DNA – Cyanobacterial Hepatotoxins Microcystin-LR and Nodularin Interaction: Electrochemical Evaluation Paulina V. F. Santos , a Ilanna C. Lopes , a, b Victor C. Diculescu, a Ana Maria Oliveira-Brett* a a Departamento de Química, Faculdade de CiÞncias e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal Tel/FAX: + 351-239835295 b Departamento de Química, Centro de CiÞncias Exatas e da Natureza, Universidade Federal da Paraíba, 58051-900, Paraíba-Brasil *e-mail: brett@ci.uc.pt Received: September 16, 2011; & Accepted: November 7, 2011 Abstract Microcystin-LR (MC-LR) and nodularin (NOD), two potent cyanotoxins with strong hepatotoxic, genotoxic and carcinogenic potential have been associated with the induction of deoxyribonucleic acid (DNA) damage in vitro and in vivo. Electrochemical studies were performed to understand the DNA interaction mechanisms with MC-LR and NOD using a dsDNA-electrochemical biosensor and incubated solutions. The decrease of the dsDNA oxidation peaks with increasing incubation time due to aggregation of DNA strands and the liberation of adenine free resi- dues, causing the occurrence of DNA abasic sites, was observed, which may introduce mutations in the dsDNA during the replication process. Keywords: Microcystin-LR, Nodularin, DNA-electrochemical biosensor, DNA oxidative damage DOI: 10.1002/elan.201100516 1 Introduction The occurrences of cyanobacterial blooms in aquatic en- vironments are of increasing concern in many lakes, rivers and brackish waters worldwide [1]. More than 40 genera of cyanobacteria are known to produce a variety of harmful compounds as secondary metabolites, called cyanotoxins [2]. Microcystin-LR (MC-LR) and nodularin (NOD) are among the most commonly reported toxins produced by cyanobacteria [3], Scheme 1. MC-LR is a heptapeptide with the chemical structure cyclo(d-alanine 1 -l-leucine 2 -d- MeAsp 3 -l-arginine 4 -Adda 5 -d-glutamate 6 -Mdha 7 ), Scheme 1A, where d-MeAsp represents the d-erythro-b-methyl aspartic acid and Mdha is N-methyldehydroalanine [3]. The designation of MC-LR arises from the two variable amino acids in positions 2, l-leucine (L), and 4, l-arginine (R). The chemical structure of NOD is very similar to the one described for MC-LR, and consists of a pentapeptide with the structure cyclo(d-MeAsp 1 -l-arginine 2 -Adda 3 -d- glutamate 4 -Mdhb 5 ), Scheme 1B, in which Mdhb is 2- (methylamino)-2-dehydrobutyric acid [4]. In the chemical structures of MC-LR and NOD, Adda corresponds to the unique C 20 b-amino acid (3-amino-9-methoxy-2,6,8-tri- methyl-10-phenyldeca-4,6-dienoic acid) [5]. MC-LR is mainly produced by Microcystis aeruginosa cyanobacteria, but may also be produced by other species, such as Anabaena, Nostoc, Phormidium and Planktothrix. On the other hand, NOD is produced only by Nodularia spumigena cyanobacteria [6]. Although MC-LR and NOD are produced by different cyanobacteria species, they are both monocyclic peptides with strong hepatotoxic activity [7], causing severe liver injury and death to humans and animals, after consump- tion of cyanobacteria-contaminated water [8, 9]. These hepatotoxins act mainly through the binding and conse- quent inhibition of serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A) inside the liver cells [10, 11]. Although MC-LR and NOD are chemically and toxico- logically very similar, NOD does not bind covalently to PP1, as in the case of MC-LR [12]. Beyond protein inhib- ition, other adverse toxicological effects have been re- ported concerning MC-LR and NOD exposure, such as intracellular glutathione alteration, reactive oxygen spe- cies production and lipid peroxidation [13, 14]. The induction of deoxyribonucleic acid (DNA) damage has also been observed [15, 16]. Furthermore, the geno- toxicity and the potential carcinogenicity of MC-LR and NOD have been extensively studied in vitro and in vivo [17]. However, some studies suggest that MC-LR and NOD genotoxicity and carcinogenicity arise mainly from the secondary effects of these toxins rather than direct toxin-DNA interactions [18–21]. The most frequent biomarker of DNA damage is 8-oxo-deoxyguanosine (8-oxoGua), which results from the addition of a hydroxyl radical to deoxyguanosine, causing misreading by DNA polymerase and G:C to T:A substitutions [22]. The detection of 8-oxoGua after expo- sure to MC-LR and NOD has been reported in vitro in cultured rat hepatocytes and in vivo in rat liver [23]. TOPICAL CLUSTER Electroanalysis 2012, 24, No. 3, 547 – 553  2012 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim 547 Full Paper