Cytotoxicity and DNA damage in the neutrophils of patients with sickle cell anaemia treated with hydroxyurea

A hidroxiuréia (HU) constitui o avanço mais importante no tratamento da anemia falciforme (AF) por prevenir complicações e aumentar a qualidade de vida dos pacientes. Entretanto, alguns aspectos do tratamento com HU permanecem obscuros, incluindo a sua ação e potencial toxicidade em outras células sanguíneas, tais como neutrófilos. Este estudo utilizou a mensuração da lactato desidrogenase (LDH) e do metil tiazoltetrazólio (MTT) e o ensaio do cometa para investigar a citotoxicidade e índice de dano (ID) ao DNA em neutrófilos de pacientes com AF em uso do medicamento. Nos ensaios de LDH e MTT, observou-se além de ausência de toxicidade, uma ação citoprotetora no grupo de pacientes tratados, Grupo SSHU (n=21, 11 mulheres e 10 homens, com idades entre 19-63 anos), quando comparados aos pacientes sem tratamento, Grupo SS (n=20, 13 mulheres e 07 homens, 18-69 anos), e grupo de indivíduos saudáveis (AA) usado como controle (n=52, 28 mulheres e 24 homens, 19-60 anos), com redução significativa (pHydroxyurea (HU) is the most important advance in the treatment of sickle cell anaemia (SCA) for preventing complications and improving quality of life for patients. However, some aspects of treatment with HU remain unclear, including their effect on and potential toxicity to other blood cells such as neutrophils. This study used the measurement of Lactate Dehydrogenase (LDH) and Methyl ThiazolTetrazolium (MTT) and the comet assay to investigate the cytotoxicity and damage index (DI) of the DNA in the neutrophils of patients with SCA using HU.In the LDH and MTT assays, a cytoprotective effect was observed in the group of patients treated, as well as an absence of toxicity. When compared to patients without the treatment, the SS group (n=20, 13 women and 07 men, aged 18-69 years), and the group of healthy individuals (AA) used as a control group (n=52, 28 women and 24 men, aged 19-60 years), The SSHU group (n=21, 11 women and 10 men, aged 19-63 years) showed a significant reduction (

Cytotoxicity and DNA damage in the neutrophils of patients with sickle cell anaemia treated with hydroxyurea

Hydroxyurea (HU) is the most important advance in the treatment of sickle cell anaemia (SCA) for preventing complications and improving quality of life for patients. However, some aspects of treatment with HU remain unclear, including their effect on and potential toxicity to other blood cells such as neutrophils. This study used the measurement of Lactate Dehydrogenase (LDH) and Methyl ThiazolTetrazolium (MTT) and the comet assay to investigate the cytotoxicity and damage index (DI) of the DNA in the neutrophils of patients with SCA using HU.In the LDH and MTT assays, a cytoprotective effect was observed in the group of patients treated, as well as an absence of toxicity. When compared to patients without the treatment, the SS group (n=20, 13 women and 07 men, aged 18-69 years), and the group of healthy individuals (AA) used as a control group (n=52, 28 women and 24 men, aged 19-60 years), The SSHU group (n=21, 11 women and 10 men, aged 19-63 years) showed a significant reduction (p20 months), demonstrating that despite the cytoprotective effects in terms of cell viability, the use of HU can induce DNA damage in neutrophils

Estudo de citotoxicidade, inflamaÃÃo e estresse oxidativo em neutrÃfilos de pacientes com anemia falciforme: influÃncia do tratamento com hidroxiurÃia

Falciform Anemia (FA) is a hereditary hemoglobinopathy resulting from a &#946;-globin gene mutation (&#945;2&#946;26 GLU&#8594; VAL) that originates a hemoglobin variant called S (HbS). Its polymerization promotes hemolytic and vaso-occlusive crises (VOC). Nowadays it is known that these reactions are initial FA events that unleash a chain reaction that ends with the generation of oxygen reactive types (ORT) nitric oxide (NO) bioavailability reduction, endothelial lesion, susceptibility to infections and a chronic inflammatory process with direct involvement of neutrophils in the development of such mechanisms. Neutrophils of FA patients, besides developing more rigid, non-deformable structures, also show alterations in the expression of adhesion molecules and the production of cytokines and other mediators that may induce or aggravate clinical events observed in the disease. Hydroxyurea (HU) is the most important improvement in FA treatment and the only medicine with a strong impact on the patientsâ quality of life, reducing the number of VOC, hospitalizations and deaths resulting from this condition. However, not much is known about the effects of this medicine on neutrophils and on the functionality of these cells. The present study aimed at investigating cytotoxicity, inflammation and oxidative stress markers in neutrophils of FA patients, as well as the effect of HU treatment over these parameters in hematology ambulatory patients from a university hospital and a blood center, both reference centers in Fortaleza â CearÃ. The sample included 101 adult patients of both sexes diagnosed with FA through molecular study and it was divided into two groups: the SS Group â composed of 47 FA patients and the SSHU Group â composed of 54 FA patients under HU treatment. A control Group AA was composed of 50 healthy individuals, voluntary blood donors matched by age and sex. Neutrophils were isolated from whole blood by differential gradient and used to measure test. In toxicity tests carried out, it was observed that HU did not have any cytotoxic effect on patientsâ neutrophils, however, was shown a cytoprotective action when compared to group AA and SS patients, with a significant reduction (p<0,001) in lactate dehydrogenase (LDH) levels and an increase in the percentage of viable cells through the metil tiazol tetrazolium (MTT) (p<0,001) and the Trypan Blue exclusion tests. Analyzing neutrophil involvement in inflammatory and oxidative stress processes and in FA, there was a significant elevation in the levels of cytokines and pro-inflammatory markers (TNF-&#945;, MPO) and reduced antiinflammatory interleukin IL-10 group SS, as well as a significant decrease in the activity of antioxidant enzymes (SOD and GSH-Px). Patients under medical treatment with the tested medicine showed similar levels to those found in group AA. Oxidative damage were analyzed through malonaldehyde measurement (MDA), which was evidenced statistical differences between all studied groups (p<0,001), however with a higher average value in the non-treated group of patients. The present study ratified the important role of neutrophils in the inflammatory response promoted by the AF, and shown in an unprecedented way, with the Northeast-BR patients, treatment with HU did not reduce the viability of neutrophils, and modulates its mechanisms pro-inflammatory and pro -oxidants at levels comparable to those of healthy individuals.Anemia Falciforme (AF) Ã uma hemoglobinopatia hereditÃria resultante de uma mutaÃÃo pontual do gene da &#946;-globina (&#945;2&#946;26 GLU&#8594; VAL), originando a hemoglobina S (HbS), cuja polimerizaÃÃo promove crises hemolÃticas e vaso-oclusivas (CVO). Atualmente, sabe-se que as mesmas sÃo eventos iniciais na AF desencadeando uma cascata de reaÃÃes que culmina com geraÃÃo de espÃcies reativas de oxigÃnio, reduÃÃo da biodisponibilidade do Ãxido nÃtrico, lesÃo endotelial, susceptibilidade Ãs infecÃÃes e processo inflamatÃrio crÃnico, com envolvimento direto dos neutrÃfilos nesses mecanismos. Os neutrÃfilos de pacientes com AF exibem estruturas mais rÃgidas e indeformÃveis e alteraÃÃes na expressÃo de molÃculas de adesÃo e produÃÃo de citocinas e outros mediadores que podem induzir ou agravar as manifestaÃÃes clÃnicas da doenÃa. A hidroxiurÃia (HU) constitui o avanÃo mais importante no tratamento da AF, sendo o Ãnico medicamento que, efetivamente, tem forte impacto na melhora da qualidade de vida dos pacientes, reduzindo o nÃmero de CVO, hospitalizaÃÃes e Ãbitos. No entanto, pouco se sabe sobre os efeitos deste medicamento sobre os neutrÃfilos e na funcionalidade dessas cÃlulas. O estudo teve como objetivo principal investigar a citotoxicidade, inflamaÃÃo e estresse oxidativo em neutrÃfilos de pacientes com AF, bem como o efeito do tratamento com HU sobre esses parÃmetros em pacientes atendidos pelos serviÃos ambulatoriais de hematologia de um hospital universitÃrio e de um hemocentro, ambos de referÃncia em Fortaleza-CearÃ. A amostra foi constituÃda por 101 pacientes adultos, de ambos os sexos, diagnosticados por estudo molecular, sendo divididos em dois grupos: Grupo SSâ formado por 47 pacientes com AF, e, Grupo SSHUâ formado por 54 pacientes em tratamento com HU. Um grupo controle, Grupo AA, foi formado por 50 indivÃduos saudÃveis, doadores voluntÃrios de sangue, com idade e sexo pareados. Os neutrÃfilos foram isolados do sangue total por diferenÃa de gradiente e utilizados para mensuraÃÃo dos testes. Nos ensaios de toxicidade, observou-se que a HU nÃo exerceu efeito citotÃxico nos neutrÃfilos dos pacientes, entretanto, foi evidenciado uma aÃÃo citoprotetora sobre os mesmos quando comparados aos pacientes SS e grupo AA, com uma reduÃÃo significativa (p<0,001) na atividade de lactato desidrogenase (LDH) e aumento no percentual de cÃlulas viÃveis pelo teste de exclusÃo por Azul de Tripan e ensaio do metil tiazol tetrazÃlio (MTT) (p<0,001). Analisando o envolvimento dos neutrÃfilos nos processos de inflamaÃÃo e estresse oxidativo na AF, constatou-se uma significativa elevaÃÃo nos nÃveis de citocinas e marcadores prÃ-inflamatÃrios (TNF-&#945;, MPO) e uma reduÃÃo da interleucina anti-inflamatÃria IL-10 no grupo SS, bem como uma diminuiÃÃo significante da atividade das enzimas antioxidantes (SOD e GSH-Px). Os pacientes em terapia com HU apresentaram nÃveis semelhantes aos encontrados no grupo AA. Danos oxidativos foram analisados pela mensuraÃÃo do malonaldeÃdo (MDA), evidenciando diferenÃa estatÃstica entre todos os grupos do estudo (p<0,001), porÃm, com valor de mÃdia superior no grupo SS. O presente estudo ratificou o papel preponderante dos neutrÃfilos na resposta inflamatÃria promovida pela AF, e mostrou de maneira inÃdita, com pacientes do Nordeste-BR, que o tratamento com HU nÃo reduziu a viabilidade de neutrÃfilos, e modulou seus mecanismos prÃ-inflamatÃrios e prÃ-oxidantes a nÃveis comparÃveis aos de indivÃduos sadios

Cytotoxicity and DNA damage in the neutrophils of patients with sickle cell anaemia treated with hydroxyurea

Hydroxyurea (HU) is the most important advance in the treatment of sickle cell anaemia (SCA) for preventing complications and improving quality of life for patients. However, some aspects of treatment with HU remain unclear, including their effect on and potential toxicity to other blood cells such as neutrophils. This study used the measurement of Lactate Dehydrogenase (LDH) and Methyl ThiazolTetrazolium (MTT) and the comet assay to investigate the cytotoxicity and damage index (DI) of the DNA in the neutrophils of patients with SCA using HU.In the LDH and MTT assays, a cytoprotective effect was observed in the group of patients treated, as well as an absence of toxicity. When compared to patients without the treatment, the SS group (n=20, 13 women and 07 men, aged 18-69 years), and the group of healthy individuals (AA) used as a control group (n=52, 28 women and 24 men, aged 19-60 years), The SSHU group (n=21, 11 women and 10 men, aged 19-63 years) showed a significant reduction (p20 months), demonstrating that despite the cytoprotective effects in terms of cell viability, the use of HU can induce DNA damage in neutrophils