DNA as molecular target of analogous palladium and platinum anti-Trypanosoma cruzi compounds: A comparative study

Journal of Inorganic Biochemistry 105 (2011) 1704–1711

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Journal of Inorganic Biochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i n o r g b i o

DNA as molecular target of analogous palladium and platinum anti-Trypanosoma cruzi compounds: A comparative study Marisol Vieites a, Pablo Smircich b, Mariana Pagano a, Lucía Otero a, Francielle Luane Fischer c, Hernán Terenzi c, María José Prieto d, Virtudes Moreno e, Beatriz Garat b,⁎, Dinorah Gambino a,⁎ a

Cátedra de Química Inorgánica, Facultad de Química, UDELAR, Gral. Flores 2124, 11800 Montevideo, Uruguay Laboratorio de Interacciones Moleculares, Facultad de Ciencias, UDELAR, Iguá 4225, 11400 Montevideo, Uruguay Centro de Biologia Molecular Estrutural, Departamento de Bioquímica CCB, Universidade Federal de Santa Catarina, 88040-900 Florianópolis SC, Brazil d Departament de Microbiología, Universitat de Barcelona, Barcelona, Spain e Departamento de Química Inorgánica, Universitat Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain b c

a r t i c l e

i n f o

Article history: Received 30 May 2011 Received in revised form 24 July 2011 Accepted 25 July 2011 Available online 30 July 2011 Keywords: Palladium Platinum 5-nitrofuryl containing thiosemicarbazones Chagas disease American Trypanosomiasis DNA interaction

a b s t r a c t In the search for drugs with anti-trypanosome activity, we had previously synthesized two series of platinum and palladium analogous compounds of the formula [M IICl2(HL)], where HL were bioactive 5-nitrofuryl or 5nitroacroleine thiosemicarbazone derivatives. In this work, we thoroughly characterized [M IICl2(HL)] complexes interaction with DNA by using different techniques: gel electrophoresis, DNA viscosity measurements, circular dichroism (CD) and atomic force microscopy (AFM). Electrophoresis results showed that all complexes induced a withdrawal of DNA superhelicity demonstrated by a decrease in electrophoretic mobility of supercoiled DNA form. This effect on migration was dependent on dose but also on the nature of both the metal and the ligand. In general, the effect produced by palladium complexes was significantly more intense than that observed for the corresponding platinum analogs. Differences between palladium and platinum complexes were also observed in CD experiments. While palladium complexes induce evident calf thymus (CT)-DNA profile changes compatible with B-DNA to Z-DNA conformational transition, no clear effect was observed for platinum ones. Additionally, AFM studies showed that changes in the shape of plasmid DNA, like supercoiling, kinks and thickness increase resulted more intense for the former. In addition, either Pd or Pt complexes increased the viscosity of CT DNA solutions in a concentration dependent manner. Although the nature of DNA interaction of both series of analogous palladium and platinum complexes seemed to be similar, an explanation for the observed differential intensity of the effect could be related to the known kinetic stability differences between palladium and platinum compounds. © 2011 Elsevier Inc. All rights reserved.

1. Introduction American Trypanosomiasis (Chagas disease) is produced by the protozoan parasite Trypanosoma cruzi. It is mainly transmitted to the mammalian host by reduviid bugs in a stercorarian mode. Although exhaustively described for the first time in 1909 by the Brazilian scientist Carlos Chagas, there are evidences demonstrating that this disease has been present in the American continent for more than 9000 years [1]. Nowadays it remains the major parasitic disease in the Americas, being endemic throughout Latin America [1–5]. Around 8– 14 million people are currently infected and this disease causes more deaths per year in this region than any other parasitic disease. In addition, the premature disability and the effect of this disease on worker productivity lead to very significant annual losses of resources ⁎ Corresponding authors. Tel.: + 598 29249739; fax: + 598 29241906. E-mail addresses: [email protected] (B. Garat), [email protected] (D. Gambino). 0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2011.07.018

and industrial productivity. Furthermore, globalization and immigration of unknowingly infected people from Latin America has also led to several infection cases in developed countries, mainly due to lack of controls and screening in blood and organ banks and to immigrant mother to child transmission during pregnancy [6]. The chemotherapy of the disease relies on two quite unspecific nitroheterocyclic drugs, nifurtimox and benznidazole, that date back over 50 years and suffer of poor efficacy in the chronic phase of the disease, severe toxicity and increasing resistance development [3,6,7]. New drugs are urgently needed. Currently, the development of bioactive metal complexes is a promising approach in the search for new potential drugs for the therapy of parasitic illnesses. Different attempts toward developing trypanocidal metal-based compounds have been described [2,4,5,8–10]. In particular, our group has been successfully working on the development of potential antitrypanosomal agents through different strategies [11–22]. One of these strategies involves the metal coordination of trypanocidal organic ligands. The obtained metal compounds

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Transformation was verified by PCR (polymerase chain reaction) and plasmid DNA was purified (Qiagen Plasmid Maxi Kit). Spectrophotometric DNA quantification was carried out at 260 nm assuming an absorptivity at 260 nm of 0.02 mL/μg cm [25].

could act through dual or even multiple mechanisms of action by combining the pharmacological properties of both the ligand and the metal, or could at least lead to additive effects. The development of agents that act against different parasitic targets could diminish host toxic effects by lowering therapeutic dose and/or could circumvent the development of drug resistance. Through this approach we have developed different series of bioactive metal compounds bearing antitrypanosomal activity. Among them we have exhaustively studied Pd(II), Pt(II), Ru(II) and Ru(III) coordination compounds of bioactive 5nitrofuryl and 5-nitroacroleine containing thiosemicarbazones [11,12,15,17,18]. These ligands (Fig. 1a) had shown higher in vitro activity against T. cruzi than nifurtimox. Their main mode of action is related to the intracellular reduction of the nitro moiety followed by redox cycling, yielding reactive oxygen species (ROS) known to cause parasite damage [22]. All performed biological experiments strongly suggest that the main mechanism underlying the trypanocidal activity of their metal complexes is the production of oxidative stress as a result of their bioreduction and extensive redox cycling [11,12,15,17,18]. Many of the Pd and Pt compounds showed increased antitrypanosomal activity in respect to the free ligands and significant interaction with DNA, suggesting this biomolecule as a second molecular target. Trying to get further insight into the mechanism of action of these metal compounds and, particularly, to characterize their interaction with DNA, in this work we present a comparative study of the interaction with DNA of analogous [MCl2(HL)] compounds, where M = Pd(II) or Pt (II) and HL= 5-nitrofuryl or 5-nitroacroleine containing thiosemicarbazones, (Fig. 1b). DNA interaction has been thoroughly studied by using different techniques: gel electrophoresis, DNA viscosity measurements, circular dichroism (CD) and atomic force microscopy (AFM).

2.2.2. Analysis of DNA–metal complex interaction 1% DMSO had to be added to achieve dissolution of the Pd complexes. For the Pt compounds, being less soluble than the others, stock solutions were prepared in net DMSO. No effect on DNA due to DMSO addition was observed even for higher concentrations than the used for dissolution purposes [25]. The purified DNA was incubated in the presence of the metal complexes for 24 h at 37 °C (final volume: 20 μL, reaction buffer: Tris 10 mM, EDTA (ethylenediaminotetraacetic acid disodium salt) 0.1 mM, pH 7.4 or HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid) 25 mM, pH 7.0). Various molar ratios (ri = mol of complex:mol of DNA base pair) and different incubation times were assayed. When corresponding, distamycin (0.1 mM) was added to the incubation reaction 10 min before the addition of the metal complex. After incubation, reactions were stopped by adding of loading buffer (25% bromophenol blue, 50% glycerol, 25 mM EDTA pH 8.0). In all cases, samples were electrophoresed in 0.7% agarose buffered with TB (90 mM Tris–borate) at 70–80 V for 2 h. The gel was subsequently stained with an ethidium bromide solution (0.5 μg/mL) for 30 min and destained in water for 20 min. Relative mobility of bands visualized under UV light was quantified using OneDSCAN. 2.3. DNA viscosity measurements Viscosity experiments were conducted at 25 °C on an automated AND viscometer model SV-10. Stock solutions of each complex were prepared by dissolution in the minimum amount of DMSO and addition of water. A 1 mM calf thymus DNA (CT DNA) solution was diluted 1:4 with Tris–EDTA (TE) buffer (pH 7.4). For each complex increasing amounts of complex stock solution were added to this DNA solution to reach complex/DNA molar ratios (ri) in the range 0–1.0. The DMSO amount in the samples never exceeded 2%. The mixtures were incubated for 24 h at 37 °C. The viscosity of each sample was repeatedly measured at 25 °C after thermal equilibrium was achieved (10–15 min). Mean values of six measurements performed at intervals of 1 min were used to evaluate the viscosity of each sample [26]. Results were expressed as relative viscosity (η/η0) where η0 is the viscosity of DNA solution (ri = 0).

2. Experimental 2.1. Preparation of the thiosemicarbazone ligands and their metal complexes All thiosemicarbazone ligands were synthesized and characterized using the previously reported methodology [23]. Palladium and platinum complexes of the formulae [MCl2(HL)] (Fig. 1b) were synthesized by ligand substitution on Na2[PdCl4] or K2[PtCl4], using a 1:1 metal to ligand molar ratio and were characterized as previously reported [12,15,17]. 2.2. Electrophoresis approach 2.2.1. Preparation of plasmid DNA Plasmid DNA (pBSK II BlueScript (Stratagene) 300 ng per reaction) was obtained and purified according to standard techniques [24]. Briefly, Escherichia coli XL1 cells were transformed with pBSK II.

2.4. Circular dichroism spectroscopy CD spectra were measured in a Jasco J-815 spectropolarimeter at 20 °C and pH 7.0 (25 mM Pipes buffer) using CT-DNA (200 μM). The

(a) S N O2N

O

N H

n

NHR

n = 0,1 n = 0,1 n = 0,1 n = 0,1

R=H R = methyl R = ethyl R = phenyl

HL1, HL5 HL2, HL6 HL3, HL7 HL4, HL8

Cl

(b) Cl

M

S

[MCl2(HL)] M = Pd(II), Pt(II)

O2N

N O

n

N H

NHR

Fig. 1. (a) Bioactive 5-nitrofuryl and 5-nitroacroleine containing thiosemicarbazones (HL) and (b) their analogous metal complexes, [MCl2(HL)], where M = Pd(II) or Pt(II).

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samples were prepared with an input molar ratio of the complex to base pairs, ri = 0.25, 0.5 and 1.0. The reactions were run at 20 °C for 5– 10 min and the spectra were registered in the 200–500 nm range.

3. Results and discussion

2.5. AFM studies

3.1.1. Effect of the complexes on migration In the assayed conditions, plasmid DNA was visualized as two forms: supercoiled DNA (form I) (higher mobility, Fig. 2) and circular relaxed DNA (form II) (lower mobility, Fig. 2). The corresponding linear DNA, not observed, would have had an intermediate mobility. The effect of the palladium and platinum [MCl2(HL)] complexes on supercoiled DNA was studied using metal complex: DNA base pairs molar ratios, ri, between 0.1 and 6.0 at 37 °C during a 24 h incubation. Since the gels were run in the absence of ethidium bromide (or other intercalating compounds), it was possible to evaluate the effect that incubation with the complexes had on DNA tertiary structure. Results for [PdCl2(HL6)], [PdCl2(HL7)], [PtCl2(HL3)] and [PtCl2(HL7)] are shown in Fig. 2. The different palladium and platinum complexes exhibited a similar behavior. The quantification of the relative amounts of both DNA forms showed that the complexes were not able to introduce scission events on supercoiled DNA (form I) that would yield circular relaxed DNA (form II). On the other hand, detailed analysis of relative mobility showed that the complexes introduced clear conformational changes. The interactions of these complexes with DNA lead to a decrease in mobility accompanied with a band dispersion of the supercoiled form. This effect can be explained by the loss of negative supercoils in the tertiary structure of DNA driving to the formation of a family of topological isomers. Although this behavior is typical of compounds that alter DNA topology such as intercalators, nonintercalator compounds could also show a similar performance [25,27]. Fig. 2 also shows that the effect on migration is dose dependent. In fact, it

3.1. Electrophoresis approach

To optimize the observation of the conformational changes in the tertiary structure of pBR322 plasmid DNA, it was heated at 60 °C for 30 min to obtain a majority of open circular form. 15 ng of pBR322 DNA was incubated in an appropriate volume with the required compound concentration corresponding to ri 1:5. Each metal complex was dissolved in a minimal amount of DMSO, and HEPES buffer pH 7.4 was then added up to the required concentration. The different solutions as well as Milli-Q® water were filtered with 0.2 μm FP030/3 filters (Schleicher & Schuell GmbH, Germany). Incubations were carried out at 37 °C for 3 and 22 h. Samples were prepared by placing a drop of DNA solution or DNA-compound solution onto mica (TED PELLA, INC. California, USA). After adsorption for 5 min at room temperature, the samples were rinsed for 10 s in a jet of deionized water (18 M Ω cm− 1 from a Milli-Q® water purification system) directed onto the surface. The samples were blow dried with compressed argon and then imaged by AFM. The samples were imaged by a Nanoscope III Multimode AFM (Digital Instrumentals Inc., Santa Barbara, CA) operating in tapping mode in air at a scan rate of 1–3 Hz. The AFM probe was 125 mm-long monocrystalline silicon cantilever with integrated conical shaped Si tips (Nanosensors GmbH Germany) with an average resonance frequency fo = 330 KHz and spring constant K = 50 N/m. The cantilever was rectangular and the tip radius given by the supplier was 10 nm, a cone angle of 35° and high aspect ratio. The images were obtained at room temperature (T = 23 ± 2 °C) and the relative humidity was usually lower than 40% [22].

(a) ri

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control [PtCl2(HL7)]

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Supercoiled relative electroforetic mobility (%)

(b)

[PdCl2(HL7)] [PtCl2(HL7)]

100 98 96 94 92 90 0

1

2

3

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5

6

ri Fig. 2. Plasmid DNA interaction with (a) [PdCl2(HL6)] and [PdCl2(HL7)] and (b) [PtCl2(HL3)] and [PtCl2(HL7)]. The different complexes studied are indicated at the bottom of each gel section. (c) Supercoiled electrophoretic mobility relative to control (ri = 0; no metal complex added) vs. ri for [PtCl2(HL7)] and [PdCl2(HL7)]. All reactions were incubated in 10 mM Tris–HCl, 0.1 mM EDTA pH 7.4 in a final volume of 20 μL, for 24 h at 37 °C. Control: Plasmid DNA incubated at 37 °C during 24 h. pBSK II BlueScript (Stratagene) DNA (I) supercoiled and (II) circular DNA. ri = metal complex:DNA base pairs molar ratio. Electrophoresis was carried out in the absence of ethidium bromide (EtBr). Gel was stained after run.

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(a)

ri

0.5

3

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Tris, compared to HEPES, to produce a similar level of DNA damage [30]. Accordingly, the effect of the [PtCl2(HL)] complexes on supercoiled DNA was also studied in HEPES buffer. Comparative results for some Pt complexes are shown in Fig. 3. An intensification of the effects of the metal complexes on the tertiary structure of plasmid DNA could be observed using HEPES buffer. Indeed, at lower ri doses, the complete loss of negative supercoils driving to a complete relaxation as well as the mobility shift of the relaxed form due to helix distortion and putative formation of positive supercoils could be observed. At the maximum ri assayed, the almost disappearance of bands, could be interpreted as due to the dispersion of the plasmid DNA forms in multiple topological isomers. Nevertheless, in these cases a degradation effect could not be excluded.

6

Form II Form I

(b) Form II Form I control

[PtCl2(HL4)

[PtCl2(HL2)]

Fig. 3. Plasmid DNA interaction with [PtCl2(HL)] complexes (HL = HL2 and HL4). The different complexes studied are indicated at the bottom of each gel section. All reactions were incubated (a) in 10 mM Tris–HCl, 0.1 mM EDTA pH 7.4 or (b) in 25 mM HEPES pH 7.0 in a final volume of 20 μL, for 24 h at 37 °C. Control: Plasmid DNA incubated at 37 °C during 24 h. pBSK II BlueScript (Stratagene) DNA (I) supercoiled and (II) circular DNA. ri = metal complex:DNA base pairs molar ratio. Electrophoresis was carried out in the absence of ethidium bromide (EtBr). Gel was stained after run.

depended on the nature of both the metal and the ligand (Fig. 2). In general, the effect produced by the palladium complexes was significantly more intense than that observed for the corresponding platinum ones. Previously performed interaction studies of Pd complexes with plasmid DNA in different buffer systems indicated that modification of DNA tertiary structure is dependent on the nature of the buffer used in the electrophoresis experiments [28,29]. For instance, a 10fold higher concentration of cisplatin was required in the presence of

3.1.2. Time course of DNA conformational changes The time course of the conformational changes introduced by the complexes on supercoiled DNA was studied. Results for some of the Pd complexes are presented in Fig. 4a and the corresponding quantification of the relative mobility change is shown in Fig. 4b. The conformational changes introduced in plasmid DNA are already evident after 15 min of incubation. The shifts on supercoiled form migration increase with time almost reaching the plateau after 2 h. Pd complexes produced a fast reduction in plasmid DNA superhelicity. The intensity of the observed effect was dependent on the nature of the HL ligand (Fig. 4). 3.1.3. Effect of the minor groove binder distamycin Distamycin is an oligopeptide antibiotic which has shown a high binding affinity for DNA minor groove, particularly in AT tract sequences [31]. In order to further analyze the interaction of the complexes with plasmid DNA the effect of this minor groove binder was studied. As shown in Fig. 5, distamycin did not inhibit the unwinding effect produced by [PdCl2(HL5)]. Indeed, the supercoiled DNA mobility shift

(a) Time (h)

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100 95 90 85 80 75 70 0

1

2

3 Time (h)

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5

Fig. 4. Time course of [PdCl2(HL)] complexes (HL = HL6 and HL7) unwinding activity on supercoiled plasmid DNA. (a) Complexes were incubated in 10 mM Tris–HCl, 0.1 mM EDTA pH 7.4 in a final volume of 20 μL with pBSK II at ri = 3.0 for the indicated periods of time. Agarose gel electrophoresis was carried out in the absence of ethidium bromide (EtBr). Gel was stained after run. pBSK II DNA (I) supercoiled and (II) circular forms are indicated. (b) % electrophoretic mobility of the supercoiled form in respect to DNA control (t= 0) vs. time.

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(a) CT-DNA r = 0.25 r = 0.5 r=1

2

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3 CD (mdeg)

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0

-1

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-2

control

control

-distamycin

250

300

+distamycin

350

400

450

Fig. 5. Effect of the minor groove binder distamycin on the interaction of [PdCl2(HL5)] with DNA. The complex was incubated in 10 mM Tris–HCl, 0.1 mM EDTA pH 7.4 with pBSK II in a final volume of 20 μL, for 24 h at 37 °C for different ri values in the presence and absence of distamycin. Agarose gel electrophoresis was carried out in the absence of ethidium bromide (EtBr). Gel was stained after run. pBSK II DNA (I) supercoiled and (II) circular forms are indicated.

(b) CT-DNA r = 0.25 r = 0.5 r=1

2

remained unchanged. However, a decrease of the topoisomer dispersion could be observed for the highest ri values.

CD (mdeg)

1

0

-1

3.2. Viscosity measurement results Viscosity measurements are sensitive to length change of DNA. Therefore, the changes on DNA viscosity value are considered conclusive for determining the binding mode of a compound to DNA in solution [32–34]. In this sense, an intercalation mode of interaction lengthens the DNA helix, as base pairs are separated to accommodate the binding ligand, leading to the increase of DNA viscosity. In contrast, other modes of interaction, including covalent binding, could bend (or kink) the DNA helix reducing its effective length and, hence, its viscosity. In order to further elucidate the binding mode of the [MCl2(HL)] complexes under study, viscosity measurements were carried out on calf thymus DNA by varying the ri values. All the studied complexes increased the viscosity of CT DNA solutions in a concentration dependent manner. Results for [PdCl2(HL1)] and [PtCl2(HL3)] complexes are depicted in Fig. 6.

-2 250

300

350

400

450

500

Wavelength (nm) Fig. 7. Circular dichroism spectra of CT-DNA (200 μM) in absence (r = 0) and presence of different concentrations of (a) [PdCl2(HL7)] and (b) [PdCl2(HL6)] (r = 0.25; r = 0.5 and r = 1.0; where r = [PdCl2(HL)]/[CT-DNA]).

3.3. Circular dichroism results Circular dichroism is a spectroscopic technique widely used to study the binding mode and interaction affinity of small molecules, like metal compounds, with biomolecules, particularly with DNA

3

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CT-DNA r=1

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ri Fig. 6. Relative viscosity vs. ri curve for [PdCl2(HL1)] and [PtCl2(HL3)] (ri = mol of complex/mol of DNA base pairs).

Fig. 8. Circular dichroism spectrum of CT-DNA (200 μM) in absence (r = 0) and presence of different concentrations of [PtCl2(HL3)] (r = 1.0; where r = [PtCl2 (HL3)]/[CT-DNA]).

M. Vieites et al. / Journal of Inorganic Biochemistry 105 (2011) 1704–1711

[35,36]. The heterocyclic bases of DNA become chiral when placed within the framework of the chiral sugar–phosphate backbone, leading to a characteristic CD spectrum in the 200–300 nm range. Modifications of the CD signals in this spectral range have shown to be

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useful to detect and follow DNA conformational changes, damage and/or cleavage upon interaction with metal complexes [36–39]. CTDNA shows the typical spectra of a right-handed B form DNA that consists of two bands: a positive one at 275 nm due to the base

pBR322 DNA control, 3h

pBR322 DNA control, 22h

[PdCl2(HL1)], 3 h

[PdCl2(HL1)], 22 h

[PtCl2(HL1)], 3 h

[PtCl2(HL1)], 22 h

Fig. 9. AFM images showing the modifications suffered by pBR322 DNA due to interaction with [PdCl2(HL1)] and [PtCl2(HL1)] complexes for different incubation times (3 and 22 h) and molar ratio compound:DNA base pairs ri = 0.2.

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stacking and a negative one at 245 nm due to the right-handed helicity. Usually, it is described that classical intercalation leads to changes in intensities of both bands due to enhancement of base stacking and stabilization of helicity, whereas simple groove binding and electrostatic interaction of small molecules produce low or no perturbation on the base stacking and helicity bands [40,41]. The conformational changes of CT-DNA induced by the Pd(II) and Pt(II) [MCl2(HL)] complexes were monitored by CD spectroscopy. Some spectral changes due to interaction were detected for the Pd(II) complexes (Fig. 7). On one hand, the intensity of both DNA bands decreased and a red shift of their maxima was observed. According to previous studies, these results suggest that both palladium complexes can interact with CT-DNA inducing conformational changes from BDNA to Z-DNA [41]. However, the changes induced by [PdCl2(HL6)] were more significant than those produced by [PdCl2(HL7)], which suggests that the former has a higher affinity for CT-DNA than the latter. These results are in agreement with those shown for both complexes in the electrophoretic approach (Figs. 2 and 4). In addition, a new negative band was observed in the range 325–350 nm after incubation of the Pd(II) complexes with CT-DNA. Each Pd(II) complex is originally in a non-chiral environment and does not show bands in CD experiments. This new signal could correspond to induced CD spectra, suggesting the binding or at least the formation of an adduct between DNA and the metal complex [36,40,41]. The incubation of CT-DNA with the Pt(II) complexes lead to a quite different effect in the CD spectrum of CT-DNA (Fig. 8). No changes were seen in the plain CT-DNA CD spectrum in the 200–300 nm range for the highest r value tested (r = 1.0). This is an indication that the conformation of the DNA chain has not been significantly changed, but it is not a proof that the Pt(II) complex does not interact with DNA in the assayed conditions. In fact, results of the other assayed techniques showed that Pt complexes do interact with DNA. Taking into account that Pt(II) are more inert than Pd(II) ones it is likely that the incubation time was not enough to allow the production of detectable interactions on CD experiments [35]. 3.4. AFM studies AFM has been used for imaging of single-stranded DNA, doublestranded DNA, DNA–protein complexes and DNA–metal complexes [42]. To provide a deeper insight into the probable mechanism of DNA interaction of [MCl2(HL)] compounds, atomic force microscopy experiments were performed. Consequently to the behavior observed in the electrophoretic experiments, all complexes modified the tertiary structure of the pBR322 DNA. AFM images of the plasmid pBR322 DNA incubated with [MCl2(HL)] complexes in concentrations corresponding to a molar ratio compound:DNA base pairs ri = 0.2 are shown in Fig. 9. After a 3 h incubation, changes in the shape of plasmid DNA, like supercoiling and kinks, could be observed. These changes were more significant after 22 h of incubation. The progressive interaction produced the supercoiling of a greater number of plasmid forms. As previously observed, effects seem to be more intense for the Pd complexes than for the Pt analogs at the same incubation time. The former also showed an increase of DNA thickness. 4. Conclusions The analyzed Pt and Pd complexes introduced anomalous structures on DNA leading to the loss of superhelicity. This effect could be unequivocally stated through the electrophoretic approach complemented with the results of the AFM studies and viscosity measurements. DNA minor groove was discarded as the main interaction site for the assayed complexes as distamycin induced minor changes in the electrophoretic behavior of the compounds and

it did not inhibit the unwinding effect produced by them. On the other hand, DNA scission events were not detected. In general, all the techniques showed that the nature of DNA interaction of both series of analogous palladium and platinum complexes seemed to be similar. However, palladium complexes produced significantly more intense and than those observed for the corresponding platinum ones. Being the isostructural Pt(II) complexes more inert than the Pd(II) ones it is likely that an initial DNA covalent interaction could mediate the final interaction leading to the DNA conformational changes observed. In particular, CD experiments uphold this hypothesis. Results showed the appearance of a new signal when DNA was incubated with the Pd compounds, suggesting the binding or at least the formation of an adduct between DNA and the metal complex. 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