This journal is c the Owner Societies 2011 Phys. Chem. Chem. Phys. Cite this: DOI: 10.1039/c1cp22158b ACMA (9-amino-6-chloro-2-methoxy acridine) forms three complexes in the presence of DNAw Natalia Busto, a Begon˜ a Garcı´a,* a Jose´ M. Leal, a Jorge F. Gaspar, b Ce´lia Martins, b Alessia Boggioni c and Fernando Secco c Received 1st July 2011, Accepted 9th September 2011 DOI: 10.1039/c1cp22158b The interaction of ACMA (9-amino-6-chloro-2-methoxy acridine) (D) with DNA (P) has been studied by absorbance, fluorescence, circular dichroism, spectrophotometry, viscometry and unwinding electrophoresis. A T-jump kinetic study has also been undertaken. The experimental data show that, totally unlike other drugs, ACMA is able to form with DNA three complexes (PD I , PD II , PD III ) that differ from each other by the characteristics and extent of the binding process. The main features of PD I fulfil the classical intercalation pattern and the formation/ dissociation kinetics have been elucidated by T-jump techniques. PD II and PD III are also intercalated species but, in addition to the dye units lodged between base pairs, they also bear dye molecules externally bound, more in PD III relative to PD II . A reaction mechanism is put forward here. Comparison between absorbance, fluorescence and kinetic experiments has enabled us to determine the binding constants of the three complexes, namely (6.5  1.1)  10 4 M 1 (PD I ), (5.5  1.5)  10 4 M 1 (PD II ) and (5.7  0.03)  10 4 M 1 (PD III ). The Comet assay reveals that the ACMA binding to DNA brings about genotoxic properties. The mutagenic potential studied by the Ames test reveals that ACMA can produce frameshift and transversion/transition mutations. ACMA also is able to produce base-pair substitution in the presence of S9 mix. Moreover, the MTT assays have revealed cytotoxicity. The biological effects observed have been rationalized in light of these features. Introduction The interaction of drugs and mutagens endowed with planar aromatic rings with double/triple-stranded nucleic acids has been extensively studied. Lerman suggested the formation of a unique type of DNA–aminoacridine complex, with acridine fully intercalated into base pairs, ruling out other modes of binding; 1 on the basis of roentgenogram and viscometric experiments, he suggested the intercalation model for the DNA/proflavine system, and since then it has been accepted that acridines fully intercalate into DNA, discarding other types of interactions. 2 Recently, we have shown that proflavine is able to display more than one mode of interaction, namely intercalated and external binding. 3 ACMA (Fig. 1) is an acridine derivative whose behaviour considerably differs from that of proflavine on its interaction with DNA. Proflavine enhances its fluorescence upon inter- calation with DNA, whereas ACMA becomes almost fully quenched. Photophysical studies have attributed this peculiarity to the electron-transfer between ACMA and guanine. 4 This divergence in the spectroscopic behaviour stems from the different nature of the substituents located around the main acridine structure. It is therefore surmised that the difference in the peripheral moiety of these drugs could affect the thermo- dynamic and kinetic aspects of the acridine binding to DNA. Intercalation into the DNA structure entails conformational changes, such as partial unwinding of the double helix, 5 an effect that may interfere with the recognition and function of DNA- associated proteins such as topoisomerases, polymerases, DNA repair systems and transcription factors, with the result of a slowing down or even inhibition of transcription and replication. 6 Information available on parent acridine derivatives and their Fig. 1 The ACMA structure (9-amino-6-chloro-2-methoxy acridine). a Universidad de Burgos, Departamento de Quı´mica, 09001 Burgos, Spain. E-mail: nataliabyv@msn.com, begar@ubu.es, jmleal@ubu.es; Fax: +34 947258800 b Universidade Nova de Lisboa, Faculdade de Cie ˆncias Me ´dicas, CIGMH, Departamento de Gene ´tica, 1349-008 Lisboa, Portugal. E-mail: jgaspar.gene@fcm.unl.pt, cmartins.gene@fcm.unl.pt c Universita ` di Pisa, Dipartimento di Chimica e Chimica Industriale, 56126 Pisa, Italy. E-mail: alessiab@ns.dcci.unipi.it, ferdi@dcci.unipi.it w Electronic supplementary information (ESI) available: See DOI: 10.1039/c1cp22158b PCCP Dynamic Article Links www.rsc.org/pccp PAPER Downloaded by Universidade Nova de Lisboa on 06 October 2011 Published on 05 October 2011 on http://pubs.rsc.org | doi:10.1039/C1CP22158B View Online