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Additional file 2: Supplementary Fig. S2. TEM analysis of left ventricular tissue from control rat. Panel a: low magnification image of a cardiomyocyte (N, nucleus) filled of mitochondria (Mit) and myofibrils (MF). In the interstitial space an endothelial cell (Ec) is lining a capillary lumen (RB, red blood cells). Panel b: image showing mitochondria (Mit) and myofribrils (MF) in a CTRL rat myocardium (N, myocyte nucleus) at higher magnification. Scale Bars: a: 5 μm; b: 2 μm.

Additional file of Cobalt oxide nanoparticles induce oxidative stress and alter electromechanical function in rat ventricular myocytes

Additional file 1: Supplementary Fig. S1. TEM analysis of left ventricular tissue from cobalt NP inhaled rat. Panel a: low magnification image of a cardiomyocyte (N, nucleus) filled of mitochondria (Mit) and myofibrils (MF). On the left, collagen bundles (Col) are present in the interstitial space. Aggregates of Co3O4-NPs (asterisk) within myofibrils and mitochondria are shown at higher magnification in (b). Panel c: low magnification image of LV myocardium showing the sarcolemma (arrow) lining the surface of a cardiomyocyte with abundant mitochondria (Mit) and myofibrils (MF). Collagen bundles (Col) are present in the interstitial space where an endothelial cell (Ec) is lining a capillary lumen (RB, red blood cells). The black rectangle inscribes an area shown at higher magnification in (d) in which the asterisks indicate NPs located in the mitochondria (Mit). Panel e: image showing a detail of mitochondria (Mit) and myofribrils (MF) in an NP-treated cardiomyocyte. The black rectangle inscribes an area shown at higher magnification in (f) in which NPs are located within the myofibrils (asterisk). Scale Bars: c: 5 μm; a,e: 2 μm; d, f: 1 μm, b: 500 nm

Epigenetic effects of environmental contaminants are currently becoming a major concern, considering their role in influencing development, adult life and etiology of disease. The field of environmental epigenetics has been growing fast in the past decade generating awareness on the need of a new approach in toxicology studies: epigenotoxicology. It is now broadly accepted that environmental pollutants may express part of their pathogenicity through epigenetic modification. Dynamic chromatin remodeling is a required step for the initial phases of gene transcription and it can occur even after complete cell differentiation in response to environmental stimuli or xenobiotic interaction. Epigenetic factors, such as DNA methylation, histone modification and micro-RNAs, have a key role in these remodeling and alterations in these mechanisms could lead to disease. DNA methylation, in particular, is tightly linked to transcriptional silencing and fundamental in gene regulation, development and pathological events. This epigenetic modification consists in the addition of a methyl group to the 5’ position of the cytosine ring catalyzed by a class of enzyme called DNA methyltransferase (DNMTs), whose primary role is the maintenance of the correct methylation pattern in the genome. Epigenetic instability and, more specifically, uncontrolled variations in the global and local methylation profile and changes in DNMTs activity have by now been recognized as a gateway for carcinogenesis. Given the need of simple and adaptive tools to assess these effects, this work proposes a revised version of a comet assay modification designed to detect global methylation changes through enzymatic digestion with two restriction enzyme (HpaII and MspI) (Perotti et al., 2015; Wentzel et al., 2010). These two enzyme are isoschizomer endonucleases that both share the same restriction site (typical of CpG islands) but one of them (HpaII) is blocked by methylation in the restriction site; HpaII amount of digestion is therefore inversely proportional to the cell global methylation level. This allows the evaluation of global methylation changes by the ratio between the two enzyme separate digestions, bypassing, in fact, the contribution given to the tail intensity due to an eventual genotoxic activity of the tested compounds (figure 1). We developed a new protocol of a methylation-sensitive (Comet Methy-sens) comet procedure and tested its repeatability on A549 cell line. We evaluated the sensitivity of the new protocol using decitabine, a well known demethylating agent that blocks cell methytransferase (DNMTs) onto the DNA molecule, and nickel chloride (NiCl2), an environmental toxicant that induces chromatine condensation and consequently global hypermethylation at sub-toxic concentrations. This procedure allows to evaluate the capability of the modified protocol to detect both hypermethylating and hypomethylating events. Enzymatic digestion showed differential variation in response to exposure to global demethylating agent and hypermethylating agent thus proving Comet Methy-sens capability of detecting both positive and negative global methylation changes, showing at the same time a good repeatability in both controls and samples. Comet Methy-sens detected a dose-dependent demethylation caused by decitabine in the analyzed A549 population while on nickel-treated A549 the technique has successfully detected nickel induced hypermethylation starting from a concentration of 500 µM. Xenobiotic-induced changes in the cell methylation pattern have a parallel influence on the main methylome maintenance system as well, thus adding a possible new biomarker of the xenobiotic-methylome interaction. To identify the presence of concomitant effects of hypo- and hyper-methylating agents at both structural level and transcriptional level we performed real-time PCR analysis of DNMTs genes transcription. Both treatments induced variations in DNMTs transcriptional levels, showing a concurrent effect on the methylation maintenance system and on the DNA structure revealed through Comet methy-sens. In conclusion, our data demonstrate that Comet Methy-sens, in combination with the analysis of transcriptional levels of DNA methyl transferases, represents a simple and multifunctional approach to implement biomonitoring studies on epigenotoxicological effects of known and unknown xenobiotics.

Gli effetti cardiotossici di alcuni farmaci antiblastici di vecchia generazione rappresentano tuttora una problema aperto nella chemioterapia oncologica. In questo contesto assume sempre più importanza l’introduzione sul mercato di nuovi chemioterapici disegnati per interagire con elevata specificità con proteine fondamentali per la tumorigenesi e quindi in grado di ridurre al minimo gli effetti di tossicità sulle cellule sane (targeted therapies). La prospettive di vita di pazienti affetti da leucemia mieloide cronica, leucemia linfoblastica acuta e da tumore stromale gastrointestinale è stata migliorata dall’introduzione di un inibitore delle tirosina chinasi cellulari, Imatinib mesylate, oggetto di questo studio. Nonostante l’indiscussa efficienza di questo farmaco, sta lentamente avanzando l’ipotesi che questa molecola possa avere effetti collaterali negativi sul sistema cardiocircolatorio. In particolare la possibilità che questo farmaco possa compromettere l’integrità delle cellule staminali e la loro capacità di rimpiazzare il tessuto danneggiato aumenta la probabilità di sviluppo di patologie cardiovascolari. Per questo motivo in questo studio sono state utilizzate cellule staminali di origine cardiaca: progenitori cardiaci di ratto (CPCs) e cellule staminali mesenchimali cardiache di origine umana (C-MSCs). Su queste linee cellulari sono state valutate la citotossicità, la genotossicità, l’attivazione del pathway autofagico e l’induzione di stress ossidativo. È stata inoltre condotta una comparazione degli effetti indotti su cellule staminali cardiache da IM con quelli dovuti al trattamento con un chemioterapico di vecchia generazione, la doxorubicina, nota per indurre tossicità cardiaca in soggetti esposti. Gli effetti indotti da questi due farmaci sono stati, inoltre, valutati sulle rispettive linee cellulari target, le K562 (linea cellulare di leucemia mieloide cronica) per IM e le MCF7 (linea cellulare di adenocarcinoma mammario) per la doxorubicina. I dati riportati in questa tesi mettono in luce che il trattamento in vitro con IM può indurre tossicità non solo in cellule target, ma anche in linee cellulari non- target. Inoltre, la comparazione con la risposta cellulare indotta dal trattamento con Doxorubicina ha confermato la maggiore specificità d’azione di IM. Allo stesso tempo si è evidenziato che un’alta concentrazione di IM, come quella che potrebbe risultare da un trattamento cronico, può indurre nelle cellule staminali cardiache una tossicità comparabile con quella osservata nelle stesse cellule trattate con Doxorubicina. Dato il crescente interesse nei confronti del pathway autofagico, come meccanismo di risposta cellulare indotto in seguito a trattamento con chemioterapici, sono stati condotti saggi al fine di comprendere il coinvolgimento di questo pathway nella risposta al trattamento con IM. Si è cercato inoltre di verificare se la modulazione della risposta autofagica potesse diminuire la tossicità indotta in cellule non- target senza compromettere l’efficacia del farmaco stesso. Cardiotoxic effects of some old generation antiblastic drugs represent an actual problem in oncologic chemotherapy. In this context it seems fundamental to introduce new chemotherapics drawn to interact with high specificity with essential proteins in tumorigenesis, decreasing toxicity in health cells (targeted therapies). The life expectancy in chronic myeloid leukemia, acute linphoblastic leukemia and gastrointestinal stromal tumor patients has been improved after the introduction of a tyrosine kinase inhibitor, Imatinib mesylate, object of this study. Despite of the effectiveness of this drug, it is advancing the hypothesis that this molecule could have side effects on cardiovascular system. In particular, this drug could compromise the integrity of cardiac stem cells and their capability to replace damaged tissues, increasing the incidence of cardiovascular diseases. In this study we used cardiac stem cells: rat cardiac progenitor cells (CPCs) and human cardiac mesenchimal stem cells (C-MSCs). Cytotoxicity, genotoxicity, autophagy activation and oxidative stress induction have been evaluated. Doxorubicin, an old generation chemotherapy, has been used to compare in vitro effects induced by IM treatment with those induced by a drug with known cardiotoxic effects. Furthermore the effects induced by IM and Doxorubicin have been evaluated on their target cell lines, K562 (chronic myelogenous leukemia cell line) for IM, and MCF7 (breast adenocarcinoma cell line) for Doxorubicin. Data reported in this thesis underline IM toxicity induction in target and non- target cell lines. Furthermore comparison with cellular response induced by Doxorubicin confirms the higher specificity of IM. On the other hand it shows that an high IM concentration, that could result from a chronic treatment, could induce in cardiac stem cells a toxicity comparable with that observed in the same cell lines treated with Doxorubicin. Given the increasing interest in autophagy, as cellular response pathway induced by chemotherapic treatment, assays have been performed to understand the role of this pathway in IM treated cell lines. Furthermore we have verified if autophagy modulation could decrease toxicity induced in non- target cell lines without affecting the drug efficacy.

Cardiovascular complications are included among the systemic effects of tyrosine kinase inhibitor (TKI)-based therapeutic strategies. To test the hypothesis that inhibition of Kit tyrosine kinase that promotes cardiac progenitor cell (CPC) survival and function may be one of the triggering mechanisms of imatinib mesylate (IM)-related cardiovascular effects, the anatomical, structural and ultrastructural changes in the heart of IM-treated rats were evaluated. Cardiac anatomy in IM-exposed rats showed a dose-dependent, restrictive type of remodeling and depressed hemodynamic performance in the absence of remarkable myocardial fibrosis. The effects of IM on rat and human CPCs were also assessed. IM induced rat CPC depletion, reduced growth and increased cell death. Similar effects were observed in CPCs isolated from human hearts. These results extend the notion that cardiovascular side effects are driven by multiple actions of IM. The identification of cellular mechanisms responsible for cardiovascular complications due to TKIs will enable future strategies aimed at preserving concomitantly cardiac integrity and anti-tumor activity of advanced cancer treatment.