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Effects of quinones and azoles on radiation-induced processes involving hydroxyl-containing carbon-centered radicals
Radiation Physics and Chemistry 144 (2018) 308–316
Contents lists available at ScienceDirect
Radiation Physics and Chemistry journal homepage: www.elsevier.com/locate/radphyschem
Effects of quinones and azoles on radiation-induced processes involving hydroxyl-containing carbon-centered radicals
T
S.N. Samovicha,b, R.L. Sverdlova,b, S.V. Voitekhovicha, Y.V. Grigorieva, O.A. Ivashkevicha,b, ⁎ O.I. Shadyroa,b, a b
Research Institute for Physical and Chemical Problems of the Belarusian State University (RI PCP BSU) Leningradskaya st., 14, 220006 Minsk, Republic of Belarus Department of Chemistry of the Belarusian State University Nezavisimosti av., 4, 220030 Minsk, Republic of Belarus
1. Introduction It has been reliably established that the free-radical processes play a crucial role in generation of radiobiological phenomena (Halliwell and Gutteridge, 2007; Joiner and van der Kogel, 2009; Von Sonntag, 1987). During the action of ionizing radiation on biosystems, various kinds of homolytic reactions may take place (Dean et al., 1997; Robbins and Zhao, 2004; Von Sonntag, 2006). However, for a long time, the attention of most researchers has been focused mainly on radiation-induced oxidation processes occurring in biomolecules, in the first place, in the cell membrane lipids, and on the ensuing pathophysiological consequences (Niki, 2009; Stark, 1991). In our studies (Shadyro, 1997), a concept was developed that the free-radical fragmentation reactions taking place in hydroxyl-containing biologically relevant substances may make a substantial contribution to the consequences resulting from irradiation of biosystems. It has been shown that the hydroxyl-containing organic compounds under radiolysis of their aqueous solutions undergo fragmentation according to the following general scheme (1):
It has been established in numerous studies that the occurrence of reactions (1) may cause damage to carbohydrates (Edimecheva et al., 2005), lipids (Shadyro et al., 2002, 2004a, 2004b), amino acids and proteins (Shadyro et al., 2000, 2003; Sladkova et al., 2012). The probability of the reaction (1) to occur depends on the structure of the initial compound. It has been established that the fragmentation rate constants for radicals (I) vary from 101 s−1 (for diol-type radicals)
(Pikaev and Kartasheva, 1975) to 105−107 s−1 (for radicals derived from organic phosphates) (Schuchmann et al., 1995). Oxygen produces a substantial influence on the probability of fragmentation reactions (1) in biomolecules, because the rate constant for the interaction of O2 with radicals of type (I) is of the order of 109 M−1 s−1. This leads to inhibition of the reaction (1). Oxygen levels in a living organism are known to vary within a considerable range: from ~ 10−4 M in well-ventilated lung cells to 10−5 M in skin cells (Carreau et al., 2011). On irradiation under conditions of low oxygen levels, the probability of reactions of type (1) grows, and hence its contribution to the development of negative consequences of the action of ionizing radiation on biomolecules increases. The aforesaid is an important incentive to perform the search for the substances capable of producing an effective regulative action on the probability of reactions of type (1) to occur. This goal can be attained by assessing the reactivity towards carbon-centered radicals of various suitable agents. Therefore, the object pursued in this study was in-
vestigation of the effects produced by quinones and azoles on formation of the end products during radiolysis of ethanol and aqueous solutions of glycerol-1-phosphate.
⁎ Corresponding author at: Research Institute for Physical and Chemical Problems of the Belarusian State University (RI PCP BSU) Leningradskaya st., 14, 220006 Minsk, Republic of Belarus. E-mail address: [email protected] (O.I. Shadyro).
http://dx.doi.org/10.1016/j.radphyschem.2017.09.004 Received 6 July 2017; Received in revised form 24 August 2017; Accepted 5 September 2017 Available online 11 September 2017 0969-806X/ © 2017 Elsevier Ltd. All rights reserved.
Radiation Physics and Chemistry 144 (2018) 308–316
S.N. Samovich et al.
Fig. 1. Structures of the quinones examined in the study.
2. Experimental section
suitable quantities of perchloric acid or sodium hydroxide. The solutions were transferred into ampoules, deaerated with high purity argon and sealed.
2.1. Chemicals
2.3. Irradiation of the samples
Methyl-p-benzoquinone (1), 2,5-dimethyl-1,4-benzoquinone (2), thymoquinone (3), coenzyme Q0 (4) and doxorubicin hydrochloride (5) (Fig. 1) produced by Sigma-Aldrich were used in this study without further purification. Azoles were represented in this study by imidazole (6), metronidazole (7) and 1,2,4-triazole (8) purchased from Sigma-Aldrich and nitrotriazoles 9–11 (Fig. 2), which were synthesized and purified according to the published procedures – 3-nitro-1,2,4-triazole (9) (Petersen et al., 2014), 1-methyl-3-nitro-1,2,4-triazole (10) (Sukhanov et al., 2005), 1-(2-hydroxyethyl)−3-nitro-1,2,4-triazole (11) (Degtyarik et al., 2012). Purity of the 1,2,4-triazoles was tested by mass spectrometry. Purity of all the compounds under study was not less than 98%. Ethanol 96% v/v was twice distilled on a rectifying column. Disodium or magnesium salts of glycerol-1-phosphate of purity not less than 95% were from Sigma-Aldrich.
Irradiation was performed on a unit MPX-γ−25 M with a Co60 source. The dose rate absorbed during the experiment was 0.22 ± 0.01 Gy/s. The absorbed dose interval varied within the range of 0.22–2.00 kGy. Fricke dosimeter was used to measure the absorbed dose rate (Fricke and Hart, 1966). 2.4. Identification of radiolysis products and determination of their concentrations 2.4.1. GC-FID analysis Products of free-radical transformations of ethanol – acetaldehyde (AA) and 2,3-butanediol (BD) – were determined on a Shimadzu chromatograph GC-17A using a silica capillary column Stabilwax-DA (l = 30 m, ID = 0.53 mm, df = 1.0 µm) against an external standard. The analysis conditions: starting temperature 40 °C, rise to 200 °C at a rate of 13 °C/min, injector temperature 220 °C, detector temperature 220 °C, carrier gas nitrogen, detector FID, injected volume 1.0 μl.
2.2. Preparation of samples for irradiation Solutions of the compounds under study were prepared by dissolving accurately weighed amounts in deaerated ethanol. Because of high volatility of the solvent, the following procedure was used to prepare solutions of the test compounds of concentrations 10−3 M. The solutions filled in densimeters were blown through with high purity argon (99.9%) for 60 min. Thereafter the volume of evaporated solvent was made up with deaerated ethanol and the densimeter content was mixed. The solutions were transferred to ampoules preliminary blown through with argon, filling not more than 60% of their fill volume, and then sealed. For preparation of solutions containing 10−1 M glycerol-1-phosphate and 10−3 М of tested compounds, twice-distilled water was used. The pH value of the solutions was adjusted to 7.0 ± 0.1 by adding
2.4.2. Spectrophotometric measurements Inorganic phosphate on a background of the organic one was determined spectrophotometrically using a modified procedure described in (Gin and Morales, 1977). To 0.5 ml of the solution to be examined were added 1 ml 1.8% solution of (NH4)2MoO4 × 4H2O in 1 M H2SO4 and 0.2 ml of 10% FeSO4 × 7H2O in 0.075 M H2SO4, and 4 ml of twicedistilled water. The solutions were stirred for 10 min on an auto-shaker. Optical density of the solutions was measured at λ = 720 nm on a Specord S600 spectrophotometer. Calibration curves were plotted using aqueous solutions of KH2PO4. The plot of optical density vs. concentration of inorganic phosphate was linear within the range of 10−5
Fig. 2. Structures of the azoles examined in the study.
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Table 1 Radiation-chemical yields of major radiolysis products of deaerated ethanol in the presence of tested quinones.
Results are expressed as means ± SD (n = 3). (a–d) – means in the same column followed by different letters are significantly different (p < 0.05).
to 10−3 M. For determination of concentrations of the compounds to be examined, measurements of optical density were performed before and after irradiation, if not stated otherwise.
2.5. Calculations of radiation-chemical yields Radiation-chemical yields (G, mol/J) were calculated from linear portions of plots of the product concentrations vs. dose absorbed. The results obtained in three independent experiments were used to calculate the yields. The error in determination of radiation-chemical yields was calculated by the least squares method using the confidence coefficient of 0.95.
2.4.3. GC–MS measurements Molecular products of interaction of α-hydroxyethyl radicals (αHER) with the quinones under study were identified using a gas-liquid chromatograph GC-2010 equipped with a column Equity-5 (l = 30 m, ID = 0.25 mm, df= 0.25 µm) and a Shimadzu mass detector GCMSQP2010 Plus. Analysis conditions: carrier gas helium, linear flow rate 1 ml/min, thermostat temperature 60 °C to 270 °C, injector temperature 270 °C, ion source temperature 280 °C, injected volume 1.0 μl.
2.6. Statistical analysis The data obtained were statistically analyzed using Statistica v. 10 software (StatSoft, Poland). The results were expressed as mean ± standard deviation (SD) from three independent parallel experiments.
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Table 2 Radiation-chemical yields for decay of the additives and formation of phenolic products in deaerated ethanol.
Results are expressed as means ± SD (n = 3). a, b – means in the same row followed by different letters are significantly different (p < 0.05).
are one of the major intermediates formed in this system under radiolysis. Therefore, in this study, we investigated effects of quinones and azoles on the yields of products formed in radiolysis of ethanol, deaerated with argon. It has been established that the following processes take place on radiolysis of ethanol (Freeman, 1982, 1987; Pikaev, 1986) (schemes 2–7):
The data were analyzed using the t-test for independent samples. The differences were considered as significant at p < 0.05.
In the absence of dissolved substances capable of oxidizing, the ethoxy radicals (CH3CH2O•) rapidly transform into α-HER (scheme 4, k4 = 1,1 × 106 M−1 s−1). It is known that in dilute solutions at a solute concentration of ≤ 10−3 M other reactions involving ethoxy radicals can be neglected (Pikaev, 1986). The CH3C•HOH radicals being formed in the absence of oxygen interact with each other giving 2,3-butanediol (BD) and acetaldehyde (AA) with approximately equal probability (k9/k8 = 0,9) (Jore, 1988) according to the schemes (8) and (9):
3. Results and discussion 3.1. Steady-state radiolysis of the compounds under study in deaerated ethanol Our studies have shown (Brinkevich et al., 2012; Samovich et al., 2014; Sverdlov et al., 2014) that radiolysis of ethanol is a convenient model to investigate interactions of α-hydroxyl-containing carboncentered radicals with various substances, because CH3C•HOH radicals 311
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Table 3 Effects of imidazole and 1,2,4-triazole nitro derivatives on radiation-chemical yields of major radiolysis products formed in deaerated ethanol.
Results are expressed as means ± SD (n = 3). a, b, c – means in the same column followed by different letters are significantly different (p < 0.05).
AA is also formed as a result of excited ethanol molecules decomposition (scheme 7). Hence, by measuring radiation-chemical yields of AA and BD in the presence and the absence of the test compounds, one may assess their reactivity towards α-HER. As follows from the data presented in Table 1, a significant decrease in radiation-chemical yields of BD was observed in the presence of quinones, including the anti-tumor agent doxorubicin 5. At the same time, the yields of AA increased, becoming almost two times greater vs. controls in the presence of 1−4.
The radiation-chemical yields for the decay of quinones and formation of the major products identified by chromato-mass spectrometry are shown in Table 2. Close values of yields for decomposition of the starting quinones and formation of the respective reduction products show that the main products of transformation of the test quinones were phenolic compounds. Hence, the test quinones can effectively oxidize α-HER to AA, themselves being transformed into semiquinone radicals (II), according to scheme (10):
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Table 4 Effects of quinones on radiation-chemical yields of inorganic phosphate.
Results are expressed as means ± SD (n = 3). a, b – means in the column followed by different letters are significantly different (p < 0.05).
The semiquinone radicals (II) can form the identified phenols through disproportionation reactions between themselves (Scheme 11) or with α-HER (Scheme 12).
addition to a delocalized semiquinone radical (or to the multiple bonds of doxorubicin) can be realized, causing a decrease in the radiationchemical yield of the BD observed in the experiment.
The lower yield values for AA observed in the case of doxorubicine as compared to those for additives 1−4 are most probably due to the low sterical accessibility of the quinoid site for the reaction with α-HER according to schemes (10) and (12). Moreover, the semiquinone radical formed from doxorubicine according to scheme (11) is more stable due to delocalization of the unpaired electron over the aromatic system of the molecule and hence less reactive towards oxidation of α-HER. Therefore, besides the oxidation reactions, the process of α-HER
Thus, the data presented above show that quinones of various structures effectively interact with α-HER by oxidizing the latter to AA. Radiation-chemical yields of AA and BD obtained in experiments on radiolysis of solutions of imidazole 6, metronidazole 7 and 1,2,4-triazole nitro derivatives 9−11 in deaerated ethanol are presented in Table 3. It follows from the data shown in Table 3 that a virtually complete suppression of BD formation was seen in the presence of nitroazoles 7 and 9−11. Besides the decrease in the yields of BD, a more
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Table 5 Effects of imidazole, 1,2,4-triazole and its nitro derivatives on radiation-chemical yields of inorganic phosphate.
Results are expressed as means ± SD (n = 3). (a–d) – means in the column followed by different letters are significantly different (p < 0.05).
The high reactivity of the test compounds towards α-HER might be evidence of their ability to affect the realization probability of reaction (1) to a significant extent. To confirm this assumption, we examined the effects of the named quinones and azoles on radiation-chemical formation of inorganic phosphate during γ-irradiation of aqueous solutions of glycerol-1-phosphate at pH 7. It has been shown (Schuchmann et al., 1995) that glycerol-1-phosphate undergoes dephosphorylation on radiolysis of its aqueous solutions. Formation of inorganic phosphate
than two-fold rise in radiation-chemical yields of AA was observed in the presence of the nitroazoles. In the presence of imidazole 6, within the experimental error limits, no changes in radiation-chemical yields of AA and BD were noted. It can be concluded that imidazole is characterized by low reactivity towards α-HER. The effects observed on adding compounds 7 and 9−11 to ethanol are associated with the presence of nitro groups in their structures, which endow them with oxidizing properties making possible scheme (13) to occur:
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occurs under the action of •OH and •H radicals on glycerol-1-phosphate followed by fragmentation of the α-HCR (III) according to scheme (14−15).
that the hydroxyl-containing lipids (phosphatidyl glycerol, phosphatidyl inositol, cardiolipin) being subjected to radiolysis undergo decomposition similar to that of glycerol-1-phosphate, giving phospha-
The obtained data show that the radiation-chemical yields of inorganic phosphate decreased significantly in the presence of quinones (Table 4) and azoles (Table 5). These findings point to the ability of the
tidic acid. For example, accumulation of phosphatidic acid in high yields was observed during radiation-induced fragmentation of cardiolipin (the main membrane lipid of mitochondria) according to the scheme similar to (15) (Shadyro et al., 2004b; Yurkova et al., 2008):
It has been found that the accumulation of phosphatidic acid in cancer cells promotes their proliferation (Foster, 2009; Toschi et al., 2009; Foster et al., 2014). Taking this into account, one can expect that the radiation-induced formation of phosphatidic acid in cancer cells should worsen the efficiency of radiotherapy in cancer patients. As quinones and azoles block these negative effects, they are expected to manifest radiosensitizing properties when introduced into cancer cells. Indeed, compounds of this type, such as metronidazole and doxorubicine are being used as radiosensitizers in radiation medicine. The data obtained in this study open up a new possibility to enhance efficiency of radiation therapy by introducing agents effectively blocking the radiation-induced processes leading to formation of products promoting cancer cell proliferation.
named compounds to block fragmentation of radicals (III) in spite of the very high rate constant for this reaction (> 106 s−1) (Schuchmann et al., 1995). Bearing in mind that during radiolysis of ethanol and of aqueous glycerol-1-phosphate solutions formation of similar α-hydroxyl-containing carbon-centered radicals is expected, a conclusion can be drawn that the mechanisms describing interaction of quinones and azoles with radicals (III) should be described by schemes analogous to (6, 8, 9). To all appearances, the inhibition of dephosphorylation results from oxidation of radicals (III) prior to their fragmentation. Hence, quinones and azoles may be promising agents for suppression of processes of type (1). It has been established earlier (Shadyro et al., 2002, 2004a, 2004b) 315
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Moscow. Robbins, M.E.C., Zhao, W., 2004. Chronic oxidative stress and radiation-induced late normal tissue injury: a review. Int. J. Radiat. Biol. 80 (4), 251–259. Samovich, S.N., Brinkevich, S.D., Edimecheva, I.P., Shadyro, O.I., 2014. Radiation-chemical transformations of coumarins in ethanolic solutions. Rad. Phys. Chem. 100 (1), 13–22. Schuchmann, M.N., Scholes, M.L., Zegota, H., Von Sonntag, C., 1995. Reaction of hydroxyl radicals with alkyl phosphates and the oxidation of phosphatoalkyl radicals by nitro compounds. Int. J. Radiat. Biol. 68 (2), 121–131. Shadyro, O.I., 1997. Radiation-induced free radical fragmentation of cell membrane components and the respective model compounds. In: Minisci, F. (Ed.), Free Radicals in Biology and Environment. Kluwer Academic Publishers, Netherlands, pp. 317–329. Shadyro, O.I., Sosnovskaya, A.A., Vrublevskaya, O.N., 2000. Deamination and degradation of hydroxyl-containing dipeptides and tripeptides in the radiolysis of their aqueous solutions. High Energ. Chem. 34 (5), 290–294. Shadyro, O.I., Yurkova, I.L., Kisel, M.A., 2002. Radiation-induced peroxidation and fragmentation of lipids in a model membrane. Int. J. Rad. Biol. 78 (3), 211–217. Shadyro, O.I., Sosnovskaya, A.A., Vrublevskaya, O.N., 2003. C-N bond cleavage reactions on the radiolysis of amino-containing organic compounds and their derivatives in aqueous solutions. Int. J. Radiat. Biol. 79 (4), 269–279. Shadyro, O.I., Yurkova, I.L., Kisel, M.A., Brede, O., Arnhold, J., 2004a. Formation of phosphatidic acid, ceramide, and diglyceride on radiolysis of lipids: identification by MALDI-TOF mass spectrometry. Free Radic. Biol. Med. 36 (12), 1612–1624. Shadyro, O.I., Yurkova, I.L., Kisel, M.A., Brede, O., Arnhold, J., 2004b. Radiation-induced fragmentation of сardiolipin in a model membrane. Int. J. Rad. Biol. 80 (3), 239–245. Stark, G., 1991. The effect of ionizing radiation on lipid membranes. Biochim. Biophis. Acta 1071 (2), 103–122. Sladkova, АА, Sosnovskaya, АА, Edimecheva, I.P., Shadyro, ОI., 2012. Radiation-induced destruction of hydroxyl-containing amino acids and dipeptides. Radiat. Phys. Сhem. 81 (12), 1896–1903. Sukhanov, G.T., Sakovich, G.V., Sukhanova, A.G., Lukin, A. Yu, 2005. Reactions of 3nitro-1,2,4-triazole derivatives with alkylating agents. 2. Alkylation of a neutral heterocycle by dimethyl sulfate. Chem. Heterocycl. Compd. 41 (8), 994–998. Sverdlov, R.L., Brinkevich, S.D., Shadyro, O.I., 2014. Effects of tryptophan derivatives and β-carboline alkaloids on radiation- and peroxide-induced transformations of ethanol. Radiat. Phys. Сhem. 98, 77–85. Toschi, A., Lee, E., Xu, L., Garcia, A., Gadir, N., Foster, D.A., 2009. Regulation of mTORC1 and mTORC2 complex assembly by phosphatidic acid: competition with rapamycin. Mol. Cell Biol. 29 (6), 1411–1420. Yurkova, I.L., Stuckert, F., Kisel, M.A., Shadyro, O.I., Arnhold, J., Huster, D., 2008. Formation of phosphatidic acid in stressed mitochondria. Arch. Biochem. Biophys. 480 (1), 17–26. Von Sonntag, C., 1987. The Chemical Basis of Radiation Biology. Taylor and Francis, London. Von Sonntag, C., 2006. Free-radical-induced DNA Damage and its Repair. SpringerVerlag, Berlin.
References Brinkevich, S.D., Ostrovskaya, N.I., Parkhach, M.E., Samovich, S.N., Shadyro, O.I., 2012. Effects of curcumin and related compounds on processes involving α-hydroxyethyl radicals. Free Radic. Res. 46 (3), 295–302. Carreau, A., El Hafny-Rahbi, B., Matejuk, A., Grillon, C., Kieda, C., 2011. Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J. Cell. Mol. Med. 15 (6), 1239–1253. Dean, R.T., Fu, S., Stocker, R., Davies, M.J., 1997. Biochemistry and pathology of radicalmediated protein oxidation. Biochem. J. 324 (Pt 1), 1–18. Degtyarik, M.M., Lyakhov, A.S., Ivashkevich, L.S., Voitekhovich, S.V., Sukhanov, G.T., Grigoriev, Y.V., 2012. 1-(2-Hydroxyethyl)-3-nitro-1,2,4-triazole and its complexes with copper(II) chloride and copper(II) perchlorate. Z. Anorg. Allg. Chem. 638 (6), 950–956. Edimecheva, I.P., Kisel, R.M., Shadyro, O.I., Kazem, K., Murase, H., Kagiya, T., 2005. Homolytic cleavage of the O-glyсoside bond in carbohydrates: a steady-state radiolysis study. J. Radiat. Res. 46 (3), 319–324. Foster, D.A., 2009. Phosphatidic acid signaling to mTOR: signals for the survival of human cancer cells. Biochim. Biophys. Acta 1791 (9), 949–955. Foster, D.A., Salloum, D., Menon, D., Frias, M.A., 2014. Phospholipase D and the maintenance of phosphatidic acid levels for regulation of mammalian target of rapamycin (mTOR). J. Biol. Chem. 289 (33), 22583–22588. Freeman, G.R., 1982. Labile species and fast processes in liquid alcohol radiolysis. In: Baxendale, J.H., Busi, F. (Eds.), The study of Fast Processes and Transient Species by Pulse Electron Radiolysis. D. Reidel Publishing Company, Dordrecht, pp. 399–416. Freeman, G.R., 1987. The radiolysis of alcohols. In: Freeman, G.R. (Ed.), Kinetics of Nonhomogeneous Processes: A Practical Introduction for Chemists, Biologists, Physicists, and Material Scientists. Wiley-Interscience, New York, pp. 73–101. Fricke, H., Hart, E.J., 1966. Chemical dosimetry. In: Attix, F.H., Roesch, W.C. (Eds.), Radiation Dosimetry. Academic press, New York, pp. 167–177. Gin, F.J., Morales, F., 1977. Application of one-step procedure of measurement of Pi in the presence of proteins actomyosin ATPase system. Anal. Biochem. 77 (1), 10–18. Halliwell, B., Gutteridge, J.M.C., 2007. Free Radicals in Biology and Medicine, 4th. ed. Oxford Univ. Press, New York. Joiner, M.C., van der Kogel, A., 2009. Basic Clinical Radiobiology, 4th. ed. Edward Arnold Publishers, London. Jore, D., Champion, B., Kaouadji, N., Jay-Gerin, J.-P., 1988. Radiolysis of ethanol and ethanol-water solutions: a tool for studying bioradical reactions. Radiat. Phys. Сhem. 32 (3), 443–448. Niki, E., 2009. Lipid peroxidation: physiological levels and dual biological effects. Free Radic. Biol. Med. 47 (5), 469–484. Petersen, R., Jensen, J.F., Nielsen, T.E., 2014. An improved protocol for the synthesis of 1(mesitylenesulfonyl)-3-nitro-1,2,4-triazole (MSNT). Org. Prep. Proced. Int. 46 (3), 267–271. Pikaev, A.K., Kartasheva, L.I., 1975. Radiolysis of aqueous solutions of ethylene glycol. Int. J. Radiat. Phys. Chem. 7 (2–3), 395–415. Pikaev, A.K., 1986. Modern Radiation Chemistry. Radiolysis of Gases and Liquids. Nauka,
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Contents lists available at ScienceDirect
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Effects of quinones and azoles on radiation-induced processes involving hydroxyl-containing carbon-centered radicals
T
S.N. Samovicha,b, R.L. Sverdlova,b, S.V. Voitekhovicha, Y.V. Grigorieva, O.A. Ivashkevicha,b, ⁎ O.I. Shadyroa,b, a b
Research Institute for Physical and Chemical Problems of the Belarusian State University (RI PCP BSU) Leningradskaya st., 14, 220006 Minsk, Republic of Belarus Department of Chemistry of the Belarusian State University Nezavisimosti av., 4, 220030 Minsk, Republic of Belarus
1. Introduction It has been reliably established that the free-radical processes play a crucial role in generation of radiobiological phenomena (Halliwell and Gutteridge, 2007; Joiner and van der Kogel, 2009; Von Sonntag, 1987). During the action of ionizing radiation on biosystems, various kinds of homolytic reactions may take place (Dean et al., 1997; Robbins and Zhao, 2004; Von Sonntag, 2006). However, for a long time, the attention of most researchers has been focused mainly on radiation-induced oxidation processes occurring in biomolecules, in the first place, in the cell membrane lipids, and on the ensuing pathophysiological consequences (Niki, 2009; Stark, 1991). In our studies (Shadyro, 1997), a concept was developed that the free-radical fragmentation reactions taking place in hydroxyl-containing biologically relevant substances may make a substantial contribution to the consequences resulting from irradiation of biosystems. It has been shown that the hydroxyl-containing organic compounds under radiolysis of their aqueous solutions undergo fragmentation according to the following general scheme (1):
It has been established in numerous studies that the occurrence of reactions (1) may cause damage to carbohydrates (Edimecheva et al., 2005), lipids (Shadyro et al., 2002, 2004a, 2004b), amino acids and proteins (Shadyro et al., 2000, 2003; Sladkova et al., 2012). The probability of the reaction (1) to occur depends on the structure of the initial compound. It has been established that the fragmentation rate constants for radicals (I) vary from 101 s−1 (for diol-type radicals)
(Pikaev and Kartasheva, 1975) to 105−107 s−1 (for radicals derived from organic phosphates) (Schuchmann et al., 1995). Oxygen produces a substantial influence on the probability of fragmentation reactions (1) in biomolecules, because the rate constant for the interaction of O2 with radicals of type (I) is of the order of 109 M−1 s−1. This leads to inhibition of the reaction (1). Oxygen levels in a living organism are known to vary within a considerable range: from ~ 10−4 M in well-ventilated lung cells to 10−5 M in skin cells (Carreau et al., 2011). On irradiation under conditions of low oxygen levels, the probability of reactions of type (1) grows, and hence its contribution to the development of negative consequences of the action of ionizing radiation on biomolecules increases. The aforesaid is an important incentive to perform the search for the substances capable of producing an effective regulative action on the probability of reactions of type (1) to occur. This goal can be attained by assessing the reactivity towards carbon-centered radicals of various suitable agents. Therefore, the object pursued in this study was in-
vestigation of the effects produced by quinones and azoles on formation of the end products during radiolysis of ethanol and aqueous solutions of glycerol-1-phosphate.
⁎ Corresponding author at: Research Institute for Physical and Chemical Problems of the Belarusian State University (RI PCP BSU) Leningradskaya st., 14, 220006 Minsk, Republic of Belarus. E-mail address: [email protected] (O.I. Shadyro).
http://dx.doi.org/10.1016/j.radphyschem.2017.09.004 Received 6 July 2017; Received in revised form 24 August 2017; Accepted 5 September 2017 Available online 11 September 2017 0969-806X/ © 2017 Elsevier Ltd. All rights reserved.
Radiation Physics and Chemistry 144 (2018) 308–316
S.N. Samovich et al.
Fig. 1. Structures of the quinones examined in the study.
2. Experimental section
suitable quantities of perchloric acid or sodium hydroxide. The solutions were transferred into ampoules, deaerated with high purity argon and sealed.
2.1. Chemicals
2.3. Irradiation of the samples
Methyl-p-benzoquinone (1), 2,5-dimethyl-1,4-benzoquinone (2), thymoquinone (3), coenzyme Q0 (4) and doxorubicin hydrochloride (5) (Fig. 1) produced by Sigma-Aldrich were used in this study without further purification. Azoles were represented in this study by imidazole (6), metronidazole (7) and 1,2,4-triazole (8) purchased from Sigma-Aldrich and nitrotriazoles 9–11 (Fig. 2), which were synthesized and purified according to the published procedures – 3-nitro-1,2,4-triazole (9) (Petersen et al., 2014), 1-methyl-3-nitro-1,2,4-triazole (10) (Sukhanov et al., 2005), 1-(2-hydroxyethyl)−3-nitro-1,2,4-triazole (11) (Degtyarik et al., 2012). Purity of the 1,2,4-triazoles was tested by mass spectrometry. Purity of all the compounds under study was not less than 98%. Ethanol 96% v/v was twice distilled on a rectifying column. Disodium or magnesium salts of glycerol-1-phosphate of purity not less than 95% were from Sigma-Aldrich.
Irradiation was performed on a unit MPX-γ−25 M with a Co60 source. The dose rate absorbed during the experiment was 0.22 ± 0.01 Gy/s. The absorbed dose interval varied within the range of 0.22–2.00 kGy. Fricke dosimeter was used to measure the absorbed dose rate (Fricke and Hart, 1966). 2.4. Identification of radiolysis products and determination of their concentrations 2.4.1. GC-FID analysis Products of free-radical transformations of ethanol – acetaldehyde (AA) and 2,3-butanediol (BD) – were determined on a Shimadzu chromatograph GC-17A using a silica capillary column Stabilwax-DA (l = 30 m, ID = 0.53 mm, df = 1.0 µm) against an external standard. The analysis conditions: starting temperature 40 °C, rise to 200 °C at a rate of 13 °C/min, injector temperature 220 °C, detector temperature 220 °C, carrier gas nitrogen, detector FID, injected volume 1.0 μl.
2.2. Preparation of samples for irradiation Solutions of the compounds under study were prepared by dissolving accurately weighed amounts in deaerated ethanol. Because of high volatility of the solvent, the following procedure was used to prepare solutions of the test compounds of concentrations 10−3 M. The solutions filled in densimeters were blown through with high purity argon (99.9%) for 60 min. Thereafter the volume of evaporated solvent was made up with deaerated ethanol and the densimeter content was mixed. The solutions were transferred to ampoules preliminary blown through with argon, filling not more than 60% of their fill volume, and then sealed. For preparation of solutions containing 10−1 M glycerol-1-phosphate and 10−3 М of tested compounds, twice-distilled water was used. The pH value of the solutions was adjusted to 7.0 ± 0.1 by adding
2.4.2. Spectrophotometric measurements Inorganic phosphate on a background of the organic one was determined spectrophotometrically using a modified procedure described in (Gin and Morales, 1977). To 0.5 ml of the solution to be examined were added 1 ml 1.8% solution of (NH4)2MoO4 × 4H2O in 1 M H2SO4 and 0.2 ml of 10% FeSO4 × 7H2O in 0.075 M H2SO4, and 4 ml of twicedistilled water. The solutions were stirred for 10 min on an auto-shaker. Optical density of the solutions was measured at λ = 720 nm on a Specord S600 spectrophotometer. Calibration curves were plotted using aqueous solutions of KH2PO4. The plot of optical density vs. concentration of inorganic phosphate was linear within the range of 10−5
Fig. 2. Structures of the azoles examined in the study.
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Table 1 Radiation-chemical yields of major radiolysis products of deaerated ethanol in the presence of tested quinones.
Results are expressed as means ± SD (n = 3). (a–d) – means in the same column followed by different letters are significantly different (p < 0.05).
to 10−3 M. For determination of concentrations of the compounds to be examined, measurements of optical density were performed before and after irradiation, if not stated otherwise.
2.5. Calculations of radiation-chemical yields Radiation-chemical yields (G, mol/J) were calculated from linear portions of plots of the product concentrations vs. dose absorbed. The results obtained in three independent experiments were used to calculate the yields. The error in determination of radiation-chemical yields was calculated by the least squares method using the confidence coefficient of 0.95.
2.4.3. GC–MS measurements Molecular products of interaction of α-hydroxyethyl radicals (αHER) with the quinones under study were identified using a gas-liquid chromatograph GC-2010 equipped with a column Equity-5 (l = 30 m, ID = 0.25 mm, df= 0.25 µm) and a Shimadzu mass detector GCMSQP2010 Plus. Analysis conditions: carrier gas helium, linear flow rate 1 ml/min, thermostat temperature 60 °C to 270 °C, injector temperature 270 °C, ion source temperature 280 °C, injected volume 1.0 μl.
2.6. Statistical analysis The data obtained were statistically analyzed using Statistica v. 10 software (StatSoft, Poland). The results were expressed as mean ± standard deviation (SD) from three independent parallel experiments.
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Table 2 Radiation-chemical yields for decay of the additives and formation of phenolic products in deaerated ethanol.
Results are expressed as means ± SD (n = 3). a, b – means in the same row followed by different letters are significantly different (p < 0.05).
are one of the major intermediates formed in this system under radiolysis. Therefore, in this study, we investigated effects of quinones and azoles on the yields of products formed in radiolysis of ethanol, deaerated with argon. It has been established that the following processes take place on radiolysis of ethanol (Freeman, 1982, 1987; Pikaev, 1986) (schemes 2–7):
The data were analyzed using the t-test for independent samples. The differences were considered as significant at p < 0.05.
In the absence of dissolved substances capable of oxidizing, the ethoxy radicals (CH3CH2O•) rapidly transform into α-HER (scheme 4, k4 = 1,1 × 106 M−1 s−1). It is known that in dilute solutions at a solute concentration of ≤ 10−3 M other reactions involving ethoxy radicals can be neglected (Pikaev, 1986). The CH3C•HOH radicals being formed in the absence of oxygen interact with each other giving 2,3-butanediol (BD) and acetaldehyde (AA) with approximately equal probability (k9/k8 = 0,9) (Jore, 1988) according to the schemes (8) and (9):
3. Results and discussion 3.1. Steady-state radiolysis of the compounds under study in deaerated ethanol Our studies have shown (Brinkevich et al., 2012; Samovich et al., 2014; Sverdlov et al., 2014) that radiolysis of ethanol is a convenient model to investigate interactions of α-hydroxyl-containing carboncentered radicals with various substances, because CH3C•HOH radicals 311
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Table 3 Effects of imidazole and 1,2,4-triazole nitro derivatives on radiation-chemical yields of major radiolysis products formed in deaerated ethanol.
Results are expressed as means ± SD (n = 3). a, b, c – means in the same column followed by different letters are significantly different (p < 0.05).
AA is also formed as a result of excited ethanol molecules decomposition (scheme 7). Hence, by measuring radiation-chemical yields of AA and BD in the presence and the absence of the test compounds, one may assess their reactivity towards α-HER. As follows from the data presented in Table 1, a significant decrease in radiation-chemical yields of BD was observed in the presence of quinones, including the anti-tumor agent doxorubicin 5. At the same time, the yields of AA increased, becoming almost two times greater vs. controls in the presence of 1−4.
The radiation-chemical yields for the decay of quinones and formation of the major products identified by chromato-mass spectrometry are shown in Table 2. Close values of yields for decomposition of the starting quinones and formation of the respective reduction products show that the main products of transformation of the test quinones were phenolic compounds. Hence, the test quinones can effectively oxidize α-HER to AA, themselves being transformed into semiquinone radicals (II), according to scheme (10):
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Table 4 Effects of quinones on radiation-chemical yields of inorganic phosphate.
Results are expressed as means ± SD (n = 3). a, b – means in the column followed by different letters are significantly different (p < 0.05).
The semiquinone radicals (II) can form the identified phenols through disproportionation reactions between themselves (Scheme 11) or with α-HER (Scheme 12).
addition to a delocalized semiquinone radical (or to the multiple bonds of doxorubicin) can be realized, causing a decrease in the radiationchemical yield of the BD observed in the experiment.
The lower yield values for AA observed in the case of doxorubicine as compared to those for additives 1−4 are most probably due to the low sterical accessibility of the quinoid site for the reaction with α-HER according to schemes (10) and (12). Moreover, the semiquinone radical formed from doxorubicine according to scheme (11) is more stable due to delocalization of the unpaired electron over the aromatic system of the molecule and hence less reactive towards oxidation of α-HER. Therefore, besides the oxidation reactions, the process of α-HER
Thus, the data presented above show that quinones of various structures effectively interact with α-HER by oxidizing the latter to AA. Radiation-chemical yields of AA and BD obtained in experiments on radiolysis of solutions of imidazole 6, metronidazole 7 and 1,2,4-triazole nitro derivatives 9−11 in deaerated ethanol are presented in Table 3. It follows from the data shown in Table 3 that a virtually complete suppression of BD formation was seen in the presence of nitroazoles 7 and 9−11. Besides the decrease in the yields of BD, a more
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Table 5 Effects of imidazole, 1,2,4-triazole and its nitro derivatives on radiation-chemical yields of inorganic phosphate.
Results are expressed as means ± SD (n = 3). (a–d) – means in the column followed by different letters are significantly different (p < 0.05).
The high reactivity of the test compounds towards α-HER might be evidence of their ability to affect the realization probability of reaction (1) to a significant extent. To confirm this assumption, we examined the effects of the named quinones and azoles on radiation-chemical formation of inorganic phosphate during γ-irradiation of aqueous solutions of glycerol-1-phosphate at pH 7. It has been shown (Schuchmann et al., 1995) that glycerol-1-phosphate undergoes dephosphorylation on radiolysis of its aqueous solutions. Formation of inorganic phosphate
than two-fold rise in radiation-chemical yields of AA was observed in the presence of the nitroazoles. In the presence of imidazole 6, within the experimental error limits, no changes in radiation-chemical yields of AA and BD were noted. It can be concluded that imidazole is characterized by low reactivity towards α-HER. The effects observed on adding compounds 7 and 9−11 to ethanol are associated with the presence of nitro groups in their structures, which endow them with oxidizing properties making possible scheme (13) to occur:
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occurs under the action of •OH and •H radicals on glycerol-1-phosphate followed by fragmentation of the α-HCR (III) according to scheme (14−15).
that the hydroxyl-containing lipids (phosphatidyl glycerol, phosphatidyl inositol, cardiolipin) being subjected to radiolysis undergo decomposition similar to that of glycerol-1-phosphate, giving phospha-
The obtained data show that the radiation-chemical yields of inorganic phosphate decreased significantly in the presence of quinones (Table 4) and azoles (Table 5). These findings point to the ability of the
tidic acid. For example, accumulation of phosphatidic acid in high yields was observed during radiation-induced fragmentation of cardiolipin (the main membrane lipid of mitochondria) according to the scheme similar to (15) (Shadyro et al., 2004b; Yurkova et al., 2008):
It has been found that the accumulation of phosphatidic acid in cancer cells promotes their proliferation (Foster, 2009; Toschi et al., 2009; Foster et al., 2014). Taking this into account, one can expect that the radiation-induced formation of phosphatidic acid in cancer cells should worsen the efficiency of radiotherapy in cancer patients. As quinones and azoles block these negative effects, they are expected to manifest radiosensitizing properties when introduced into cancer cells. Indeed, compounds of this type, such as metronidazole and doxorubicine are being used as radiosensitizers in radiation medicine. The data obtained in this study open up a new possibility to enhance efficiency of radiation therapy by introducing agents effectively blocking the radiation-induced processes leading to formation of products promoting cancer cell proliferation.
named compounds to block fragmentation of radicals (III) in spite of the very high rate constant for this reaction (> 106 s−1) (Schuchmann et al., 1995). Bearing in mind that during radiolysis of ethanol and of aqueous glycerol-1-phosphate solutions formation of similar α-hydroxyl-containing carbon-centered radicals is expected, a conclusion can be drawn that the mechanisms describing interaction of quinones and azoles with radicals (III) should be described by schemes analogous to (6, 8, 9). To all appearances, the inhibition of dephosphorylation results from oxidation of radicals (III) prior to their fragmentation. Hence, quinones and azoles may be promising agents for suppression of processes of type (1). It has been established earlier (Shadyro et al., 2002, 2004a, 2004b) 315
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