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THE EFFECT OF CULTURE CONDITIONS ON TOXICITY OF 6-MERCAPTOPURINE TO CHLORELLA VULGARIS Department of General and Analytical Chemistry, Medical University of Silesia, Faculty of Pharmacy, Jagiellonska 4, 41-200 , Sosnowiec, Poland e-mail: [email protected] The thiopurine antimetabolite 6-Mercaptopurine (6-MP) is an analogue of the purine base hypoxanthine and is indicated for remission induction and maintenance therapy of acute lymphatic leukemia. The active metabolites of 6-MP alter cellular metabolism in a number of ways, including inhibition of purine biosynthesis de novo and incorporation into cellular RNA and DNA [1]. 6-MP is practically insoluble in water, it dissolves in dilute solutions of alkali hydroxides and hydrochloric acid. The ionization constants, pK, for anion formation is 7.7 (in pyrimidine ring) and 11.8 (in imidazole ring) and for cation formation < 2.5 (in imidazole ring) [2]. The partition coefficient octanol-water, log Pow, is -0.19 [3]. 6-MP can exist in six tautomeric forms because of the protropic tautomerism of the imidazole ring and thione-thiol tautomerism of the pyrimidine ring. 6-MP in aqueous solution may exist in the equilibrium of tautomeric forms, but in alkaline medium is practically Figure 1. Structures of the neutral and anionic forms of 6-Mercaptopurine. An adsorption and distribution of the drug are determined by its physicochemical properties e.g. its ionization and a partition coefficient between lipid and aqueous phases corresponding to drug lipophilicity. Non-ionized form of the drug is most often preferably dissolved in lipids and thus is easily transported through lipid In recent studies about toxicity of new synthesized purine thioderivatives relative to Chlorella vulgaris, in which 6-MP was treated as reference compound, cultures were processed in culture media with pH of 6.7 – 7.0 [4, 5]. However C. vulgaris exhibit high resistance to pH changes of culture medium (even to 11.0). In this work the effect of increased pH on 6-MP toxicity was examined. Experiments were carried out with unicellular green alga Chlorella vulgaris, Beij., strain 264, Boehm and Borns 1972/1 CCALA (Czechoslovak Academy of Science). The cultures were conducted in 250 ml Erlenmeyer flasks with 50 ml of modified Kuehl-Lorenzen [6] liquid medium (enriched with 4% CO2 and 3.85 mmol/l HCO - 3 ) at 24 ± 2oC and with continuous mixing using a magnetic stirrer. The cultures were synchronized by light-dark cycles (12-h light and 12-h dark period). The 6- Mercaptopurine (Sigma Aldrich) was added to algal medium as a solution in DMSO or 0.1 mol/l NaOH to obtain the required concentration (48.0, 96.0 and 144.0 mg/l). The final concentration of DMSO and NaOH did not inhibit nor stimulated the growth of C. vulgaris. The initial pH of the liquid medium was 6.7, 7.7 or 9.0. The tested cultures and control cultures (without the 6-MP) were conducted in parallel. Three replicates of each examined concentration and six replicates of the controls were examined. A growth of the cultures was monitored after 24 and 48 hs by measuring spectrophotometrically the optical density of the cell suspension at 680 nm. Growth inhibition and EC50 were calculated according to ISO 8692 [7]. The distribution coefficient, log D, which is a function of both its lipophilicity when non-ionized and the degree of ionization and is defined as the effective lipophilicity of a compound at a given pH, was calculated using eq. (1) [8]: log (P/D) – 1 = pK – pH (1) where: pK = 7.72 [5], log P = 0.62 is the value calculated for neutral form of 6-MP from experimental values obtained with RPTLC method [9]. The amount (%), of 6-MP ionized form were calculated using the eq. (2) [10]: % ionized = 100/[1 + 10(pK – pH)] (2) Absorption spectra of the 6-MP in standard solutions of pH 5.0 – 13.0 and in liquid media (after the end of the cycle) were recorded between 220 and 380 nm using Jasco (V-530) UV VIS Spectrophotometer. The calculated distribution coefficient (log D) and the percentage of 6-MP ionized form in solutions of pH range 5.0 – 9.0 are presented in Table 1. The presented results show that effective lipophilicity of 6-MP is depended to solution pH and will decrease if pH and amount of ionized form increases. Table 1. The effect of pH on the physicochemical properties of 6-MP The λmax values of 6-MP standard solutions in a pH range of 5.0 – 13.0 determined according to UV spectra calibration curves (ultraviolet spectra) are also presented in Table 1. The changes of UV maximum absorption and hypsochromic shift of λmax of about 13 nm for 6-MP solutions are related to formation of monoanionic form from neutral one and dianionic form from monoanionic one. The characteristic of C. vulgaris culture growth is presented in Table 2 as percentage of growth inhibition related to control culture. In the same table the λmax values determined for 6-MP in a culture media are presented that confirms various ionization state of this compound. The determined values of 6-MP toxic concentration related to C. vulgaris are expressed as EC50 (mg/l). These results indicate that toxicity of 6-MP is depended to pH of culture media. The EC50 value for culture media of pH 9.0 was twice higher than one determined for culture media of pH 6.7. Table 2. EC50 values characterizing toxicity of 6-MP to C. vulgaris % inhibitiona λmaks [nm] of 6-MP in the liquid medium a inhibition percentage related to the control culture Obtained results indicate that pH of culture media influences the toxicity of 6- MP and the ionized forms of 6-MP were significantly less toxic for C. vulgaris. This phenomen may be caused by decreased lipophilicity of anionic form relative to neutral one. Lipophilicity is an important property affecting the bioactivity of drugs and increasing lipophilicity usually correlates with increasing biological activity. The transport rate and the amount of transported substance into the cell is positively correlated with a partition coefficient log P. The log P depends on pH and degree of dissociation, which directly corresponds to the amount of dissociated form. Non-ionized and lipophilic substance is easily transported into the cell because the cell membrane is a selectively permeable lipid bilayer. However, it has only a very low permeability to ionic molecules and thus their concentration inside the cell will be lower. The other cause of decreased toxicity of dissociated 6-MP might be its impaired interaction with DNA. 6-MP is an antimetabolite of natural purine base and may be incorporated into DNA as deoxy-6-thioguanosine (S6G) instead of guanine and thus modify structural properties of DNA duplex. Bohon J. [11] and Somerville L. [12] have shown, that active metabolite S6G (in keto form) was incorporated into DNA. This required formation of weakened Watson- Crick hydrogen bonds which led to S6G-C and S6G-T pairs (Fig. 2). However, S6G-C is highly favoured because of triple hydrogen-bond. It can be concluded, that 6-MP monoanionic form generated during N(1)-H dissociation at pH 7.7 and 9.0 may not combine with natural base by forming Watson- Crick hydrogen bonds. This is a reason why this form S6G does not incorporate into DNA duplex and, in consequence, does not show cytotoxic activity to C. vulgaris. . Figure 2. Base pair configuration for S6G-C, S6G-T and G-T and canonical G-C [11]. REFERENCES [1] J.E. Polifka, J.M. Friedman, Teratology, 65 (2002) 240. [2] D.J. Brown, S.F. Mason, J. Chem. Soc., (1957) 682. [3] P.N. Craig, in: Comprehensive Medicinal Chemistry, C. Hansch (ed.), vol. 6, [4] A. Kowalska, J. Sochacka, Acta Polon. Pharm – Drug Res., 60 (2003) 144. [5] J. Sochacka, A. Kowalska, Ann. Pol. Chem. Soc., 3(2) (2004) 735. [6] A. Kuehl, H. Lorenzen, in: Methods and Cell Physiology, D. M. Prescott (ed.), N. Y. [7] ISO 8692, International Organization for Standarization, Geneve Switzerland 1989. [8] B. Malawska, K. Kulig, M. Wiśniewska, J. Planar Chromatogr., 13 (2000) 187. [9] J. Sochacka, A. Kowalska, J. Planar Chromatogr., 19 (2006) 320. [10] A. Albert, E.P. Serjant, The Determination of Ionization Constants, 3rd edn, Chapman and Hall, New York 1984, p. 203. [11] J. Bohon, C. Santos, Nucl. Acids Res., 31 (2003) 1331. [12] L. Somerville, E. Krynecki, J. Biol. Chem., 278 (2003) 1005.

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