DNA-Metallodrugs Interactions Signaled by Electrochemical Biosensors: An Overview
The interaction of drugs with DNA is an important aspect in pharmacology. In recent years, many important technological advances have been made to develop new techniques to monitor biorecognition and biointeraction on solid devices. The interaction between DNA and drugs can cause chemical and conformational modifications and, thus, variation of the electrochemical properties of nucleobases. The propensity of a given compound to interact with DNA is measured as a function of the decrease of guanine oxidation signal on a DNA electrochemical biosensor. Covalent binding at N7 of guanine, electrostatic interactions, and intercalation are the events that this kind of biosensor can detect. In this context, the interaction between a panel of antitumoral Pt-, Ru-, and Ti-based metallodrugs with DNA immobilized on screen-printed electrodes has been studied. The DNA biosensors are used for semiquantitative evaluation of the analogous interaction occurring in the biological environment.
1. INTRODUCTION<!>2.1. Preparation of the screen-printed electrodes (SPEs)<!>2.2. Preparation of the biosensor<!><!>2.2. Preparation of the biosensor<!>2.3. Platinum complexes [64, 65]<!>2.4. Ruthenium complexes: NAMI-A<!>2.5. Titanium complexes<!>3. CONCLUDING REMARKS
<p>Chemotherapy is an important weapon for combating
cancers. Numerous compounds have been developed as potential candidates for
anticancer drugs, but only a handful of them have become effective in clinical
protocols. The need of developing new drugs in order to effectively treat
various forms of cancer is widely recognized. The development of new drugs
requires that the underlying mechanism of action at the cellular and molecular
levels has been completely understood.</p><p>The potential targets for anticancer drugs are
essentially four: nucleic acids, specific enzymes, microtubules, and
hormone/growth factor receptors. When nucleic acids are the target, the DNA
damage causes cell death (cytotoxic and genotoxic drugs).</p><p>There are several mechanisms by which drugs can bind
DNA [1], the most well
understood being alkylation of nucleophilic sites within the double helix. Most
clinically effective alkylating agents have two moieties capable of developing
transient carbocations that bind covalently to the electron-rich sites on DNA
such as the N7 position of guanine (electrophilic agents). The cross-linking of
the two strands of DNA produced by the bifunctional alkylating agents prevents
the use of that DNA as a template for further DNA and RNA synthesis leading to
inhibition of replication and transcription and, then, to cell death. A large
number of chemical compounds are alkylating agents under physiologic
conditions, and a variety of such compounds have exhibited antitumor activity.
To this category belong "nitrogen mustards" (mechlorethamine, the original
"nitrogen mustard," cyclophosphamide, ifosfamide, melphalan, and chlorambucil),
aziridines and epoxides (thiotepa, mitomycin C, and diaziquone), alkyl
sulfonates (like busulfan and its analogues), nitrosoureas (carmustine,
lomustine, and semustine, above all), triazenes, hydrazines, and related
compounds. Moreover, also cisplatin and its congeners are traditionally, albeit
improperly, considered alkylating drugs.</p><p>A second mechanism of drug binding to nucleic acids is
intercalation, that is, the insertion of a planar (generally aromatic) ring
molecule between two adjacent nucleotides of DNA. This mechanism is
characteristic of many antitumor antibiotics, such as daunorubicin and
doxorubicin. The antibiotic molecule is noncovalently, although firmly, bound
to DNA and distorts the shape of the double helix resulting in inhibition of
DNA replication and RNA transcription.</p><p>Finally, a third mechanism of DNA damage is
illustrated by bleomycins. These glycopeptides intercalate between
guanine-cytosine base pairs of DNA. The end of the peptide binds Fe(II), able
to catalyze the reduction of molecular oxygen to superoxide or hydroxyl
radicals, that produce DNA strand scission by oxidative stress [2].</p><p>Due to the stringent relationship between DNA-drug
interaction and potential antitumor effect, molecular recognition of
nucleic acids by low molecular weight compounds is an area of fundamental
interest. A detailed understanding of the interaction of small molecules with
DNA is very important in pharmacology.</p><p>In this context, cisplatin (cis -diamminedichloroplatinum(II),
[Pt(NH3)2Cl2]) probably represents a milestone in the antitumor
chemotherapy. The activity of cisplatin was serendipitously discovered in 1969
by Rosenberg and colleagues while studying the effects of electric current on
bacterial growth. Cisplatin entered clinical trials in the early 1970s and was
found to have significant antitumor activity against testicular, ovarian,
bladder, and head and neck cancer. Because of the nephro- and neuro-toxicity of
cisplatin, there have been intensive efforts to devise analogues with similar
or improved pharmacological characteristics. Carboplatin, cis -diammine-(1,1-cyclobutanedicarboxylato)platinum(II)
(approved worldwide in 1992), shows an antitumor activity similar to that of
cisplatin, but with reduced systemic toxicity (better therapeutic index), while
oxaliplatin (1R,2R-diaminocyclohexane)oxalatoplatinum(II) (approved for
clinical use worldwide in 2003) is effective against colorectal tumors, which
are nonsensitive to cisplatin. The consciousness that cisplatin readily reacts
with DNA, and that this reaction is crucial in the antitumor activity, focused
a great attention in the field of the interaction between metal complexes and
biomolecules. Today, forty years after the discovery of the properties of
cisplatin, there is no other metal, that is, better understood in its
reactivity toward DNA than platinum. Moreover, despite the enormous amount of
other metal complexes tested, cancer chemotherapy using metallopharmaceuticals
is still largely dominated by platinum compounds [3]. The exploration of
nonplatinum metal complexes for use as anticancer agents was initiated in
attempts to find less toxic and more specific drugs. In this framework, some
ruthenium and titanium complexes have offered the most encouraging results
[4]. The imidazolium trans -[tetrachlorodimethylsulfoxideimidazoleruthenate(III)]
(NAMI-A, [ImH], [trans-RuCl4(DMSO)Im], Im = imidazole) failed the
classical screens of putative anticancer agents, but prevented the development
and growth of pulmonary metastases in solid tumors [5–8], and recently has successfully completed Phase I Clinical Trials [9]. The closely related compound, indazolium trans-[tetrachlorobis(1H -indazole)ruthenate(III)]
(KP1019, [IndH], [trans-RuCl4Ind2], Ind = indazole) induces apoptosis in
colorectal carcinoma cells and it is currently in Phase I Clinical Trials
[10]. Two titanium
compounds [11], namely
titanocene dichloride [TiCl2Cp2][12, 13] and budotitane, a β -diketonate
derivative, [cis-diethoxybis(1-phenylbutane-1,3-dionato)titanium
(IV)], [14], reached
Phase II and Phase I Clinical Trials in Germany, respectively. The general
mechanism of action of these nonplatinum compounds is not completely understood
yet, but many observations point out that DNA may not be the primary target of
these classes of compounds [15, 16].</p><p>Characterization of DNA-adducts generally requires a
combination of chemical and biological techniques to obtain structural
information and to assess the extent and the nature of specific type of binding
to DNA, in terms of dissociation constant, stoichiometry, and kinetic constant.
Methods able to evaluate the presence of any interaction and, in some cases, to
calculate the binding parameter can be classified as mixture- and
separation-based methodology. The mexture-based type includes UV absorption and
fluorescence [17],
nuclear magnetic resonance (NMR, [18]),
and Raman spectroscopy [19], mass spectrometry (MS, [20]), calorimetry [21], and surface plasma
resonance [22]. The
separation-based methods include dialysis, ultrafiltration,
ultracentrifugation, chromatography (liquid chromatography and thin-layer
chromatography), and electrophoresis (planar and capillary electrophoresis
[23, 24]). The last two separation methods
are generally combined with sensitive detection techniques (hyphenated
techniques), such as MS.</p><p>Among the physicochemical techniques, there has been a
growing interest in electrochemical investigations. Compared to other methods,
electrochemical techniques are characterized by simplicity and reliability and
require small amounts of sample, thus offering advantages over biological and
chemical assays. Since many small molecules exhibit redox activity,
electrochemical method should provide a useful complement to the previously
listed methods of investigation.</p><p>The electrochemical method is mainly based on the
differences in the redox behavior of the nucleic acid-binding molecules in the
absence and presence of DNA—including the shifts of the formal potential
of the redox couple and the decrease of the peak current resulting from the
dramatic decrease in the diffusion coefficient after association with DNA
(solution electrochemical methods) (see [25] for a recent review).</p><p>On the other hand, since the discovery of the
electrochemical activity of nucleic acids by Paleček at the end
of the 1950s [26], also DNA has been on the focus of the electrochemical techniques. The binding of drugs to DNA has been described by means of the variation of the oxidation
peak current of the electroactive nucleobases, such as guanine and adenine, in
the presence of the interacting species.</p><p>According to a recent IUPAC document [27], a biosensor is defined as
a specific type of chemical sensor comprising a biological recognition element
and a physicochemical transducer. The biological element is capable of recognizing
the presence, activity, or concentration of a specific analyte in solution. The
recognition may be either a binding process (affinity ligand-based biosensor,
when the recognition element is, e.g., an antibody, a DNA segment, or a cell
receptor) or a biocatalytic reaction (enzyme-based biosensor). The interaction
of the recognition element with a target analyte results in a measurable change
in a given property. The transducer converts the change in solution property
into a quantifiable signal. The mode of transduction may employ several
techniques, including electrochemical, optical, and mass or heat measurements.
In our case, the electrochemical DNA biosensor consists of a nucleic acid
recognition layer, that is, immobilized over an electrochemical transducer
[28]. The signal
transducer monitors the change that has occurred as a consequence of the
binding, converting this into an electronic signal [29]. Observing the pre- and
postelectrochemical signals of DNA-drug interaction provides good evidence for
the event. The reproducibility of the experiment is strictly related to the
history of the electrode surface. In particular, the preparation of the
electrode surface influences the final response. For this reason, the use of
disposable, low-cost electrode characterized by high reproducibility overcomes
the problem, as far as a new, fresh surface is used in each experiment. Various
planar technologies are employed for developing solid-state sensors having the
above-said characteristics [30]. Screen printing is especially suitable due to its
simplicity, low-cost, high reproducibility, and efficiency in large-scale
production. This technology enables the deposition of a thick layer of
conductive ink on inexpensive substrates and allows precise pattern control.</p><p>Although systematic research in this field started
recently, several seminal review articles have already been focused on this
topic [31–44].</p><p>The use of DNA-based biosensors is not limited to the
study of interaction between drugs and DNA, but many other applications have
been reported. On one hand, DNA- iosensors have been used to test water, food,
soil, fish bile [45],
and plant samples for the presence of mutagenic pollutants with binding
affinities for the structure of DNA [33, 45–54]; on the other hand,
DNA-based affinity biosensors have been used to detect specific oligonucleotide
sequences in order to find the presence of genes (or mutant genes) associated
with particular human diseases [55]. Both aspects are beyond the focus of this paper and
will not be discussed further.</p><p>However, specific oligonucleotide sequences may be
related to the protective cell mechanisms that act against anticancer drugs
(drug resistance). One of the main obstacles in the use of metallodrugs in
clinical treatment is the development of resistance. In the case of platinum
drugs, many mechanisms have been proposed to explain resistance, suggesting
that this phenomenon is multifactorial: decrease of intracellular drug
accumulation, faster repair of DNA adducts, and increased activity of
intracellular pathways of thiol production, in particular glutathione,
metallothionein, and thioredoxin, known to be involved in the detoxification of
metals. For these reasons, any information on DNA-binding modes, recognition,
and repair of DNA damage may be helpful not only to understand the molecular
basis of the repair mechanisms, but also to develop new classes of compounds
with improved pharmacological properties [56, 57].</p><!><p>Screen printing is a technique conventionally used in
the graphics industry, in the production of circuit boards, or in printing
t-shirt designs. When inexpensive, easily mass produced, and, therefore,
disposable electrodes for the development of electrochemical biosensors are
required, screen-printing is a viable production method. Single use sensors
assure avoidance of contamination between samples and constant sensitivity of
the different printed sensors. A wide range of different inks (carbon or noble
metals-based) and base materials (ceramics or plastic base materials) can be
combined to produce electrode systems to suit specific applications.</p><p>The planar SPEs used in our laboratories have a
three-electrode configuration (Figure 1). They are printed by using inks
consisting in finely divided particles of different materials in a blend of
thermoplastic resins (silver ink for the reference electrode, graphite ink for
working and counter electrodes, while titanium dioxide ink was used for
insulating the electrodes).</p><p>The sensors were produced in sheets of 80 electrodes.
To facilitate handling, the screen-printed electrochemical cells were stuck on
a rigid polycarbonate-based support. Each (disposable) electrode can be easily
cut by scissors and fits a standard electrical connector [30, 58, 59].</p><!><p>As already mentioned in the Introduction, a biosensor
is defined as an analytical device, which is capable of providing quantitative
or semiquantitative analytical information using a biological recognition
element either integrated within or intimately associated with a
physicochemical transducer [27].
In our case, the transducer is the SPE while the oxidation peak of guanines is
used as the transduction signal for recognize DNA interactions.</p><p>The preparation and the following measurement of the
interaction at the DNA-modified SPEs include four steps [50]</p><!><p>Electrode activation: a (mild) surface conditioning step is necessary to oxidize the
graphite impurities and to obtain a more hydrophilic surface to favor DNA
immobilization (+1.6 V versus Ag-pseudoreference for 120 seconds and +1.8 V for 60 seconds
in 0.25 M acetate buffer, containing 10 mM KCl, pH 4.75, under stirred
conditions).</p><p>DNA immobilization: in this step DNA is electrochemically accumulated and adsorbed onto the
electrode surface by applying a positive potential able to attract negative
charged groups of DNA (activated SPE is dipped in a solution of 50ppm calf
thymus ds-DNA in 0.25 M acetate buffer with 10 mM KCl, applying a potential of +0.5 V versus Ag-SPE
for 5 minutes, under stirred conditions).</p><p>Blank or sample interaction: in this step, the response of the guanine before
(reference signal) or after interaction is evaluated (the DNA-modified SPE is
dipped for 2 minutes in a solution containing the interacting molecule dissolved
in a suitable buffer/saline solution or in the same buffer saline solution without
any analyte, to obtain the reference).</p><p>Measurement: a square wave voltammetric (SWV) scan is carried out to evaluate the oxidation
of guanine residues on the electrode surface (the height of the guanine peak at +0.95 V versus Ag-SPE
was measured in 0.25 M acetate buffer, containing 10 mM KCl).</p><!><p>The four-step protocol is the result of a series of experiments aimed to optimize the final response, in terms of peak height and
reproducibility of the signal [50, 60]. The results showed an increase of the sensitivity increasing DNA concentration (until a saturation phenomenon occurred) and the
immobilization time (similar results were obtained by other authors. See [61]). The 50 ppm
ds-DNA concentration and an immobilization time of 5 minutes (step 2) were chosen as the best compromise for further experiments.</p><p>DNA biosensor performances were strongly influenced by the physical properties of DNA (i.e., purity, average chain length, presence of ss-DNA) [48].
Moreover, the solution where the final measurement is performed influences the
signal aspect: the acetate buffer gives the best results [48, 50], and this choice was
reported also by other authors [62].</p><p>We usually estimate the DNA modification due to the
interaction with the analyte with the value of the percentage of signal
decrease (S%). This value is the ratio of the guanine peak height
after the interaction of the DNA adsorbed onto the SPE with the analyte (S
sample) and the guanine peak height of the DNA in the buffer
solution without drug (S
blank), S%=(S
sample/S
blank) × 100. A typical voltammogram is shown in 
Figure 2.</p><p>It must be noted that the two curves of Figure 2 have
been obtained from two different SPEs. In fact, only one SWV scan is allowed on
each biosensor. If a second SWV is performed, no peak can be observed because
of the complete oxidation of the guanine of the immobilized DNA [48]. For this reason, the final S% values are
expressed as a mean of (at least) three independent measurements. With this
procedure, the "memory effect" between one sample and another is avoided. The
phenomenon referred to as "electrode fouling," which is one of the main
drawbacks of the electrochemical sensors, is overcome and no calibration is
required. Furthermore, the reproducibility of the guanine peak height,
calculated over three or more scans on different electrodes is very high, and
the standard deviation was estimated to be less than 10%.</p><p>It is also possible to study the adenine oxidation
peak, but in this case less reproducible signals are obtained (see error bars
in Figure 2) [54].
Moreover, this peak is sometimes obscured by the solvent discharge.</p><p>Covalent binding with one or both grooves of the
double helix, hydrogen and/or van der Waals bonds and intercalation of planar
condensed aromatic ring systems between adjacent base pairs (π -stacking) are the perturbations that the
electrochemical DNA biosensor can detect [63].</p><!><p>Cisplatin, 1 (Figure 3), is administered
intravenously for clinical use. In the extracellular environment, the platinum
compound experiences high chloride concentration (∼ 100 mM) and does not undergo appreciable hydrolysis. When cisplatin passes the cell membrane, the reduced intracellular chloride
concentration (∼ 5−10 mM) allows the chloro ligands to be replaced in
a stepwise manner by water molecules to form cis-[Pt(H2O)(NH3)2Cl]+ and cis-[Pt(H2O)2(NH3)2]2+ [66]. It is generally accepted that these two ions are
much more reactive than cisplatin and, therefore, react with N-donor ligands,
such as DNA nucleobases (the preferred target on DNA is recognized as the
guanines having the highest electron density of all four nucleobases), leading
to the bending of the DNA structure by 35−40° [67–69]. This key reaction is responsible for the anticancer effect of cisplatin which is able to induce
apoptosis/necrosis of the cancer cell [70]. Carboplatin, 2 , undergoes much slower hydrolysis than cisplatin. Since the DNA reactions are primarily limited by the hydrolytic pathways, the reaction between carboplatin and DNA is extremely slow under physiological conditions. For example, the half life of carboplatin
reaction with DNA is estimated to be several days [71].</p><p>The behavior of these two complexes was compared with
that of [Pt(bpy)(py)2][PF6]2, 3 (bpy = bipyridyl, py = pyridine). This
complex lacks appropriate leaving groups, so that 3 is devoid of any
alkylating properties, but is able to intercalate DNA [72].</p><p>Figure 4 shows the trend of S% values
resulting from the interaction between DNA biosensor and a 0.1 mM solution of 1 in 5 mM (intracellular conditions) and 100 mM (extracellular conditions) NaCl,
respectively. As expected, the behavior of 1 strictly depends on the
concentration of the NaCl and on the aging time of the solution: high
concentrations of chlorides inhibit the aquation of cisplatin and, hence, its
interaction with DNA.</p><p>As far as 1 becomes, after hydrolysis, doubly
positive charged species, we have checked whether a simple long-range
electrostatic interaction in lieu of an effective coordination to DNA is able
to affect the oxidation signal of guanine. For this purpose, we have tested the
interaction between the biosensor and solutions containing divalent cations
Zn(II) and Cu(II). For both solutions, no variation in the guanine signal was
observed (S% = 100%).</p><p>Figure 5 shows the S% values
resulting from increasing concentrations of metal complexes 1 – 3 in 0.25 M phosphate buffer (PB, pH = 7.4), containing 5 mM NaCl (intracellular conditions).</p><p>The interaction increases in a dose-dependent manner,
and is stronger for 1 and softer for 2 . Compound 3 shows
an initial strong interaction, overimposable to that of 1 , but a minor
one at higher concentrations, probably because of the saturation of the
intercalating sites on DNA.</p><p>Figure 6 compares the behavior of the three metal
complexes in identical experimental conditions (in particular at the same
concentration) when the solution aging time is varied. As expected, a stronger
effect of solution aging time on S% is observed for 1 , while, in the case of 3 , hydrolysis is not required. In fact,
this complex does not need to dissociate any ancillary ligand to exert its
activity.</p><p>Compounds 1 and 2 produce the same
electrophilic agent upon hydrolysis, namely [Pt(H2O)2(NH3)2 ]2+, nevertheless, Figures 5 and 6 reveal that the interaction of carboplatin is much lower than that of cisplatin because of the different rate of hydrolysis (t
1/2 in chloride-free phosphate at 37°C is about 450 hours for 2 [71] compared with 2 hours for cisplatin [73]).</p><p>In the case of 2 , the rate of hydrolysis, and
hence the interaction with DNA, is increased in the presence of chlorides. In
fact, an exchange between the 1,1-cyclobutanedicarboxylato ligand and chlorides
in solution is able to transform 2 in 1 [74] that, in turn, undergoes quick activation by aquation. This effect is negligible in the presence of
weaker Lewis bases, for instance perchlorates (Figure 7). The exchange reaction
is time-dependent as observed in both in 2 and in its malonato-analogue
(cis-diamminomalonatoplatinum, [Pt(NH3)2(malonato)], 4), and
increases with [Cl−] (Figure 8).</p><p>The biosensor may also be used to differentiate the
intercalating from the covalent interactions. In fact, by using the same
experimental procedures previously described, it is possible to adsorb
single-stranded DNA onto the SPE [48]. Similar concentrations of compound 3 gave
lower S% values (i.e.,
higher interaction) on the ds-DNA- versus the ss-DNA-based sensor (S% = 65 ± 3 versus 92 ± 3, resp.), enforcing the experimental data that
identify this complex as an intercalator in lieu of a coordinating agent.</p><!><p>The complex NAMI-A, (H2Im)[trans-Ru(III)Cl4(DMSO)-(Im)],
is a pseudo-octahedral complex with four equatorial chloride ligands and the
heterocyclic bases and DMSO as axial ligands (Figure 9).</p><p>The complex loses its chloride ligands and transforms
into the corresponding, more reactive, aquated species [4] able to bind irreversibly to
DNA, albeit this binding is weaker than for similar platinum complexes
[75]. In fact,
Gallori et al. showed that NAMI-A interacts
with DNA at concentrations significantly higher than those at which cisplatin
produces similar effects [76].
On the other hand, tight binding of NAMI-A to proteins has been described
[64, 77, 78] and it is likely to
conceive that the mechanism underlying the antimetastatic activities of NAMI-A
does not involve DNA binding as the most significant process, but, perhaps the
inhibition of the matrix metallo-proteinases MMP-2 and MMP-9 [8].</p><p>Also in the case of NAMI-A, S% value decreases
as concentration increases (Figure 9), but the concentration of the supporting
electrolyte plays minor roles (Figures 9 and 10). In fact, NaCl, that should
exert mass effect, and NaClO4, that produces the noncoordinating perchlorate
anion, gave similar results (Figure 9) [64]. Indeed, it is known in literature that chlorides
have a minor effect over NAMI-A aquation [79]. NAMI-A shows higher S% values in
comparison with 1 , especially at low chloride concentration. These
experimental data further reinforce the hypothesis that DNA is not the
preferential targets of NAMI-A.</p><!><p>Unlike the very well-studied platinum complexes,
interactions of Ti complexes with DNA are poorly understood. It seems that
titanocene dichloride TiCp2Cl2 (Figure 11) is able to interact with
transferrin, the protein associated with iron transport. In this form, titanium
active species could cross the cell membrane, but the nature of the actual
cytotoxic species remains unknown [15].</p><p>In literature, there are conflicting results about
whether titanocene dichloride binds DNA or not. Some reports have suggested
that TiCp2Cl2 does not bind nucleotides and oligonucleotides at physiological
pH [80–82], but there is experimental
evidences of titanium being accumulated in the cellular nucleic acid-rich
regions, particularly in the chromatin [83]. Recently, it has been shown that TiCp2Cl2
interacts weakly with nucleotides at neutral pH through the phosphoesters most
probably as bare Ti(IV) species [84].</p><p>These conflicting results about titanocene dichloride
binds DNA prompted us to test if the biosensor were able to give some
information about the degree of interaction.</p><p>Figures 12 and 13 show, unequivocally, that TiCp2Cl2 has a lower degree of interaction with DNA biosensor than cisplatin [65]. The trend of S% with solution
aging time is almost constant. These two points fit with the literature data
showing that the hydrolysis of TiCp2Cl2 proceeds much faster than cisplatin:
the half-life of the first aquation of chloride ligand is too fast to be
measured and the second aquation step has a t
1/2 ≈ 50 minutes
[80]. Therefore, we
expect that both the active species [TiCp2(H2O)Cl]+ and [TiCp2(H2O)2]2
+
are present in solution just at the beginning of the experiment. If we accept
that the DNA binding occurs at the phosphate groups level, it is evident that
an ionic interaction between Ti cation and external phosphate backbone produces
a minor effect on the oxidation of G with respect to the direct coordination of
N7.</p><!><p>In the field of environmental sciences it has been
demonstrated the good relationship between genotoxicity of a sample (measured
by specific assays like Toxalert) and the presence of substances with high
affinity for DNA (measured by the DNA biosensor) [48, 50, 51], but the use of biosensors
in pharmacokinetic studies deserves some caution.</p><p>It is generally accepted that a direct relationship between
cytotoxicity and DNA-bound Pt exists [85–87], but there are also many factors that hamper the DNA
platination. The DNA biosensors do not give an "absolute" measure of the
genotoxic power of a potential drug as it uses DNA free of histones, not
organized in superior structures, and nuclear and cellular membranes are
missing. Furthermore, in a cell-free system, the cellular thiols (glutathione
and metallothioneins) able to intercept the platinum complexes are not present
and other repair mechanisms are missing.</p><p>Moreover, the ratio between metal drug and DNA is far
from real pharmacological conditions. In fact, in the case of Pt drugs, it has
been measured that cytotoxicity occurs when there are around 2−10 nmoles of
Pt/g DNA [88]. Our determinations revealed that about 3 × 10−9 g of DNA coated the biosensor [65, 89]. This means that the ratio between metal complex and immobilized DNA is incredibly high and in these conditions a large number of
compounds could interact with guanine, even without being active antitumor
drugs.</p><p>However, this procedure can be very useful for a rapid
screening of the samples and may be of interest in studying (i) the possible
reaction of the metal complex in solution and hence the formation of DNA-active
or inactive species by reaction with water or other molecules acting as ligand
(i.e., chlorides), and (ii) the strength of perturbation caused directly or
through the DNA chain by such metallodrugs on the electron density of N7-G,
that is, the real observable in such a measurements.</p><p>For the above reasons, the DNA biosensor could give
useful and quick information and could be integrated in a panel of tests in
order to quickly evaluate and quantifies the affinity of low-weight molecules
with DNA.</p>