The modulation of the DNA-damaging effect of polycyclic aromatic agents by xanthines

Abstract

Recently, accumulated statistical data indicate the protective effect of caffeine consumption against several types of cancer diseases. There are also reports about protective effect of caffeine and other xanthines against tumors induced by polycyclic aromatic hydrocarbons. One of the explanations is based on biological activation of such carcinogens by cytochromes that are also known for metabolism of caffeine. However, there is also numerous data indicating reverse effect on cytotoxicity of anticancer drugs that inhibit the action of topoisomerase I (e.g. Camptothecin or Topotecan) and topoisomerase II inhibitors (e.g. Doxorubicin, Mitoxantrone or mAMSA). In this work we tested the hypothesis that the caffeine protective effect is the result of sequestering of aromatic mutagens by formation of stacking (π–π) complexes. As the models for the study we have chosen two well-known mutagens, that do not require metabolical activation: quinacrine mustard(QM, aromatic, heterocyclic nitrogen mustard) and mechlorethamine (NM2, aliphatic nitrogen mustard). The flow cytometry study of these agents’ action on the cell cycle of HL-60 cells indicated that caffeine prevents the cytotoxic action of QM, but not that of NM2. The formations of stacking complexes of QM with caffeine were confirmed by light absorption, calorimetric measurements and by molecular modeling calculation. Using the statistical thermodynamics calculations we calculated the “neighborhood” association constant (K_AC=59±2 M⁻¹) and enthalpy change ($\text{Δ}\text{H}{}^{\mathrm{0\prime }}\text{=−116}\text{cal}\text{mol}{}^{\mathrm{-1}}$); the favorable entropy change of complex formation ($\text{Δ}\text{S}{}^{\mathrm{0\prime }}\text{=7.72}\text{cal}\text{mol}{}^{\mathrm{-1}}\text{K}{}^{\mathrm{-1}}$, due to release of several water molecules, associated with components in the process of complex formation). The Gibbs’ free energy change of QM–CAF formation is $\text{Δ}\text{G}{}^{\mathrm{0\prime }}\text{=−2.41}\text{kcal}\text{mol}{}^{\mathrm{-1}}$. We were unable to detect any interaction between NM2 and caffeine either by spectroscopic or calorimetric measurement. In order to establish, whether the intercalation of QM plays any role in cytotoxic effect we tested, as a control, non-alkylatiatig, but also intercalating QM derivative—quinacrine (Q). The later had no cytostatic effect on HL-60 cell even at there order of higher concentration than QM or NM2 but, similar to QM forms (which we demonstrated) stacking complexes with caffeine (K_AC=75±3 M⁻¹). These results strongly indicate, that the attenuating effect of caffeine on cytotoxic or mutagenic effects of some mutagens, is not the results of metabolic processes in the cells, but simply the physicochemical process of sequestering of aromatic molecules (potential carcinogens or mutagens) by formation of stacking complexes with them. The caffeine may then act as the “interceptor” of potential carcinogens (especially in the upper part of digesting track where its concentration can reach the concentration of mM level). There is, however, no indication either in the literature or in our experiments that xanthines can reverse the damage to nucleic acids when the damage to DNA has already occurred.

Introduction

Caffeine (CAF) has a myriad of effects on living organisms. In oncology, most studies have focused on the propensity of CAF to potentate the cytological effects of variety of DNA damaging agents, such as an ionizing radiation, alkylating compounds, cis-platinium and cyclophosphamide, hydroxyurea and others [2], [3], [4], [5], [6], [7]. The mechanism by which CAF enhances the toxicity of these agents is believed to involve suppression of DNA repair and/or DNA synthesis [8], [9], [10]. Because the CAF is probably the most commonly used alkaloid worldwide, these findings intensified the cancer epidemiological study on the adverse effects, the consumption of the CAF containing beverages, i.e. coffee, tea and cocoa. The recent reviews of medical literature on this subject suggest [11] that coffee does not have appreciable effect on a number of diseases but the CAF has not been shown to be teratogenic [12].

Surprisingly, there were certain published studies which indicate that CAF consumption was inversely associated with the cancer risk of a digestive tract [13], [14], [15], breast [16], [17], pancreas [18] or thyroid [19]. A biologic mechanism for such risk reduction has not been established [20]. There is very interesting study by Rothwell which describes a dose-related inhibition of chemical carcinogenesis (cigarette smoke condensate) in mouse skin by CAF [21]. The effect of CAF on cyclic AMP synthesis was considered as a possible explanation of this phenomenon.

Caffeine consumption can also significantly reduce mammary carcinoma multiplicity induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rats [22]. Recently published study on a benzo[a]pyrene (BP) induced lung tumor in mice confirmed that CAF is among cancer preventive agents [23]. Both DMBA and BP are procarcinogens and require biological activation by cytochromes P450 [24], [25]. These enzymes also metabolize CAF and it was proposed that protective effect of CAF is a result of competitive interaction with cytochromes [26], [27]. However, similar protective effect of CAF has been observed for several polycyclic aromatic compounds that do not require cytochromes for their biological activity. Intercalating anticancer drugs Doxorubicin, Mitoxantrone and mAMSA [28], [29], [30], [31], [32], which are believed to be the topoisomerase II blockers, and non-intercalators, topoisomerase I inhibitors, Camptothecin and Topotecan [33] belong to a group of such agents. There is a possibility these phenomena may be a result of CAF having an effect on topoisomerases action, or inhibition of RNA synthesis [32]. However, we believe that formation of stacking (π–π) complexes [34] between CAF and polycyclic aromatic drugs is responsible for the reduction toxicity of these drugs. Our studies confirmed the formation of such complexes with several anticancer drugs [31], [33] and the fluorescence DNA ligands DAPI and ethidium bromide [35]. We also proposed statistical thermodymical models that describe the formation of these complexes [36]. According to our hypothesis, the mixed aggregation with the CAF reduces free drug concentration in solution, which is then reflected as diminished pharmacological activity. To confirm this hypothesis we have chosen two well-known mutagenic agents as a subject of this study: QM and NM2. Both these compounds are derivatives of nitrogen mustard (Fig. 1) and in an aqueous solution spontaneously form reactive arizidinium ions that can alkylate nucleic acids and proteins [37], [38]. The reason for the selection of QM and NM2 for our studies is that they do not require any enzymatic activation. QM, however, can bind to DNA by intercalation [39] while NM2, as a aliphatic compound, can not.

To test whether this process has any effect on cell cytotoxity we included the quinacrine (Q, Fig. 1) to our study; this compound intercalates, but does not alkylate nucleic acids.