- Email: [email protected]
Effect of hydroxy substituent on the prooxidant action of naphthoquinone compounds
Toxicology in Vitro 24 (2010) 905–909
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Effect of hydroxy substituent on the prooxidant action of naphthoquinone compounds Keiko Murakami a, Miyako Haneda a, Shouko Iwata b, Masataka Yoshino a,c,* a
Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan Nagoya University Bioscience and Biotechnology Center, Laboratory of Animal Cell Function, Chikusa-ku, Nagoya 464-8601, Japan1 c Department of Food and Nutritional Environment, Kinjo Gakuin University, Omori 2-1723, Moriyama-ku, Nagoya 463-8521, Japan b
a r t i c l e
i n f o
Article history: Received 27 July 2009 Accepted 26 November 2009 Available online 2 December 2009 Keywords: Naphthoquinone Juglone Lawsone Lipid peroxidation 8-Hydroxy-20 -deoxyguanosine Ferrous ion oxidation ESR spectra
a b s t r a c t Prooxidant activity of naphthoquinone compounds was analyzed by lipid peroxidation, and the formation of base adduct in DNA. Naphthoquinones with electron-repelling hydroxyl group in the benzene moiety such as juglone and shikonin of lower concentrations stimulated the microsomal lipid peroxidation, but lawsone and lapachol with hydroxyl group in the quinone moiety did not enhance the formation of lipid peroxides. Naphthoquinone-dependent lipid peroxidation was closely related to the enhancement of Fe2+ autooxidation. Treatment of DNA with juglone a representative of 5-hydroxylated naphthoquinone stimulated the formation of 8-hydroxy-20 -deoxyguanosine, whereas lawsone and lapachol showed negligible formation of DNA base adduct. ESR spectra showed that juglone can form semiquinone radical in the presence of ferrous ion, but lawsone cannot. Biological toxicity of juglone with the potent electron-repelling group at 5-position may be due to the reactive oxygen species formed by semiquinone radical, but naphthoquinone compounds with an electron-repelling group in the quinone moiety, lawsone shows weak toxicity with only a little ability producing reactive oxygen species by the negligible formation of semiquinone. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Naphthoquinone compounds, widely distributed in plants have pharmacological and toxicological importance, and are often used as cancer chemotherapeutic drugs, antimalarials, antibacterial agents and fungicides (O’Brien, 1991; Monks et al., 1992). Cytotoxic action of naphthoquinones varies depending on the structure of the compounds: juglone, 5-hydroxy-1,4-naphthoquinone causes cell death of some types of cells, whereas lawsone, 2-hydroxy-1,4napthoquinone shows little or no cytotoxicity (Kumbhar et al., 1996) (Fig. 1). These cytotoxic effects have been explained by the decrease in glutathione due to the enzymatic redox cycling of naphthoquinones (Ross et al., 1986; Öllinger and Brunmark, 1991), and by the formation of adducts to DNA and protein (Rossi et al., 1986). In the present work, we analyzed the direct prooxidant action of naphthoquinone compounds with a focus on the lipid peroxidation, and the formation of DNA base adduct, 8-hydroxy-20 -deoxyguanosine. Prooxidant properties of naphthoquinones were closely related to the enhancement of autooxidation of reduced iron. Juglone with the electron-repelling hydroxyl group in the benzene moiety was suggested to form semiquinone radical
* Corresponding author. Present address: Department of Food and Nutritional Environment, Kinjo Gakuin University, Omori 2-1723, Moriyama-ku, Nagoya 4638521, Japan. Fax: +81 52 798 4927. E-mail address: [email protected] (M. Yoshino). 1 Present address. 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.11.018
causing potent oxidative damage of DNA and lipids, whereas naphthoquinones with hydroxyl group in the quinone moiety showed a weak cytotoxicity that was ascribed to the reduction of transition metals forming oxygen radicals. 2. Materials and methods 2.1. Materials Juglone, plumbagin, shikonin, menadione, lawsone, lapachol, DMPO (5,50 -dimethyl-1-pyrroline N-oxide), 8-hydroxy-20 -deoxyguanosine, enzymes for DNA hydrolysis, and bathophenanthroline disulfonate were obtained from Sigma–Aldrich Chemicals (Tokyo, Japan). NADP-Isocitrate dehydrogenase is a product of Oriental Yeast Co. (Osaka, Japan). Other chemicals were obtained from commercial sources. 2.2. Lipid peroxidation of liver microsomes Microsomes were prepared from the livers of adult male rats by standard differential centrifugation techniques (Quinlan et al., 1988). Reaction mixture of 1 ml contained 60 mM Tris–HCl buffer (pH 7.5), 10 lM FeCl3, 0.5 mM ascorbic acid, microsomal fraction of 0.2 mg protein, and naphthoquinones. The mixture was incubated at 37 °C for 20 min, and the reaction was stopped by addition of 100% trichloroacetic acid. Lipid peroxides produced were deter-
906
K. Murakami et al. / Toxicology in Vitro 24 (2010) 905–909
Fig. 1. Structure of naphthoquinone compounds.
mined as the thiobarbituric acid-reactive substances (absorbance at 532 nm) (Draper and Hadley, 1990). 2.3. Autooxidation of Fe2+ ion
Fig. 2. Effect of naphthoquinone compounds on the iron-mediated lipid peroxidation of rat liver microsomes. Lipid peroxidation was expressed as the production of thiobarbituric acid-reactive substances (TBARS) (Draper and Hadley, 1990), which was induced by 10 lM FeCl3 and 0.5 mM ascorbic acid. Naphthoquinone compounds were added at the indicated concentrations. Absorbance at 532 nm was measured after the incubation at 37 °C for 20 min. Data represent mean ± SD with three different determinations. d, juglone; D, shikonin; h, plumbagin; N, lapachol; j, lawsone.
Fe2+ autooxidation was evaluated by determining the Fe2+ concentration on the basis of the bathophenanthroline disulfonate method (Mahler and Elowe, 1954) with a slight modification described previously (Yoshino and Murakami, 1998). 2.4. Quantitation of 8-hydroxy-20 -deoxgyguanosine (8-OHdG) in calf thymus DNA treated with naphthoquinone compounds Calf thymus DNA was treated with naphthoquinone compounds in the presence of CuCl2. Incubation conditions and the analytical method for determination of 8-OHdG were similar to those described previously (Yoshino et al., 1999; Yoshino et al., 2002) based on HPLC equipped with electrochemical detector (Homma et al.,1994). 2.5. ESR analysis Reaction mixture containing 50 lM naphthoquinone, 0.1 mM DMPO and potassium phosphate buffer (pH 7.5) in the absence and presence of iron, superoxide dismutase or catalase was mixed aerobically for 15 s, and ESR spectra were recorded on a JEOL JESTE200 with the following instrument settings: modulation; 100 kHz, microwave frequency; 9.4 GHz, power; 1.5 mW, magnetic field; 336 ± 5 mT, sweep time; 4 min, modulation amplitude; 0.2 mT, time consist; 0.3 s, receiver gain; 1600. 2.6. Statistics Statistical analysis was performed by Student’s t-test. 3. Results 3.1. Effect of napththoquinone compounds on the microsomal lipid peroxidation We examined the effect of naphthoquinone compounds (Fig. 1) on the lipid peroxidation of microsomes from rat liver (Fig. 2). Naphthoquinone compounds with 5-hydroxyl group enhanced lipid peroxidation. Juglone and shikonin of lower concentration markedly increased the thiobarbituric acid-reactive substances as a marker of lipid peroxidation, but these compounds of higher concentrations rather tended to decrease the formation of thiobarbitu-
Fig. 3. Effect of naphthoquinone compounds on the autooxidation of ferrous ion. Iron autooxidation was followed by determining the ferrous ion concentration according to the bathophenanthroline disulfonate method (Yoshino and Murakami, 1998). Reaction mixture of 2 ml containing 0.15 mM naphthoquinone compounds in 10 mM Tris–HCl buffer (pH 7.25) was incubated with 0.1 mM FeSO4. Aliquots of 0.2 ml were mixed with 0.1 ml of 1 mM bathophenanthroline disulfonate at appropriate intervals, and the absorbance at 540 nm was measured. Each point represents the mean ± SD of three independent experiments. Open circle, no addition; filled diamond, menadione. Other symbols are identical to those of Fig. 2.
ric acid-reactive substances. Plumbagin at its higher concentration also increased the lipid peroxidation. On the contrary, lawsone and lapachol with 2-hydroxyl group in the benzene moiety did not stimulate lipid peroxidation. 3.2. Effects of naphthoquinone compounds on the autooxidation of Fe2+ ion The effect of naphthoquinone compounds on the rate of Fe2+ autooxidation was examined. Juglone and shikonin increased the rate of the Fe2+ oxidation effectively, and plumbagin enhanced the autooxidation of Fe2+ ion to a lesser extent. Lawsone, menadi-
K. Murakami et al. / Toxicology in Vitro 24 (2010) 905–909
907
Fig. 4. Formation of 8-hydroxy-20 -deoxyguanosine in DNA treated with naphthoquinone compounds. Calf thymus DNA was treated with naphthoquinone compounds or ascorbic acid in the presence of CuCl2. The reaction mixture of 4 ml contained 100 lg of calf thymus DNA, 0.2 mM naphthoquinone compounds or 10 mM ascorbic acid and 0.2 mM CuCl2 in 10 mM Tris–HCl buffer (pH 7.4). The mixture was incubated at 37 °C for 60 min, and used for the determination of 8hydroxy-20 -deoxyguanosine (8-OHdG) and deoxyguanosine (dG). 8-OHdG was detected using ESA Neurochem electrochemical detector (Homma et al., 1994), and unmodified deoxyguanosine was detected by UV absorption. The amount of 8OHdG present in the DNA samples was calculated by measuring the area of the peaks obtained from both electrochemical and UV traces and comparing those obtained from DNA samples with those obtained from the standards. Data are represented as the ratio of 8-OHdG/dG. Points indicate mean ± SD of three different determinations. Asterisks indicate significant differences between the control and the naphthoquinones added: p < 0.001; N.S., no significance.
one and lapachol showed little or no stimulating effect on Fe2+ oxidation (Fig. 3). 3.3. Oxidative damage and formation of 8-OHdG in calf thymus DNA When calf thymus DNA was treated with ascorbic acid and CuCl2, 8-OHdG was effectively formed (Yoshino et al., 1999). We examined the formation of 8-OHdG by naphthoquinone compounds with 5-hydroxyl and 2-hydroxyl groups in the presence of copper. Juglone markedly increased the 8-OHdG, the concentration of which reached the one second to third of the levels observed in DNA treated with ascorbate/copper. Of the naphthoquinones with 5-hydroxyl group tested, plumbagin tended to increase the 8-OHdG formation, but did not show significant difference against control group. Lawsone and lapachol, naphthoquinones with 2-hydroxyl group in the quinone moiety showed little or no production of 8-OHdG (Fig. 4). 3.4. Formation of semiquinone radical Formation of semiquinone radical was examined with ESR spectroscopy. We analyzed the ESR spectra of juglone, a representative with 5-hydroxyl group and lawsone with 2-hydroxyl group in the quinone moiety. Juglone showed generation of a semiquinone radical when ferrous ion added (Fig. 5), whereas lawsone did not produce a semiquinone radical in the presence of ferrous ion (Fig. 6). 4. Discussion Quinones are widely distributed in nature and play an essential biological role in mitochondrial respiration and photosynthesis. Some quinone compounds such as benzoquinone and naphthoquinones show potent cytotoxic and anti-proliferative effects on various cells and organisms (Öllinger and Brunmark, 1991; Kumbhar et al., 1996; Inbaraj and Chignell, 2004), and have been used for
Fig. 5. ESR spectra of juglone in the absence and presence of superoxide dismutase and catalase. Reaction mixture contained: (A) No addition (50 lM juglone and 0.1 mM DMPO); (B) A plus 50 lM FeSO4; (C) B + superoxide dismutase; (D) B + superoxide dismutase + catalase. Arrows indicate the signals of semiquinone radicals.
topical treatment of certain diseases (O’Brien, 1991; Monks et al., 1992). Quinone toxicity has been studied extensively with isolated hepatocytes and keratinocytes (Öllinger and Brunmark, 1991; Inbaraj and Chignell, 2004), and was assumed to be due to two basic mechanisms: (a) the capacity of quinines to produce reactive oxygen species, followed by GSH oxidation to GSSG (Ross et al., 1986; Öllinger and Brunmark, 1991) and (b) the electrophilicity of quinones, which enables them to form adducts to cellular constituents (Rossi et al., 1986). Production of free radical by quinones has been thought to be a consequence of dioxygen reduction by
908
K. Murakami et al. / Toxicology in Vitro 24 (2010) 905–909
In this work we analyzed the direct prooxidant action of naphthoquinone compounds with a focus on the ability of the formation of reactive oxygen species. Naphthoquinone compounds with 5hydroxyl group in the benzene moiety such as juglone, shikonin and plumbagin markedly stimulated the lipid peroxidation. These results were in good agreement with the finding that naphthoquinones with 5-hydroxyl group in the benzene moiety have potent cytotoxicity on hepatocytes (Öllinger and Brunmark, 1991) and keratinocytes (Inbaraj and Chignell, 2004). 5-Hydroxylated naphthoquinones-mediated lipid peroxidation was closely related to the oxidation of Fe2+ ion. Stimulation of ferrous ion oxidation by juglone due to its electron-attracting activity was accompanied with the formation of semiquinone radical, which can reduce dioxygen molecule forming superoxide radical. Lipid peroxidation is considered to be initiated by the formation of perferryl ion or Fe[II]–O2–Fe[III] complex (Svingen et al. 1971; Miller and Aust, 1989; Yoshino and Murakami, 1998). Stimulation by 5-hydroxynaphthoquinone of Fe2+ oxidation can form perferryl ion or the complex, resulting in the enhancement of lipid peroxidation. On the other hand, naphthoquinones with 2-hydroxyl or 2-methyl group in the quinone moiety such as lawsone and lapachol showed no stimulation of lipid peroxidation. Lawsone and lapachol further did not stimulate Fe2+ ion oxidation. These effects of 2-substituted naphthoquinones were well agreed with little cytotoxicity (Öllinger and Brunmark, 1991; Inbaraj and Chignell, 2004). Of particular interest is the finding that the juglone, 5-hydroxynaphthoquinone could form 8-OHdG in DNA, whereas lawsone the naphthoquinone with hydroxyl or methyl group in quinone ring did not produce 8-OHdG in DNA. Introduction of electron-donating hydroxyl group lowers redox potentials of the naphthoquinone compound. In particular 2-hydroxyl or methyl group attached to quinone moiety of the naphthoquinone compound decreases the potential effectively, but 5-hydroxyl group in the benzene moiety of the naphthoquinone causes only a little effect on the potential. Juglone with higher reduction potential is in equilibrium between quinone and quinol forms: production of semiquinone radical may result from disproportionation of the parent quinone and hydroquinone forms (Bothner-By, 1953).
Q þ HQ ¡ 2SQ The quinol form can reduce copper, and generate superoxide radical resulting in the formation of hydroxyl radical, which can produce 8OHdG in DNA. However, lawsone with lowered reduction potential did not affect transition metals, and thus did not form lipid peroxides and of DNA base adduct. Cytotoxicity and chemotherapeutic effects of naphthoquinone compounds can be explained by the position of side chains. Introduction of electron-donating or electron-attracting groups to the benzene or quinone moieties determines the prooxidant properties. References
Fig. 6. ESR spectra of lawsone in the absence and presence of superoxide dismutase and catalase. Reaction mixture was similar to that of Fig. 5 except that juglone was replaced by lawsone. Symbols were identical to those of Fig. 5.
semiquinone intermediates, which are formed via one-electron reduction by enzymes such as microsomal NADPH-cytochrome P450 reductase or mitochondrial NADH-ubiquinone oxidoreductase (Inbaraj and Chignell, 2004).
Bothner-By, A.A., 1953. Quinone—hydroquinone exchange reactions. II. Duroquinone and durohydroquinone exchange in buffered methanol solution. Journal of American Chemical Society 75, 728–730. Draper, H.H., Hadley, M., 1990. Malondialdehyde determination as index of lipid peroxidation. Methods in Enzymology 186, 421–431. Homma, Y., Tsunoda, M., Kasai, H., 1994. Evidence for the accumulation of oxidative stress during cellular ageing of human diploid fibroblasts. Biochemical and Biophysical Research Communications 203, 1063–1068. Inbaraj, J.J., Chignell, C.F., 2004. Cytotoxic action of juglone and plumbagin: a mechanistic study using HaCaT keratinocytes. Chemical Research in Toxicology 17, 55–62. Kumbhar, A., Padhye, S., Ross, D., 1996. Cytotoxic properties of ironhydroxynaphthoquinone complexes in rat hepatocytes. BioMetals 9, 235–240. Mahler, H.R., Elowe, D.G., 1954. Studies on metalloflavoproteins. II. The role of iron in diphosphopyridine nucleotide cytochrome c reductase. Journal of Biological Chemistry 210, 165–179. Miller, D.M., Aust, S.D., 1989. Studies of ascorbate-dependent, iron-catalyzed lipid peroxidation. Archives of Biochemistry and Biophysics 271, 113–119.
K. Murakami et al. / Toxicology in Vitro 24 (2010) 905–909 Monks, T.J., Hanzlik, R.P., Cohen, G.M., Ross, D., Graham, D.G., 1992. Quinone chemistry and toxicity. Toxicology and Applied Pharmacology 112, 2–16. O’Brien, P.J., 1991. Molecular mechanisms of quinone cytotoxicity. ChemicoBiological Interactions 80, 1–41. Öllinger, K., Brunmark, A., 1991. Effect of hydroxy substituent position on 1,4naphthoquinone toxicity to rat hepatocytes. Journal of Biological Chemistry 266, 21496–21503. Quinlan, G.J., Halliwell, B., Moorhouse, C.P., Gutteridge, J.M.C., 1988. Action of lead(II) and aluminium (III) ions on iron-stimulated lipid peroxidation in liposomes, erythrocytes and rat liver microsomal fractions. Biochimica et Biophysica Acta 962, 196–200. Ross, D., Thor, H., Threadgill, M.D., Sandy, M.S., Smith, M.T., Moldéus, P., Orrenius, S., 1986. The role of oxidative processes in the cytotoxicity of substituted 1,4naphthoquinones in isolated hepatocytes. Archives of Biochemistry and Biophysics 248, 460–466.
909
Rossi, L., Moore, G.A., Orrenius, S., O’Brien, P.J., 1986. Quinone toxicity in hepatocytes without oxidative stress. Archives of Biochemistry and Biophysics 251, 25–35. Svingen, B.A., Buege, J.A., O’Neal, F.O., Aust, S.D., 1971. The mechanism of NADPHdependent lipid peroxidation. The propagation of lipid peroxidation. Journal of Biological Chemistry 254, 5892–5899. Yoshino, M., Murakami, K., 1998. Interaction of iron with polyphenolic compounds. Application to antioxidant characterization. Analytical Biochemistry 257, 40–44. Yoshino, M., Haneda, M., Naruse, M., Murakami, K., 1999. Prooxidant activity of flavonoids: copper-dependent strand breaks and the formation of 8-hydroxy20 -deoxyguanosine in DNA. Molecular Genetics and Metabolism 68, 468–472. Yoshino, M., Haneda, M., Naruse, M., Htay, H.H., Iwata, S., Tsubouchi, R., Murakami, K., 2002. Prooxidant action of gallic acid compounds: copper-dependent stand breaks and the formation of 8-hydroxy-20 -deoxyguanosine in DNA. Toxicology In Vitro 16, 705–709.
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Effect of hydroxy substituent on the prooxidant action of naphthoquinone compounds Keiko Murakami a, Miyako Haneda a, Shouko Iwata b, Masataka Yoshino a,c,* a
Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan Nagoya University Bioscience and Biotechnology Center, Laboratory of Animal Cell Function, Chikusa-ku, Nagoya 464-8601, Japan1 c Department of Food and Nutritional Environment, Kinjo Gakuin University, Omori 2-1723, Moriyama-ku, Nagoya 463-8521, Japan b
a r t i c l e
i n f o
Article history: Received 27 July 2009 Accepted 26 November 2009 Available online 2 December 2009 Keywords: Naphthoquinone Juglone Lawsone Lipid peroxidation 8-Hydroxy-20 -deoxyguanosine Ferrous ion oxidation ESR spectra
a b s t r a c t Prooxidant activity of naphthoquinone compounds was analyzed by lipid peroxidation, and the formation of base adduct in DNA. Naphthoquinones with electron-repelling hydroxyl group in the benzene moiety such as juglone and shikonin of lower concentrations stimulated the microsomal lipid peroxidation, but lawsone and lapachol with hydroxyl group in the quinone moiety did not enhance the formation of lipid peroxides. Naphthoquinone-dependent lipid peroxidation was closely related to the enhancement of Fe2+ autooxidation. Treatment of DNA with juglone a representative of 5-hydroxylated naphthoquinone stimulated the formation of 8-hydroxy-20 -deoxyguanosine, whereas lawsone and lapachol showed negligible formation of DNA base adduct. ESR spectra showed that juglone can form semiquinone radical in the presence of ferrous ion, but lawsone cannot. Biological toxicity of juglone with the potent electron-repelling group at 5-position may be due to the reactive oxygen species formed by semiquinone radical, but naphthoquinone compounds with an electron-repelling group in the quinone moiety, lawsone shows weak toxicity with only a little ability producing reactive oxygen species by the negligible formation of semiquinone. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Naphthoquinone compounds, widely distributed in plants have pharmacological and toxicological importance, and are often used as cancer chemotherapeutic drugs, antimalarials, antibacterial agents and fungicides (O’Brien, 1991; Monks et al., 1992). Cytotoxic action of naphthoquinones varies depending on the structure of the compounds: juglone, 5-hydroxy-1,4-naphthoquinone causes cell death of some types of cells, whereas lawsone, 2-hydroxy-1,4napthoquinone shows little or no cytotoxicity (Kumbhar et al., 1996) (Fig. 1). These cytotoxic effects have been explained by the decrease in glutathione due to the enzymatic redox cycling of naphthoquinones (Ross et al., 1986; Öllinger and Brunmark, 1991), and by the formation of adducts to DNA and protein (Rossi et al., 1986). In the present work, we analyzed the direct prooxidant action of naphthoquinone compounds with a focus on the lipid peroxidation, and the formation of DNA base adduct, 8-hydroxy-20 -deoxyguanosine. Prooxidant properties of naphthoquinones were closely related to the enhancement of autooxidation of reduced iron. Juglone with the electron-repelling hydroxyl group in the benzene moiety was suggested to form semiquinone radical
* Corresponding author. Present address: Department of Food and Nutritional Environment, Kinjo Gakuin University, Omori 2-1723, Moriyama-ku, Nagoya 4638521, Japan. Fax: +81 52 798 4927. E-mail address: [email protected] (M. Yoshino). 1 Present address. 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.11.018
causing potent oxidative damage of DNA and lipids, whereas naphthoquinones with hydroxyl group in the quinone moiety showed a weak cytotoxicity that was ascribed to the reduction of transition metals forming oxygen radicals. 2. Materials and methods 2.1. Materials Juglone, plumbagin, shikonin, menadione, lawsone, lapachol, DMPO (5,50 -dimethyl-1-pyrroline N-oxide), 8-hydroxy-20 -deoxyguanosine, enzymes for DNA hydrolysis, and bathophenanthroline disulfonate were obtained from Sigma–Aldrich Chemicals (Tokyo, Japan). NADP-Isocitrate dehydrogenase is a product of Oriental Yeast Co. (Osaka, Japan). Other chemicals were obtained from commercial sources. 2.2. Lipid peroxidation of liver microsomes Microsomes were prepared from the livers of adult male rats by standard differential centrifugation techniques (Quinlan et al., 1988). Reaction mixture of 1 ml contained 60 mM Tris–HCl buffer (pH 7.5), 10 lM FeCl3, 0.5 mM ascorbic acid, microsomal fraction of 0.2 mg protein, and naphthoquinones. The mixture was incubated at 37 °C for 20 min, and the reaction was stopped by addition of 100% trichloroacetic acid. Lipid peroxides produced were deter-
906
K. Murakami et al. / Toxicology in Vitro 24 (2010) 905–909
Fig. 1. Structure of naphthoquinone compounds.
mined as the thiobarbituric acid-reactive substances (absorbance at 532 nm) (Draper and Hadley, 1990). 2.3. Autooxidation of Fe2+ ion
Fig. 2. Effect of naphthoquinone compounds on the iron-mediated lipid peroxidation of rat liver microsomes. Lipid peroxidation was expressed as the production of thiobarbituric acid-reactive substances (TBARS) (Draper and Hadley, 1990), which was induced by 10 lM FeCl3 and 0.5 mM ascorbic acid. Naphthoquinone compounds were added at the indicated concentrations. Absorbance at 532 nm was measured after the incubation at 37 °C for 20 min. Data represent mean ± SD with three different determinations. d, juglone; D, shikonin; h, plumbagin; N, lapachol; j, lawsone.
Fe2+ autooxidation was evaluated by determining the Fe2+ concentration on the basis of the bathophenanthroline disulfonate method (Mahler and Elowe, 1954) with a slight modification described previously (Yoshino and Murakami, 1998). 2.4. Quantitation of 8-hydroxy-20 -deoxgyguanosine (8-OHdG) in calf thymus DNA treated with naphthoquinone compounds Calf thymus DNA was treated with naphthoquinone compounds in the presence of CuCl2. Incubation conditions and the analytical method for determination of 8-OHdG were similar to those described previously (Yoshino et al., 1999; Yoshino et al., 2002) based on HPLC equipped with electrochemical detector (Homma et al.,1994). 2.5. ESR analysis Reaction mixture containing 50 lM naphthoquinone, 0.1 mM DMPO and potassium phosphate buffer (pH 7.5) in the absence and presence of iron, superoxide dismutase or catalase was mixed aerobically for 15 s, and ESR spectra were recorded on a JEOL JESTE200 with the following instrument settings: modulation; 100 kHz, microwave frequency; 9.4 GHz, power; 1.5 mW, magnetic field; 336 ± 5 mT, sweep time; 4 min, modulation amplitude; 0.2 mT, time consist; 0.3 s, receiver gain; 1600. 2.6. Statistics Statistical analysis was performed by Student’s t-test. 3. Results 3.1. Effect of napththoquinone compounds on the microsomal lipid peroxidation We examined the effect of naphthoquinone compounds (Fig. 1) on the lipid peroxidation of microsomes from rat liver (Fig. 2). Naphthoquinone compounds with 5-hydroxyl group enhanced lipid peroxidation. Juglone and shikonin of lower concentration markedly increased the thiobarbituric acid-reactive substances as a marker of lipid peroxidation, but these compounds of higher concentrations rather tended to decrease the formation of thiobarbitu-
Fig. 3. Effect of naphthoquinone compounds on the autooxidation of ferrous ion. Iron autooxidation was followed by determining the ferrous ion concentration according to the bathophenanthroline disulfonate method (Yoshino and Murakami, 1998). Reaction mixture of 2 ml containing 0.15 mM naphthoquinone compounds in 10 mM Tris–HCl buffer (pH 7.25) was incubated with 0.1 mM FeSO4. Aliquots of 0.2 ml were mixed with 0.1 ml of 1 mM bathophenanthroline disulfonate at appropriate intervals, and the absorbance at 540 nm was measured. Each point represents the mean ± SD of three independent experiments. Open circle, no addition; filled diamond, menadione. Other symbols are identical to those of Fig. 2.
ric acid-reactive substances. Plumbagin at its higher concentration also increased the lipid peroxidation. On the contrary, lawsone and lapachol with 2-hydroxyl group in the benzene moiety did not stimulate lipid peroxidation. 3.2. Effects of naphthoquinone compounds on the autooxidation of Fe2+ ion The effect of naphthoquinone compounds on the rate of Fe2+ autooxidation was examined. Juglone and shikonin increased the rate of the Fe2+ oxidation effectively, and plumbagin enhanced the autooxidation of Fe2+ ion to a lesser extent. Lawsone, menadi-
K. Murakami et al. / Toxicology in Vitro 24 (2010) 905–909
907
Fig. 4. Formation of 8-hydroxy-20 -deoxyguanosine in DNA treated with naphthoquinone compounds. Calf thymus DNA was treated with naphthoquinone compounds or ascorbic acid in the presence of CuCl2. The reaction mixture of 4 ml contained 100 lg of calf thymus DNA, 0.2 mM naphthoquinone compounds or 10 mM ascorbic acid and 0.2 mM CuCl2 in 10 mM Tris–HCl buffer (pH 7.4). The mixture was incubated at 37 °C for 60 min, and used for the determination of 8hydroxy-20 -deoxyguanosine (8-OHdG) and deoxyguanosine (dG). 8-OHdG was detected using ESA Neurochem electrochemical detector (Homma et al., 1994), and unmodified deoxyguanosine was detected by UV absorption. The amount of 8OHdG present in the DNA samples was calculated by measuring the area of the peaks obtained from both electrochemical and UV traces and comparing those obtained from DNA samples with those obtained from the standards. Data are represented as the ratio of 8-OHdG/dG. Points indicate mean ± SD of three different determinations. Asterisks indicate significant differences between the control and the naphthoquinones added: p < 0.001; N.S., no significance.
one and lapachol showed little or no stimulating effect on Fe2+ oxidation (Fig. 3). 3.3. Oxidative damage and formation of 8-OHdG in calf thymus DNA When calf thymus DNA was treated with ascorbic acid and CuCl2, 8-OHdG was effectively formed (Yoshino et al., 1999). We examined the formation of 8-OHdG by naphthoquinone compounds with 5-hydroxyl and 2-hydroxyl groups in the presence of copper. Juglone markedly increased the 8-OHdG, the concentration of which reached the one second to third of the levels observed in DNA treated with ascorbate/copper. Of the naphthoquinones with 5-hydroxyl group tested, plumbagin tended to increase the 8-OHdG formation, but did not show significant difference against control group. Lawsone and lapachol, naphthoquinones with 2-hydroxyl group in the quinone moiety showed little or no production of 8-OHdG (Fig. 4). 3.4. Formation of semiquinone radical Formation of semiquinone radical was examined with ESR spectroscopy. We analyzed the ESR spectra of juglone, a representative with 5-hydroxyl group and lawsone with 2-hydroxyl group in the quinone moiety. Juglone showed generation of a semiquinone radical when ferrous ion added (Fig. 5), whereas lawsone did not produce a semiquinone radical in the presence of ferrous ion (Fig. 6). 4. Discussion Quinones are widely distributed in nature and play an essential biological role in mitochondrial respiration and photosynthesis. Some quinone compounds such as benzoquinone and naphthoquinones show potent cytotoxic and anti-proliferative effects on various cells and organisms (Öllinger and Brunmark, 1991; Kumbhar et al., 1996; Inbaraj and Chignell, 2004), and have been used for
Fig. 5. ESR spectra of juglone in the absence and presence of superoxide dismutase and catalase. Reaction mixture contained: (A) No addition (50 lM juglone and 0.1 mM DMPO); (B) A plus 50 lM FeSO4; (C) B + superoxide dismutase; (D) B + superoxide dismutase + catalase. Arrows indicate the signals of semiquinone radicals.
topical treatment of certain diseases (O’Brien, 1991; Monks et al., 1992). Quinone toxicity has been studied extensively with isolated hepatocytes and keratinocytes (Öllinger and Brunmark, 1991; Inbaraj and Chignell, 2004), and was assumed to be due to two basic mechanisms: (a) the capacity of quinines to produce reactive oxygen species, followed by GSH oxidation to GSSG (Ross et al., 1986; Öllinger and Brunmark, 1991) and (b) the electrophilicity of quinones, which enables them to form adducts to cellular constituents (Rossi et al., 1986). Production of free radical by quinones has been thought to be a consequence of dioxygen reduction by
908
K. Murakami et al. / Toxicology in Vitro 24 (2010) 905–909
In this work we analyzed the direct prooxidant action of naphthoquinone compounds with a focus on the ability of the formation of reactive oxygen species. Naphthoquinone compounds with 5hydroxyl group in the benzene moiety such as juglone, shikonin and plumbagin markedly stimulated the lipid peroxidation. These results were in good agreement with the finding that naphthoquinones with 5-hydroxyl group in the benzene moiety have potent cytotoxicity on hepatocytes (Öllinger and Brunmark, 1991) and keratinocytes (Inbaraj and Chignell, 2004). 5-Hydroxylated naphthoquinones-mediated lipid peroxidation was closely related to the oxidation of Fe2+ ion. Stimulation of ferrous ion oxidation by juglone due to its electron-attracting activity was accompanied with the formation of semiquinone radical, which can reduce dioxygen molecule forming superoxide radical. Lipid peroxidation is considered to be initiated by the formation of perferryl ion or Fe[II]–O2–Fe[III] complex (Svingen et al. 1971; Miller and Aust, 1989; Yoshino and Murakami, 1998). Stimulation by 5-hydroxynaphthoquinone of Fe2+ oxidation can form perferryl ion or the complex, resulting in the enhancement of lipid peroxidation. On the other hand, naphthoquinones with 2-hydroxyl or 2-methyl group in the quinone moiety such as lawsone and lapachol showed no stimulation of lipid peroxidation. Lawsone and lapachol further did not stimulate Fe2+ ion oxidation. These effects of 2-substituted naphthoquinones were well agreed with little cytotoxicity (Öllinger and Brunmark, 1991; Inbaraj and Chignell, 2004). Of particular interest is the finding that the juglone, 5-hydroxynaphthoquinone could form 8-OHdG in DNA, whereas lawsone the naphthoquinone with hydroxyl or methyl group in quinone ring did not produce 8-OHdG in DNA. Introduction of electron-donating hydroxyl group lowers redox potentials of the naphthoquinone compound. In particular 2-hydroxyl or methyl group attached to quinone moiety of the naphthoquinone compound decreases the potential effectively, but 5-hydroxyl group in the benzene moiety of the naphthoquinone causes only a little effect on the potential. Juglone with higher reduction potential is in equilibrium between quinone and quinol forms: production of semiquinone radical may result from disproportionation of the parent quinone and hydroquinone forms (Bothner-By, 1953).
Q þ HQ ¡ 2SQ The quinol form can reduce copper, and generate superoxide radical resulting in the formation of hydroxyl radical, which can produce 8OHdG in DNA. However, lawsone with lowered reduction potential did not affect transition metals, and thus did not form lipid peroxides and of DNA base adduct. Cytotoxicity and chemotherapeutic effects of naphthoquinone compounds can be explained by the position of side chains. Introduction of electron-donating or electron-attracting groups to the benzene or quinone moieties determines the prooxidant properties. References
Fig. 6. ESR spectra of lawsone in the absence and presence of superoxide dismutase and catalase. Reaction mixture was similar to that of Fig. 5 except that juglone was replaced by lawsone. Symbols were identical to those of Fig. 5.
semiquinone intermediates, which are formed via one-electron reduction by enzymes such as microsomal NADPH-cytochrome P450 reductase or mitochondrial NADH-ubiquinone oxidoreductase (Inbaraj and Chignell, 2004).
Bothner-By, A.A., 1953. Quinone—hydroquinone exchange reactions. II. Duroquinone and durohydroquinone exchange in buffered methanol solution. Journal of American Chemical Society 75, 728–730. Draper, H.H., Hadley, M., 1990. Malondialdehyde determination as index of lipid peroxidation. Methods in Enzymology 186, 421–431. Homma, Y., Tsunoda, M., Kasai, H., 1994. Evidence for the accumulation of oxidative stress during cellular ageing of human diploid fibroblasts. Biochemical and Biophysical Research Communications 203, 1063–1068. Inbaraj, J.J., Chignell, C.F., 2004. Cytotoxic action of juglone and plumbagin: a mechanistic study using HaCaT keratinocytes. Chemical Research in Toxicology 17, 55–62. Kumbhar, A., Padhye, S., Ross, D., 1996. Cytotoxic properties of ironhydroxynaphthoquinone complexes in rat hepatocytes. BioMetals 9, 235–240. Mahler, H.R., Elowe, D.G., 1954. Studies on metalloflavoproteins. II. The role of iron in diphosphopyridine nucleotide cytochrome c reductase. Journal of Biological Chemistry 210, 165–179. Miller, D.M., Aust, S.D., 1989. Studies of ascorbate-dependent, iron-catalyzed lipid peroxidation. Archives of Biochemistry and Biophysics 271, 113–119.
K. Murakami et al. / Toxicology in Vitro 24 (2010) 905–909 Monks, T.J., Hanzlik, R.P., Cohen, G.M., Ross, D., Graham, D.G., 1992. Quinone chemistry and toxicity. Toxicology and Applied Pharmacology 112, 2–16. O’Brien, P.J., 1991. Molecular mechanisms of quinone cytotoxicity. ChemicoBiological Interactions 80, 1–41. Öllinger, K., Brunmark, A., 1991. Effect of hydroxy substituent position on 1,4naphthoquinone toxicity to rat hepatocytes. Journal of Biological Chemistry 266, 21496–21503. Quinlan, G.J., Halliwell, B., Moorhouse, C.P., Gutteridge, J.M.C., 1988. Action of lead(II) and aluminium (III) ions on iron-stimulated lipid peroxidation in liposomes, erythrocytes and rat liver microsomal fractions. Biochimica et Biophysica Acta 962, 196–200. Ross, D., Thor, H., Threadgill, M.D., Sandy, M.S., Smith, M.T., Moldéus, P., Orrenius, S., 1986. The role of oxidative processes in the cytotoxicity of substituted 1,4naphthoquinones in isolated hepatocytes. Archives of Biochemistry and Biophysics 248, 460–466.
909
Rossi, L., Moore, G.A., Orrenius, S., O’Brien, P.J., 1986. Quinone toxicity in hepatocytes without oxidative stress. Archives of Biochemistry and Biophysics 251, 25–35. Svingen, B.A., Buege, J.A., O’Neal, F.O., Aust, S.D., 1971. The mechanism of NADPHdependent lipid peroxidation. The propagation of lipid peroxidation. Journal of Biological Chemistry 254, 5892–5899. Yoshino, M., Murakami, K., 1998. Interaction of iron with polyphenolic compounds. Application to antioxidant characterization. Analytical Biochemistry 257, 40–44. Yoshino, M., Haneda, M., Naruse, M., Murakami, K., 1999. Prooxidant activity of flavonoids: copper-dependent strand breaks and the formation of 8-hydroxy20 -deoxyguanosine in DNA. Molecular Genetics and Metabolism 68, 468–472. Yoshino, M., Haneda, M., Naruse, M., Htay, H.H., Iwata, S., Tsubouchi, R., Murakami, K., 2002. Prooxidant action of gallic acid compounds: copper-dependent stand breaks and the formation of 8-hydroxy-20 -deoxyguanosine in DNA. Toxicology In Vitro 16, 705–709.