Bioelectrochemistry 76 (2009) 201–207
Contents lists available at ScienceDirect
Bioelectrochemistry
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 / b i o e l e c h e m
In situ electrochemical and AFM study of thalidomide–DNA interaction
S.C.B. Oliveira a, A.M. Chiorcea-Paquim a, S.M. Ribeiro a,b, A.T.P. Melo a,b, M. Vivan c, A.M. Oliveira-Brett a,⁎
a
b
c
Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal
Departamento de Bioquímica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal
Hospital da Universidade de Coimbra, 3000 Coimbra, Portugal
a r t i c l e
i n f o
Article history:
Received 19 December 2008
Received in revised form 5 March 2009
Accepted 10 March 2009
Available online 18 March 2009
Keywords:
Thalidomide
Thalidomide–DNA interaction
DNA-damage
Voltammetry
AFM
a b s t r a c t
The interaction of thalidomide (TD) with double-stranded DNA (dsDNA) was studied using atomic force
microscopy (AFM) at highly oriented pyrolytic graphite (HOPG), differential pulse voltammetry (DPV) at
glassy carbon electrodes (GCE), UV–Vis and electrophoresis. After incubation of dsDNA with different
concentrations of TD, the AFM images show the formation of thin and incomplete TD–DNA network films
with a number of embedded molecular aggregates and regions of uncovered HOPG. Both the TD–dsDNA
aggregates and network thickness directly depended on the TD concentration and incubation time. The
voltammetric data also showed that the modifications caused by TD to the DNA double helical structure are
time-dependent. In agreement with AFM, DPV, UV–Vis and electrophoresis results, a model is proposed for
the TD–DNA interaction, considering that TD intercalates into the dsDNA, causing defects in the dsDNA
secondary structure and DNA double helix unwinding. Moreover, both AFM and DPV show that condensation
is caused to DNA by TD and occurs until 24 h of incubation, as well as DNA oxidative damage, detected
electrochemically by the appearance of the 8-oxoGua and/or 2,8 oxoAde oxidation peak.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
In recent years increased attention has been focused on the ways in
which drugs interact with DNA, with the goal of understanding the
toxic as well as chemotherapeutic effects of these small molecules [1–
3]. Generally, there are two well-characterized binding modes for
small molecules, including molecular drugs, with dsDNA: covalent
(chemical modification of various DNA constituents) and noncovalent (outer electrostatic binding, groove binding, or intercalation)
[3]. Intercalation and groove binding are the two most common
modes by which small molecules bind directly and selectively to
double-stranded DNA (dsDNA). Intercalation, which is an enthalpically driven process, results from insertion of a planar aromatic ring
system between dsDNA base pairs with concomitant unwinding and
lengthening of the DNA helix. In contrast, groove binding, which is
predominantly entropically driven, involves covalent and noncovalent (electrostatic) interactions that do not perturb the duplex
structure to any great extent [4].
Thalidomide (TD) [1,3-dioxo-2-(2′,6′-dioxopiperidin-3-y1)-isoindol or ±phthalimidoglutarimide], Scheme 1, was originally developed
as a sedative and anti-emetic drug to combat morning sickness during
pregnancy. However, TD was removed from the market when its teratogenic side effects, best described as heavy dysmelia (stunted limb
⁎ Corresponding author. Tel./fax: +351 239 835295.
E-mail address: [email protected] (A.M. Oliveira-Brett).
1567-5394/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bioelechem.2009.03.003
growth), appeared in newborn children. In fact, inadequate tests were
performed before its release to assess the drug's safety, with catastrophic results for the children of women who had taken TD during
their pregnancies. In recent years, TD has regained scientific interest
because of its potential for treating a number of otherwise intractable
inflammatory skin diseases: erythema nodosum leprosum (ENL), a
complication of leprosy, graft versus host disease, weight loss in tuberculosis, aphthous ulcers, wasting; and human immunodeficiency virus
replication in acquired immune deficiency syndrome and cancer [5].
Concerning TD teratogenicity, more than 30 hypotheses have been
proposed and conflicting results have been reported [6–10]. One of the
proposed mechanisms of the teratogenic action of TD is that it
intercalates into dsDNA. It was proposed that a stacked complex is
formed between the flat double phthalimide rings of TD and the
deoxyguanosine. Also, it has been stated that the TD in solution
interacts with purines but not with pyrimidines, and that it has a
greater affinity for guanine than for adenine [6].
In the present paper, a systematic study to elucidate the
mechanism of interaction of TD with dsDNA was carried out on two
different types of carbon electrode, glassy carbon using differential
pulse voltammetry (DPV) and highly oriented pyrolytic graphite
(HOPG) using magnetic AC mode atomic force microscopy (MAC
Mode AFM). DPV can be successfully employed for the rapid detection
of small perturbations of the double helical structure and DNA
oxidative damage, due to its high sensitivity and selectivity when
compared with other methods reported in the literature, while AFM
can give important information concerning DNA structural modifications with extraordinary resolution and accuracy [2,3,11–14]. Non-
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integration time 25 ms and accumulation 1000 points. All UV–Vis
spectra were measured from 190 nm to 400 nm.
2.4. DNA gel electrophoresis
Scheme 1. Chemical structure of TD.
electrochemical methods such as gel electrophoresis and UV–Vis
spectroscopy also were used to investigate the interaction of TD with
dsDNA.
2. Experimental
2.1. Materials and reagents
Calf thymus dsDNA, polyguanylic (poly[G]), polyadenylic (poly[A])
acids and (±) thalidomide were obtained from Sigma-Aldrich and
used without further purification. All solutions were prepared using
analytical grade reagents and purified water from a Millipore Milli-Q
system (conductivity ≤0.1 μS cm− 1).
The supporting electrolyte used in all experiments was pH 4.5
0.1 M acetate buffer. The TD solubility in aqueous solution is very low
and was reported to be ~50 µM [5]. Consequently the supernatant of a
TD saturated aqueous solution was used throughout this study, being
prepared by shaking 30 mg of TD with 5 mL of buffer supporting
electrolyte and filtering. Stock solutions of 300 µg mL− 1 dsDNA, poly
[G] and poly[A] were prepared in deionised water and kept at 4 °C. The
solutions were diluted to the desired concentration by mixing buffer
supporting electrolyte.
Microvolumes were measured using EP-10 and EP-100 Plus
Motorized Microliter Pippettes (Rainin Instrument Co. Inc., Woburn,
USA). The pH measurements were carried out using a Crison micropH
2001 pH-meter with an Ingold combined glass electrode. All
experiments were done at room temperature (25 ± 1 °C).
2.2. Atomic force microscopy
HOPG, grade ZYB of 15 × 15 × 2 mm3 dimensions, from Advanced
Ceramics Co., USA, was used as a substrate in the MAC Mode AFM
study. The HOPG was freshly cleaved with adhesive tape prior to each
experiment and imaged by AFM in order to establish its cleanliness.
AFM was performed with a PicoSPM controlled by a MAC mode
module and interfaced with a PicoScan controller from Agilent
Technologies, Tempe, AZ, USA. All the AFM experiments were performed with a CS AFM S scanner with a scan range of 6 µm in x–y and
2 µm in z, from Agilent Technologies. Silicon type II MAClevers of
225 µm length, 2.8 N m− 1 spring constants and 60–90 kHz resonant
frequencies in air (Agilent Technologies) were used. All AFM images
were topographical and were taken with 256 samples/line × 256 lines
and scan rates of 0.8–2.0 lines s− 1. When necessary, MAC Mode AFM
images were processed by flattening in order to remove the background slope and the contrast and brightness were adjusted. Section
analyses were performed with PicoScan software version 5.3.3,
Agilent Technologies, and with Origin version 6.0, Microcal Software,
Inc., USA.
Nondenaturing agarose (0.5%, ultrapure DNA grade from Sigma)
gels were prepared in TA buffer. 40 μL dsDNA and TD–dsDNA samples
aliquots (with 0.25% bromophenol blue in water) were loaded into
wells, and electrophoresis was carried out in TAE buffer for 1 h at
100 V. After 2% ethidium bromide (EtBr) stained DNA was visualized
and photographed.
2.5. Voltammetric parameters and electrochemical cells
Voltammetric experiments were carried out using a µAutolab
running with GPES 4.9 software, Eco-Chemie, Utrecht, The Netherlands. The experimental conditions for DPV were: pulse amplitude
50 mV, pulse width 70 ms and scan rate 5 mV s− 1. Measurements
were carried out using a glassy carbon electrode (GCE) (d = 1.5 mm),
with a Pt wire counter electrode, and an Ag/AgCl (3 M KCl) electrode
as reference, in a 0.5 mL one-compartment electrochemical cell.
The GCE was polished using diamond spray (particle size 1 μm)
before every electrochemical assay. After polishing, the electrode was
rinsed thoroughly with Milli-Q water. Following this mechanical
treatment, the GCE was placed in buffer supporting electrolyte and
various DP voltammograms were recorded until a steady state
baseline voltammogram was obtained. This procedure ensured very
reproducible experimental results.
All the voltammograms presented were background-subtracted
and baseline-corrected using the moving average application with a
step window of 5 mV, included in GPES version 4.9 software. This
mathematical treatment improves the visualization and identification
of peaks over the baseline without introducing artefacts, although the
peak current intensity is in some cases reduced (b10%) relative to that
of the untreated curve.
2.6. Samples preparation
TD–dsDNA, TD-poly[G] and TD-poly[A] were prepared by incubation at room temperature of dsDNA, poly[G] and poly[A] in pH 4.5
0.1 M acetate buffer with different concentrations of the desired TD
solution, during different periods of time. Control solutions of dsDNA,
poly[G] and poly[A] in pH 4.5 0.1 M acetate buffer were also prepared
and stored during the same periods of time in similar conditions as the
TD–dsDNA, TD-poly[G] and TD-poly[A] incubated solutions.
For the AFM experiments, the concentration of dsDNA was 10 μg mL− 1
and the incubation times were: 10 min, 5 h and 24 h. The dsDNA control,
TD and TD–dsDNA modified HOPG surfaces were obtained by depositing
200 µL samples of the appropriate solution, dsDNA, TD or TD–DNA, onto
the freshly cleaved HOPG surface, during 3 min. The excess of solution was
gently cleaned with a jet of Millipore Milli-Q water, and the HOPG with
adsorbed molecules was then dried in a sterile atmosphere and imaged
by MAC Mode AFM in air.
For the voltammetric, electrophoresis and UV–Vis studies, the
concentrations of dsDNA, poly[G] and poly[A] were in the range
50–250 μg mL− 1 and the incubation times were: 5 min, 10 min, 15 min,
30 min, 1 h, 5 h, 24 h and 72 h.
3. Results and discussion
3.1. Atomic force microscopy evaluation of TD–DNA interaction
2.3. UV–Vis absorption
Absorption spectra were recorded using a UV–Vis spectrophotometer SPECORD S100 from Carl Zeiss Technology with Win-Aspect
software. The experimental conditions for absorption spectra were:
The mechanism of interaction of TD with dsDNA was first
investigated by AFM in air. For a correct evaluation of the dsDNA
conformational modifications after interaction with TD, in the AFM
study small concentrations of 10 μg mL− 1 dsDNA and 1 μM, 4 μM and
S.C.B. Oliveira et al. / Bioelectrochemistry 76 (2009) 201–207
9 μM TD, and an atomically smooth HOPG electrode, with less than
0.06 nm of root-mean-square (r.m.s.) roughness for a 1000 × 1000 nm2
surface area, were used. The GCE used for the voltammetric
characterization was much rougher, with 2.10 nm r.m.s. roughness
for the same surface area, therefore unsuitable for AFM surface
characterization [15]. Control experiments using GCE and HOPG
electrodes showed similar electrochemical behaviour.
First, AFM was employed to study the dsDNA spontaneous
adsorption from a control solution of 10 μg mL− 1 dsDNA, Fig. 1A,
showing coiled dsDNA molecules of 1.6 ± 0.4 nm height.
The TD adsorbs spontaneously onto HOPG, leading to the formation of
different morphological films depending on the solution concentration.
AFM images of a TD modified HOPG surface obtained from a solution of
1 μM TD, show only a few molecules assembled as small, 1.7±0.4 nm
height, spherical aggregates, Fig. 1B. For concentrations of 4 μM TD,
larger aggregates are observed, forming an incomplete network of
1.6 ± 0.4 nm height, Fig. 1C. The HOPG coverage and the height of the
TD network film increases with increasing the TD concentration to
9 μM. The molecules form looped filaments of 1.9 ± 0.3 nm height
that still do not cover completely the electrode, Fig. 1D.
The TD–dsDNA modified HOPG surfaces were obtained as described
in Section 2.6, by spontaneous adsorption from incubated solutions of
10 μg mL− 1 dsDNA with 1 μM (Fig. 2A), 4 μM (Fig. 2B–D) and 9 μM
(Fig. 2E and F) of TD, during several periods of time. This procedure led to
co-adsorption of TD–dsDNA, free dsDNA and free TD molecules. As
described below, the AFM images always show the formation of a thin
and incomplete network film, which presents a number of molecular
aggregates embedded into its structure, and regions of uncovered HOPG.
Both the TD–dsDNA network thickness and aggregate height are directly
depending on the TD concentration and incubation time.
203
The TD–dsDNA layer adsorbed onto HOPG from an incubated
solution of dsDNA with 1 μM TD during 10 min (data not show)
presents an average height and standard deviation of 1.7 ± 0.1 nm, and
very small aggregates of 2–5 nm height. A similar TD–dsDNA lattice of
1.6 ± 0.3 nm height is observed after a long 24 h incubation time, but
larger 4–11 nm height aggregates are formed, Fig. 2A.
A more complex situation was observed when dsDNA was incubated
with more concentrated solutions of TD. AFM images of the TD–dsDNA
film formed with 4 μM TD, during 10 min, Fig. 2B, show molecules selfassembled in a more densely packed lattice of 1.6 ± 0.3 nm height, with a
large number of 2–10 nm height aggregates. Increasing the incubation
time to 5 h, Fig. 2C, and 24 h, Fig. 2D, the TD–dsDNA network became
thinner, 1.2 ± 0.2 nm after 5 h and 1.3 ± 0.2 nm height after 24 h, while
the molecular aggregates decrease in number and become higher, up to
15 and 20 nm height, respectively.
The same effect of decreasing the thickness of the TD–dsDNA
network, decreasing the number of molecular aggregates and increasing
the aggregate height, with increasing incubation time from 10 min to
24 h, was observed for the TD–dsDNA films formed by incubation of
dsDNA with 9 μM TD. The TD–dsDNA lattice formed from solutions
incubated during 10 min is 1.8 ± 0.3 nm thick and the aggregates are less
than 4 nm height, Fig. 2E, while the film formed after 24 h incubation is
1.1 ± 0.2 nm height with 3–20 nm height aggregates, Fig. 2F.
The AFM images showed that the TD–dsDNA films obtained
immediately after the preparation of the incubated solutions (10 min)
always presented similar thicknesses to those of both control dsDNA
and TD films formed under the same experimental conditions.
Additionally, a large number of small aggregates are observed, related
with condensed DNA molecules formed after interaction with TD.
Increasing the incubation time, a decrease of the TD–dsDNA film
Fig. 1. AFM images of (A) dsDNA and (B–D) TD modified HOPG obtained from solutions in pH 4.5 0.1 M acetate buffer: (A) 10 μg mL− 1 dsDNA and (B) 1 μM, (C) 4 μM and (D) 9 μM TD.
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Fig. 2. AFM images of TD–dsDNA modified HOPG obtained from incubated solutions in pH 4.5 0.1 M acetate buffer of 10 μg mL− 1 dsDNA with (A) 1 μM TD, 24 h; (B–D) 4 μM TD:
(B) 10 min, (C) 5 h, (D) 24 h; (E, F) 9 μM TD: (E) 10 min, (F) 24 h.
thickness is observed, when compared with the control dsDNA and
TD, due to structural modifications of the DNA double helix and
reorganization of the DNA self-assembled on the HOPG surface. The
number of TD–dsDNA aggregates decreases with the incubation time,
but they get larger, suggesting that TD induces large morphological
changes in the dsDNA structure.
3.2. UV–Vis evaluation of TD–DNA interaction
To determine whether TD influences the dsDNA conformation, the
differences in the absorption spectra of dsDNA were monitored in the
absence and presence of TD. The absorption spectra of 20 µg mL− 1
dsDNA and 50 µM TD are shown in Fig. 3.
A typical main absorption band at 260 nm is observed in dsDNA,
while TD shows three bands, the first at 220 nm followed by two other
bands at 242 nm, and at 300 nm. When dsDNA and TD were mixed, after
10 min of incubation the spectra exhibited only two bands at 220 nm and
242 nm and the absorption intensity diminished. The absorption bands
at 260 nm and 300 nm nearly disappeared. This is a strong indication
that there was a DNA conformational transition during incubation with
TD, which leads to the condensation of the DNA double helix structure.
3.3. Electrophoresis evaluation of TD–DNA interaction
Gel retardation has been widely used to monitor DNA structural
change [11,16]. Compact DNA is resistant to intercalating dyes and
S.C.B. Oliveira et al. / Bioelectrochemistry 76 (2009) 201–207
205
excluded from binding sites due to the TD intercalation in the double
helix structure of DNA and/or that TD–DNA was in a condensed form
with a lower electrophoretic mobility into the gel, when compared
with the control dsDNA.
The sample containing 100 µg mL− 1 dsDNA incubated with 50 µM
TD during 72 h (lane 3) presented a slightly longer migration and the
intensity of the bands located near the loaded well increased when
compared with the results obtained with the control dsDNA solution
(lane 1). Consequently, it can be concluded that, for the experimental
conditions used, a DNA conformational transition occurred during
incubation with TD, which led to the modification of the DNA
secondary structure [11].
3.4. Voltammetric evaluation of TD–DNA interaction
Fig. 3. Absorption spectra of: (▬) 20 µg mL− 1 dsDNA; (•••) 50 µM TD and (▪▪▪) 50 µg mL− 1
dsDNA incubated with 50 µM TD during 10 min. Inset. Insert shows the absorption
spectra between 240 and 330 nm.
several DNA intercalators such as ToTo, YoYo, syber Gold and EB, were
being used as fluorescent quenching assay to examine DNA condensation [17].
Gel electrophoresis was performed as described in Section 2.4 to
examine conformation changes in dsDNA during incubation with TD.
The electrophoretic control experiments were of crucial importance to
confirm by another method the detection and correct interpretation of
the effects caused by TD on dsDNA containing solutions.
The electrophoretic migration profile of the incubated solutions; of
100 µg mL− 1 dsDNA with 50 µM TD during 10 min and 72 h (lanes 2
and 3, respectively) and of 250 µg mL− 1 dsDNA with 25 µM TD during
10 min and 72 h (lanes 5 and 6, respectively) is shown in Fig. 4. The
control solutions of 100 and 250 µg mL− 1 dsDNA were analyzed after
72 h whereas the TD–dsDNA incubated solutions (lanes 1 and 4,
respectively) and the two bands observed for dsDNA in the absence of
TD correspond to different-sized length fragments present in the
loaded samples [11].
In all DNA samples that were incubated with TD, except line 3, the
EB intensity decreased when compared with the results obtained from
the control DNA solutions (lanes 1 and 4). This indicates that EB is
Fig. 4. Nondenaturing agarose (0.5%) gel electrophoresis of 100 and 250 µg mL− 1
dsDNA control (lanes 1 and 4) and incubated solutions during 10 min and 72 h of:
100 µg mL− 1 dsDNA with 50 µM TD (lanes 2 and 3) and 250 µg mL− 1 dsDNA with
25 µM TD (lanes 5 and 6).
The oxidation behaviour of TD [5], poly[G], poly[A] and dsDNA in
pH 4.5 0.1 M acetate buffer was briefly revisited using DPV, Fig. 5, in
order to make it easier to identify the peaks occurring after the TD–
dsDNA, TD-poly[G] and TD-poly[A] interactions. The DP voltammogram of TD is shown in Fig. 5A and only one oxidation peak occurs at
Epa = + 0.80 V, due to oxidation of phthalimide ring, where one
electron is removed, following deprotonation and direct nucleophilic
attack by water with the production of 5-hydroxythalidomide,
Scheme 2.
The oxidation of dsDNA shows two small oxidation peaks, Fig. 5B,
corresponding to the oxidation of desoxyguanosine (dGuo), at
Epa = + 1.03 V [18], and desoxyadenosine (dAdo), at Epa = +1.30 V [19],
residues in the polynucleotide chain. The DP voltammogram of
dsDNA shows small oxidation peaks due to the difficulty of the
electron transfer from the inside of the double-stranded rigid form of
DNA to the electrode surface.
The DP voltammograms of poly[G] and poly[A] show only one
oxidation peak, Fig. 5B, the poly[G] homopolynucleotide contains only
guanine residues and the oxidation occurs at the desoxyguanosine
(dGuo) residue at Epa = +1.03 V, while the poly[A] contains only
adenine residues and the oxidation occurs at desoxyadenosine (dAdo)
at Epa = + 1.30 V.
The interaction between dsDNA and TD was studied using DPV. In
all experiments, different concentrations of TD were incubated for
different periods of time with 100 µg mL− 1 dsDNA and their
interaction was followed by detection at CGE directly in solution.
The CG surface was cleaned between each measurement to avoid
Fig. 5. Background-subtracted DP voltammograms of (A) ~50 µM TD and (B) 100 µg mL− 1
of: (▬) dsDNA, (•••) poly[G] and (▪▪▪) poly[A], in pH 4.5 0.1 M acetate buffer.
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S.C.B. Oliveira et al. / Bioelectrochemistry 76 (2009) 201–207
Scheme 2. Oxidation mechanism of TD in pH 4.5.
current decrease due to strong absorption of both TD and dsDNA after
successive scans.
The effects of the TD–dsDNA interaction were followed by
comparing the changes of the TD oxidation peak in the absence/
presence of the DNA, the changes of the DNA oxidation peaks,
desoxyguanosine (dGuo) and desoxyadenosine (dAdo) in the
absence/presence of TD and monitoring the appearance of the
guanine and/or adenine oxidation product peak at Epa = +0.45 V,
vs. Ag/AgCl, in pH 4.5 0.1 M acetate buffer, as an indicator of oxidative
damage caused to DNA [20,21]. A control solution of 100 µg mL− 1
dsDNA was also prepared in buffer and analyzed after the same
periods of time as the TD–dsDNA incubated solutions.
The interaction between 100 µg mL− 1 dsDNA and ~40 µM TD was
initially studied after 10 min of incubation. The DP voltammogram
recorded does not show any peaks when compared with the results
obtained from the control dsDNA solution, Fig. 6A. This indicates that
dsDNA became condensed after the TD–DNA interaction and the DNA
Fig. 6. Background-subtracted DP voltammograms of ( ) control 100 µg mL− 1 dsDNA
and (▬) incubated solutions in pH 4.5 0.1 M acetate buffer of: (A) 100 µg mL− 1 dsDNA
with ~40 µM TD during 10 min, 1 h and 5 h, and (B) 100 µg mL− 1 dsDNA with ~ 40 µM
TD during 72 h, in pH 4.5 0.1 M acetate buffer.
bases and the intercalated TD are hindered inside the compact structure,
which makes the electron transfer to the electrode surface difficult and
explains the disappearance of the TD, dGuo and dAdo oxidation peaks.
Moreover, the absence of the TD oxidation peak confirmed that the
disappearance of the dsDNA oxidation peaks is indeed due to the dsDNA
condensation, and is not caused by blocking of the GCE by TD oxidation
products. Furthermore, since no peaks correspond to 8-oxoGua or
2,8-oxoAde, the oxidation products of dGuo or dAdo, were observed
in the DP voltammogram, it was concluded that, for the experimental
conditions used, there was no oxidative damage.
On the other hand, the DP voltammograms obtained after 1 h of
incubation showed four small well-defined oxidation peaks Fig. 6A.
The two oxidation peaks of dGuo and dAdo were observed, although
they showed a large decrease of the oxidation current due to the TD–
DNA condensation, when compared with the results obtained from the
control dsDNA solution. The oxidation peak found at Epa = +0.45 V is
attributed to 8-oxoGua and/or 2,8 oxoAde oxidation, and the peak
observed at Epa = + 0.80 V is due to the oxidation of TD intercalated in
the dsDNA and/or the oxidation of free guanine released from dsDNA
after 1 h of incubation, since both the oxidation of TD and of free
guanine occurs at exactly the same value of potential.
When TD–dsDNA was investigated after 5 and 24 h (data not
shown) incubation, Fig. 6A, the DP voltammograms showed again four
well-defined small oxidation peaks and an increase in all oxidation
peak currents was observed when compared with the results obtained
after 1 h of incubation, although the dGuo and dAdo oxidation peaks
were still smaller than the ones observed for control dsDNA, which
demonstrates that condensation of TD–DNA is still occurring.
The DP voltammograms obtained after 72 h again showed four
well-defined oxidation peaks and a large increase in all oxidation peak
currents was observed, Fig. 6B, when compared with the results
obtained after incubation during 1 and 5 h and with the control
dsDNA, and no condensation was observed.
Similar experiments were carried out for different incubation
times and concentrations of TD (not shown). The increase in current
of the dGuo and dAdo oxidation peaks and of the peaks observed at
Epa = + 0.45 V and Epa = + 0.80 V was proportional to the increase in
concentration and incubation time.
In order to obtain more information about the interaction TD–
dsDNA, several experiments were performed using polynucleotides of
known sequences, namely poly[G] and poly[A] (not shown). The
interaction between TD-poly[G] and TD-poly[A] was studied after
10 min and 72 h of incubation, as described in Section 2.6. The
voltammograms recorded showed a large decrease in dGuo and dAdo
oxidation peak currents, when compared with the results obtained
from the control poly[G] and poly[A] solution, and no oxidation peaks
were found at Epa = +0.45 V and Epa = +0.80 V. The experiments
showed that TD has affinity for guanine and adenine, intercalating and
condensating the quadruple and/or double helix structure of poly[G]
and the double helix structure of poly[A] formed in acid media, at
pH 4.5 [22–24]. As no peaks corresponding to 8-oxoGua and/or
2,8-oxoAde were observed, it was concluded that, for the experimental conditions used, there was no oxidative damage. This can be
explained by the greater stability of the poly[G] and poly[A] complex
secondary structures formed at pH 4.5 that make the ability of TD to
S.C.B. Oliveira et al. / Bioelectrochemistry 76 (2009) 201–207
cause oxidative damage difficult. However, due to the structural difference between poly[G] and poly[A], it was not possible to compare
the affinity of TD for each of these molecules.
The voltammetric data, which agreed well with the AFM, UV–Vis
and electrophoresis results, are consistent with a model proposed to
describe the TD interaction with the dsDNA, in which TD leads to
modifications in the DNA double helical structure in a time-dependent
manner. After 10 min incubation time the TD intercalates into the
double-stranded rigid form of DNA and also induces condensation,
confirmed by the disappearance of the dGuo and dAdo oxidation
peaks and by the formation of a large number of spherical TD–DNA
aggregates observed by AFM.
The intercalation, with the formation of a stacked complex
between the flat double phthalimide rings of thalidomide and
desoxyguanosine, was previously described in the literature [6]. This
first step of TD–DNA intercalation changes the physical properties of
the DNA double helix: the base pairs become unstuck vertically to
allow TD intercalation, the sugar-phosphate backbone is distorted and
the helical structure is destroyed [22]. Consequently, the TD–DNA
intercalation is followed by unwinding of the DNA double helix, which
leaves the DNA bases and the intercalated TD more exposed to the
electrode surface, thus facilitating their oxidation. This explains the
increase of the TD, dGuo and dAdo oxidation peak currents and the
decrease of the TD–DNA film thickness observed in the AFM images,
when increasing the incubation time. However, both AFM and DPV
show that condensation of the TD–DNA still occurs until 24 h of
incubation. Moreover, the oxidative damage caused to DNA by TD was
also detected electrochemically by the appearance of the 8-oxoGua
and/or 2,8 oxoAde oxidation peak.
4. Conclusion
Voltammetric, AFM, UV–Vis and electrophoresis results demonstrated that TD interacts specifically with the dsDNA by intercalation,
inducing structural changes in B-DNA, in a time-dependent manner.
A reorganization of the self-assembled network on the surface of
the HOPG electrode was observed after incubation of dsDNA with
different concentrations of TD, resulting in a decrease of the TD–DNA
network film thickness, decrease of the number of molecular TD–DNA
aggregates and increase of the TD–DNA aggregate height, with
increasing incubation time. The voltammetric study showed that TD
has affinity for both guanine and adenine, and the interaction between
TD and dsDNA induced major changes in the oxidation peaks of the
TD, dGuo and dAdo, depending directly on the dsDNA and TD
concentrations and incubation time. Oxidative damage caused to DNA
by TD was also detected electrochemically by the appearance of the
8-oxoGua and/or 2,8-oxoAde oxidation peak.
Multiple time and concentration dependent effects on the dsDNA
structure are induced by TD: the aggregation and the initial
disappearance of the dGuo and dAdo oxidation peaks related to the
TD induced DNA condensation; the formation of thinner TD–DNA
films and the increase of the dGuo and dAdo peaks with increasing the
incubation time related to the DNA double helix unwinding that
follows the TD intercalation into the dsDNA.
Consequently, the toxic effects of TD caused to dsDNA, i.e. DNA
condensation, intercalation followed by DNA double helix unwinding
and oxidative damage, are explained.
207
Acknowledgements
Financial support from Fundação para a Ciência e Tecnologia (FCT),
SFRH/BD/27322/2006 (S.C.B. Oliveira) and SFRH/BPD/27087/2006
(A.M. Chiorcea-Paquim), project PTDC/QUI/65255/2006, POCI (cofinanced by the European Community Fund FEDER), and CEMUC-R
(Research Unit 285), is gratefully acknowledged.
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