Syzygium cumini COMO AGENTE ANTIGLICANTE, ANTI-

Dentre os compostos estudados com o objetivo de neutralizar os danos causados pelo MG, a aminoguanidina (Amg) recebeu grande destaque (KLAASSEN et al., 2013). A Amg é um composto que reage diretamente com o grupo aldeído do MG, prevenindo a ligação deste aos grupos animo livres dos aminoácidos e das proteínas (THEODORE et al., 1994). Porém, por não ser um bom antioxidante e ser descrito como indutor da formação de peróxido de hidrogênio in vitro (CHEN et al, 2003; OU e WOLFF, 1993), compostos naturais mais eficazes vem sendo estudados. Desta forma, a busca por agentes terapêuticos naturais vem crescendo bastante, principalmente por plantas utilizadas popularmente para reduzir os níveis de hiperglicemia (SABU, SMITHA e KUTTAN, 2002). No sul do Brasil e da Ásia (Índia, Bangladesh, Myanmar, Nepal, Paquistão, Sri Lanka e Indonésia), o uso da planta Syzygium

cumini (L.) Skeels (Sc), da família Mirtácea, para tratar diabetes mellitus é comum na medicina

tradicional. A planta também é conhecida como jambolão, ameixa preta, jamun, ameixa java, amora indiana, ameixa malabar, ameixa roxa e Jamaica (AYYANAR e SUBASH-BABU, 2012). Etnofarmacologicamente, as pessoas dessas regiões utilizam as folhas na forma de infusão ou decocção, em concentrações que variam de 0,2 a 6,9 g/L (TEIXEIRA e FUCHS, 2006).

A partir da medicina popular uma série de estudos investigando a possível capacidade antiglicêmica e antioxidante de extratos da planta S. cumini foram realizados. A maioria das pesquisas investiga sua capacidade de reduzir a glicemia (TROJAN-RODRIGUES et al., 2012). Porém, vários estudos sugerem que o extrato das folhas da S. cumini também pode reduzir danos induzidos por radiação ao DNA de cultura de células (JAGETIA AND BALIGA, 2002), é um bom agente antinociceptivo em roedores (QUINTANS et al., 2014) e pode prevenir o aumento da atividade da adenosina desaminase sob condições hiperglicêmicas (DE BONA et al., 2014). Além disso, nosso grupo demonstrou em estudo recente que o extrato aquoso das folhas da S.

ocasionados pelo Ca2+, por estresse oxidativo e peroxidação lipídica (ECKER e VIEIRA et al., 2015). Embora efeitos antidiabéticos promissores sejam observados em indivíduos diabéticos, há necessidade de mais estudos in vitro e in vivo que investiguem as propriedades toxicológicas/farmacológicas da espécie. Seus possíveis efeitos protetores contra os danos induzidos por MG permanecem praticamente inexplorados.

3. JUSTIFICATIVA

Apesar de ser fisiologicamente formado e metabolizado, altos níveis do composto dicarbonila MG estão relacionados com diversas patologias crônicas e sistêmicas (MA et al., 2017; RODRIGUES et al., 2017; MÜNCH et al., 2003). Embora várias pesquisas sobre a citotoxicidade mediada pelo MG apontem o mecanismo de glicação, a formação de AGES, a indução de estresse oxidativo e a morte celular como eventos importantes, ainda há a necessidade de identificar alvos moleculares envolvidos em tais processos, bem como ampliar os estudos toxicológicos em termos de célula/tecidos/órgãos alvos. O conhecimento mais detalhado dos efeitos tóxicos do MG em tecidos específicos contribuirá também de forma significativa para uma análise mais completa dos insultos teciduais gerados pela hiperglicemia, especialmente no tecido sanguíneo. Além do aspecto fisiológico, o uso de eritrócitos, plaquetas e leucócitos para se avaliar a toxicidade mediada pelo MG representa um bom modelo experimental para a investigação de compostos com potencial tóxico ou farmacológico (DOS SANTOS et al., 2009). Isso porque o conjunto das diferentes funções celulares fornecerá um amplo espectro de dados que incluem desde alterações morfológicas e enzimáticas até efeitos genotóxicos, os quais podem ser avaliados por metodologias relativamente simples, de baixo custo, eficazes e que não infringem qualquer tipo de dano ou sofrimento animal. Desta forma, considerando o potencial tóxico do MG em diferentes modelos experimentais, justifica-se a execução de estudos que procurem identificar células e alvos moleculares do composto. O estudo do papel de plantas com potencial antihiperglicêmico e antioxidante como a Syzygium

cumini sobre os efeitos tóxicos do composto, além de ser inédito, poderá ser promissor como

estratégia terapêutica para prevenir/atenuar os efeitos deletérios da hiperglicemia via formação excessiva de MG e AGES.

4. OBJETIVOS

4.1 OBJETIVO GERAL

Investigar os mecanismos e alvos moleculares envolvidos na citotoxicidade do MG em células sanguíneas humanas, bem como o papel do extrato aquoso de folhas da planta Syzygium

cumini sobre algumas alterações causadas pelo composto em eritrócitos, leucócitos e plaquetas.

4.2 OBJETIVOS ESPECÍFICOS

- Explorar e aumentar o conhecimento acerca dos efeitos exercidos pelo MG em eritrócitos, leucócitos e plaquetas humanas após exposição aguda, verificando a hemólise e fragilidade osmótica nos eritrócitos, dano ao DNA e viabilidade celular em leucócitos, e a atividade de ectonucleotidases purinérgicas em plaquetas;

- Avaliar os efeitos citotóxicos induzidos pelo MG em leucócitos após exposição aguda, medindo como parâmetros de toxicidade alguns marcadores de estresse oxidativo e morte celular programada, bem como alterações na expressão de alvos moleculares envolvidos com estes eventos;

- Investigar o papel do extrato aquoso das folhas de Syzygium cumini, uma planta utilizada popularmente como agente antidiabético, sobre a citotoxicidade mediada pelo MG em células sanguíneas humanas in vitro.

5. ARTIGOS CIENTÍFICOS

Os resultados desta tese estão dispostos na forma de artigo científico (1) e manuscritos (2). Os itens referentes a materiais e métodos, resultados, discussão e referências bibliográficas encontram-se nos próprios artigo e manuscritos. O artigo está disposto na forma que foi publicado na revista Toxicology Mechanism and Methods. O manuscrito (1) está na forma que será submetido para a revista Toxicology in vitro, enquanto o manuscrito (2) está na forma em que será submetido para a revista BBA General Subjects.

5.1 ARTIGO

EVALUATION OF METHYLGLYOXAL TOXICITY IN HUMAN

ERYTHROCYTES, LEUKOCYTES AND PLATELETS

ALESSANDRO DE SOUZA PRESTES, MATHEUS MÜLLING DOS SANTOS, ASSIS ECKER, DANIELA ZANINI, MARIA ROSA CHITOLINA SCHETINGER, DENIS BROOCK ROSEMBERG, JOÃO BATISTA TEIXEIRA DA ROCHA AND NILDA

VARGAS BARBOSA

5.2 MANUSCRITO (1)

METHYLGLYOXAL TRIGGERS PROGRAMMED CELL DEATH IN

HUMAN LEUKOCYTES, MODULATING THE EXPRESSION OF

ANTIOXIDANT AND APOPTOTIC RESPONSIVE GENES

ALESSANDRO DE SOUZA PRESTES, MATHEUS MULLING DOS SANTOS, ASSIS ECKER, EMILY P. WACZUK, JOÃO BATISTA TEIXEIRA DA ROCHA, NILDA

Abstract

Methylglyoxal (MG) is a -dycarbonyl compound derived mainly from glycolysis, whose accumulation triggers deleterious effects on cells and tissues. Here, we evaluated the cytotoxic effects induced by MG in leucocytes after an acute exposure, measuring as endpoints of toxicity some markers of oxidative stress and programmed cell death. Human leukocytes were isolated and incubated with MG at concentrations ranging from 0.1 to 10 mM, for 3h, and subsequently prepared for assays based in flow cytometry and gene expression profile. The cells exposed to highest concentrations of MG (5 and 10mM) had significant loss of viability, increased reactive species (RS) production and apoptosis/necrosis rate. These phenomena were accompanied by morphological changes (increased size and granularity) and disruption in mRNA expression of antioxidant, apoptotic and glycation-responsive genes, particularly: Nrf2 (Nuclear factor (erythroid-derived 2)-like 2), SOD1 (CuZn-superoxide dismutase), SOD2 (Mn- superoxide dismutase), GSR (glutathione-S-reductase), BAX (BAX-associated X protein), BCL-2 (BCL-2-associated X protein), AIF (apoptosis inducing factor), GLO-1 (glyoxalase-1) and RAGE (receptor for advanced glycation end products). Positive correlations were found among size/granularity, early apoptosis and RS overproduction. Our data provide evidences that MG activates programmed cell death pathways in leukocytes and this effect involve the modulation of expression of antioxidant, apoptotic and glycation responsive genes.

1. Introduction

Methylglyoxal (MG) is a dycarbonyl compound normally produced as a by-product of glycolysis. Although MG is efficiently detoxified by glyoxalase system, high concentrations of MG are found in subjects with chronic hyperglycemia, Alzheimer disease and in aging (Shamsaldeen YA et al., 2016; Rabbani N, Xue M and Thornalley PJ, 2016). Dycarbonyl compounds have reactive aldehyde groups that can react with the amine groups from proteins. This process called glycation is crucial in the toxicity of MG. The final stage of these reactions is generally the formation of advanced glycation end-products (AGES), which can interact with RAGES receptors (Buschmann K et al., 2014). The activation of RAGES is involved in the pathogenesis of many diseases, including diabetes mellitus, atherosclerosis and Alzheimer disease (Leung SS et al., 2016; Byun K et al., 2017; Cai Z et al., 2017).

It is known that dicarbonyl stress can disrupt the redox state of cells (Xue M et al, 2012; Rabbani N et al., 2016). In eukaryotic cells, the adaptive responses to redox unbalance involve the activation of multiple genes encoding antioxidant enzymes, including the nuclear erythroid- related factor 2 (Nrf2). Nrf2 is an important transcription factor that regulates the genes of enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) isoforms, among others (Motohashi and Yamamoto, 2004; Stacey et al., 2013; Uruno et al., 2015). Glyoxalase-1 (Glo-1), enzyme that converts MG to D-lactic acid, is also controlled by Nrf2.

Apoptosis and necrosis have been well accepted as programmed forms of cell death triggered by events that disturb the cell redox signaling (Boujrad H et al., 2007; Alberts, 2010). These cell death pathways might work in parallel or sequentially, exhibiting multiple common points and using similar effectors (Boujrad H et al., 2007), and have emerged as important markers of cell injury mediated by MG (Tajes M et al., 2014; Fan et al., 2003). Related studies

also have pointed MG as inductor of eryptosis, anoikis and necroptosis (Nicolay et al., 2006; Chan et al., 2008; Kang et al., 1996; Conroy, 1979). However, the key steps and/or molecular targets initiated by MG towards the signal transduction pathways that lead to cell death are still not fully understood.

Oxidative stress and cell death have been explored as endpoints of MG toxicity in various cell types. Focusing in leukocytes, herein we performed a battery of tests to identify molecular targets involved in these events. Our results showing that the programmed cell death elicited by MG in human leukocytes encompass disruption in mRNA expression of antioxidant and apoptotic responsive genes provide a mechanistic overview about the toxicology of this aldehyde.

2. Materials and Methods

2.1. Chemicals

Dihydrorhodamine-123 (DHR-123), propidium iodide, annexin V/Pi kit, agarose, methylglyoxal, ethidium bromide solution and primers used for RT-qPCR analysis were purchased from Sigma-Aldrich (St. Louis, MO, USA). DNase I, TRIZOL reagent and DNase- free RNase were purchased from Invitrogen (Carlsbad, California, EUA). All other chemicals were of analytical reagent grade and purchased from Merck (Rio de Janeiro, RJ, Brazil).

2.2. Leukocytes isolation and Treatments

Leukocytes were isolated from venous blood following the methodology described by Santos et al. (2009) and Bueno et al. (2013), with minor modifications. Venous blood was obtained from healthy volunteers (5 men, 4 women, mean age of 30 ± 10) under overnight fasting state (12 hours). Exclusion criteria included exposure to alcohol or nicotine, diagnosis of acute or chronic diseases and any drug treatment for a period of 90 days. The blood collection

protocol used was conducted according to the principles expressed in Declaration of Helsinki, under supervision and specific approval of the Research Ethics Committee of Federal University of Santa Maria, Santa Maria, RS, Brazil. All subjects were properly instructed and provided a personal written informed consent to the experimental protocol before participating of the study. The protocol was approved at number 0089.0.243.000-07/2007.

The method for leukocytes separation consisted of differential erythrocytes sedimentation with dextran 5%, dissolved in PBS buffer (136 mM NaCl, 2.68 mM KCl, 1.47 mM KH2PO4

and 8.1 mM Na2HPO4, pH 7.4). Thus, 2 ml of dextran 5% was added in the total collected

blood. After, the tube was gently homogenized and left to stand 45 minutes at room temperature (35 minutes at an inclination of 45° and 10 minutes at an inclination of 90°). The pellet was suspended into erythrocyte lysis solution (150 mM NH4Cl, 10 mM NaHCO3, 1mM EDTA) and

centrifuged (480 × g, 2 min). The supernatant was discarded and the pellet washed twice with 1 ml erythrocyte lysis solution. After second centrifugation, the pellet was suspended in 2 ml Hank’s buffer solution (HBSS) (5.4 mM KCl; 0.3 mM Na2HPO4; 0.4 mM KH2PO4; 4.2 mM

NaHCO3, 0.5 mM MgCl2, 122.6 mM NaCl, 10 mM D-glucose, 10 mM Tris-HCl, 1.3 mM

CaCl2; pH 7.4), and the concentration of leukocytes was adjusted to 2 x 106 leukocytes/ml per

group with HBSS.

For treatments, the isolated leukocytes (2 x 106 leukocytes/ml per sample) were

incubated in a medium containing only PBS buffer (control) or PBS buffer plus different concentrations of MG (0.1, 0.5, 1, 2, 5 and 10 mM) for 2.5 hours at 37 °C in the dark.

2.3. Measurement of Reactive Species (RS)

RS levels measurement was performed using the probe dihydrorhodamine 123 (DHR- 123), as described by Gomes, 2005. DHR-123 can passively diffuse across membranes where is oxidized to rhodamine 123 that displays green fluorescence. After treatments, leukocytes

were incubated with DHR-123 (final concentration 15 µM) for 60 minutes at 37 °C in dark. The samples were read in a flow cytometry apparatus (BD Accury C6 Cytometer), where the fluorescence intensity was collected using FL1 filter (530/30 nm, standard FITC filter set). A life-gate based on forward scatter (FSC) and side scatter (SSC) parameters were made to analyze only leukocytes and exclude cell debris. A total of 100.000 events was collected for each analysis.

2.4. Morphological parameters analysis

Changes in size and lumpiness of cells (increased size and granularity) might be an early indicator of apoptosis. After treatments, the size and granularity of leukocytes were analyzed through the digital signals of Forward Scatter (FSC) and Side Scatter (SSC) recorded in a flow cytometry apparatus (BD Accury C6 Cytometer). A total of 100.000 events was acquired for each sample.

To confirm the morphological changes induced by treatments, leukocytes were also photographed in a confocal microscopy under contrast phase (Fluoview FV10i-LIV Olympus confocal microscopy).

2.5. Measurement of apoptotic/necrotic indexes

Cell apoptosis/necrosis indexes were measured using a commercial kit containing propidium iodide (PI) and annexin V reagents (Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit). After treatments, cell samples were processed as indicated by the manufacturer and the signals generated by annexin V and PI were analyzed immediately on flow cytometer (BD Accury C6 Cytometer), using FL1 and FL3 channels. The percentage of viable cells (events no marked by FL3 or FL1), cells in early apoptosis (events marked only by FL1) or cells with advanced apoptosis/necrosis (events marked by both FL3 and FL1) were quantified

and analyzed. Appropriate fluorescence compensation was manually set for FL1 (annexin) and FL3 channel (PI) to avoid signal overlap. A total of 80.000 events was collected for each sample.

2.6. RNA extraction and degradation assays

Total RNA was extracted with Trizol reagent (Invitrogen Technology, Carlsbad, CA, USA) following the manufactures protocol. The isolated RNA was submitted to electrophoresis (120 V, 500 mM and 150 W, for 20 minutes), in ethidium bromide agarose gel (0.5 μg/mL EtBr in 1% agarose gel) containing Tris-acetate buffer medium. Images were recorded by a high- resolution camera (Canon EOS Rebel T3) under UV light. The quantification of RNA was performed by image analysis using the Image J software (data not shown).

2.7. Analysis of gene expression by real-time reverse transcriptase polymerase chain reaction

(RT-PCR)

Gene specific primer sequences were based on published sequences in GenBank Overview (http://www.ncbi.nlm.nih.gov/genbank/) designed with Primer3 program version 0.4.0 (http://frodo.wi.mit.edu/primer3/) and custom made by Invitrogen® (Table 1). The primers used in the present study are listed in Table 1. GAPDH was applied as reference gene. Total RNA was isolated as previously described (section “RNA extraction and degradation

assay”). RT-PCR was performed in the samples treated with MG at concentrations of 0.5, 1

and 2.5 mM, which did not cause significant degradation in RNA (data not shown). The cDNA of each sample was synthesized with M-MLV reverse transcriptase enzyme (Invitrogen Technology, Carlsbad, CA, USA), using total RNA and random primers (Sigma-Aldrich, St. Louis, Missouri, EUA) according to the manufacturer’s suggested protocol (Invitrogen Technology, Carlsbad, CA, USA). For the quantification of mRNA expression, we used

forward and reverse primers depicted in Table 1. Quantitative RT-PCR was performed in 20 μl PCR mixtures containing 1× PCR Buffer, 0.2 mM dNTPs, 0.2 μM of each primer,1.5-3.0 mM of MgCl2 (see Table 1), 0.01× SYBR Green I (Molecular Probes, Millipore, Bedford, MA,

USA) and 0.5 U Platinum Taq DNA polymerase (Invitrogen Technology, Carlsbad, CA, USA). The thermal cycle was carried out in StepOne™ Real-Time PCR System (StepOne™ Software Version 2.3) and the following protocol was used: activation of Taq DNA polymerase at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 58-60°C for 15 seconds and extension at 72°C for 15 seconds. SYBR fluorescence was analyzed by StepOne™ Software Version 2.3, and the CT (cycle threshold) value for each sample was calculated and reported using the 2−ΔΔCT method. For each well, analyzed after six independent

experiments (n=6), ΔCT value was obtained by subtracting the GAPDH CT value from the CT value of the other genes. The ΔCT mean value obtained from the control group of each gene was used to calculate the ΔΔCT of the respective gene (2−ΔΔCT), according to Bustin et al.

(2009).

2.8. Statistical Analysis

Statistical analyses were performed using one-way ANOVA followed by Tukey multiple test when appropriate. Results are expressed as mean + SEM of 4-6 independent experiments. GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla California USA) was used to calculate linear regression among the following parameters: RS levels x morphological changes x cell death. Correlation analysis were performed using the Pearson product-moment coefficient (r). Differences were considered statistically significant when p≤0.05.

3.1. MG increases RS levels in leukocytes

The exposure of leukocytes to 5 and 10 mM MG increased significantly the levels of RS when compared to the control (Fig. 1A and 1B). Different, the treatment of cells with 0.1, 0.5, 1 and 2 mM MG for 2.5 h did not modify the levels of RS (Fig. 1A and 1B).

3.2. MG induces morphological changes in leukocytes

The scatter parameters show that the exposure of leukocytes to 5 and 10 mM MG caused a marked increase) in their size (~1,45x and ~1,56x, respectively; Fig. 2A and 2B). Concomitantly, an increased granularity was detected in the leukocytes treated with these concentrations of MG (~3,78x and ~5,04x for MG e and 10 mM, respectively; Fig. 2A and 2B). These morphological changes (size and granularity) were also observed in the images of leukocytes recorded by confocal microscopy (Fig. 3). Leukocytes exposed to MG from 0.1 mM up to 2 mM did not exhibit these profiles of alterations.

3.3. MG decreases cell viability and increases apoptosis/necrosis rate in leukocytes

The indexes of viable cells, cells in early apoptosis and cells in advanced apoptosis/necrosis were estimated by flow cytometry (Fig. 4). Exposure to MG from 1 mM caused a significant decrease in the viability of cells (around 22% less than control; while MG at 2, 5 and 10 mM decreased the cell viability around 28%, 35% and 34%, respectively). Advanced apoptosis/necrosis rate was also increased in leukocytes exposed to MG from 1 mM (around 30% higher than control) (Fig. 4A and 4B). The number of cells in early apoptosis did not differ among treatments.

We performed correlations among RS production, morphological changes and cell death parameters. We found that the increased size and lumpiness of leukocytes exposed to MG was

positively correlated with RS overproduction caused by MG 5 and 10 mM (r = 0.9915 and 0.9917 for size and lumpiness, respectively, both with p<0.001) and rate of cells on early apoptosis (r = 0.9605 and 0.0.8293, with p<0.001 and p<0.005 for size and lumpiness, respectively; Fig. 5). There was also a positive correlation between RS overproduction and early apoptosis (r = 0.9330, with p<0.005, Fig. 5). The other parameters analyzed did not exhibit significant correlation (data not shown).

3.4. MG modulates mRNA expression of antioxidant, apoptotic and glycation-responsive genes

Here we investigated the effect of MG exposure on mRNA levels of some genes responsive to oxidative stress, apoptosis and MG signaling pathways in leukocytes. For oxidative stress, RT-qPCR analyses were performed using the primers sequences for enzymes SOD1, SOD2, GPx1, GPx2, GPx4, CAT, GSR, MPO and nuclear transcription factor Nrf2. The primers sequences of BAD, BAX, BCL-2, CASP 3, CASP 9, AIF and AGE, GLO1, GLO2 were used for the analyses of apoptotic and MG signaling responsive genes, respectively.

In this set of experiment, firstly we verified, by agarose gel electrophoresis with ethidium bromide, that the cells exposed to 10 mM MG had a pronounced degradation in their total RNA content when compared to the control (data not shown). Based in the curve carried out for RNA degradation, mRNA expression was not determined in leukocytes exposed to highest concentrations of MG (5 and 10 mM).

3.5. mRNA expression of oxidative stress responsive genes

Figure 6 show that mRNA encoding the nuclear factor Nrf2 was significantly decreased in the cells exposed to 2.5 mM MG (~85,1%) compared to the control. A similar effect was observed in SOD1 and SOD2 mRNA expression from leukocytes exposed to 2.5 mM MG (~86,1% and 90,05%, respectively). Different, exposure of cells to 1 and 2.5 mM MG caused

an up-regulation in the levels of mRNA that encode the glutathione-S-reductase (GSR) (~166,8% and 130,9%, respectively). MG did not induce changes in the relative transcript levels of genes GPx1, GPx2, GPx4, CAT and MPO (Fig. 6).

3.6. mRNA expression of apoptotic and MG signaling pathways responsive genes

Exposure of leukocytes to 2.5 mM MG caused both a significant increase in the transcript levels of mRNA that encode the pro-apoptotic gene BAD (~150,5%) and a decrease in mRNA expression of anti-apoptotic gene BCL-2 (~66,4%) when compared to the control (Fig. 7). AIF was also overexpressed after treatment with 2.5 mM MG (~49,7%). No significant difference among the groups was found in the transcript levels of BAX, CASP 3 and CASP 9. Concerning MG responsive genes, leukocytes exposed to 2.5 and 1 mM MG had an increase in