Abstract

Introduction

Hemoglobin S (Hb S) is produced by the substitution of thymine by adenine (GAG to GTG) in the sixth codon of the β globin gene, which results in the production of valine rather than glutamic acid. In homozygous form (Hb SS), this mutation is known as sickle cell anemia (SCA), and in heterozygous form (Hb AS) as sickle cell trait (Frenette and Atweh, 2007Frenette PS and Atweh GF (2007) Sickle cell disease: Old discoveries, new concepts, and future promise. J Clin Invest 117:850–858.). SCA is characterized by chronic inflammation and recurrent ischemic-reperfusion events that may lead to an excess of free radicals and oxidative stress (Souza, 2001Souza PC (2001) Avaliação dos produtos de degradação oxidativa da Hb S em eritrócitos de doentes falcêmicos. Rev Bras Hematol Hemoter 23:53–54.;Kaul et al., 2004Kaul DK, Liu X, Choong S, Belcher JD, Vercellotti GM and Hebbel RP (2004) Anti-inflammatory therapy ameliorates leukocyte adhesion and microvascular flow abnormalities in transgenic sickle mice. Am J Physiol Heart CircPhysiol 287:H293–H301.; Dasgupta et al., 2006Dasgupta T, Hebbel RP and Kaul DK (2006) Protective effect of arginine on oxidative stress in transgenic sickle mouse models. Free Radic Biol Med 41:1771–1780.). A number of studies suggest that oxidative stress plays an important role in the pathophysiology of SCA (Hebbel et al., 1988Hebbel RP, Morgan WT, Eaton JW and Hedlund BE (1988) Accelerated autoxidation and heme loss due to instability of sickle hemoglobin. Proc Natl Acad SciUSA 85:237–241.; Naoum, 2000Naoum PC (2000) Interferentes eritrocitários e ambientais na anemia falciforme. Rev Bras Hematol Hemoter 22:5–22.; de Oliveira Filho et al., 2013de Oliveira Filho RA, Silva GJ, de Farias Domingos I, Hatzlhofer BL, da Silva Araujo A, de Lima Filho JL, Bezerra MA, Martins DB and de Araujo RF (2013) Association between the genetic polymorphisms of glutathione S-transferase (GSTM1 and GSTT1) and the clinical manifestations in sickle cell anemia. Blood Cells Mol Dis 51:76–79.). Due to its greater tendency for self-oxidation and oxidant generation, Hb S leads to increased lipid peroxidation (LPO) and lysis in the sickle cell membrane. The intensity of LPO and the reduction in antioxidant defense both appear to be related to the clinical severity of SCA (Dasgupta et al., 2006Dasgupta T, Hebbel RP and Kaul DK (2006) Protective effect of arginine on oxidative stress in transgenic sickle mouse models. Free Radic Biol Med 41:1771–1780.; Shimauti et al., 2010Shimauti EL, Silva DG, de Almeida EA, Zamaro PJ, Belini Junior E and Bonini-Domingos CR (2010) Serum melatonin level and oxidative stress in sickle cell anemia. Blood Cells Mol Dis 45:297–301.).

Melatonin, a potential antioxidant and anti-inflammatory agent, has been implicated in a number of pathological processes because of its ability to detoxify reactive oxygen species (ROS) and nitrogen species (RNS), and to stimulate antioxidant enzymes (Reiter et al., 2000Reiter RJ, Tan DX, Osuna C and Gitto E (2000) Actions of melatonin in the reduction of oxidative stress. A review. J Biomed Sci 7:444–458.; Cuzzocrea and Reiter, 2002Cuzzocrea S and Reiter RJ (2002) Pharmacological actions of melatonin in acute and chronic inflammation. Curr Top Med Chem 2:153–165.; Allegra et al., 2003Allegra M, Reiter RJ, Tan DX, Gentile C, Tesoriere L and Livrea MA (2003) The chemistry of melatonin’s interaction with reactive species. J Pineal Res 34:1–10.; Mayo et al., 2005Mayo JC, Sainz RM, Tan DX, Hardeland R, Leon J, Rodriguez C and Reiter RJ (2005) Anti-inflammatory actions of melatonin and its metabolites, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), in macrophages. J Neuroimmunol 165:139–149.; Tan et al., 2007Tan DX, Manchester LC, Terron MP, Flores LJ and Reiter RJ (2007) One molecule, many derivatives: A never-ending interaction of melatonin with reactive oxygen and nitrogen species? J Pineal Res 42:28–42.; Shimauti et al., 2010Shimauti EL, Silva DG, de Almeida EA, Zamaro PJ, Belini Junior E and Bonini-Domingos CR (2010) Serum melatonin level and oxidative stress in sickle cell anemia. Blood Cells Mol Dis 45:297–301.), thereby exerting a protective effect against oxidative damage in different biological systems. The trolox equivalent antioxidant capacity (TEAC), which assesses the response to free radical attack (Re et al., 1999Re R, Pellegrini N, Proteggente A, Pannala A, Yang M and Rice-Evans C (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26:1231–1237.), and the level of thiobarbituric acid reactive species (TBARS), a reliable indicator of membrane lipid peroxidation or oxidative stress (Block et al., 2002Block G, Dietrich M, Norkus EP, Morrow JD, Hudes M, Caan B and Packer L (2002) Factors associated with oxidative stress in human populations. Am J Epidemiol 156:274–285.), are some of the most important and widely used biomarkers to assess oxidative damage.

The clinical course of SCA is heterogeneous and the clinical expression of the condition appears to be influenced by genetic features such as alpha thalassemia, the haplotypes in the beta globin gene cluster and Hb F levels (Steinberg and Embury, 1986Steinberg MH and Embury SH (1986) Alpha-thalassemia in blacks: Genetic and clinical aspects and interactions with the sickle hemoglobin gene. Blood 68:985–990.; Zago and Pinto, 2007Zago MA and Pinto ACS (2007) The pathophysiology of sickle cell disease: From the genetic mutation to multiorgan disfunction. Rev Bras Hematol Hemoter 29:207–214.; Silva and Gonçalves, 2010Silva LB and Gonçalves RP (2010) Phenotypic characteristics of patients with sickle cell anemia related to βS-globin gene haplotypes in Fortaleza, Ceara. Rev Bras Hematol Hemoter 32:40–44 [in Portuguese with Abstract in English].; Shimauti et al., 2011Shimauti EL, Zamaro PJ and Bonini-Domingos CR (2011) Interaction between Hb SS and alpha thalassemia (3.7 kb deletion): A familial study. Rev Bras Hematol Hemoter 33:244–245.). Alpha thalassemia can be caused by the deletion of one or both alpha globin genes (α₁ and α₂) (Harteveld and Higgs, 2010Harteveld CL and Higgs DR (2010) Alpha-thalassaemia. Orphanet J Rare Dis 5:13.), and the most common cause of the condition in Brazil is the deletion of the −α^3.7kbgene as a result of homologous recombination between misaligned chromosomes (Sonati et al., 1991Sonati MF, Farah SB, Ramalho AS and Costa FF (1991) High prevalence of alpha-thalassemia in a black population of Brazil. Hemoglobin 15:309–311.; Wagner et al., 2010Wagner SC, de Castro SM, Gonzalez TP, Santin AP, Filippon L, Zaleski CF, Azevedo LA, Amorin B, Callegari-Jacques SM and Hutz MH (2010) Prevalence of common alpha-thalassemia determinants in south Brazil: Importance for the diagnosis of microcytic anemia. Genet Mol Biol 33:641–645.). Polymorphisms in the β^S globin gene cluster have been associated with at least five haplotypes that are classified according to the African and Middle Eastern regions where the genes are most commonly found. The Benin haplotype is associated with West Africa, while Bantu is most commonly seen in Eastern and south-central Africa. The Senegal and Cameroon haplotypes are associated with Atlantic West Africa and the African West Coast, respectively, while the Arab-Indian haplotype is found in India and the East Arabian Peninsula (Chebloune et al., 1988Chebloune Y, Pagnier J, Trabuchet G, Faure C, Verdier G, Labie D and Nigon V (1988) Structural analysis of the 5′ flanking region of the beta-globin gene in African sickle cell anemia patients: Further evidence for three origins of the sickle cell mutation in Africa. Proc Natl Acad Sci USA 85:4431–4435.; Nagel and Ranney, 1990Nagel RL and Ranney HM (1990) Genetic epidemiology of structural mutations of the beta-globin gene. Semin Hematol 27:342–359.; Elion et al., 1992Elion J, Berg PE, Lapoumeroulie C, Trabuchet G, Mittelman M, Krishnamoorthy R, Schechter AN and Labie D (1992) DNA sequence variation in a negative control region 5′ to the beta-globin gene correlates with the phenotypic expression of the beta s mutation. Blood 79:787–792.; Lapoumeroulie et al., 1992Lapoumeroulie C, Dunda O, Ducrocq R, Trabuchet G, Mony-Lobe M, Bodo JM, Carnevale P, Labie D, Elion J and Krishnamoorthy R (1992) A novel sickle cell mutation of yet another origin in Africa: The Cameroon type. Hum Genet 89:333–337.). The Senegal and Arab-Indian haplotypes are associated with increased Hb F levels (> 15%) and less severe SCA. The Benin haplotype, on the other hand, is associated with intermediate Hb F levels (5% to 15%) and a severe clinical course, while individuals with the Bantu haplotype presenting lower Hb F levels (< 5%) and a worse clinical evolution (Powars, 1991Powars DR (1991) βs gene-cluster haplotypes in sickle cell anemia. Clinical and hematologic features. Hematol Oncol Clin North Am 5:475–493.; Elion et al., 1992Elion J, Berg PE, Lapoumeroulie C, Trabuchet G, Mittelman M, Krishnamoorthy R, Schechter AN and Labie D (1992) DNA sequence variation in a negative control region 5′ to the beta-globin gene correlates with the phenotypic expression of the beta s mutation. Blood 79:787–792.; Powars and Hiti, 1993Powars D and Hiti A (1993) Sickle cell anemia. βs gene cluster haplotypes as genetic markers for severe disease expression. Am J Dis Child 147:1197–1202.; Galiza Neto and Pitombeira, 2003Galiza Neto GC and Pitombeira MS (2003) Molecular aspects for sickle cell anemia. J Bras Patol Med Lab 39:51–56 [in Portuguese with Abstract in English].).

The intense miscegenation of the Brazilian population has resulted in regional differences in ethnic composition that have led to a heterogeneous distribution of the Hb S gene (Wenning et al., 2000Wenning MR, Kimura EM, Costa FF, Saad ST, Gervasio S, de Jorge SB, Borges E, Silva NM and Sonati MF (2000) Alpha-globin genes: Thalassemic and structural alterations in a Brazilian population. Braz J Med Biol Res 33:1041–1045.; Chinelato-Fernandes and Bonini-Domingos, 2005Chinelato-Fernandes AR and Bonini-Domingos CR (2005) The contribution of molecular studies of S-like hemoglobins to knowledge of the genetic diversity of the Brazilian population.Rev Bras Hematol Hemoter 27:208–210 [in Portuguese with Abstract in English].; Seixas et al., 2008Seixas FAV, Silva CD, Tominaga J, Ferro OC and Nilson LG (2008) Incidence of hemoglobinopathies in Northwest Paraná, Brazil. Rev Bras Hematol Hemoter 30:287–291.; Silva Filho et al., 2010Silva LB and Gonçalves RP (2010) Phenotypic characteristics of patients with sickle cell anemia related to βS-globin gene haplotypes in Fortaleza, Ceara. Rev Bras Hematol Hemoter 32:40–44 [in Portuguese with Abstract in English].). Paraná state, located in southern Brazil, has a 1.52% prevalence for the heterozygous S gene (Watanabe et al., 2008Watanabe AM, Pianovski MA, Zanis Neto J, Lichtvan LC, Chautard-Freire-Maia EA, Domingos MT and Wittig EO (2008) Prevalence of hemoglobin S in the State of Parana, Brazil, based on neonatal screening. Cad Saude Pública 24:993–1000 [in Portuguese with Abstract in English].). Hb S was introduced in Brazil during the African slave trade from the 16^th to 19^th centuries. Historical records state that most slaves from Benin went to the port of Salvador, in the state of Bahia, while Rio de Janeiro received slaves from the Angola region, where the Bantu haplotype was the most prevalent (Silva Filho et al., 2010Silva LB and Gonçalves RP (2010) Phenotypic characteristics of patients with sickle cell anemia related to βS-globin gene haplotypes in Fortaleza, Ceara. Rev Bras Hematol Hemoter 32:40–44 [in Portuguese with Abstract in English].). After arriving in Rio de Janeiro, slaves were redistributed to other regions in the country, and studies show that most states in Brazil, especially those in the southeast and southern regions of the country, received slaves from Rio de Janeiro (Fleury, 2007Fleury MK (2007) Haplotipos do cluster de globina beta em pacientes com anemia falciforme no Rio de Janeiro: Aspectos Clinicos e laboratoriais. Rev Bras Anal Clin 39:89–93.). There are also records of slave trading in the state of Paraná in the 17^th and 18^th centuries, and starting from the second half of the 18^th century, internal migration of former slaves was observed from other regions of Brazil to Paraná (Luna and Klein, 2004Luna FV and Klein HS (2004) Economia e sociedade escravista: Minas Gerais e São Paulo em 1830. Rev Bras Est Pop 21:173–193.). Paraná has a multiethnic population, a large percentage of which is of European origin. The percentage of Afro-Brazilians in Paraná was thought to be quite small, until a survey by the Brazilian Institute of Geography and Statistics revealed that these individuals made up 24% of the state’s population (IBGE, 2013). These findings showed that Paraná is the southern Brazilian state with the largest proportion of Afro-Brazilian individuals (Gomes Júnior et al., 2008Gomes Júnior J, da Silva GL and Costa PAB (2008) Paraná Negro. UFPR/PROEC, Curitiba, 104 pp.).

The goal of the present study was to analyze the frequency of β^S gene haplotypes and alpha thalassemia (−α^3.7kb) and to verify their association with oxidative stress biomarkers and the antioxidant defense system, in addition to establishing the clinical and hematological profile of β^Sglobin gene carriers in Paraná.

Subjects and Methods

Subjects

Seventy-seven individuals, regardless of gender, were randomly selected from the northwest region of the state of Paraná in southern Brazil. Of these participants, 17 were Hb SS carriers (aged 10 to 39 years) in a stable phase of the disease selected from outpatient clinics, 30 were carriers of Hb AS (14 to 53 years) selected from voluntary blood donors and 30 were healthy Hb AA carriers (11 to 55 years) who had total Hb levels within normal limits for their gender and age and were recruited as a control group (Lewis et al., 2006Lewis SM, Bain BJ and Bates I (2006) Hematologia Prática de Dacie e Lewis. 9th edition. Artmed, Porto Alegre, 571 pp.). Eligible participants were non-smokers, not pregnant, non-alcoholics, in the stable phase of the disease, and had not received blood transfusions in the three months prior to participating in the study. Data regarding medication use, clinical events and blood transfusions were obtained by evaluating the subjectsmedical records and by applying questionnaires at the time of blood collection. The study protocol was approved by the Research Ethics Committee of the Department of Life Sciences, Linguistics and Exact Science of the State University of São Paulo (UNESP), Brazil (protocol number 0025.0.229.000-07).

Biological samples

After informed consent, 20 mL of peripheral blood was collected and distributed into the following tubes: a) a tube with K3EDTA (4 mL) for hematological, chromatographic and molecular analyses, b) a tube with heparin (7 mL) for determination of plasma biomarkers of oxidative stress and antioxidant capacity, after centrifugation at1500 rpm for 20 min, and c) a tube without anticoagulant (9 mL) to obtain serum for the quantification of melatonin. All biological samples were collected at 6:30 a.m. to prevent the inhibition of melatonin secretion caused by exposure to daylight.

Hemoglobin profile and hematological parameters

Hematological parameters were obtained using an Auto Hematology Analyzer (BC-300 PLUS - Mindray, China) and microscopic analysis was done using blood smears stained according to the May-Grunwald-Giemsa method (Lewis et al., 2006Lewis SM, Bain BJ and Bates I (2006) Hematologia Prática de Dacie e Lewis. 9th edition. Artmed, Porto Alegre, 571 pp.). The tests used to screen for hemoglobinopathy included hemoglobin electrophoresis at pH 8.4 (Marengo-Rowe, 1965Marengo-Rowe AJ (1965) Rapid electrophoresis and quantitation of hemoglobin on cellulose acetate. J ClinPathol 18:790–792.) and pH 6.2 (Vella, 1968Vella F (1968) Acid agar gel electrophoresis of human hemoglobins. Am J Clin Pathol 49:440–442.). The quantification of hemoglobin fractions was performed by high-performance liquid chromatography (HPLC) using a Variant chromatography system (Bio-Rad) (Instruction Manual, 2006).

Identification of Hb S genotypes, β^S gene haplotypes and −α^3.7kb thalassemia

The Hb S genotypes were confirmed by molecular analysis with polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP). The segment that encodes the β^S gene was amplified using specific primers and the amplified segment was cleaved with DdeI restriction endonuclease (New England Biolabs, MA, USA) (Bonini-Domingos, 2006Bonini-Domingos CR (2006) Metodologias Laboratoriais para o Diagnóstico de Hemoglobinopatias e Talassemias. HN Editora, São José do Rio Preto, 122 pp.). The β^S globin haplotypes were determined by PCR-RFLP with an assessment of six polymorphic restriction sites: 5′γ^G (XmnI), γ^G(HindIII), γ^A (HindIII), ψβ (HincII), 3′ψβ (HincII) and 5′β (HinfI), according to the method reported by Sutton et al. (1989)Sutton M, Bouhassira EE and Nagel RL (1989) Polymerase chain reaction amplification applied to the determination of beta-like globin gene cluster haplotypes. Am J Hematol 32:66–69.. The −α^3.7 deletion was identified by multiplex PCR as described by Chong et al.(2000)Chong SS, Boehm CD, Higgs DR and Cutting GR (2000) Single-tube multiplex-PCR screen for common deletional determinants of alpha-thalassemia. Blood 95:360–362..

Biochemical analysis

Lipid peroxidation was assessed based on the plasma TBARS levels, according to the method described by Mihara and Uchiyama (1978)Mihara M and Uchiyama M (1978) Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 86:271–278.. Plasma antioxidant capacity was determined by the TEAC method, which involves the use of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, product no. 23881-3; Aldrich Chemical Co.), a synthetic water-soluble antioxidant analogous to vitamin E (Miller et al., 1993Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V and Milner A (1993) A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates.ClinSci 84:407–412.; Re et al., 1999Re R, Pellegrini N, Proteggente A, Pannala A, Yang M and Rice-Evans C (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26:1231–1237.).The serum melatonin concentration was assessed using HPLC coupled to a coulometric electrochemical detector (Coulochem III ESA model 526, Bedford, MA, USA), as previously reported (Shimauti et al., 2010Shimauti EL, Silva DG, de Almeida EA, Zamaro PJ, Belini Junior E and Bonini-Domingos CR (2010) Serum melatonin level and oxidative stress in sickle cell anemia. Blood Cells Mol Dis 45:297–301.).

Statistical analysis

Statistical analyses were done using Statistica software, version 8.0. The data were assessed for normality and homoscedasticity, and were analyzed using the non-parametric Mann-Whitney and Kruskal-Wallis tests, complemented by Dunn’s test. Correlation analyses were done using Spearman’s test. The significance level for the tests was set at 5% (α = 0.05).

Results

Nine (52.9%) individuals with SCA (Hb SS) used hydroxyurea (HU). However, as there were no significant differences in Hb F levels (p = 0.199), TEAC (p = 0.832), melatonin (p = 0.962) and TBARS (p = 0.835) between HU users and nonusers, these individuals were combined into a single group. Analyses also showed that the age and gender of the individuals in the Hb SS, Hb AS and Hb AA groups did not influence the levels of redox status biomarkers (p > 0.05).

Alpha thalassemia was analyzed in 47 individuals (17 Hb SS and 30 Hb AS), eight of whom (17%) had the −α^3.7kb mutation. Seven (14.9%) individuals were heterozygotes (−α/αα) and one (2.1%) was homozygous (−α/−α) for the mutation (Table 1).The β^S gene polymorphisms were analyzed in 17 Hb SS patients. The Bantu haplotype was the most frequent in these individuals and was found in 21 (62%) chromosomes, while the Benin haplotype was found in 11 (32%) chromosomes (Table 2). The Senegal and Arab-Indian haplotypes were not detected in the sample.

The subjects were divided into Bantu/Bantu and Bantu/Benin groups in order to analyze the influence of haplotypes on the oxidative and hematologic parameters and on phenotypic expression.Table 3 shows the redox profile and hematologic characteristics of the entire sample. The serum melatonin concentrations, TEAC and LPO in Hb AS individuals were not statistically different from those in subjects with Hb AA genotypes (p > 0.05). However, a marked reduction in serum melatonin levels (p < 0.001) and an elevation in TEAC (p ≤ 0.007) and LPO (p < 0.001) were observed in Hb SS subjects as compared to Hb AS and Hb AA individuals.The antioxidant/oxidant and hematologic profiles did not differ significantly between the Bantu/Bantu and Bantu/Benin groups (p > 0.05), except for the monocyte levels (p = 0.03) (Table 4).When grouped together, the Bantu and Benin haplotypes showed significant increases in LPO and TEAC levels, and a significant reduction in melatonin levels (p < 0.05) as compared to the control group (Hb AA) (Table 5). There were no significant differences in the clinical characteristics of the Bantu/Bantu and Bantu/Benin groups, or between HbSS/−α^3.7kb patients and those without thalassemia (p > 0.05) (Table 6).

Spearman correlation analyses revealed positive correlations between the TBARS and HCM values in the Bantu/Bantu group (r = 0.84; p = 0.03). There were also negative correlations between TEAC and melatonin levels (r=−0.91; p = 0.01), and between Hb F and reticulocytes (r=−0.9; p < 0.001) in this group. A strong correlation trending towards significance was also found between monocytes and reticulocytes (r = 0.77; p = 0.07). In the Bantu/Benin group, there were positive correlations between TEAC and reticulocytes (r = 0.73; p = 0.05), TBARS and reticulocytes (r = 0.82; p = 0.02), TBARS and monocytes (r = 0.73; p = 0.05) and Hb F and total Hb (r = 0.83; p = 0.01). The data suggested that LPO was directly associated with the degree of hemolysis. In the SS/−α^3.7 group, there were positive correlations between TBARS and TEAC (r = 0.9; p < 0.001) and between TBARS and reticulocytes (r = 0.9; p < 0.001), and negative correlations between TBARS and total Hb (r=−0.9; p < 0.001). In the Hb SS/(αα/αα) group, there was a negative correlation between Hb F and Hb S (r=−0.64; p = 0.012).

Discussion

This is the first report on haplotype frequency in Hb SS individuals in the state of Paraná. The results indicate that the Bantu haplotype, followed by the Benin haplotype, was the most frequent in the northwest region of Paraná. The most frequent genotype was Bantu/Benin. The haplotype frequency identified here was similar to that reported for the states of São Paulo (Figueiredo et al., 1996Figueiredo MS, Kerbauy J, Gonçalves MS, Arruda VR, Saad ST, Sonati MF, Stoming T and Costa FF (1996) Effect of alpha-thalassemia and beta-globin gene cluster haplotypes on the hematological and clinical features of sickle-cell anemia in Brazil. Am J Hematol 53:72–76.) and Rio de Janeiro (Silva Filho et al., 2010Silva LB and Gonçalves RP (2010) Phenotypic characteristics of patients with sickle cell anemia related to βS-globin gene haplotypes in Fortaleza, Ceara. Rev Bras Hematol Hemoter 32:40–44 [in Portuguese with Abstract in English].). For many years, slaves arrived in Brazil on forced migration routes between the Benin bay in Ghana and Nigeria to the northeastern region of Brazil, and from Congo and Angola to Rio de Janeiro (Silva Filho et al., 2010Silva LB and Gonçalves RP (2010) Phenotypic characteristics of patients with sickle cell anemia related to βS-globin gene haplotypes in Fortaleza, Ceara. Rev Bras Hematol Hemoter 32:40–44 [in Portuguese with Abstract in English].). Rio de Janeiro was a major slave distribution center, sending slaves especially to the states of São Paulo and Rio Grande do Sul. The high frequency of Bantu followed by Benin haplotypes identified in this study agrees with historical records of the slave trade and internal migration between states in Brazil.

The present findings also agree with the scientific literature, as other studies have reported similar results for the association between the Bantu/Bantu and Bantu/Benin haplotypes, increased LPO and reduced antioxidant defense (Rusanova et al., 2010Rusanova I, Escames G, Cossio G, de Borace RG, Moreno B, Chahboune M, Lopez LC, Diez T and Acuna-Castroviejo D (2010) Oxidative stress status, clinical outcome, and beta-globin gene cluster haplotypes in pediatric patients with sickle cell disease. Eur J Haematol 85:529–537.). The Bantu haplotype is associated with a lower concentration of Hb F and greater clinical severity than the Benin haplotype (Powars, 1991Powars DR (1991) βs gene-cluster haplotypes in sickle cell anemia. Clinical and hematologic features. Hematol Oncol Clin North Am 5:475–493.). However, our data showed no significant differences in the Hb F values, global redox status or clinical manifestations between homozygous Bantu haplotypes and heterozygous Bantu/Benin haplotypes. These results corroborate data from other studies (Rieder et al., 1991Rieder RF, Safaya S, Gillette P, Fryd S, Hsu H, Adams 3rd JG and Steinberg MH (1991) Effect of beta-globin gene cluster haplotype on the hematological and clinical features of sickle cell anemia. Am J Hematol 36:184–189.; Figueiredo et al., 1996Figueiredo MS, Kerbauy J, Gonçalves MS, Arruda VR, Saad ST, Sonati MF, Stoming T and Costa FF (1996) Effect of alpha-thalassemia and beta-globin gene cluster haplotypes on the hematological and clinical features of sickle-cell anemia in Brazil. Am J Hematol 53:72–76.; Silva and Gonçalves, 2010Silva LB and Gonçalves RP (2010) Phenotypic characteristics of patients with sickle cell anemia related to βS-globin gene haplotypes in Fortaleza, Ceara. Rev Bras Hematol Hemoter 32:40–44 [in Portuguese with Abstract in English].). Based on our results, it is possible that neither the Bantu/Benin nor the Bantu/Bantu haplotypes protect against oxidative damage and clinical features of SCA. If the Senegal haplotype had been present in the sample, it would have been possible to draw more accurate conclusions regarding the effect of these polymorphisms on oxidative and clinical-laboratorial characteristics.

The significantly reduced serum melatonin concentration in patients with SCA corroborates a previous study by Shimauti et al. (2010)Shimauti EL, Silva DG, de Almeida EA, Zamaro PJ, Belini Junior E and Bonini-Domingos CR (2010) Serum melatonin level and oxidative stress in sickle cell anemia. Blood Cells Mol Dis 45:297–301. that also found an association between low melatonin concentrations and this illness. The decrease in serum melatonin together with the elevated TEAC and LPO levels observed here agree with results from other investigations (Gitto et al., 2001Gitto E, Tan DX, Reiter RJ, Karbownik M, Manchester LC, Cuzzocrea S, Fulia F and Barberi I (2001) Individual and synergistic antioxidative actions of melatonin: Studies with vitamin E, vitamin C, glutathione and desferrioxamine (desferoxamine) in rat liver homogenates. J Pharm Pharmacol 53:1393–401.; Reiter et al., 2005Reiter RJ, Manchester LC and Tan DX (2005) Melatonin in walnuts: Influence on levels of melatonin and total antioxidant capacity of blood. Nutrition 21:920–924.). Although serum melatonin levels tend to decrease as age increases (Waldhauser et al., 1988Waldhauser F, Weiszenbacher G, Tatzer E, Gisinger B, Waldhauser M, Schemper M and Frisch H (1988) Alterations in nocturnal serum melatonin levels in humans with growth and aging. J Clin Endocrinol Metab 66:648–652.; Zhdanova et al., 1998Zhdanova IV, Wurtman RJ, Balcioglu A, Kartashov AI and Lynch HJ (1998) Endogenous melatonin levels and the fate of exogenous melatonin: Age effects. J Gerontol A Biol Sci Med Sci 53:B293–B298.), melatonin concentrations in individuals with SCA (Hb SS) have been found to be equally low in all age-groups (Shimauti et al., 2010Shimauti EL, Silva DG, de Almeida EA, Zamaro PJ, Belini Junior E and Bonini-Domingos CR (2010) Serum melatonin level and oxidative stress in sickle cell anemia. Blood Cells Mol Dis 45:297–301.). Studies have shown that, in conditions of intense oxidative stress, interactions with ROS lead to the metabolization of melatonin and subsequent reductions in its serum concentration (Tan et al., 2007Tan DX, Manchester LC, Terron MP, Flores LJ and Reiter RJ (2007) One molecule, many derivatives: A never-ending interaction of melatonin with reactive oxygen and nitrogen species? J Pineal Res 42:28–42.). Melatonin also increases the total antioxidant capacity, possibly by acting in synergy with classic antioxidants.

In SCA, monocytes are responsible for the activation of endothelial cells, and act as a trigger for the translocation of the nuclear transcription factor NF-kB (Belcher et al., 2000Belcher JD, Marker PH, Weber JP, Hebbel RP and Vercellotti GM (2000) Activated monocytes in sickle cell disease: Potential role in the activation of vascular endothelium and vaso-occlusion. Blood 96:2451–2459.). The activated vascular endothelium expresses the adhesion molecules ICAM-I, VCAM-I, P-selectin and E-selectin, and the procoagulant tissue factor, all which play an important role in the physiopathology of vascular occlusion in Hb SS individuals. The strong positive correlation between monocytes and reticulocytes in the Bantu/Bantu group, and between TBARS, reticulocytes and monocytes in the Bantu/Benin group suggests that higher hemolytic rates are associated with increased LPO and inflammatory responses. These data suggest a vulnerability of these genotypes to vasculopathy.

The frequency of coinheritance of Hb S with −α^3.7kb observed here was similar to that reported for the states of São Paulo and Rio de Janeiro (Figueiredo et al., 1996Figueiredo MS, Kerbauy J, Gonçalves MS, Arruda VR, Saad ST, Sonati MF, Stoming T and Costa FF (1996) Effect of alpha-thalassemia and beta-globin gene cluster haplotypes on the hematological and clinical features of sickle-cell anemia in Brazil. Am J Hematol 53:72–76.; Silva Filho et al., 2010Silva LB and Gonçalves RP (2010) Phenotypic characteristics of patients with sickle cell anemia related to βS-globin gene haplotypes in Fortaleza, Ceara. Rev Bras Hematol Hemoter 32:40–44 [in Portuguese with Abstract in English].). In these individuals, the most commonly observed haplotype was Bantu/Benin, as reported in studies done in the city of Salvador, in the state of Bahia, and in the state of São Paulo (Lyra et al., 2005Lyra IM, Gonçalves MS, Braga JA, GesteiraMde F, Carvalho MH, Saad ST, Figueiredo MS and Costa FF (2005) Clinical, hematological, and molecular characterization of sickle cell anemia pediatric patients from two different cities in Brazil. Cad Saude Pública 21:1287–1290.). Investigations of the interaction between Hb SS and alpha thalassemia (−α^3.7kb) and its effect on clinical and hematological factors have produced conflicting results. Some studies have reported reduced hemolytic rates, increased total Hb concentration and a protective effect against clinical manifestations of the disease (Higgs et al., 1982Higgs DR, Aldridge BE, Lamb J, Clegg JB, Weatherall DJ, Hayes RJ, Grandison Y, Lowrie Y, Mason KP, Serjeant BE, et al. (1982) The interaction of alpha-thalassemia and homozygous sickle-cell disease. N Engl J Med 306:1441–1446.; Powars, 1991Powars DR (1991) βs gene-cluster haplotypes in sickle cell anemia. Clinical and hematologic features. Hematol Oncol Clin North Am 5:475–493.; Belisario et al., 2010Belisario AR, Rodrigues CV, Martins ML, Silva CM and Viana MB (2010) Coinheritance of alpha-thalassemia decreases the risk of cerebrovascular disease in a cohort of children with sickle cell anemia. Hemoglobin 34:516–529.; Fertrin and Costa, 2010Fertrin KY and Costa FF (2010) Genomic polymorphisms in sickle cell disease: Implications for clinical diversity and treatment. Expert Rev Hematol 3:443–458.), while other studies have found no differences between individuals with and without the −α^3.7 mutation (αα/αα) (Mouele et al., 1999Mouele R, Boukila V, Fourcade V, Feingold J and Galacteros F (1999) Sickle-cell disease in Brazzaville, Congo: Genetical, hematological, biochemical and clinical aspects. Acta Haematol 101:178–184.).

Previous studies have reported on the importance of alpha thalassemia in the modulation of SCA (Steinberg, 2005Steinberg MH (2005) Predicting clinical severity in sickle cell anaemia. Br J Haematol 129:465–481.; Belisario et al., 2010Belisario AR, Rodrigues CV, Martins ML, Silva CM and Viana MB (2010) Coinheritance of alpha-thalassemia decreases the risk of cerebrovascular disease in a cohort of children with sickle cell anemia. Hemoglobin 34:516–529.); however, our study failed to demonstrate significant differences in the frequencyof clinical complications between groups with and without −α^3.7 thalassemia coinheritance, possibly because of our small sample size. Nonetheless, the inverse correlation detected between TBARS and total Hb and the positive correlation between reticulocytes and TBARS suggest that −α^3.7 thalassemia may decrease hemolysis and attenuate lipid peroxidation. It is also interesting to note that, in the present sample, the use of HU, a chemotherapeutic agent used to increase the Hb F concentration and attenuate hemolysis, was more common in SS individuals without the −α^3.7kb mutation and in patients with the Bantu/Bantu genotype, which is generally associated with a worse clinical outcome.

The ability of HU to increase Hb F levels varies among patients and the duration of treatment with this chemotherapeutic agent influences the increase in Hb F. In order to achieve the expected benefits, the treatment should last for at least two years (Davies and Gilmore, 2003Davies SC and Gilmore A (2003) The role of hydroxyurea in the management of sickle cell disease. Blood Rev 17:99–109.). As shown here, most patients (66.6%) with Hb SS who were receiving HU had been under treatment for less than two years.Therefore, treatment duration may be one of the determinant factors for the lack of significant differences in Hb F levels between patients receiving HU or not.

In conclusion, the results of this study provide a relevant contribution to our understanding of the pattern of colonization and to the anthropological and historical background of the Afro-Brazilian population in the state of Paraná. Our data suggest that the antioxidant defense in SCA patients with the Bantu/Bantu or Bantu/Benin genotype is insufficient to control oxidative stress. Melatonin, a potent antioxidant and anti-inflammatory agent, is a possible treatment option for SCA patients with low serum melatonin levels. However, the protective effect of melatonin against oxidative stress in individuals with sickle cell disease requires further study. More comprehensive investigations using data from other regions in the state of Paraná must be done to better characterize the frequency of β^S gene haplotypes and of −α^3.7kb thalassemia coinheritance, as well as their effects on oxidative stress and phenotypic expression in patients with Hb SS.

Acknowledgments

The authors thank the following foundations for financial support: Brazilian Ministry of Health (grant no. 3072/2007), Paraná State Ministry of Science, Technology and Higher Education (grant no. 5636/2009), Araucária Foundation for Scientific and Technological Development of Paraná (grant no. 322/2009), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number 409691/2006-2).

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