Dolores Marín

Cyclic voltammetry of mitomycin C and DNA

Abstract Cyclic voltammetry (CV) in connection with the hanging mercury drop electrode was used to study mitomycin C (MC) and its mixtures with DNA. CV of the MC solution (without DNA) revealed that (a) at low MC concentrations (around 10 μM) the voltammetric cathodic peaks increase with consecutive cycles in the repeated cycle mode, (b) the CV responses of MC (both in the single-sweep and repeated cycle modes) depend strongly on the potential E A co which MC adsorbed at the electrode, (c) if the adsorption is performed at E A corresponding to the potentials of MC reduction, MC is so strongly bound to the electrode that the MC layer can be transferred to the electrolyte (not containing any dissolved MC) where it produces a response not substantially different from that obtained with the electrode immersed in the MC solution. These results have been tentatively explained by the reduction-initiated polymerization and/or insolubilization of MC at the electrode. Easy preparation of the MC-modified electrode demonstrated in this paper may find practical applications, namely in MC analysis. Preliminary results are reported on the behaviour of MC + DNA mixtures. The addition of double-stranded or single-stranded (denatured) DNAs into the MC solution results in suppression of the MC CV peaks in the first cycle. In the second cycle a new reversible couple appears which increases with the number of cycles. The anodic peak G of DNA (due to guanine) decreases simultaneously. These preliminary results suggest that some potential-controlled interactions of MC with DNA might occur at the electrode.

INTERACTIONS OF SURFACE-CONFINED DNA WITH ACID-ACTIVATED MITOMYCIN C

The anti-cancer drug mitomycin C (MC) was acid-activated and its interaction with single-stranded calf thymus DNA, immobilized at the surface of the hanging mercury drop electrode (DNA-modified HMDE) was studied by cyclic voltammetry. It was found that immersion of the DNA-modified electrode in a solution of acid-activated MC (at pH 3.9) for a short time (usually 1 min) at open current circuit, followed by transfer of the electrode in a neutral blank background electrolyte, resulted in a decrease of the anodic peak G (due to guanine residues in DNA) and in the formation of a reversible couple at approx. -0.44 V. The potential of the cathodic peak was approx. 50 mV more negative than the cathodic peak of the acid-activated MC obtained under the same conditions in the absence of DNA. No changes of peak G occurred and only a very small cathodic peak appeared if the DNA-modified electrode was immersed in an MC solution not exposed to acid pH. On the basis of these results and additional experiments, including dependence on concentration, time and pH during the interaction of MC with DNA at the electrode surface, we concluded that acid-activated MC is covalently bound to guanine residues in DNA immobilized at the electrode surface and that the quinone group in the DNA-MC adduct is reversibly reduced at the electrode.

Electrochemical Analysis of Anthramycin: Hydrolysis, DNA‐Interactions and Quantitative Determination

In aqueous solution, anthramycin methyl ether (AME), anhydroanthramycin (an imino form) and anthramycin are in equilibrium. It is found that anhydroanthramycin is electrochemically active. The AME hydrolysis reaction is studied by cyclic voltammetry. The kinetics of the DNA-drug reaction is followed by AdTSV and it is found that the slowness of the interaction is due to anthramycin changes which take place in solution independently of the DNA presence. DPP is used as a sensitive method for the quantitative determination of the drug and a detection limit of 70 nM is obtained.

Interactions of surface-confined DNA with electroreduced mitomycin C comparison with acid-activated mitomycin C.

The anticancer activity of the antineoplastic drug mitomycin C (MC) was investigated using transfer stripping cyclic voltammetry (TSCV) with single-stranded DNA-modified hanging mercury drop electrode (HMDE). Reductive activation of MC is necessary for drug covalent binding to DNA, and we have found that some potential-controlled interactions of MC with DNA occur at the electrode, i.e. MC can be activated by electroreduction. Acid and electroreductive MC activations were compared and different adducts were subsequently generated, suggesting that the drug can bind to DNA in more than one way. Under conditions of acid activated MC, a monofunctional adduct between C-1 of MC and N-7 of guanine was formed on the electrode surface, reduced at - 0.44 V (vs. SCE). However, when the DNA-modified electrode was immersed in a MC solution and potentials corresponding to the quinone moiety reduction (- 0.3 V or more negative vs. SCE) were applied, an intrastrand bifunctional adduct between C-1 and C-10 of MC and two N-7 of a pair of adjacent guanines in ssDNA were formed at the electrode, reduced at - 0.49 V, i.e. 50 mV more negative than the monoadduct. The results presented in this paper show for the first time electrochemical detection of DNA-MC adducts at the hanging mercury drop electrode.

Electrochemical study of antineoplastic drug thiotepa hydrolysis to thiol form and thiotepa—DNA interactions

Abstract Thiotepa (TT) is an alkylating antineoplastic drug used in the chemotherapy of bladder carcinoma among other uses. In acetate buffer pH 4.2 and 37°C, TT is hydrolyzed to a thiol compound (TT-SH) and we have found that the conversion percentage of TT into TT-SH depends on TT concentration. In DC polarography the TT is inactive and the TT-SH produces a diffusion controlled anodic wave. In stripping voltammetry the TT presents a poorly reproducible adsorption peak and the TT-SH two cathodic peaks. In this paper, the interactions between TT and DNA have been studied using voltammetric techniques and agarose gel electrophoresis. The results show that aziridine moiety is liberated from the TT molecule and binds covalently to nitrogens in the DNA bases, introducing strand breaks into DNA.

Entropy decrease associated to solute compartmentalization in the cell.

We have deduced equations to quantify the entropy associated to the compartmentalization of components in eukaryotic cells as a function of cell and compartment volumes, and of the concentration of solutes. On the basis of known and plausible values of volume and solute concentrations and the deduced equations, we estimate that the contribution of compartmentalization to the decrease of entropy is approximately -14.4 x 10(-14)JK(-1)cell(-1) (-0.7 J K(-1)L(-1)) in the case of Saccharomyces cerevisiae, a typical eukaryotic cell, and approximately -49.6 x 10(-14)JK(-1)cell(-1) (-1.0 J K(-1)L(-1)) in the more complex Chlamydomonas reinhardtii. When compared with other potential contributing factors, such as the informational entropy of DNA and the conformational entropy of proteins, compartmentalization appears as an essential development that significantly decreased the entropy of living cells during biological evolution.

Thermodynamic balance of photosynthesis and transpiration at increasing CO2 concentrations and rapid light fluctuations

Experimental and theoretical flux models have been developed to reveal the influence of sun flecks and increasing CO2 concentrations on the energy and entropy balances of the leaf. The rapid and wide range of fluctuations in light intensity under field conditions were simulated in a climatic gas exchange chamber and we determined the energy and entropy balance of the leaf based on radiation and gas exchange measurements. It was estimated that the energy of photosynthetic active radiation (PAR) accounts for half of transpiration, which is the main factor responsible for the exportation of the entropy generated in photosynthesis (Sg) out of the leaf in order to maintain functional the photosynthetic machinery. Although the response of net photosynthetic production to increasing concentrations of CO2 under fluctuating light is similar to that under continuous light, rates of transpiration respond slowly to changes of light intensity and are barely affected by the concentration of CO2 in the range of 260-495 ppm, in which net photosynthesis increases by more than 100%. The analysis of the results confirms that future increases of CO2 will improve the efficiency of the conversion of radiant energy into biomass, but will not reduce the contribution of plant transpiration to the leaf thermal balance.

The cancer Warburg effect may be a testable example of the minimum entropy production rate principle

Cancer cells consume more glucose by glycolytic fermentation to lactate than by respiration, a characteristic known as the Warburg effect. In contrast with the 36 moles of ATP produced by respiration, fermentation produces two moles of ATP per mole of glucose consumed, which poses a puzzle with regard to the function of the Warburg effect. The production of free energy (ΔG), enthalpy (ΔH), and entropy (ΔS) per mole linearly varies with the fraction (x) of glucose consumed by fermentation that is frequently estimated around 0.9. Hence, calculation shows that, in respect to pure respiration, the predominant fermentative metabolism decreases around 10% the production of entropy per mole of glucose consumed in cancer cells. We hypothesize that increased fermentation could allow cancer cells to accomplish the Prigogine theorem of the trend to minimize the rate of production of entropy. According to the theorem, open cellular systems near the steady state could evolve to minimize the rates of entropy production that may be reached by modified replicating cells producing entropy at a low rate. Remarkably, at CO2 concentrations above 930 ppm, glucose respiration produces less entropy than fermentation, which suggests experimental tests to validate the hypothesis of minimization of the rate of entropy production through the Warburg effect.

Methods for direct determination of mitomycin C in aqueous solutions and in urine

Stripping voltammetry (SV) is used to quantitatively determine concentrations of the anti-neoplastic drug mitomycin C (MMC) alone and in mixtures with 5-fluorouracil and cisplatin, both of which are used in combined chemotherapy withMMC. If the accumulation is performed at the potentials of MMC reduction (−0.35Vvs. SCE), reduced MMC is strongly adsorbed at the electrode. It is possible to prepare a MMCmodified electrode, which, after a washing step, is transferred to the background electrolyte to determine MMC by voltammetry. This procedure, which is termed transfer stripping voltammetry (TSV), helps to eliminate interferences and can be applied for a direct determination ofMMCalone or in mixtures with other drugs in urine.

Electrochemical behaviour and micromolar determination of the antineoplastic agent azaribine and its mixtures with cytidine

Abstract Azaribine, an antineoplastic drug of the pyrimidinic antimetabolites group, has been studied by means of polarography, cyclic voltammetry, coulometry and spectrophotometry in buffered aqueous Britton-Robinson medium at pH ranged between 2.5 and 12.0, at room temperature (25°C) and ionic strength 0.1 M. An acid-base equilibrium involving several proton donors present in the medium precedes the electrodic process, which is an overall two-electron process where the electroactive species is the azaribine acid form (neutral molecule) and the reducible group is the C(5)=N(6) double bond. The quantitative analysis of azaribine has been carried out using DPP at the optimum basal conditions. A detection limit of 0.2 μM corresponding to azaribine has been estimated. Quantitative determination of cytidine + azaribine mixtures is undertaken as well, since both compounds can appear together in biological samples. The investigation is carried out at a micromolar level and a mean error of about 2% has been detected.