................................................................................................................47 Figure 22 - Plasma levels of CK-MB in 7.0 mg/kg and 9.0 mg/kg MTX-treated animals. (A and B) CK-MB levels in plasma of adults after cumulative administration of 7.0 mg/kg MTX and 9.0 mg/kg MTX, respectively. (C and D) CK-MB levels in plasma of pediatrics after administration of 7.0 mg/kg MTX and 9.0 mg/kg MTX, respectively. Results, in units per liter (U/L), are presented as means ± standard deviation (SD), and were obtained from 5-8 animals from each treatment group. Statistical comparisons were made using the MannWhitney Rank Sum test (*p < 0.05, treatment vs. control).....................................................50 Figure 23 - (A and B) GSHt and GSSG levels in the heart, respectively, after cumulative 7.0 mg/kg MTX administration in adult mice. (C and D) GSHt and GSSG levels in the heart, respectively, after cumulative 7.0 mg/kg MTX administration in pediatric mice. Results are presented as means ± standard deviation (SD), and were obtained from 7-8 animals from each treatment. Statistical comparisons were made using the Mann-Whitney Rank Sum test between the treated group and respective control (*p < 0.05, treatment vs. control). ............51 Figure 24 - (A and B) GSHt and GSSG levels in the heart, respectively, after cumulative 9.0 mg/kg MTX administration in adult mice. (C and D) GSHt and GSSG levels in the heart, respectively, after cumulative 9.0 mg/kg MTX administration in pediatric mice. Results are presented as means ± standard deviation (SD), and were obtained from 7-8 animals from each treatment. Statistical comparisons were made using the Mann-Whitney Rank Sum test between the treated group and respective control (**p < 0.05, treatment vs. control). ..........52 List of figures xxiii Figure 25 - The spectrum of MDA(TBA) 2 adduct from 500 to 600 nm (6 µM MDA standard). .............................................................................................................................................54 Figure 26 - (A) Chromatograms of a 7.0 mg/kg MTX-treated adult liver sample, before and after TBA derivatization (dashed and continuous line, respectively). (B) Chromatogram from a 6 µM MDA standard after TBA derivatization and the chemical structure of the MDA(TBA) 2 adduct. .................................................................................................................................54 Figure 27 - ATP levels in the liver of mice exposed to cumulative dose of 7.0 mg/kg MTX and 9.0 mg/kg MTX. (A and B) ATP levels in the liver of adults after cumulative dose of 7.0 mg/kg MTX and 9.0 mg/kg MTX, respectively. (C and D) ATP levels in liver of pediatrics after cumulative dose of 7.0 mg/kg MTX and 9.0 mg/kg MTX, respectively. Results, in nmol / mg protein, are presented as means ± standard deviation (SD), and were obtained from 7-8 animals from each treatment group. Statistical comparisons were made using the MannWhitney Rank Sum (*p < 0.05, **p < 0.01, ***p < 0.001 treatment vs. control). .....................56 Figure 28 - Cardiac histopathology by light microscopy from MTX-treated animals. (A) Light micrograph from the control of 9.0 mg/kg MTX of adult mice, showing normal morphology and structure; (B) Light micrograph from pediatric mice injected with cumulative dose of 9.0 mg/kg MTX. Vacuolization (green arrow) and inflammatory infiltration (blue arrow) are shown. (C) Light micrograph from pediatric mice injected with cumulative dose of 7.0 mg/kg MTX. This treated group presents large and uncondensed nucleus. (D) Light micrograph from adult mice injected with cumulative dose of 9.0 mg/kg MTX. The cardiomyocytes present high degree of cellular edema with minor structure density, irregular nucleus and vacuolization (green arrow) were observed. (E) Light micrograph from adult mice injected with cumulative dose of 9.0 mg/kg MTX. Cellular edema and necrotic zones are evident. (F) Light micrograph from pediatric mice injected with cumulative dose of 7.0 mg/kg MTX. The inflammatory status of the tissue is clear, as indicated by blue arrows showing the presence of infiltrative inflammatory cells. These results evidence that cardiotoxicity occurred in all MTX-treated animals. Pediatric mice seem to be more protected from damage than adult mice, since pediatric mice had less cardiac histological damage. ...........................................................59 xxiv List of tables LIST OF TABLES Table 1 - Classification of agents regarding their cardiac side effects. .................................. 3 Table 2 - Risk factors for anthracycline and MTX-induced cardiotoxicity. .............................. 5 Table 3 - Details of therapeutics and common toxicities of DOX and MTX ...........................18 Table 4 - Plasma creatine kinase-MB (CK-MB) levels, heart weight / body weight ratio, and liver weight / body weight ratio of the MTX-treated and control mice. ...................................41 Table 5 - Total glutathione (GSHt) and oxidized glutathione (GSSG) cellular levels in the heart and GSHt in the kidneys, in MTX-treated (cumulative dose of 4.5 mg/kg and 9.0 mg/kg) and control mice, in adult and pediatric populations. ............................................................42 Table 6 - Total glutathione (GSHt) and GSSG cellular levels in liver, in MTX-treated (cumulative dose of 4.5 mg/kg and 9.0 mg/kg) and control mice, in adult and pediatric populations. ..........................................................................................................................43 Table 7 - Free equivalents of malondialdehyde (MDA) levels in liver and kidneys in MTXtreated (cumulative dose of 4.5 mg/kg and 9.0 mg/kg) and control mice, in adult and pediatric populations. ..........................................................................................................................44 Table 8 - Ratio of heart weight / body weight of the MTX-treated and control mice. .............48 Table 9 - Ratio of liver weight / body weight ratio of the MTX-treated and control mice. .......48 Table 10 - Lymphocytes changes in MTX-treated and control mice. ....................................48 Table 11 - Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and AST/ALT ratio of the MTX-treated and control mice. ............................................49 Table 12 - Total CK (creatine kinase) in plasma of MTX-treated and control mice. ..............49 Table 13 - Total glutathione (GSHt) and GSSG cellular levels in liver and kidneys in MTXtreated (cumulative dose of 7.0 mg/kg and 9.0 mg/kg) and control mice, in adult and pediatric populations. ..........................................................................................................................53 Table 14 - Malondialdehyde (MDA) levels in liver and kidneys in MTX-treated (cumulative dose of 7.0 mg/kg and 9.0 mg/kg) and control mice, in adult and pediatric populations. .......55 Table 15 - ATP levels in the heart and kidneys of mice exposed to cumulative dose of 9.0 mg/kg MTX and 7.0 mg/kg MTX. ..........................................................................................56 Table 16 - Caspase-3, -8 and -9 activities in the heart of mice treated with total cumulative doses of 7.0 mg/kg MTX or 9.0 mg/kg MTX. .........................................................................57 Table 17 - Semi-quantitative analysis of the morphological injury parameters of MTX-treated and controls groups, in adult and pediatric populations. .......................................................58 Abbreviations xxv ABBREVIATIONS ATP – adenosine 5′-triphosphate BHT – butylated hydroxytoluene BNP – B-type (or brain) natriuretic peptide BSA – body surface area CHF – congestive heart failure CK – creatine kinase CK-MB – isoenzyme MB of creatine kinase cTnI – troponin I cTnT – troponin T DNA – deoxyribonucleic acid DNR – daunorubicin DNROL – daunorubicinol DOX – doxorubicin DOXOL – doxorubicinol ECG – electrocardiogram EPIOL – epirubicinol FDA – U.S Food and Drug Administration γ-GCS – γ-glutamylcysteinesynthase GSH – reduced glutathione GSHt – total glutathione GSSG – oxidized glutathione HO ● – hydroxyl radical H 2 O 2 – hydrogen peroxide HPLC – high-performance liquid-chromatography i.p. – intraperitoneal i.v. – intravenous h – hour HER2 – human epidermal growth factor receptor 2 LVEF – left ventricular ejection fraction 6 1. Introduction meters (m 2 ). The calculation can be done using the following expression, which relates height and weight from the patient: Surfacearea=Height(cm)×weigh(kg) 3600 Figure 2 - Mathematical formula to calculate body-surface area (BSA). There is a dose relationship between cardiac toxicity and the cumulative administered dose of anthracyclines and MTX (Figure 3 and Table 2). Although there is little concern about cardiotoxicity at low cumulative doses, the cardiotoxicity relation curve grows rapidly as the cumulative dose increases. In a retrospective analysis, Von Hoff et al. reported that there is a continuous increasing risk with the increase of total cumulative dose of those anticancer agents, estimating the cumulative percentage of patients who developed congestive heart failure (CHF) at a cumulative dose of DOX of 400 mg/m 2 to be 3%, 7% at 550 mg/m 2 , and 18% at 700 mg/m 2 . Others authors discuss that Von Hoff study had some restraint and perhaps underestimated the incidence. In a study by Swain et al. the estimated cumulative percentage of patients with CHF associated with DOX reaches 5% of patients at a cumulative dose of 400 mg/m 2 , rising to 26% at a dose of 550 mg/m 2 , and 48% at a dose of 700 mg/m 2 (Figure 3) (Swain et al., 2003). The usual administered dose of DOX depends of the type of tumor, although the most common dose is 60-75 mg/m 2 every 3 weeks (Seiter, 2005). It should be noted that late asymptomatic cardiomyopathy can occur at any dose, even as low as 100 mg/m 2 (Menna et al., 2012). Figure 3 - Congestive heart failure (CHF) versus cumulative dose for doxorubicin, adapted from Ewer & Suter, 2010. CHF vs. Cumulative Dose of DOX 1. Introduction 7 Age is also a crucial factor for the development of cardiotoxicity in type I anticancer drugs. The cardiac damage that occurs in childhood may appear early after cancer treatment, or, more frequently, it is diagnosed years later. In general, the younger the patient, the greater the susceptibility and risk for developing delayed cardiac damage, worsening when there are synergistic and additive factors as concomitant cardiotoxic therapies. In the literature, it is stated that DOX-induced cardiomyopathy impairs myocardial growth as the younger patients mature, leading to a possible development of CHF during early childhood (Adams and Lipshultz, 2005, Seiter, 2005). In a study from the Childhood Cancer Survivor Study on children diagnosed with cancer in the 70s and 80s, they observed that childhood survivors were at 15.1-fold higher rate of developmental CHF and 10.4-fold higher rate of developmental cardiovascular disease, compared to siblings (Oeffinger et al., 2006). This topic will be further addressed later on in this dissertation in an independent section. The cardiotoxic effects of anthracyclines are also higher in women, black children and patients with trisomy 21, as well as in the presence of concomitant diabetes, obesity, kidney dysfunction, and others (Krischer et al., 1997) (Table 2). One possible explanation for the increased susceptibility to myocardial damage in females resulting from anthracycline therapy is the highest percentage of body fat compared to males. As anthracyclines are poorly absorbed in fat, cellular concentrations tend to be higher in non-adipose tissue in patients that receive doses calculated by BSA or weight (Krischer et al., 1997). Another possible reason may be related to sex differences in the expression of multidrug-resistance genes, which control cellular excretion of anthracyclines; the sex-related differences in the pharmacokinetics of anthracyclines demonstrate that females have a lower clearance rate (Krischer et al., 1997). Treatment duration may also influence the risk of developing cardiotoxicity during or after oncologic therapy, since prolonged administration has been known to reduce the severity of cardiac damage (Hortobagyi et al., 1989). Other risk factors include the existence of previous cardiac disease at the beginning of the treatment or electrolyte disturbances, namely of calcium and magnesium (Adão et al., 2013). 1.4. Cardiotoxicity detection Some guidelines exist for interruption of anticancer treatment when unacceptable toxicity is observed (Bovelli et al., 2010, Eschenhagen et al., 2011). Thus, it is vital to implement cardioprotective strategies and monitor cardiac function during therapy. It is possible to evaluate some toxic effects using cardiac imaging studies such as echocardiography to 8 1. Introduction identify myocardial dysfunction, or even endomyocardial biopsy for measuring the presence and extent of fibrosis (Schimmel et al., 2004). A decline of left ventricular ejection fraction (LVEF) higher than 10% associated with an absolute value of less than 50% is usually a clinical setting to stop the cardiotoxic treatment (Colombo and Cardinale, 2013). However, these detection methods have low sensitivity for detecting cardiac damage at an early stage, since no considerable change in ventricular systolic function occurs until a critical myocardial damage has been made. Furthermore, cardiotoxicity is commonly detected only after functional impairment has already occurred, therefore excluding its prevention. In fact, a normal LVEF does not exclude per se the possibility of occurrence of late cardiotoxicity (Colombo and Cardinale, 2013). Simplest minimally invasive and low cost methods such plasma or serum cardiac markers have been investigated to monitor the anthracycline-induced cardiomyopathy, making them sensitive tools for detection of early damage, with high prognostic value (Colombo and Cardinale, 2013). A known marker for heart damage is the cardiac troponins I and T (cTnI and cTnT), which are elevated in the serum in case of myocyte injury, even when a subclinical alteration occurs with minor anthracyclines exposure (Franco et al., 2011, Colombo and Cardinale, 2013). Most of the troponins are present in the cardiac myocyte sarcomere, attached to actin and are released slowly, allowing the detection of acute damage, as well as ongoing injury (Lipshultz et al., 2008, Scully and Lipshultz, 2010). The monitoring of troponin levels is, thus, useful for prophylactic cardioprotective therapy before occurrence of irreversible damage. Another well-known cardiac marker for CHF is serum Btype (or brain) natriuretic peptide (BNP) hormone that is secreted by cardiomyocytes due to overload pressure in the heart, as well as left ventricular dysfunction. It is useful as early stress indicator before irreversible damage occurs (Schimmel et al., 2004, Ewer and Ewer, 2010, Scully and Lipshultz, 2010, Franco et al., 2011). High sensitivity serum C reactive protein (HsCRP) is also a cardiac marker and evidences generalized inflammation being a predictor of ischemic and non-ischemic cardiomyopathy (Lipshultz et al., 2008, Franco et al., 2011). Other conventional biomarkers used for assessing myotoxicity in humans and experimental animals for decades include aspartate aminotransferase (AST) and creatine kinase (CK) in the plasma, but they have some drawbacks regarding tissue specificity (Tonomura et al., 2012). The myocardial isoenzyme of CK, namely CK-MB, is a cardiospecific biomarker that represents up to 30% of total-CK in heart and is released when cardiomyocytes are injured (Adams et al., 1993, Lewandrowski et al., 2002, Horacek et al., 2007, O'Brien, 2008). 1. Introduction 9 1.5. Anticancer drugs and their cardiotoxicity 1.5.1. Anthracyclines Anthracyclines are considered a class of highly effective anticancer drugs with the widest spectrum of activity in human cancers. The skeleton of anthracyclines is composed of a tetracyclic ring with adjacent quinone-hydroquinone moieties, an aminosugar attached by a glycosidic bond to C-7, and a side chain with a carbonyl group at C-13 (Figure 4) (Menna et al., 2012). There is no safe dose of anthracyclines. During the last years, there have been several attempts to find new anthracyclines with better activity and less toxicity to cardiac tissue than DOX and DNR (Minotti et al., 2004, Salvatorelli et al., 2013). Thus, hundreds of analogs have arisen with small modifications that can be placed in the various available locations in the molecule, but few analogs reached the stage of clinical development and approval: the popular epirubicin and idarubicin as substitutes for DOX and DNR, respectively. Beyond these, only a few more anthracyclines reached clinical approval: among them, MTX, an anthracenedione, was used in attempt to overcome anthracyclines cardiotoxicity (Minotti et al., 2004). The first anthracyclines, isolated from Streptomyces peucetius, were developed in the early 1950s: DOX and DNR (Minotti et al., 2004). This last molecule differs from DOX only by not having a hydroxyl group in the side chain (Figure 4), which contributes for the different spectrum activity of DOX and DNR. DOX is clinically and widely used in the treatment of breast cancer, childhood solid tumors, soft tissue sarcomas and lymphomas, while DNR exhibits activity in acute lymphoblastic or myeloblastic leukemia’s (Minotti et al., 2004). Anthracyclines have cytostatic effects in tumor cells related to their multiple pharmacological mechanisms of action: intercalation in deoxyribonucleic acid (DNA), inhibition of topoisomerase II, and prevention of synthesis of nucleic acids [(DNA and ribonucleic acid (RNA)] and proteins (Ewer and Suter, 2010). These cytotoxic drugs are well known for their cardiotoxicity (Schimmel et al., 2004). More than ever, cardiovascular diseases in cancer patients are a matter of concern for cardiologists and oncologists. The use of anticancer drugs, in particular cytotoxic agents, has raised several problems since they trigger multiple adverse effects in healthy tissues, namely the heart, with cardiotoxicity being the dose limiting factor in cancer treatment (Schimmel et al., 2004, Ewer and Ewer, 2010). The heart is an organ with limited potential for regeneration and, therefore, its damage is considered permanent and with long-term importance (Ewer and Ewer, 2010). 10 1. Introduction O O OH OHO CH3 O O CH3 OH NH2 OH O OH O O OH OHO CH3 O O CH3 OH NH2 CH3 O OH Figure 4 - Chemical structure of doxorubicin (DOX) and daunorubicin (DNR). 1.5.1.1. Cardiotoxicity of anthracyclines The anthracycline-induced cardiotoxicity can be divided into two categories: acute and chronic. The acute-induced cardiotoxicity symptoms occur within a week after administration (<1%) (Chen et al., 2011), are dose-independent and are characterized by sudden alterations of ventricular repolarization, electrocardiographic alterations, ventricular and supraventricular arrhythmias, acute coronary syndrome, pericarditis, and myocarditis (Adão et al., 2013). The chronic induced cardiotoxicity is divided in early-onset when it occurs within 1 year after completing treatment and it is related to myocyte damage or death (<2.1%) (Krischer et al., 1997, Chen et al., 2011); and in late-onset, when it occurs after the first year and is related to depressed contractility and inappropriately thin left ventricular wall (5-50%) (Schimmel et al., 2004, Chen et al., 2011, Adão et al., 2013). The late chronic cardiotoxicity of anthracyclines is dose-dependent (Adão et al., 2013) and can be manifested years after the discontinuation of treatment (Ewer and Suter, 2010, Scully and Lipshultz, 2010). It is also important to refer that the late-onset form may remain asymptomatic for years (Colombo and Cardinale, 2013). In all these categories, electrophysiological changes, decrease of LVEF, reduced exercise ability, and CHF may occur and are dependent of the rate and schedule of therapy (Schimmel et al., 2004, Adams and Lipshultz, 2005, Ewer and Ewer, 2010, Scully and Lipshultz, 2010). Besides cardiomyocyte damage, the anthracyclines are also able to cause damage to endothelial cells (Octavia et al., 2012). The clinical manifestations of late-onset toxicity often appear years after therapy cessation. Compensatory mechanisms are activated in the heart after injury, including DOX DNR 1. Introduction 11 adrenergic nervous system, renin-angiotensin system, survival factors, and adaptive myocardial hypertrophy (Mann and Bristow, 2005, Lipshultz et al., 2008, Ewer and Suter, 2010). In fact, these systems are able to restore hemodynamic cardiovascular function to a normal homeostatic range and the side effects in patients may remain asymptomatic. Unfortunately, these compensatory mechanisms may become exhausted over time and the heart will begin to fail due to the sustained activation of these systems. When compensating mechanisms are overcome, patients may exhibit progressive tachycardia, fatigue, and difficulties in breathing, then undergoing from asymptomatic to symptomatic heart failure (Figure 5) (Mann and Bristow, 2005, Ewer and Suter, 2010). Figure 5 - Progression of the myocardial injury to congestive heart failure (CHF) caused by anthracyclines. Adapted from Mann & Bristow (2005) and Ewer & Suter (2010). 1.5.1.2. Anthracyclines and their mechanisms of cardiotoxicity The underlying mechanisms of anthracyclines cardiotoxicity are complex and still being explored (Ewer and Suter, 2010). Currently, it is thought that anthracyclines may become cardiotoxic after one or two electron reductive activation via enzymes, or through the formation of anthracycline-iron complexes (Minotti et al., 2004, Lipshultz et al., 2008, Montaigne et al., 2012). The predominant mechanism elicited by DOX involves oxidative damage: the one electron reduction in the quinone moiety of DOX results in the formation of the semiquinone free radical. This semiquinone free radical forms a complex with iron (Fe 2+ ) and is regenerated to quinone again by reduction of molecular oxygen to O 2 •  and hydrogen peroxide (H 2 O 2 ) (Minotti et al., 2004, Montaigne et al., 2012), molecules belonging to the 12 1. Introduction family of ROS. This cycle is supported by a number of NADPH-oxidoreductases: cytochrome P-450 or b5 reductases, mitochondrial nicotinamide adenine dinucleotide (NADH) dehydrogenase, xanthine dehydrogenase, and endothelial nitric oxide synthase (Minotti et al., 2004, Montaigne et al., 2012, Costa et al., 2013b). The superoxide anion radical may react with nitric oxide ( • NO), resulting in peroxynitrite (ONOO - ), highly reactive nitrogen species (RNS) (Menna et al., 2012), and DOX semiquinone can react with H 2 O 2 to yield hydroxyl radical (HO ● ) (Costa et al., 2013b). These redox active molecules are responsible for dysregulation of energy metabolism, damage of cardiac mitochondrial DNA and membrane integrity in these vital cells (Schimmel et al., 2004, Scully and Lipshultz, 2010, Menna et al., 2012). The ROS and RNS formed can also interact with the calcium release channels, by altering calcium stores in sarcoplasmic reticulum (Pai and Nahata, 2000). Cardiac mitochondria are main targets for anthracyclines cardiotoxicity, since they have high affinity to a mitochondrial lipid, cardiolipin, and the mitochondrial NADH dehydrogenase intervenes in the one electron reduction (Costa et al., 2013b). During the redox cycle, the anthracycline semiquinone can also be a target for oxidation, losing the sugar moiety, and leading to the formation of anthracycline aglycones. These are more lipophilic molecules that easily intercalate in the mitochondrial membranes (Costa et al., 2013b). Cardiac mitochondria seem to be susceptible to aglycones, since they can cause mitochondrial dysfunction by modifying sulfhydryl groups and induce calcium-independent oxidation of mitochondrial NADPH, leading to O 2●  production. Furthermore, the accumulation of the lipophilic aglycones in the inner mitochondrial membrane diverts electrons from the normal pathways, affecting the energy metabolism and cellular respiration (Costa et al., 2013b). The two electron reduction of the carbonyl side chain of anthracyclines is characterized by a conversion of the anthracyclines into secondary alcohol metabolites, such as doxorubicinol (DOXOL) or daunorubicinol (DNROL) and this conversion is catalyzed by cytoplasmic cardiac aldo/keto or carbonyl reductases (Menna et al., 2008b, Costa et al., 2013b). These alcohol metabolites are less active in redox cycle than the original quinones but are more potent in dysregulating calcium and iron homeostasis (Figure 6) (Menna et al., 2008b, Ewer and Ewer, 2010, Scully and Lipshultz, 2010). The increased available “free” iron in cells promotes Fenton reactions and consequently further oxidative stress (Minotti et al., 2004). 1. Introduction 13 Figure 6 - Anthracyclines, such as doxorubicin (DOX), enter easily in cardiomyocytes through passive diffusion and become cardiotoxic after oneor two-electron reductive activation. The cardiotoxicity of anthracyclines is multifactorial. The cardiac damage caused by oxidative stress has probably two phases: early cardiotoxicity characterized by formation of semiquinones (one electron reduction) or aglycones that trigger an initial high oxidative stress burst and the late/chronic cardiotoxicity characterized by the less redox active but highly toxic secondary alcohols (two electron reduction), forming doxorubicinol (DOXOL) which forms a long-lived toxic reservoir due to its high hydrophilicity. See text for details. The secondary alcohols have increased polarity and then remain more time with the cells and, therefore, their cardiac elimination is reduced when compared to anthracyclines. Hence, this cardiac accumulation in the heart forms a long-lived anthracycline reservoir, which may explain the reason why anthracyclines cause chronic cardiac toxicity (Figure 6) (Menna et al., 2008b, Menna et al., 2012). Moreover, DOXOL and DNROL are 30-40 times more potent than DOX and DNR in inactivating calcium-handling ATPases, ion channels or membrane ion exchangers (Menna et al., 2012). These alcohol metabolites are known to interact with thiol groups on proteins, leading to cell damage (Octavia et al., 2012). These alterations result in ROS that are responsible for lipid peroxidation and DNA, RNA, and protein synthesis damage, with alterations in calcium transport (e.g. sarco/endoplasmic reticulum Ca 2+ -ATPase, SERCA) and reduced glutathione reductase gene expression. These changes result in tissue injury, cell death, and impaired cardiac contraction (Schimmel et al., 2004, Lipshultz et al., 2008, Menna et al., 2008b, Costa et al., 2013b). Apart from its limited 14 1. Introduction regenerative capacity, cardiomyocytes present a high susceptibility to oxidative damage due to their limited biochemical antioxidant reserves, namely ROS-detoxifying enzymes such as catalase and glutathione peroxidase (Pai and Nahata, 2000, Schimmel et al., 2004). The oxidative stress hypothesis for the anthracycline-induced cardiotoxicity was also demonstrated through the use of transgenic mice with overexpression of antioxidant defense systems. In fact, high resistance to DOX-induced cardiac lipid peroxidation was observed in transgenic overexpressing catalase mice (Kang et al., 1996) and the detoxification by scavenging superoxide radicals produced by DOX occurred in transgenic overexpressing mitochondrial manganese-dependent superoxide dismutase (Yen et al., 1996). Anthracyclines accumulate in the heart at concentrations 10to 500-times higher than their extracellular concentrations. Moreover, cardiolipin, a polyunsaturated fatty acid-rich phospholipid found in high concentrations in the inner mitochondrial membrane, has high affinity for anthracyclines, promoting high intramitochondrial levels of these drugs (Lipshultz et al., 2008). Therefore, in the mitochondria, the respiratory chain is inhibited when anthracyclines bind to cardiolipin or interact with mitochondrial DNA, favoring energy metabolism impairment and enhancing oxidative stress. The higher levels of cardiolipin within the mitochondria, the greater the susceptibility of cardiac cells damage by anthracyclines (Lipshultz et al., 2008, Scully and Lipshultz, 2010, Franco et al., 2011). Anthracyclines decrease ATP production by disrupting the cardiac specific gene expression of critical enzymes involved in energetics, as well as by disrupting structural gene products (cardiac troponins, myosin light chains, and CK) (Lipshultz et al., 2008). Decreases in ATP levels can be also due to the activation of apoptotic signaling and calcium-dependent proteases, which consume ATP and compromise protein reparation; in general, fix and replace damaged proteins represents a high energy expenditure (Octavia et al., 2012). The decrease in protein expression and the degradation of myofilaments lead to a negative balance of cardiac structural sarcoplasmic proteins (Dillenburg et al., 2013), such as titin, a very large myofilament protein, which aids the sarcomere return to the resting state during diastole as well as it regulates the initiating contraction during systole; and dystrophin, another large protein of the sarcomere, which acts as an anchor of the extracellular matrix to the cytoskeleton via actin (Chen et al., 2011). This cardiac contractility impairment can also be due to the direct inhibition and/or reduced expression of the calcium-ATPase that sequesters calcium in the sarcoplasmic reticulum (Lipshultz et al., 2008, Chen et al., 2011, Montaigne et al., 2012). Decreased energy levels impair the ability of cardiomyocytes to contract correctly, leading, in some cases, to cell death (Lipshultz et al., 2008). Complementary mechanisms of anthracycline cardiotoxicity involve cardiac cells death by apoptosis or necrosis (Adão et al., 2013). It is usually accepted that the apoptosis signaling is activated by the oxidative stress caused by anthracyclines (Octavia et al., 2012). 1. Introduction 15 Anthracycline-induced apoptosis in the heart appears to be linked to the mitochondrial pathways, requiring Bax, cytochrome c and caspase-3 activation (Figure 7) (Montaigne et al., 2012). Anthracycline increases mitochondrial oxidative stress and disrupts calcium levels, altering membrane permeability and its potential, and resulting in opening of the transition pore (mPTP), which is associated with the release of apoptotic factors such as cytochrome c from mitochondria to cytosol. In the cytosol, cytochrome c forms a complex with the adaptor protein apoptosis protease activator protein-1 (Apaf-1) and procaspase-9, named apoptosome, which activates caspase-9 (Montaigne et al., 2012). Anthracyclines also induce apoptosis by activation of p38 mitogen-activated protein kinases (MAPK), stress-activated protein kinase (SAPK), and c-Jun N-terminal kinases (JNK) through oxidative stress mechanisms (ROS and RNS) (Minotti et al., 2004, Montaigne et al., 2012), as well as by disrupting / down regulating the expression and activity of the transcription factor GATA-4 (Montaigne et al., 2012). GATA-4 is a member of the zinc finger transcriptional factor family and it has an important role in regulating differentiation, sarcomere synthesis, and survival signaling, and also in promoting an anti-apoptotic response in the heart (Montaigne et al., 2012). Anthracyclines-induced apoptosis of cardiac cells is prevented by several survival factors, namely by phosphoinositide kinase (PI3K) / Akt and neuregulin/HER2 activation (Minotti et al., 2004, Montaigne et al., 2012). Figure 7 - Signaling pathways involved in anthracycline-induced toxicity. The principal mechanism of anthracycline damage is via the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), leading to lipid peroxidation and membrane damage. In mitochondria, ROS and calcium overload lead to the release of cytochrome c, which activates caspases and leads to apoptosis. Other mechanisms include damage to nuclear DNA, disturbance of energetic metabolism, disruption of sarcomere, and suppression of transcription factors, namely GATA-4 that regulates cell survival. 22 1. Introduction leukemia and solid tumors (Kremer et al., 2002) and it is estimated that more than 50% of all childhood cancer survivors have been treated with anthracycline-based therapy (Chen et al., 2011, Dillenburg et al., 2013). Although cancer recurrence and secondary malignancies are concerning problems in children cancer survivors, children have a long life-expectancy after a successful antineoplastic treatment and thus it is important to have a follow-up protocol for pediatric patients. Cardiovascular diseases are important risk factors influencing long-term survival, having children higher mortality risk when compared to the general population (Lawless et al., 2007, Dillenburg et al., 2013). As stated before, in a study from the Childhood Cancer Survivor Study in 10397 children diagnosed with cancer in the 70s and 80s and treated with anthracyclines or non-anthracyclines based regimen, it was observed that childhood survivors were at 15.1-fold higher rate of developmental of CHF and 10.4-fold higher rate of developmental of cardiovascular disease when compared with siblings (Oeffinger et al., 2006). 1.6.1. Cardiotoxicity in pediatrics with anthracyclines Late cardiotoxicity is more frequent than early cardiotoxicity in children treated with anthracyclines (Krischer et al., 1997, Seiter, 2005). In general, the younger the patient, the greater the susceptibility to develop cardiac damage. The age when patients receive treatment with anthracyclines seems to be a crucial factor for the development of cardiotoxic events, since inadequate ventricular growth occurs in time (Krischer et al., 1997). The risk of developing anthracycline-induced CHF is 9.8% if children are treated with a cumulative anthracycline dose of 300 mg/m 2 or more, 20 years after the start of anthracycline therapy (van Dalen et al., 2006). Children receiving cumulative doses of anthracyclines >550 mg/m 2 have a cardiotoxicity risk 5 times higher of developing cardiotoxicity compared to the risk among those receiving lower cumulative doses (Krischer et al., 1997). Abnormalities of cardiac structure and function have been registered in 65% of survivors of childhood acute lymphoblastic leukemia, 6 years after the end of therapy with anthracyclines (Lipshultz et al., 1991). Although adults usually develop chronic dilated cardiomyopathy (significantly reduced left ventricular fractional shortening and contractility with left ventricular dilation) after anthracycline therapy, children typically develop dilated cardiomyopathy and/or restrictive cardiomyopathy (Lipshultz et al., 2008), characterized by diastolic dysfunction and normal to reduced left ventricular dimension with significantly reduced left ventricular thickness, fractional shortening, and contractility (Lipshultz et al., 2005). Late symptomatic 1. Introduction 23 cardiomyopathy in 15 of 300 patients that ended anthracycline-based treatment with a median of 11 years-old were also reported (Steinherz et al., 1995). These same patients had a median of 22 years-old at the time of late cardiac decompensating and one with symptomatic anthracycline cardiotoxicity had heart transplantation. When looking at subclinical / asymptomatic cardiotoxicity, namely abnormal systolic function and/or increased afterload, the reported frequency ranged between 0-57% in a review of studies between 1966 and 2001 including children (newborn infant to 18 years-old) treated with anthracyclines (Kremer et al., 2002). The exact incidence percentage is difficult to determine because of the lack of standardized detection and reporting of cardiovascular events, as well as of insufficient long-term follow-up (Chen et al., 2011). Anthracyclines in growing children seem to alter heart growth potential and its compensatory mechanisms (Urbanova et al., 2010). These same mechanisms were already demonstrated in laboratory with juvenile mice, which showed depletion of the cardiac progenitor cells that are essential to regeneration and blood vessel formation, when exposed to anthracyclines (Huang et al., 2010). A cumulative dose of 3 mg/kg of DOX in postnatal mice reduced the size of the cardiac progenitor pool, impaired their ability to differentiate into cardiac and vascular cells lineage and affected vascular development in the heart (Huang et al., 2010). It was possible to show, in rats, that DOX impaired progenitor cells and caused heart failure. The exogenous delivery of progenitor cells counteracted the progression of DOX-cardiotoxicity: cardiac progenitor cells were injected in the failing myocardium and this treatment promoted regeneration of cardiomyocytes and vascular structures, improving ventricular performance, and animal survival (De Angelis et al., 2010). In a recent study, CHF was the most common cardiac disturbance (54% of the total cardiac events), followed by cardiac arrhythmia, ischemia/myocardial infarction, valvular disease and pericarditis in childhood cancer survivors that were treated with anthracyclines and/or cardiac irradiation (van der Pal et al., 2012). 1.6.2. Cardiotoxicity in pediatrics with mitoxantrone MTX is used in pediatric population, either in cancer treatment as a second line therapy of multiple sclerosis (van Dalen et al., 2004, Kornek et al., 2011). MTX-pediatric users and survivors will largely increase in the coming decades with increased incidence of MTXinduced cardiotoxicity (Ungerleider et al., 1985, van Dalen et al., 2004). The younger age (< 4 years old), concomitant cardiotoxic therapy, and other cardiac comorbidities largely increase MTX-cardiotoxicity incidence, but, so far no mechanisms have been discovered (Chen et al., 2011). 24 1. Introduction In a review of 17 studies done in 2004 by van Dalen and co-authors, they estimated that MTX-related symptomatic cardiotoxicity / clinical heart failure varies between 0 and 6.7% and asymptomatic cardiac damage varies between 0 and 80% in children (van Dalen et al., 2004). However, the exact cumulative incidence is difficult to estimate due to the low methodological quality, namely definitions of subclinical cardiotoxicity and differences in the clinical data, namely regarding to the patients, to the dose of the drugs, which does not permit to know if MTX is less cardiotoxic than anthracyclines in children (van Dalen et al., 2004, Ojha et al., 2013). Developmental of irreversible cardiac dysfunction in one child with acute leukemia requiring heart transplant was described several years after therapy with an anthracycline and anthracenedione (Urbanova et al., 2010). The 4 year-old boy was treated with DNR and MTX and 12 years after the diagnosis, he developed symptoms of cardiac dysfunction, including decreased LVEF, mitral insufficiency, dilated cardiomyopathy, and CHF. He required a heart transplant 19 years after the diagnosis (Urbanova et al., 2010). In the treatment of advanced acute leukemia and solid tumors in 84 children, 6 developed evidence of cardiac dysfunction, including 3 occurrences of CHF (Ungerleider et al., 1985). In a study with 101 patients with advanced pediatric malignant solid tumors, 2 died due to CHF after receiving 54 and 90 mg/m 2 of MTX, and previous DOX treatment, and 3 had changes in cardiac function (Pratt et al., 1986). In another study of the same author, 2 children receiving MTX that were previously treated with DOX and cisplatin, were reported to develop CHF. One of the patients died two weeks later (Pratt et al., 1983). A report by O’Brien et al. that included children with Down syndrome and newly diagnosed with acute myeloid leukemia (n = 57) treated concomitantly with DNR and MTX, indicate 10 cases of development of symptomatic cardiomyopathy during or soon after completion of treatment and, of these, 3 deaths resultant of CHF (O'Brien et al., 2008). Also following the concomitant treatment (cytosine-arabinoside and MTX) of 19 children with acute myeloid leukemia, one died after further MTX treatment due to toxic cardiomyopathy (Ritter et al., 1987). In 2007, in a study with 34 children with acute myeloid leukemia ranging from 2 months to 15 years-old, two that received cumulative doses of at least 400 mg/m 2 of anthracycline equivalents (DNR and MTX) experienced symptomatic heart failure; and 5 from other dose protocols experienced asymptomatic left ventricular systolic dysfunction (Tan et al., 2007). Also in children treated for acute myeloid leukemia, Dahl et al. reported cardiotoxicity manifested by decreased LVEF or shortening fraction in 15 of a total 66 patients, with 3 episodes of CHF occurrence, when treated with combination of MTX, etoposide, and cyclosporine (Dahl et al., 2000). At this point, no mechanisms were attributed to the MTX cardiotoxicity in young patients. 2. Aims of the study 25 2. AIMS OF THE STUDY Is the anti-tumor drug mitoxantrone more cardiotoxic in pediatric than in adult mice? The cardiotoxicity induced by MTX has long been described. Furthermore, the quality of life of cancer survivors is a matter of concern (van Dalen et al., 2004, Seiter, 2005). Despite the widespread use of MTX and the fact that it presents a toxic cardiac clinical profile similar to DOX, their mechanisms of toxicity towards non-target tissues, namely heart, are different and the mechanisms of MTX-induced cardiotoxicity remain largely unknown (Menna et al., 2008b). Cardiac damage promoted by MTX is a serious life-threatening issue as it increases mortality and morbidity, and the heart is very susceptible to this chemotherapeutics agent (Seiter, 2005). Additionally, MTX is used for the treatment of several tumors, including childhood cancers such as lymphoma and leukemia, as well as multiple sclerosis, which makes MTX-cardiotoxicity a concerning issue also in this population. Children that survive cancer can potentially have a long life-expectancy after treatment, but little is known about the risk of this population to develop heart toxicity after MTX treatment. Only a better understanding of the mechanisms involved in the injury caused by chemotherapeutics to normal tissues will allow the designing of better therapies. In this context, the present study was undertaken to investigate what is the impact of MTX-induced cardiotoxicity in childhood and which parameters are associated to this toxicity. So far, no laboratory studies were done with MTX in pediatric populations. This dissertation aims to seek plausible explanations for MTX-induced cardiotoxicity and susceptibilities in young animals. Since the cumulative dose is the most predictive risk factor for cardiotoxicity, different MTX cumulative dose were used. Moreover, multiple administration of MTX, interrupted by free-drug periods, was given to mimic the human MTX-therapy in pediatric and adult CD-1 mice, which were sacrificed weeks after the last administration in order to evaluate cumulative toxicity. Additionally, liver and kidneys were also studied. To achieve the aims, several techniques were used, namely enzymatic assays to evaluate the redox status (GSH/GSSG), bioenergetics (ATP) and apoptotic events in tissues. Plasma clinical parameters (AST, ALT, total-CK and CK-MB) in plasma and lipid peroxidation in tissues, as well as histopathological examination of the heart were also performed. 3. Materials and methods 27 3. MATERIALS AND METHODS 3.1. Chemicals Sodium phosphate monobasic was purchased from Panreac (Barcelona, Spain), potassium sodium tartrate to Fluka (Buchs SG, Switzerland), and HEPES and sodium chloride to VWR (Leuven, Belgium). Ammonium acetate, dimethyl sulfoxide (DMSO), EDTA, Folin–Ciocalteu reagent, Histosec paraffin pastilles, magnesium chloride, perchloric acid, trichloroacetic acid (TCA), sodium hydroxide, copper (II) sulfate, sodium carbonate, disodium phosphate, were purchased from Merck (Darmstadt, Germany). Methanol, acetonitrile, and xylene were purchased from Fisher Scientific (Loughborough, UK). The peptide substrate for caspase-3 (Ac-DMQD-AMC), the peptide substrate for caspase-8 (Ac-IETD-AMC), and the peptide substrate for caspase-9 (Ac-LEHD-AMC) were obtained from Peptanova (Sandhausen, Germany). Eosin 1% aqueous was obtained from Biostain (Traralgon, Australia), Harris hematoxylin was from Harris Surgipath (Richmond, IL, USA) and Histofluid from Marienfeld (Lauda-Königshofen, Germany). All the other reagents used in this dissertation were purchased from Sigma-Aldrich at the highest purity available (St. Louis, MO, USA). 3.2. Animals Male CD-1 mice weighing 10-12g and 38-40g were obtained from Charles River Laboratories (L'Arbresle, France) in experiment 1 and from Harlan (Udine, Italy) in experiment 2 and kept in the vivarium of Instituto Superior de Ciências da Saúde – Norte (ISCS-N) – CESPU (Paredes, Portugal). They were let to adjust to the environmental conditions for 4 days, before experiments began. The animal weighing 10-12g, corresponding to a pediatric population had approximately 3-weeks old and the mice with 3840g were approximately 8 to 10 weeks-old. According to the literature available, the first group did not enter puberty (child) while the latter is in adulthood, being considered a suitable approach to perform the experiments and mimic human MTX-therapy in pediatric and adult populations (Eisen, 1976, Østergaard et al., 2011). All animal procedures were performed focusing on improving animal welfare. The laboratory animal research was performed with awareness to the various issues regarding the three Rs and the present legislation, which meet the research ethics concepts of Replacement, Refinement, and Reduction (Costa and Antunes, 2011, Olsson et al., 2011). The animals were housed in a cage 1290D Eurostandard Type III (425 x 266 x 155 mm - floor area 820 cm 2 ) in a temperature (22 ± 2ºC) and humidity-controlled environment and a 28 3. Materials and Methods 12h light-dark cycle. Standard rodent chow 4RF21 GLP certificate diet (Mucedola, Settimo Milanese, Italy) and water were provided ad libitum. Housing and experimental treatment of the animals were in accordance with the Guide for the Care and Use of Laboratory Animals from the Institute for Laboratory Animal Research (ILAR 1996) and with the guidelines defined by the European Council Directive (86/609/EEC). The number of animals per cage was according to European Union recommendations and revision of Appendix A of the European Convention ETS 123 (Figure 9). Considering the time of the animal experiments and the probable growth of the animals, a maximum of 8 animals per cage was determined. This allowed socialization and acceptable welfare conditions. Moreover, animal experiments were licensed by the Portuguese General Directory of Veterinary Medicine (reference number 0421/000/000/2013) and approved by the Ethical Committee of Faculdade de Farmácia da Universidade do Porto (protocol number 7/03/2013). Figure 9 - Number of animals per cage according to EU recommendations and Revision of Appendix A (ETS 123) (Tecniplast, 2009). 3.3. Study design The administration schedule of MTX was given in order to mimic the human MTX-therapy that consists of multiple administration in separated time-points (Vorobiof et al., 1987, Paul et al., 2007). All injections were given at afternoon to improve tolerability of high-dose of MTX, as seen in the literature (Levi et al., 1994). Consumption of food and water, and animal weight were recorded along the experiments, twice a week. Animals were kept in a social environment as a group (Curfs et al., 2011) and, therefore, food and water consumption were assessed for the entire group and afterwards calculated in function of body weight of each animal. 3. Materials and methods 29 week 1 week 2 week 3 week 4 week 5 week 6 Development of cumulative toxicity Injection of MTX Sacrifice 3.3.1. Experiment 1 The animals were divided into three groups of pediatric mice (A, B, C) and three groups of adult mice (D, E, F), of 6 animals each (Figure 10). Animals in groups B and E were subjected to a total cumulative dose of 4.5 mg/kg MTX as a result of 6 intraperitoneal (i.p.) injections (2 per week). To animals in groups C and F was given a total cumulative dose of 9.0 mg/kg MTX as a result of 6 i.p. injections (2 per week). Animals in groups A and D were the respective controls and were given NaCl 0.9 % (saline solution) in the same schedule (Figure 10). In the cumulative dose of 9.0 mg/kg MTX, treated mice received each time 1.5 µg/g body weight of MTX dose solution (0.1 mg/mL MTX) and in the cumulative dose of 4.5 mg/kg MTX, treated mice received, each time, 0.75 µg/g body weight of MTX solution (0.05 mg/mL). To control animals was given 0.9% saline solution in the same equivalent volume of both treated-conditions. The injections were given on the left side of the peritoneum in the 1 st , 2 nd and 5 th injection days, and on the right side in the 3 rd , 4 th and 6 th , using disposable 1 mL sterile U-100 insulin syringes and a 25 gauge (G) Neolus needle (Terumo Europe España, Madrid, Spain). MTX dihydrochloride was dissolved in sterile 0.9% saline solution. After the last MTX administration, the animals were maintained in a drug-free period for 20 days to allow the development of cumulative toxicity, before sacrifice. Figure 10 - Schematic representation of the distribution of the animals by age group and doses (control, cumulative doses of 4.5 mg/kg or 9.0 mg/kg of MTX), as well as the timeline of the administration of MTX and time to sacrifice. 3.3.2. Experiment 2 The animals were divided into four groups of pediatric mice (A, B, C, D) and four groups of adult mice (E, F, G, H), of 8 animals each. All animals were given 6 i.p. injections (2 per week) following the same schedule and procedure as described in experiment 1. Animals in 30 3. Materials and Methods week 1 week 2 week 3 Injection of MTX Sacrifice week 1 week 2 week 3 week 4 week 5 week 6 Development of cumulative toxicity 7.0 mg/kg MTX 9.0 mg/kg MTX groups B and F were given a total cumulative dose of 7.0 mg/kg MTX. Animals in groups D and H were given a cumulative dose of 9.0 mg/kg MTX. Animals in groups A/E and C/G were the controls of the dose of 7.0 mg/kg MTX and 9.0 mg/kg MTX, respectively, and were given NaCl 0.9 % (Figure 11). The cumulative dose of 9.0 mg/kg MTX-treated mice received 1.5 µg/g body weight of MTX dose solution each day (0.1 mg/mL MTX) and the cumulative dose of 7.0 mg/kg MTX-treated mice received 1.17 µg/g body weight of MTX solution (0.08 mg/mL). To control animals were given 0.9% saline solution in the same equivalent volume. After MTX administration, the animals dosed 9.0 mg/kg of MTX were sacrificed 24h after the 6 th and last injection, whereas the animals of cumulative dose 7.0 mg/kg of MTX were maintained in a drug-free period, in order to allow the development of cumulative toxicity, before sacrifice. According to animal welfare and data gathered in experiment 1, these animals were sacrificed at day 14 after the last administration to avoid further suffering and loss of animal samples. Figure 11 - Schematic representation of the distribution of the animals by age group and cumulative concentrations (cumulative doses of 7.0 mg/kg or 9.0 mg/kg of MTX and respective controls), as well as the timeline of the administration of MTX. Oppositely to the cumulative dose of 7.0 mg/kg of MTX, no time was given to the dose of 9.0 mg/kg of MTX-treated animals for development of cumulative toxicity and sacrifice was done 24h after the last administration. 3.4. Blood and tissue collection The animals were anesthetized with isoflurane inhalation and sacrificed by exsanguination, collecting the blood in inferior vena cava into EDTA-containing tubes. This blood was used for the determination of plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine-kinase MB (CK-MB) and total creatine kinase (total-CK). 3. Materials and methods 31 Hematological parameters (neutrophils, eosinophils, basophils, lymphocytes, monocytes, red blood cells, hemoglobin, and platelets) of two mice of each group were also determined. These determinations were performed in an external laboratory as described in the following section. Immediately after sacrifice, the heart was excised, separated from the pericardium, and weighed. In experiment 2, a transverse section (approximately 2mm from the apex) on the heart of three mice of each group was collected and fixed in 4% paraformaldehyde (diluted in PBS 1X, 2.5% sucrose, 0.1% glutaraldehyde, pH 7.2-7.4), for examination of cardiac damage by light microscopy. The same section from the remaining animals was collected and frozen at -80ºC for future western blot analysis. Furthermore, approximately 40mg of the heart was collected in complete lysis buffer (25 mM HEPES, 5 mM MgCl 2 , 1 mM EGTA, 0.5% Triton X-100, 1 mM pefabloc, 5 mM DTT, pH 7.4) to measure the activities of caspases 3, 8, and 9 and frozen a -80 ºC until the assays. The remaining heart was homogenized with a Potter homogenizer in a phosphate buffer KH 2 PO 4 100mM (pH=7.4). The animals’ liver and kidneys were also removed, weighed, and homogenized with an Ultra-Turrax homogenizer in the same phosphate buffer. A homogenate aliquot of the organs was treated with 10% perchloric acid 1:1 (5% final acid concentration) to precipitate proteins and centrifuged at 13000 rpm for 10 min at 4ºC. The resulting supernatant was separated and frozen at -80ºC for ATP determination and at -20ºC for GSHt/GSSG determinations. Another aliquot of the homogenates was mixed with 10% trichloroacetic acid 1:1 (5% final acid concentration), supplemented with 10 µL of 5% of antioxidant butylated hydroxytoluene (BHT) (diluted in methanol) for each mL of the mix and immediately frozen at -80ºC (for a maximum one month) until determination of malondialdehyde (MDA) levels. The remaining homogenates were frozen at -20ºC for protein determination. All these procedures were performed in icecold tubes. 3.5. Measurement of hematological parameters, aminotransferases, totalCK and CK-MB After blood collection in EDTA-containing tubes, the hematological parameters were quantified in the day of the experiments using an automatic blood cell counter ABX Micros 60 (Horiba ABX, Amadora, Portugal). The remaining blood was centrifuged at 920g for 10 min for separation of plasma. The plasma was frozen at -20ºC until determination of AST, ALT, CK-MB and total-CK. These determinations in the mice plasma were done through enzymatic assays in the apparatus ABX Pentra 400 with ABX Pentra reagents (Horiba ABX, Amadora, Portugal), according to the manufacturer’s instructions. The hematological 38 4. Results 0 3 7 10 14 17 21 24 28 31 35 37 30 35 40 45 50 ** ** *** 0.75 2.25 3.00 3.75 4.501.50 1.50 3.00 4.50 6.00 7.50 9.00 Time (days) Cumulative dose of MTX (mg/kg) Cumulative dose of MTX (mg/kg) A Average body weight (g) 0 3 7 10 14 17 21 24 28 31 35 37 10 20 30 40 50 1.50 3.00 4.50 6.00 7.50 9.00 Time (days) Cumulative dose of MTX (mg/kg) Cumulative dose of MTX (mg/kg) 0.75 1.50 2.25 3.00 3.75 4.50 B Average body weight (g) Figure 13 - Average body weight in MTX-treated (exposed to cumulative dose of 4.5 mg/kg MTX and 9.0 mg/kg MTX) and control mice, in adult (A) and pediatric (B). Results in grams (g) are presented as mean ± standard deviation (SD), from six animals in each group, excepting after the 21 st day after which the percentage of survival was different from 100%. Light blue open squares (□) represent cumulative dose of 4.5 mg/kg MTX, dark blue triangles (∆) represent cumulative dose of 9.0 mg/kg MTX and black solid circles (●) represent saline-control. Statistical comparisons were made using two-way ANOVA followed by the Bonferroni post hoc test (*p < 0.05, **p < 0.01 and ***p < 0.001, treatment vs. control). In 9.0 mg/kg MTX-treated animals, it was not possible to make any statistical comparisons after day 21, since the number of the animals is different from controls. Regarding food consumption, adults receiving cumulative dose of 4.5 mg/kg MTX had lower consumption since the very beginning of experiment 1, comparing to saline-treated mice. In adult mice receiving cumulative dose of 9.0 mg/kg, food consumption seems to be less constant: in these animals a significant decrease in food consumption after the last administration (17 th day) occurred (Figure 14A). In the pediatric mice population, 4.5 mg/kg MTX group had significant lower food consumption comparing to saline-control population. In 9.0 mg/kg MTX group, the difference was more notorious during the MTX administrations; in fact after the last administration the food consumption increased (Figure 14B). Water consumption data show a decreased intake in adults receiving both cumulative doses of MTX since the beginning of the experiment, but more prominent in highest dose of MTX as time elapsed (Figure 15A). The same occurred in pediatric mice in the 4.5 mg/kg MTX dose. However, in the highest dose group a lower water intake occurred in the 17 th and 21 st days while afterwards water intake increased, demonstrating a similar profile to food intake (Figure 15B). As it is impossible to know the food and water consumption of each animal, all consumptions were normalized to their weights as proportions, assuming that all animals had intake rates proportional to their current weights. 4. Results 39 3 7 10 14 17 21 24 28 31 35 0 5 10 15 ** *** ** *** *** *** *** *** **** *** Time (days) Food intake / da y / a nim al (g) A 3 7 10 14 17 21 24 28 31 35 4 6 8 10 *** *** ** * *** *** *** Time (days) Food inta ke / day / anima l (g) B 3 7 10 14 17 21 24 28 31 35 2 4 6 8 *** *** *** *** *** *** *** *** *** *** ** *** *** *** A Time (days) Water intake / day / animal (mL) 3 7 10 14 17 21 24 28 31 35 2 4 6 8 *** *** *** *** *** *** * ** B Time (days) Water intake / day / animal (mL) Figure 14 - Food consumption in MTX-treated (exposed to 4.5 mg/kg and 9.0 mg/kg cumulative dose) and control mice, in adult (A) and pediatric (B). Results in g/day/animal are presented as means ± standard deviation (SD), from six animals in each group up to day 21. Light blue open squares (□) represent cumulative dose of 4.5 mg/kg MTX, dark blue triangles (∆) represent cumulative dose of 9.0 mg/kg MTX and black solid circles (●) represent saline-control. Statistical comparisons were made using two-way ANOVA followed by the Bonferroni post hoc test (*p < 0.05, **p < 0.01 and ***p < 0.001, treatment vs. control). Figure 15 - Water consumption in MTX-treated (exposed to 4.5 mg/kg and 9.0 mg/kg cumulative dose) and control mice, in adult (A) and pediatric (B). Results in mL/day/animal are presented as means ± standard deviation (SD), from six animals in each group up to day 21. Light blue open squares (□) represent cumulative dose of 4.5 mg/kg MTX, dark blue triangles (∆) represent cumulative dose of 9.0 mg/kg MTX and black solid circles (●) represent saline-control. Statistical comparisons were made using two-way ANOVA followed by the Bonferroni post hoc test (*p < 0.05, **p < 0.01 and ***p < 0.001, treatment vs. control). 4.1.2. Plasma AST, ALT and CK-MB levels, and heart weight / body weight and liver weight / body weight ratios Plasma levels of AST and ALT were altered in some groups. AST and ALT levels were significantly decreased in 4.5 mg/kg MTX-treated adult mice compared to levels in the control 40 4. Results Control 4.5 mg/kg 0 10 20 30 40 50 ** C Control 4.5 mg/kg 0 1 2 3 4 E Control 4.5 mg/kg 9.0 mg/kg 0 1 2 3 4 * F AST / ALT Ratio mice (Figure 16A and C). In the pediatric population, AST levels were significantly elevated in 9.0 mg/kg MTX-treated animals, compared to the levels in the control mice (Figure 16B and D). The AST/ALT ratio is significantly increased in 9.0 mg/kg MTX of the surviving pediatric group (Figure 16F). Figure 16 - Plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and its ratio in mice exposed to cumulative dose of 4.5 mg/kg MTX and 9.0 mg/kg MTX. Results, in units per liter (U/L), are presented as means ± standard deviation (SD). The number of animals varied between 2 to 6. (A and B) Plasma AST levels after MTX administration in adult and pediatric mice, respectively. Statistical comparisons were made using the t-test when two groups were considered and One-Way ANOVA, followed by the Student-Newman-Keuls post hoc test, when three groups were considered (*p < 0.05 and **p < 0.01, treatment vs. control). (C and D) Plasma ALT levels after MTX administration in adult and pediatric mice, respectively. Statistical comparisons were made using the t-test when two groups were considered and Kruskal-Wallis ANOVA on Ranks when three groups were considered. (E and F) AST/ALT ratio in adult and pediatric mice, respectively. Statistical comparisons were made using the t-test when two groups were considered and One-Way ANOVA, followed by the StudentNewman-Keuls post hoc test, when three groups were considered (*p < 0.05 treatment vs. control). ADULT PEDIATRIC 4. Results 41 No significant differences were observed in CK-MB levels between MTX-treated and control mice, as well as in the percentage of heart weight / heart weight ratio of the animals in this experiment in both treatment groups. The percentage of liver weight / body weight ratio was higher in 9.0 mg/kg MTX-treated pediatric mice, showing higher liver mass in that group (Table 4). Table 4 - Plasma creatine kinase-MB (CK-MB) levels, heart weight / body weight ratio, and liver weight / body weight ratio of the MTX-treated and control mice. ADULT Parameter Control 4.5 mg/kg 9.0 mg/kg CK-MB (U/L) 98 ± 45 89 ± 37 - Heart weight / body weight ratio (%) 0.52 ± 0.08 0.49 ± 0.04 - Liver weight / body weight ratio (%) 4.84 ± 0.60 5.06 ± 0.50 PEDIATRIC Parameter Control 4.5 mg/kg 9.0 mg/kg CK-MB (U/L) 97 ± 32 118 ± 72 104 ± 11 Heart weight / body weight ratio (%) 0.52 ± 0.04 0.50 ± 0.08 0.53 ± 0.01 Liver weight / body weight ratio (%) 5.38 ± 0.55 4.86 ± 0.37 6.46 ± 0.20* Data are presented as means ± standard deviation (SD). The number of animals varied between 2 to 6. Statistical comparisons were made using the t-test when two groups were considered and One-Way ANOVA, followed by the Student-Newman-Keuls post hoc test, when three groups were considered (*p < 0.05 treatment vs. control). 4.1.3. GSHt and GSSG cellular levels in heart, liver, and kidneys The levels of GSHt and GSSG in the heart and kidneys in 4.5 mg/kg MTX and 9.0 mg/kg MTX-treated animals can be observed in Table 5. There are no significant absolute differences in the two organs between the MTX-treated groups and controls expressed by nmol / mg protein. However, the MTX triggered alterations in redox status as seen in the GSH/GSSG ratio (Figure 17). In the heart, the pediatrics that received the cumulative dose of 9.0 mg/kg MTX had significant increased GSH/GSSH ratio when compared to control. 42 4. Results Control 4.5 mg/kg 9.0 mg/kg 0 10 20 30 40 * B Table 5 - Total glutathione (GSHt) and oxidized glutathione (GSSG) cellular levels in the heart and GSHt in the kidneys, in MTXtreated (cumulative dose of 4.5 mg/kg and 9.0 mg/kg) and control mice, in adult and pediatric populations. HEART (nmol / mg protein) Control 4.5 mg/kg 9.0 mg/kg Adults GSHt 5.40 ± 1.75 4.89 ± 0.48 - GSSG 0.38 ± 0.13 0.35 ± 0.13 - Pediatrics GSHt 6.41 ± 0.64 6.48 ± 0.64 8.75 ± 0.37 GSSG 0.42 ± 0.10 0.34 ± 0.08 0.31 ± 0.11 KIDNEYS (nmol / mg protein) Control 4.5 mg/kg 9.0 mg/kg Adults GSHt 3.07 ± 1.04 4.28 ± 2.45 - Pediatrics GSHt 2.67 ± 0.58 3.32 ± 1.67 1.97 ± 0.69 Data of GSHt and GSSG levels, in nanomol per mg of protein (nmol / mg protein), are presented as means ± standard deviation (SD), and were obtained from 2-6 animals from each treatment group. Statistical comparisons were made using the t-test when two groups were considered and Kruskal-Wallis ANOVA on Ranks, when three groups were considered. Figure 17 - (A and B) GSH / GSSG ratio in heart after MTX administration in adult and pediatric mice, respectively. Results are presented as means ± standard deviation (SD), and were obtained from 2-6 animals from each treatment group. Statistical comparisons were made using the t-test when two groups were considered and One-Way ANOVA, followed by the StudentNewman-Keuls post hoc test, when three groups were considered (*p < 0.05, treatment vs. control). The levels of GSHt and GSSG in liver in 4.5 mg/kg MTX and 9.0 mg/kg MTX-treated animals can be observed in Table 6. There are no significant absolute differences in the liver between the MTX-treated groups and controls when expressed by nmol / mg protein. Similarly to the heart, the MTX triggers alterations in redox status as seen in the GSH/GSSG ratio (Figure 18). Oppositely to heart, in the liver, the pediatric group had significant decreased GSH/GSSG, whereas the 4.5 mg/kg MTX-treated adults group showed a significant higher ratio than controls. 4. Results 43 GSH / GSSG ratio in liver Table 6 - Total glutathione (GSHt) and GSSG cellular levels in liver, in MTX-treated (cumulative dose of 4.5 mg/kg and 9.0 mg/kg) and control mice, in adult and pediatric populations. LIVER (nmol / mg protein) Control 4.5 mg/kg 9.0 mg/kg Adults GSHt 43.60 ± 14.10 40.63 ± 8.83 - GSSG 1.42 ± 0.53 1.12 ± 0.22 - Pediatrics GSHt 48.23 ± 11.27 45.79 ± 8.39 47.28 ± 9.38 GSSG 1.16 ± 0.30 1.37 ± 0.31 2.64 ± 1.18 Data of GSHt and GSSG levels, in nanomol per mg of protein (nmol / mg protein), are presented as means ± standard deviation (SD), and were obtained from 2-6 animals from each treatment group. Statistical comparisons were made using the t-test when two groups were considered and Kruskal-Wallis ANOVA on Ranks, when three groups were considered. Figure 18 - (A and B) GSH/GSSG ratio in the liver after MTX administration in adult and pediatric mice, respectively. Results are presented as means ± standard deviation (SD), and were obtained from 2-6 animals from each treatment group. Statistical comparisons were made using the t-test when two groups were considered and Kruskal-Wallis ANOVA on Ranks, followed by the Dunn’s post hoc test, when three groups were considered (*p < 0.05, treatment vs. control). 4.1.4. Lipid peroxidation levels MDA can be unbound (free MDA) or bound (total MDA) to proteins and other matrix molecules. In this work, results of lipid peroxidation represent free equivalents of MDA. MTX administration in animals did not induce significant changes in MDA (indicator of lipid peroxidation) in liver and kidneys (Table 7). No measurable levels of MDA were observed in the heart. In this experiment, the MDA levels were measured just by fluorescence reading in microplate reader. 44 4. Results Table 7 - Free equivalents of malondialdehyde (MDA) levels in liver and kidneys in MTX-treated (cumulative dose of 4.5 mg/kg and 9.0 mg/kg) and control mice, in adult and pediatric populations. LIVER (nmol / g protein) Control 4.5 mg/kg 9.0 mg/kg Adults 27.60 ± 4.15 22.45 ± 2.54 - Pediatrics 34.37 ± 8.15 41.08 ± 3.14 30.28 ± 6.60 KIDNEYS (nmol / g protein) Control 4.5 mg/kg 9.0 mg/kg Adults 39.52 ± 6.08 51.94 ± 15.18 - Pediatrics 43.04 ± 17.68 45.76 ± 13.19 48.91 ± 15.69 Data of MDA levels, in nanomol per g of protein (nmol / g protein) are presented as means ± standard deviation (SD), and were obtained from 2-6 animals from each treatment group. Statistical comparisons were made using the t-test when two groups were considered and One-Way ANOVA when three groups were considered. 4.2. Experiment 2 4.2.1. Body weight and daily food / water consumption As in experiment 1 the survival percentage in the groups that received the highest cumulative dose of 9.0 mg/kg MTX was reduced (0% for adults and 33% for pediatrics), this dose was maintained but animals were sacrificed 24h after the last administration to avoid animal suffering, loss of biological sample, and assess whether MTX-induced damage had already occurred in an early stage. Additionally, instead of the cumulative dose of 4.5 mg/kg MTX administration, the mice received a cumulative dose of 7.0 mg/kg MTX twice a week for three weeks, and were maintained in a drug-free period for developmental of cumulative toxicity. However, to maintain the survival percentage at 100%, the animals of dose 7.0 mg/kg MTX were not sacrificed in 37 th day of the experiment as in the experiment 1: the animals showed signs of toxicity as in experiment 1 and in order to minimize the suffering, humane endpoints were taken into account and the animals were sacrificed one week earlier (day 31). In the adult population, the average body weight was different in the 7.0 mg/kg MTX and 9.0 mg/kg MTX groups (Figure 19A and C). The 9.0 mg/kg MTX-treated mice had similar average body weight compared to their controls during the course of the experiment, whereas the 7.0 mg/kg MTX-treated mice had lower average body weight than their controls after reaching the maximum cumulative dose. Significant differences in the gain body weight occurred after the 21 st day, and loss of weight in the 24 th day, in this group. In the pediatric 4. Results 45 ***** *** Weight (g) population, the 7.0 mg/kg MTX-treated animals had significant less body weight gain compared to controls after the 17 th day. The 9.0 mg/kg MTX-treated animals in the last evaluation also showed significant loss of body weight (Figure 19B and D). Figure 19 - (A and B) Average body weight in 7.0 mg/kg MTX-treated adult and pediatric mice, respectively. (C and D) Average body weight in 9.0 mg/kg MTX-treated adult and pediatric mice, respectively. Results in grams (g) are presented as mean ± standard deviation (SD), from eight animals in each group, excepting in 7.0 mg/kg MTX-treated pediatrics (n = 7). Light blue open squares (□) represent cumulative dose of 7.0 mg/kg MTX treatment, dark blue triangles (∆) represent cumulative dose of 9.0 mg/kg MTX treatment and black solid circles (●) represent saline-control treatment. Statistical comparisons were made using two-way ANOVA followed by the Bonferroni post hoc test (*p < 0.05, **p < 0.01 and ***p < 0.001, treatment vs. control). Differences in food consumption were more significant in the case of the 7.0 mg/kg MTX-treated pediatrics (Figure 20B), with significant lower consumption from 14 th day until the end of the experiment, compared to the control group. The adults that received the same dose had significant different food consumption only in the week following the last MTX administration, with a higher consumption compared to the control group (Figure 20A). The adult animals that received a cumulative dose of 9.0 mg/kg MTX had two different situations regarding their food consumption: significant lower consumption at the beginning of the *** ** *** *** Weight (g) Weight (g) * Weight (g) 46 4. Results 3 7 10 14 17 5.0 6.0 7.0 8.0 9.0 *** *** C Time (days) Food intake / day / animal (g) 3 7 10 14 17 4 5 6 7 ** ** D Time (days) Food intake / day / animal (g) 3 7 10 14 17 21 24 28 31 4.0 5.0 6.0 7.0 8.0 *** ** A Time (days) Food intake / day / animal (g) 3 7 10 14 17 21 24 28 31 4.0 5.0 6.0 7.0 8.0 **** ** *** *** *** * B Time (days) Food intake / day / animal (g) experiment and higher at the end, compared to control (Figure 20C). The pediatric mice, in a general manner, had lower consumption during all the period of the experiment, compared to control in both concentrations (Figure 20B and D), evidencing a similar consumption behavior with the 7.0 mg/kg MTX-treated pediatrics. Figure 20 - (A and B) Food consumption in 7.0 mg/kg MTX-treated adult and pediatric mice, respectively. (C and D) Food consumption in 9.0 mg/kg MTX-treated adults and pediatrics, respectively. Results in grams (g) are presented as mean ± standard deviation (SD), from eight animals in each group, excepting 7.0 mg/kg MTX-treated pediatrics (n = 7). Light blue open squares (□) represent cumulative dose of 7.0 mg/kg MTX treatment, dark blue triangles (∆) represent cumulative dose of 9.0 mg/kg MTX treatment and black solid circles (●) represent saline-control treatment. Small vertical line in 17 th day (x axis) indicates the last MTX administration. Statistical comparisons were made using two-way ANOVA followed by the Bonferroni post hoc test (*p < 0.05, **p < 0.01 and ***p < 0.001, treatment vs. control). Data of water consumption are presented in Figure 21 and a significant lower consumption in all groups is observed, excepting in 9.0 mg/kg MTX-treated pediatric mice, compared to respective control groups. In the adults, before reaching the total cumulative dose, the water intake was significantly impaired. In the 7.0 mg/kg-treated pediatric mice, only in the last administrations and after, changes were seen. 4. Results 47 3 7 10 14 17 21 24 28 31 2 4 6 8 *** *** *** *** *** *** *** *** A Time (days) Water intake / day / animal (mL) 3 7 10 14 17 21 24 28 31 2 4 6 8 *** *** *** *** *** *** Time (days) Water intake / day / animal (mL) B 3 7 10 14 17 5 6 7 8 9 *** *** *** Time (days) Water intake / day / animal (mL) C 3 7 10 14 17 5 6 7 8 9 * D Time (days) Water intake / day / animal (mL) Figure 21 - (A and B) Water consumption in 7.0 mg/kg MTX-treated adult and pediatric mice, respectively. (C and D). Water consumption in 9.0 mg/kg MTX-treated adults and pediatrics, respectively. Results in grams mL/day/animal are presented as mean ± standard deviation (SD), from eight animals in each group, excepting 7.0 mg/kg MTX-treated pediatrics (n = 7). Light blue open squares (□) represent cumulative dose of 7.0 mg/kg MTX treatment, dark blue triangles (∆) represent cumulative dose of 9.0 mg/kg MTX treatment and black solid circles (●) represent saline-control treatment. Small vertical line in 17 th day (x axis) indicates the last MTX administration. Statistical comparisons were made using two-way ANOVA followed by the Bonferroni post hoc test (*p < 0.05, **p < 0.01 and ***p < 0.001, treatment vs. control). 4.2.2. Ratios of heart weight / body weight and liver weight / body weight In table 8, the percentage of ratios of heart weight / body weight can be observed. The cumulative dose of 7.0 mg/kg MTX induced a significant difference in the ratio in the adults group, which presented a decreased heart weight compared to control. In this same dose group, MTX induced a significant decrease in the percentage of liver weight / body weight ratio compared to control (Table 9). No other significant differences were observed for these parameters. 54 4. Results N N OH OH S H N N OH OH S 0123456 0.000 0.001 0.002 0.003 0.004 0.005 Without TBA derivatization With TBA derivatization 3.488 A Time (min) AU Figure 25 - The spectrum of MDA(TBA) 2 adduct from 500 to 600 nm (6 µM MDA standard). Figure 26 - (A) Chromatograms of a 7.0 mg/kg MTX-treated adult liver sample, before and after TBA derivatization (dashed and continuous line, respectively). (B) Chromatogram from a 6 µM MDA standard after TBA derivatization and the chemical structure of the MDA(TBA) 2 adduct. 4. Results 55 No free equivalents of MDA in heart are presented in this study, since the all values were below of the lowest standard used. Regarding to the liver, in the 9.0 mg/kg MTX-treated pediatric group, lower lipid peroxidation levels were observed when compared to control. No differences were found in the liver of the 7.0 mg/kg MTX pediatric group, neither in all the adults groups. In kidneys, high standard deviations and no differences between populations were observed (Table 14). Table 14 - Malondialdehyde (MDA) levels in liver and kidneys in MTX-treated (cumulative dose of 7.0 mg/kg and 9.0 mg/kg) and control mice, in adult and pediatric populations. LIVER nmol / g protein Control 7.0 mg/kg 7.0 mg/kg Control 9.0 mg/kg 9.0 mg/kg Adult 8.48 ± 4.61 6.27 ± 8.70 11.07 ± 11.47 16.24 ± 3.86 Pediatric 4.22 ± 4.13 4.76 ± 3.44 13.40 ± 3.34 7.12 ± 3.40 ** KIDNEYS nmol / g protein Control 7.0 mg/kg 7.0 mg/kg Control 9.0 mg/kg 9.0 mg/kg Adult 61.26 ± 34.28 84.81 ± 72.33 53.97 ± 33.33 41.89 ± 26.26 Pediatric 59.93 ± 28.74 42.91 ± 15.80 41.20 ± 20.92 40.55 ± 12.51 Data of MDA levels, in nanomol per g of protein (nmol / g protein) are presented as means ± standard deviation (SD), and were obtained from 4-8 animals from each treatment group. Statistical comparisons were made using the Mann-Whitney Rank Sum test (**p < 0.01, treatment vs. control). 4.2.8. ATP levels To understand if MTX has different chronic effects in cellular energetics of the two populations, intracellular ATP levels were measured in the heart, liver, and kidneys of the animals exposed to MTX. Alterations in hepatic ATP levels are evident in Figure 27. In adult mice, an approximately 3-fold and 2-fold significant decrease in ATP levels occurred, in 9.0 mg/kg MTX and 7.0 mg/kg MTX treatment doses, respectively (Figure 27A and B). In the pediatric population, changes also occurred in ATP, although in a lower extent. A significant decrease in the hepatic ATP levels occurred in 9.0 mg/kg group when compared to control (Figure 27C). In the 7.0 mg/kg MTX tested concentration, no significance changes were detected. Regarding heart and kidney, no significant differences were observed any treatment group as can be seen in Table 15. 56 4. Results Control 9.0 mg/kg 0.0 0.2 0.4 0.6 0.8 *** B Control 7.0 mg/kg 0.0 0.2 0.4 0.6 0.8 ** nmol ATP / mg protein in liver A nmol ATP / mg protein in liver nmol ATP / mg protein in liver Figure 27 - ATP levels in the liver of mice exposed to cumulative dose of 7.0 mg/kg MTX and 9.0 mg/kg MTX. (A and B) ATP levels in the liver of adults after cumulative dose of 7.0 mg/kg MTX and 9.0 mg/kg MTX, respectively. (C and D) ATP levels in liver of pediatrics after cumulative dose of 7.0 mg/kg MTX and 9.0 mg/kg MTX, respectively. Results, in nmol / mg protein, are presented as means ± standard deviation (SD), and were obtained from 7-8 animals from each treatment group. Statistical comparisons were made using the Mann-Whitney Rank Sum (*p < 0.05, **p < 0.01, ***p < 0.001 treatment vs. control). Table 15 - ATP levels in the heart and kidneys of mice exposed to cumulative dose of 9.0 mg/kg MTX and 7.0 mg/kg MTX. HEART nmol /mg protein Control 7.0 mg/kg 7.0 mg/kg Control 9.0 mg/kg 9.0 mg/kg Adult 3.92 ± 1.34 3.23 ± 1.39 2.85 ± 0.95 3.33 ± 1.32 Pediatric 3.94 ± 1.97 3.77 ± 1.42 2.99 ± 1.19 2.96 ± 1.23 KIDNEYS nmol /mg protein Control 7.0 mg/kg 7.0 mg/kg Control 9.0 mg/kg 9.0 mg/kg Adult 0.20 ± 0.02 0.20 ± 0.02 0.24 ± 0.06 0.19 ± 0.03 Pediatric 0.19 ± 0.02 0.18 ± 0.03 0.24 ± 0.03 0.28 ± 0.09 Results, in nmol per mg of protein (nmol / mg protein), are presented as means ± standard deviation (SD) and were obtained from 7-8 animals from each treatment group. Statistical comparisons were made using the Mann-Whitney Rank Sum between treated groups and the respective controls. ADULT PEDIATRIC 4. Results 57 4.2.9. Caspase-3, -8 and -9 activities To assess whether MTX induced cell death via apoptosis, the activities of caspase-3, -8 and -9 were measured through a method based in the cleavage of the respective caspase substrates. Caspase-3 activity was significantly decreased in pediatric mice population treated with cumulative dose of 9.0 mg/kg MTX (*p < 0.05) (Table 16). No other differences were observed in the activity of caspase-3 in other populations. Regarding the activity of caspase-8 and caspase-9, no significant differences were observed in either MTX-cumulative doses or populations. Table 16 - Caspase-3, -8 and -9 activities in the heart of mice treated with total cumulative doses of 7.0 mg/kg MTX or 9.0 mg/kg MTX. Parameter ADULT (Fluorescent units/ mg protein) Control 7.0 mg/kg 7.0 mg/kg Control 9.0 mg/kg 9.0 mg/kg Caspase-9 92 ± 96 81 ± 132 153 ± 118 254 ± 118 Caspase-8 788 ± 259 747 ± 261 1605 ± 193 1697 ± 442 Caspase-3 587 ± 131 597 ± 197 1861 ± 324 2281 ± 702 Parameter PEDIATRIC (Fluorescent units / mg protein) Control 7.0 mg/kg 7.0 mg/kg Control 9.0 mg/kg 9.0 mg/kg Caspase-9 115 ± 80 131 ± 116 364 ± 149 461 ± 283 Caspase-8 980 ± 300 998 ± 292 2310 ± 429 2190 ± 558 Caspase-3 685 ± 186 621 ± 155 2771 ± 200 1953 ± 563 * Data of caspase 9, 8 and 3 activities, in fluorescent units / mg protein, are presented as means ± standard deviation (SD), and were obtained from 4-8 animals from each treatment group. Statistical comparisons were made using the Mann-Whitney Rank Sum test (*p < 0.05, treatment vs. control). 4.2.10. Structural examination of heart Histologic examination by light microscopy of cardiac morphology in all groups of MTXtreated mice was performed. Lesions in the cardiac tissue were microscopically characterized by cellular degeneration, interstitial inflammatory cell infiltration, and necrotic zones. Results of semi-quantitative analysis of the MTX-treated and controls groups are 58 4. Results presented in Table 17. Major qualitative and representative structural alterations are depicted in Figure 28. The two cumulative doses of MTX provoked cardiac damage to both populations. In MTX-treated adults, the presence of cellular edema, cytoplasmic vacuolization of cardiomyocytes interstitial inflammatory cell infiltration, as well as some necrotic zones was evident. Similarly, pediatric group showed interstitial inflammatory cell infiltration but in a lower extent when compared to adults, showing less signs of cytoplasmic vacuolization and necrotic zones. In 7.0 mg/kg MTX-treated animals, namely in adults, the cardiac damage is well visible in the endocardium towards to pericardium zone, while in 9.0 mg/kg MTX-treated animals the lesions appeared essentially in the endocardium. The pediatric animals of the 7.0 mg/kg MTX dose presented a well conserved periphery. In the 7.0 mg/kg MTX-treated pediatric population, the existence of sporadic cardiomyocytes showing large nuclei and more than one nucleolus was well observed, being presumably in activity. All groups showed myocardium with preserved structure (score = 0), therefore data regarding tissue disorganization were omitted from the table. Table 17 - Semi-quantitative analysis of the morphological injury parameters of MTX-treated and controls groups, in adult and pediatric populations. ADULT Control 7.0 mg/kg 7.0 mg/kg Control 9.0 mg/kg 9.0 mg/kg Cellular degeneration 0.64 ± 0.63 1.42 ± 0.71 **** 0.50 ± 0.67 1.47 ± 0.76 **** Necrosis 0.00 ± 0.00 0.23 ± 0.43 *** 0.00 ± 0.00 0.31 ± 0.47 **** Inflammatory activity 0.30 ± 0.46 1.02 ± 0.49 **** 0.26 ± 0.45 0.91 ± 0.70 **** PEDIATRIC Control 7.0 mg/kg 7.0 mg/kg Control 9.0 mg/kg 9.0 mg/kg Cellular degeneration 0.50 ± 0.54 0.82 ± 0.63 ** 0.38 ± 0.49 0.98 ± 0.74 **** Necrosis 0.00 ± 0.00 0.04 ± 0.20 0.00 ± 0.00 0.13 ± 0.34 * Inflammatory activity 0.22 ± 0.42 0.85 ± 0.52 **** 0.25 ± 0.44 0.81 ± 0.50 **** Results, given in scores, are presented as means ± standard deviation (SD) and were obtained from 3 animals from each treatment group, excepting the control of 7.0 mg/kg MTX-treated pediatric group (n = 2). Statistical comparisons were made using the Mann-Whitney Rank Sum (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, treatment vs. control). 4. Results 59 Figure 28 - Cardiac histopathology by light microscopy from MTX-treated animals. (A) Light micrograph from the control of 9.0 mg/kg MTX of adult mice, showing normal morphology and structure; (B) Light micrograph from pediatric mice injected with cumulative dose of 9.0 mg/kg MTX. Vacuolization (green arrow) and inflammatory infiltration (blue arrow) are shown. (C) Light micrograph from pediatric mice injected with cumulative dose of 7.0 mg/kg MTX. This treated group presents large and uncondensed nucleus. (D) Light micrograph from adult mice injected with cumulative dose of 9.0 mg/kg MTX. The cardiomyocytes present high degree of cellular edema with minor structure density, irregular nucleus and vacuolization (green arrow) were observed. (E) Light micrograph from adult mice injected with cumulative dose of 9.0 mg/kg MTX. Cellular edema and necrotic zones are evident. (F) Light micrograph from pediatric mice injected with cumulative dose of 7.0 mg/kg MTX. The inflammatory status of the tissue is clear, as indicated by blue arrows showing the presence of infiltrative inflammatory cells. These results evidence that cardiotoxicity occurred in all MTX-treated animals. Pediatric mice seem to be more protected from damage than adult mice, since pediatric mice had less cardiac histological damage. 60 4. Results 5. Discussion 61 5. DISCUSSION The cumulative dose-dependent cardiotoxicity of MTX remains a major clinical concern. Although MTX shows a clinical profile similar to DOX, their mechanisms of cardiotoxicity differ and MTX-induced cardiotoxicity mechanisms remain largely unknown. At present, animal models allow to understand the molecular mechanistic basis by which anticancer drugs cause cardiotoxicity, including progressive cardiac dysfunction (Bovelli et al., 2010). However, many studies evaluate the toxic profile of anticancer drugs using acute administration conditions, which does not represent the cancer therapy reality and of the late cardiotoxicity that is the main safety concern of anthracyclines and antracenediones. In fact, their cardiotoxicity might appear as a late event, occurring even years after the completion of therapy (Seiter, 2005). In the present study, a model of multiple MTX administrations was used in male CD-1 mice, to whom was administered MTX via i.p. twice a week for three weeks. Multiple administrations in different time-points were given to mice in order to mimic the human MTX-therapy (Vorobiof et al., 1987, Paul et al., 2007). These multiple administrations aim to gradually elicit a cumulative cardiotoxic dose: the use of an acute high dose would concur to acute MTX-cardiotoxicity that is not the main objective of this dissertation. In accordance, the animals were also maintained for several weeks without further MTX-administrations to allow the development of symptomatology and observe the effects of the previously accumulated MTX. The fast growing animal model used allows the study of several parameters and late effects in a small period of time. In all the experiments, the MTX administration was given in the afternoon, as it was reported that the three pharmacokinetic half-lives of MTX are shorter in mice in this period of the day, maintaining highest anti-tumor activity with lower toxicity. Thus, this time period was assumed to be more tolerable to MTX administration, mainly when a high-dose delivery is contemplated (Levi et al., 1994). 5.1. General welfare of the MTX-treated animal model In both experiments, the toxicity of MTX was evident by the observation of the general conditions of the pediatric and adult animals, namely external signs and physiological parameters. In experiment 1, no adult mice survived to the highest dose of 9.0 mg/kg MTX, while in the pediatric population the percentage of survival was 33% at that same high cumulative dose. Adult mice had a significant decrease in body weight, which was not observed in the pediatric groups. A higher toxicity is therefore apparent to the general well- 62 5. Discussion being of the adult populations. Although the adults of the 7.0 mg/kg group in experiment 2 had decreased body weight, compared to controls, the pediatric animals also showed significant differences in body weight, namely with to less gain of body weight. Altogether, pediatric population appeared to present a higher resistance to the cumulative toxicityinduced by MTX than the adult population. During MTX administrations no changes were observed and these effects on body weight resulted from the MTX total cumulative dose. In fact, in experiment 2, the highest dose of 9.0 mg/kg of MTX did not cause abrupt changes in the weight of the animals sacrificed 24h after the last administration (excepting last administration day, in pediatric mice), in opposition to the 7.0 mg/kg MTX condition, where animals were allowed to develop cumulative toxicity. The loss of body weight in mice has been reported as common for MTX (Levi et al., 1994, Raghunand et al., 2003, Keese et al., 2009, Niang et al., 2011, Yagublu et al., 2013), but so far no data regarding pediatric population existed. Considering the possibility that the significant differences in body weight might be related to differences in food / water consumption, the food and water intakes were recorded twice a week. Normalization to each day and each animal weight was performed, assuming that each animal ate in a proportional manner relatively to its body weight. Comparing to the saline-treated controls, consumptions from MTX-treated animals were not very consistent during the time of the experiments. In general, food and water intake were markedly reduced in the following days after MTX-administration and, together with body weight loss, these data were related to the morbidity caused by MTX. An apparent exception occurred in the 7.0 mg/kg MTX-treated adults, which had higher food consumptions in the week after the last injection accompanied with decrease body weight. However, loss of body weight might not be exclusively due to lower food consumption. Gastrointestinal disturbances have been reported in patients as a common complication of cytotoxic cancer chemotherapy, specifically systemic treatments, as cytotoxic agents do not distinguish between cancer cells and normal cells undergoing rapid division like the ones that exist in the gastrointestinal tract (Di Fiore and Van Cutsem, 2009, Boussios et al., 2012). Therefore, poor absorption may have occurred as a result of the damaging effects of MTX on the epithelial lining of the intestine and, thus, contributed to a significant reduction in body weight in MTX-treated mice. Energy imbalance due to metabolic changes, involving factors such as reduced food intake (imbalances between orexigenic and anorexigenic signals), physical activity (decreased muscle mass and function) and resting energy expenditure are common in chemotherapy regimens (Gadea et al., 2012, Nicolini et al., 2013). Moreover, younger age may favor weight gain (Gadea et al., 2012), which is in concordance to the present study. The overall condition was also compromised in MTX-treated animals. The necropsy made to the animals that did not survive in experiment 1 showed damage in various organs. Liver presented abnormal external signs of damage, presenting in some cases dark color 5. Discussion 63 while blue stains were common in the peritoneum of MTX-treated animals. Signs of injury were also present in the remaining pediatric animal survivors in the highest 9.0 mg/kg MTX in experiment 1 and in the 7.0 mg/kg MTX dose, namely inertia and absence of diary hygiene, demonstrating that MTX has a time-dependent late toxic profile, an observation that was possible because these groups were not sacrificed immediately after the last MTX administration. 5.2. Biochemical blood analysis and organ damage Several parameters were determined to assess MTX-induced toxicity in the heart and other organs and whether the different aged populations present different susceptibilities. Plasma AST, ALT, and CK-MB levels were measured in both studies (in experiment 2, totalCK was also measured). Both AST and ALT are present within the hepatocytes and are released into the blood stream when the membranes of hepatic cells are damaged. AST and ALT increase in plasma can result of inflammatory processes, being also sensitive indicators of necrotic hepatic lesions (Rej, 1989, Duong and Loh, 2006). ALT is a more sensitive and specific for liver damage, namely to monitor the effects of hepatotoxic drugs (Wilson, 2007), whereas AST is found predominately in tissues of high metabolic activity, such as heart, skeletal muscle, kidneys, as well as liver (Rej, 1989, Duong and Loh, 2006, Wilson, 2007). Although MTX-treated patients have been documented with transient elevations in plasma AST and ALT levels (Paciucci and Sklarin, 1986), the AST and ALT levels in experiment 1 were decreased in the 4.5 mg/kg MTX-treated adults. Biological explanations to decreased blood aminotransferase activities are difficult to characterize and may be caused by druginduced metabolic impairment, such as vitamin B 6 (a cofactor in aminotransferase reactions) or zinc deficiency (Waner and Nyska, 1991). These decreased levels are likely associated with metabolic conditions, such anorexia and severe weight loss (Waner and Nyska, 1991). MTX may be indirectly associated with this decrease in aminotransferase activities, since severe loss of body weight due to toxic effect of drugs may possibly lead to the reduction in their activities. Moreover, the gastrointestinal disturbances caused by MTX may contribute to a malabsorption syndrome that decreases the availability of the cofactors required for the activity of the aminotransferases (Di Fiore and Van Cutsem, 2009). Renal failure can also cause decrease aminotransferase activities. Patients with chronic renal failure have shown low plasma AST and ALT that may be or not related to deficiency in vitamin B 6 (Warnock et al., 1974, Waner and Nyska, 1991, Yasuda et al., 1995). Other reasonable explanations may be: suppression of AST and ALT synthesis in the hepatocyte, inhibition of their release from 70 5. Discussion compromised. Moreover, it is possible that other sources of energy allow the maintenance of ATP levels and only at the end-stage heart failure, the bioenergetics of the heart fails completely. However and although CK-MB represents up to 30% of the total-CK in heart (Adams et al., 1993), the decrease in plasma CK-MB levels may be correlated with an early impairment in the energetic balance of the heart. Hepatocellular bioenergetics was significantly impaired in MTX-treated mice. An approximately 3-fold and 2-fold decrease in ATP levels occurred in adult mice at 9.0 mg/kg MTX and 7.0 mg/kg MTX, respectively. In pediatric mice, decrease in ATP values also occurred, but only in the 9.0 mg/kg MTX-treated group. Depletion of ATP, together with oxidative stress, is one of the most common causes of liver damage (Bantel and SchulzeOsthoff, 2012). Necrosis is typically characterized and accompanied by severe ATP depletion, whereas apoptosis is an ATP-dependent cell death program (Ferrari et al., 1998). Thus, depletion in ATP reserves in all MTX-treated groups, (with the exception of the 7.0 mg/kg MTX-treated pediatric mice), could in some way be an indicator of cell death by necrosis in liver, as already demonstrated by Ehninger et al. in isolated perfused rat liver (Ehninger et al., 1984). However, a same drug can induce cell death by necrosis depending of the dose and time of administration (Bantel and Schulze-Osthoff, 2012) and ALT values showed no differences in these animals, thus not confirming the MTX-induced necrosis in the liver. The ATP depletion may be related to other phenomena, namely MTX metabolism. It is described that MTX suffers metabolism in liver (Ehninger et al., 1984, Ehninger et al., 1990, Mewes et al., 1993). The complete loss of the cytotoxicity of MTX in HepG2 cells when cytochrome P-450 is inhibited, suggests that MTX is cytotoxic when it is metabolized and oxidized in the liver (Mewes et al., 1993). The liver is the most important organ of metabolization and any xenobiotic that suffers metabolic metabolization can be a potential hepatotoxic. MTX has been shown to be bioactivated by hepatic cytochrome P-450 super family (Blanz et al., 1991, Mewes et al., 1993). The metabolites formed, namely naphthoquinoxaline cause ATP depletion in cardiac cell models (Shipp et al., 1993), however those data are not yet confirmed in hepatic cellular models. In rats, the administration of a cumulative dose of 7.5 mg/kg of MTX and sacrificed 22 days after the last MTX administration leads to decrease in hepatic ATP levels (Rossato et al., 2013a). Even so, no further mechanisms were exploited. The determination of ROS and calcium levels might help to further understand the responsible mechanisms for depletion of ATP, as they are responsible for membrane mitochondrial disruptions and ATP production impairment (Brookes et al., 2004). 5. Discussion 71 5.5. Lymphocytes and MTX MTX has known immunosuppressant activity (Fox, 2004). In experiment 2, the 9.0 mg/kg MTX-treated pediatrics and adults presented a decline in lymphocyte number. These facts suggest that the highest dose of 9.0 mg/kg has a high impact in the immune system of the animals. In a study in MTX-treated mice with cumulative dose of 7.0 mg/kg for 14 days, MTX exerted a suppressive influence on the humoral immune system (Fidler et al., 1986). Low lymphocyte levels in rats treated with a cumulative dose of 7.5 mg/kg of MTX and sacrificed 2 days after the last administration were previously reported (Rossato et al., 2013c). No major inferences can be done between pediatrics and adults about lymphocytes and the effect of MTX in these populations since data were scarce as only representative animals were taken. 5.6. Histopathological examination of cardiac damage Histopathological changes in the heart evidenced the toxic profile of MTX in experiment 2. Both MTX-treated pediatric and adult mice showed inflammatory activity by the presence of mild leukocyte infiltration (1 to 3 cells by visual field) in the interstitial space of cardiomyocytes. Indeed, myocardial inflammation is a common consequence of myocardial injury (González et al., 2011). Evaluating cellular degeneration, adult mice showed higher degree of damage compared to pediatric mice, showing a greater extent of cellular vacuolization and cellular edema, as well as some sporadic zones of necrosis. The pediatrics group treated with 9.0 mg/kg MTX dose, although sacrificed one day after the last MTX administration, presented higher damage regarding to necrosis events (statistically different) compared to it control group, than animals administered 7.0 mg/kg MTX, evidencing that necrosis may be occurring in a dose-dependent manner. Vacuolization events were also described, by transmission electron microscopy, in MTX-treated myocytes from neonatal rats after 2 µm/mL exposure to MTX (Shipp et al., 1993), and in a endomyocardial biopsy section from a patient treated with 204 mg/m 2 of MTX (Aapro et al., 1983). In dogs that received 6 courses of 0.25 mg/kg of MTX after 7 weeks of initial DOX treatment (4 courses of 1.64 mg/kg DOX), moderate myocytic vacuolization, as well as occasionally interstitial edema were seen (Tham et al., 1987). Vacuolization was also seen in anthracyclines-based regimen in the heart of patients studied at necropsy (Isner et al., 1983) and in mice (Rahman et al., 1982, Desai et al., 2013), as well inflammatory infiltration, edema and necrosis (Dudka et al., 2012), thus demonstration that histological damage is similar in anthracyclines and MTX, while the involved mechanisms seem to differ. Comparing necrosis to CK-MB or AST levels, 72 5. Discussion the results are contradictory to what was discussed in the CK-MB section, which make us to conclude that it is necessary to carefully analyze the data, since a correlation between necrosis and CK-MB or AST is not always valid, depending on the extent of the injury and the elapsed time since the damage occurred. As CK-MB peaks in plasma at early 24h after cardiac injury and rapid decline (Jaffe et al., 2006), it is reasonable to conclude that CK-MB is not a good predictor of cardiac injury in cases of very early or late cumulative cardiotoxicity. Indeed, significant elevations of troponins are thought to better reflect myocardial necrosis, as patients with detectable troponins, but no plasma CK-MB, may reveal microscopic myocardial necrosis events (Lewandrowski et al., 2002). A way to solve this question is to analyze the cardiac tissues by immunohistochemistry with specific markers of necrotic cells. In the semi-quantitative histological evaluation, pediatric mice appear to be more protected than adult mice, since necrosis and cellular degeneration occurred at a lower extent than in adult mice, as demonstrated by the scores. Likewise, the cardiomyocytes of pediatric mice sporadically showed large nuclei and more than one nucleolus, which might be indicative of higher nuclear activity for synthesizing protein products or other cellular metabolism. The pediatric population had a better conserved periphery than adults. It was not possible to distinguish whether MTX provoked cardiotoxicity in a concentration-dependent manner because the groups that received the two cumulative doses were sacrificed at a different time-point and MTX causes cumulative damage. Thus, the assessment of damage would be erroneously rated. However, a striking evidence was observed: the animals of 9.0 mg/kg MTX, namely adults, developed a greater cardiac damage in the endocardium zone, while the damage in 7.0 mg/kg MTX animals was significant in endocardium but towards to pericardium zone. This effect demonstrates that the MTX causes toxicity not only during the treatment, but also after its administration, corroborating the characteristic of chronic cardiotoxicity in this type of anticancer therapy and its large accumulation in the heart (Ehninger et al., 1990). Other evaluations in these groups of animals have to be made, to better understand what mechanisms are implicated in the cardiotoxicity observed, such as the evaluation of conjunctive tissue or specific markers by immunohistochemistry, like damage in progenitor cells. 5.7. Apoptosis in the heart Caspases are main components in the mechanisms responsible for apoptosis, representing a regulated molecular process that removes excess or unwanted cells by controlled autodigestion from organism, and can be activated by exogenous stimuli such as 5. Discussion 73 inflammation, hypoxia, radiation, and chemotherapeutic drugs (Thompson, 1995, Shi, 2002). Therefore, the possibility of cell death by apoptosis was assessed in experiment 2, measuring caspase activities: the initiators caspase-8 and caspase-9, and the downstream effector caspase-3. MTX did not promote increase in the caspase activities in the MTXtreated groups. In fact, a decrease in caspase-3 activity in 9.0 mg/kg MTX-treated pediatric animals was observed. This result has to be further investigated, since it was already shown that MTX activates caspase-3 in H9c2 cells incubated with 100nM and 1µM MTX (Rossato et al., 2013c) and with 1.60 µM MTX (Kluza et al., 2004), although this fact was only demonstrated in in vitro cellular models. Some studies reported decreased caspase-3 activities when exposed to oxidative environment, such superoxide and ● NO. High concentrations of ● NO are proapoptotic, whereas low concentrations have been shown to be protective against apoptosis (Jiang et al., 2009). The mechanism by which apoptosis is decreased could to be related to the S-nitrosylation of caspase-3, as demonstrated in primary cultures of fibroblasts from a patient treated with peroxynitrite (Jiang et al., 2009) and in neonatal cardiomyocytes from rats treated with DOX (previously treated with ● NO) (Maejima et al., 2005). Thus, further investigation (namely protein nitration data) regarding this matter is need be fully understand the mechanisms implicated in the caspase-3 activity inhibition verified in the 9.0 mg/kg MTX-treated pediatric mice and whether ● NO is involved in this issue or if the redox state, MTX concentration, exposure time and combination with oxygen, superoxide and other molecules in this group are involved in rescuing cardiomyocytes from apoptosis (Maejima et al., 2005). 74 5. Discussion 6. Conclusions 75 6. CONCLUSIONS The main objective of this dissertation was to assess if pediatric population was more resistant or prone to MTX toxicity. The general welfare of the animals showed that the pediatric population is more resilient to MTX-induced toxicity as, in experiment 1, some of these animals survived with the 9.0 mg/kg MTX cumulative dose. In both experiments, the average body weight of pediatric mice almost did not vary when compared to controls and, when changes occurred, they were lower when compared to adult mice. Assessment of plasma aminotransferases, as well as the cardiac marker CK-MB revealed different results in the MTX-treated animals compared to controls. At a first sight, they seemed sensitive indicators of the MTX-induced damage. Elevations of AST in 9.0 mg/kg MTX-treated pediatric animals in experiment 1 may be the result of heart damage since no changes in ALT levels were seen. The decreases in AST and ALT in 4.5 mg/kg MTX-treated adults may be related to their body weight decreases or to metabolic changes. Regarding to the data obtained in plasma CK-MB levels in pediatric mice, the increase observed in 7.0 mg/kg treated group and the decrease in the 9.0 mg/kg treated group revealed that this parameter is timeand injury-dependent. Lipid peroxidation was not altered in any groups and organs, excepting in the liver of 9.0 mg/kg MTX-treated pediatric mice in experiment 2, in which lipid peroxidation was decreased. MTX is known to have low ability to cause lipid peroxidation. In experiment 1, the evaluation of glutathione in the heart showed that 9.0 mg/kg MTX-treated pediatric mice had increased GSH/GSSG ratio, indicating that they may have adapted increasing their antioxidant defenses over time towards MTX-induced toxicity. Oppositely, the 9.0 mg/kg MTX-treated adults, which were not allowed to develop cumulative toxicity (experiment 2), showed higher values of cardiac GSSG. Herein, we conclude that pediatric mice developed with the elapsed time compensatory mechanisms against the cardiotoxic potential of MTX increasing their cardiac GSH levels. The heart and liver showed dissimilar characteristics towards MTX. Adults of 4.5 mg/kg MTX dose had an increased hepatic GSH/GSSG ratio, while the 7.0 mg/kg MTX dose had a decrease in GSHt values. These data suggest that adults developed mechanisms in the liver to compensate the damage induced by MTX, through GSH synthesis or by drug conjugation/export. The liver of the 9.0 mg/kg MTX-treated pediatrics showed low GSH/GSSG ratio in experiment 1, not revealing this adaptation mechanism. The lower dose of 7.0 mg/kg in adults showed similar results in the liver of the 9.0 mg/kg-surviving pediatric animals regarding GSH/GSSG, revealing that adults are more sensitive to the toxicity of MTX. These results of oxidative stress suggest that biological age of the mice has influence in the redox defense potential and detoxification. 76 6.Conclusions Hepatic energetic impairment occurred in all MTX-treated animals in experiment 2, excepting in 7.0 mg/kg MTX-treated pediatric mice, which proves that this younger population is more resilient to MTX-induced toxicity. Furthermore, this conclusion was corroborated in the histological study of the heart: pediatric population had less cardiac damage. Both populations showed inflammatory activity, cellular degeneration, with cellular edema and vacuolization, as well as some sporadic zones of necrosis, however in a lower degree in pediatric mice. Time is a major handicap to appraise the mechanisms involved in the cardiotoxicity of MTX, since the animals may have develop time-dependent compensatory mechanisms and, thus, give different results of a same parameter, depending of the elapsed time. Investigation should be made comparing several parameters, doses and time, allowing understanding and corroborating the different mechanisms associated to the observed toxicity. The results presented demonstrate that evaluating a toxic profile of a drug in a living system requires a multifactorial analysis. Elapsed time after MTX administration and cumulative dose are the main factors to consider when studying MTX toxicity, but the age of the animal models should also be taken into account. More time should be given to assess whether the late cardiotoxicity would develop in non-lethal doses. Moreover, the determination of phosphocreatine levels and the evaluation of fibrotic tissue in the heart will allow a better understanding of the real cardiac status, as a functionally working myocardium is crucial. 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