Effect of Nonsteroidal Anti-Inflammatory Drugs on Lipid Peroxidation by Hydroxyl Radical

ISSN 0306-3623/98 $19.00 1 .00 PII S0306-3623(98)00044-5 All rights reserved

Gen. Pharmac. Vol. 31, No. 3, pp. 405–408, 1998 Copyright  1998 Elsevier Science Inc. Printed in the USA.

Effect of Nonsteroidal Anti-Inflammatory Drugs on Lipid Peroxidation by Hydroxyl Radical Yasuhisa Tsujimoto,*1 Kazuo Saitoh,1 Midori Kashima,1 Atsushi Shiozawa,1 Masahiro Kozuka,1 Hideki Hashizume,1 Koh Kimura,1 Muneyoshi Yamazaki1 and Akira Fujii2 Departments of 1Endodontics and 2Pharmacology, Nihon University School of Dentistry at Matsudo, 870-1 Sakaecho, Nishi-2, Matsudo, Chiba 271-8587, Japan [Tel: 81-47-360-9371; Fax: 81-47-360-9370] ABSTRACT. 1. Effects of acetaminophen and sodium salicylate on hydoxyl radical elimination were studied using an electron-spin-resonance spin-trapping method. The effects of these agents on lipid peroxidation of the human erythrocyte membrane were also investigated by the thiobarbituric acid method. 2. Acetaminophen and sodium salicylate depressed hydroxyl radical generated by Cu21/H2O2. 3. Acetaminophen inhibited Cu21/H2O2-dependent lipid peroxidation; however, sodium salicylate enhanced Cu21/H2O2-dependent lipid peroxidation. gen pharmac 31;3:405–408, 1998.  1998 Elsevier Science Inc. KEY WORDS. NSAIDs, hydroxyl radical, lipid peroxidation, ESR, TBA

INTRODUCTION Lipid peroxidation of the biomembrane is mediated mainly by hydroxyl radical (•OH). Excessive peroxidation of unsaturated fatty acids in biomembranes is thought to be related to several abnormal states. Because phenolic compounds have •OH scavenging ability (Hashizume, 1993), it may easily be assumed that 2OH-bearing compounds such as acetaminophen and sodium salicylate (compounds of nonsteroidal anti-inflammatory drugs [NSAIDs]) act as scavengers of •OH; however, no published work has reported on the relationship between •OH and 2OH-bearing NSAIDs. In a previous study, we investigated a lipid peroxidation system by •OH in a reaction system of Cu21/H2O2 in the presence of human erythrocyte membranes (Hashizume, 1993; Miyazawa et al., 1995; Nagashima, 1989). The aim of this study is to clarify the characteristics of 2OHbearing NSAIDs on lipid peroxidation via •OH. We first measured •OH that was generated in a Cu21/H2O2 reaction system using an electron spin resonance (ESR) spin-trapping method, previously reported by Hashizume (1993). Then, NSAIDs (acetaminophen and sodium salicylate) were added to the Cu21/H2O2 reaction system to investigate their •OH elimination abilities. NSAIDs were also added to the lipid peroxidation reaction system in the presence of human erythrocyte membrane, and lipid peroxidation was examined by the thiobarbituric acid (TBA) method (Wilbur et al., 1949). MATERIALS AND METHODS

were obtained from local sources and were of the highest grade available.

Determination of •OH Hydroxyl radical was measured by the ESR spin-trapping method. The quantity of •OH was normalized relative to the standard signal intensity of a manganese oxide marker (MnO) as: •OH relative intensity5•OH signal intensity (second peak)/Mn21 marker intensity (Ueta et al., 1988). The ESR spectrum was recorded using a JEOL JES FR 80 apparatus.

Measurement of •OH The reaction mixture (200 ml) contained 18.4 mmol of DMPO, 200 mmol of CuCl2 and 20 nmol of H2O2. After rapid stirring, the reaction mixture was placed into an ESR flat cell. Recording of the ESR spectrum was started 40 sec after the addition of DMPO.

Measurement condition ESR measurement conditions were as follows: microwave power, 8.0 mW; magnetic field, 335.465 mT; sweep time, 2 min; modulation frequency, 100 kHz; modulation amplitude, 0.1 mT; time constant, 0.1 sec.

Materials

Evaluation of elimination ability of •OH

TBA was obtained from Tokyo Kasei (Japan). The spin-trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), was obtained from Dojin Chemical, Japan. l-Ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate potassium salt (EPC-K1), an •OH scavenger, was obtained from Senju Pharmaceutical Co. (Japan). Acetaminophen and sodium salicylate were from Wako Pure Chemicals (Japan). Other chemicals

Various concentrations of NSAIDs and EPC-K1 were added to the reaction mixture with •OH generation. Elimination ability of •OH was determined from the change in signal intensity of the ESR spintrapping method (Ueta et al., 1988). EDTA was added to the reaction mixture to reconfirm Cu21 requirement on •OH generation.

*To whom correspondence should be addressed. Received 4 September 1997.

Healthy male volunteers (20 to 30 years old) provided informed consent and then gave 25 ml of blood from antecubital vein. Ghost

Preparation of human erythrocyte membranes

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FIGURE 1. ESR spectra of •OH trapped by DMPO in the reaction system. (a) Control. (b–e) Effect of addition of EPC-K1 on the reaction system. (f–h) Effect of addition of EDTA on the reaction system. *DMPO-OH spin adduct.

solutions were prepared according to the method of Dodge et al. (1963). Thus, human blood cells were hemolyzed and washed with an excess of 10 mM Tris-HCl buffer (pH 7.4) to avoid contamination of hemoglobin. The final precipitate was suspended in the same buffer, and the suspension was used as the ghost solution. All procedures were performed at below 48C except where noted.

Formation and determination of the TBA-reactive substance

Y. Tsujimoto et al.

FIGURE 2. ESR spectra of •OH trapped by DMPO in the reaction system. (a) Control. (b–d) Effect of addition of sodium salicylate on the reaction system. (e–g) Effect of addition of acetaminophen on the reaction system. *DMPO-OH spin adduct.

reaction system, the amount of DMPO-OH spin adduct decreased in a concentration-dependent manner (Figs. 1 and 2). Figure 3 shows the relationship between the signal intensity of the DMPOOH spin adduct and concentrations of EPC-K1, EDTA and NSAIDs. The 50% inhibitory doses (ID50) were: EPC-K1, 4.131026 M; EDTA, 1.831025 M; acetaminophen, 3.731024 M; sodium salicylate, 8.531024 M (Table 1).

Lipid peroxidation experiment In the lipid peroxidation experiment, when various concentrations of EPC-K1, EDTA and acetaminophen were added to lipid peroxida-

Lipid peroxidation was determined by measuring the formed TBAreactive substance. A standard reaction mixture for the formation of TBA-reactive substance contained 1.3 mmol of Tris-Hcl buffer (pH 7.4), 50 nmol of CuCl2, 10.3 mmol of H2O2 and 300 ml of the ghost solution in a final volume of 500 ml. The reaction was initiated by addition of ghost solution. After incubation at 378C for the indicated period of time, the reaction was terminated by the addition of 500 ml of 10% trichloroacetic acid solution. The TBA-reactive substance was then determined by the method of Wilbur et al. (1949), in which the absorbance at 530 nm was determined with a Hitachi 624 spectrophotometer. RESULTS

ESR experiment Figure 1a shows a typical adduct of DMPO-OH that was trapped by DMPO. This spin adduct pattern was used as a control, and the second peak of DMPH-OH spin adduct was used as the relative intensity. When various concentrations of EPC-K1 (•OH scavenger), EDTA (chelating agent) and NSAIDs were added to the Cu21/H2O2

FIGURE 3. Effect of EDTA, EPC-K1 and NSAIDs on the formation of DMPO-OH adduct. 1: EPC-K1; h: EDTA; n: acetaminophen; s: sodium salicylate.

NSAIDs on Lipid Peroxidation

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TABLE 1. ID50 and sample for the •OH generation system ID50 (M) EPC-K1 EDTA Acetaminophen Sodium salicylate

4.1 1.8 3.7 8.5

3 3 3 3

1026 1025 1024 1024

ID50 was obtained from the regression line of the least squares method (see Fig. 3).

tion reaction system, lipid peroxidation was decreased by addition of each sample in a concentration-dependent manner (Figs. 4 to 6). However, when various concentrations of sodium salicylate were added to the reaction system, lipid peroxidation was increased 1.5 to 2.0 times than that of control (data not shown).

FIGURE 5. Concentration-dependent effect of EDTA on lipid peroxidation.

Recently, the active oxygen species have been reported to cause various diseases. McCord (1974) suggested that inhibition of active oxygen showed an anti-inflammatory effect. Kimura (1997) reported that naproxen and flurbiprofen inhibited the formation of superox2 ide (O2 2•), but sodium salicylate acted to capture O2• directly, and acetaminophen seemed to show a similar activity toward superoxide dismutase, converting O2 2• to H2O2. Kimura concluded that NSAIDs have inhibitory effects on the generation system of O2 2•, and also scavenging effects on the O22• generated. However, there is no report on the relationship between •OH scavenging ability and antilipid peroxidation for acetaminophen and sodium salicylate. In the ESR experiment, •OH was detected in the Cu21/H2O2 reaction system. When various concentrations of EDTA, EPC-K1 and NSAIDs were added to the Cu21/H2O2 reaction system, the amount of •OH was decreased in a concentration-dependent manner. In the case of EDTA, •OH was inhibited by chelation. In the case of EPCK1, •OH was eliminated by scavenging. The amount of •OH was also decreased by addition of NSAIDs; however, it cannot be concluded that there was a scavenging or chelating effect of NSAIDs on •OH generation in the Cu21/H2O2 reaction system. Chemical structures of acetaminophen and sodium salicylate are shown in Figure 7. They have a hydroxyl group in the structure. Hashizume (1993) has reported that the phenolic compounds, phenol and guaiacol, have •OH scavenging ability, suggesting the occurrence of nucleophilic aromatic substitution. One possible course for decreasing of •OH, using the present experimental conditions, might be via nucleophilic aromatic substitution by acetaminophen and sodium salicylate.

In the lipid peroxidation experiment, EDTA, EPC-K1 and acetaminophen inhibited lipid peroxidation in the Cu21/H2O2 reaction system. This inhibition was thought to be caused by the chelating effect by EDTA and •OH elimination effects by EPC-K1 and acetaminophen. However, sodium salicylate did not inhibit lipid peroxidation. This suggests that the addition of sodium salicylate to the reaction system with the cell component would change the property of sodium salicylate, resulting in lipid peroxidation. It has been believed, until recently, that stomach mucous membrane obstacles were caused by sodium salicylate, related to the depression of cyclooxygenase-1. The present results indicate that sodium salicylate has the elimination ability of active oxygen; however, it failed to prevent the development of a mucous membrane obstacle by lipid peroxidation. Sodium salicylate may change its structural character after elimination of •OH, thus lipid peroxidation would occur. Schapira et al. (1995) used sodium salicylate as an •OH trapper for rat acute lung inflammation caused by •OH, and detected the formulation of 2,3- or 2,5-dihydroxybenzoic acid after salicylate hydroxylation. It is well known that the phenol coefficient changes by replacement of phenol’s hydrogen to the other functional groups. However, the relation between salicylate hydroxylation and lipid peroxidation has not been clarified. Further detailed studies of these aspects should be conducted in the future. The results of this study suggest that acetaminophen had •OH elimination and lipid peroxidation inhibitory abilities, but sodium salicylate had only •OH elimination ability. It must be emphasized that the relationship between •OH elimination and lipid peroxidation inhibitory abilities is important for controlling diseases. The results of the present study have clarified some of the mechanisms of NSAIDs with respect to active oxygen, some of which were not known until recently, and have contributed to the study of drugs controlling active oxygen and lipid peroxidation.

FIGURE 4. Concentration-dependent effect of EPC-K1 on lipid peroxidation.

FIGURE 6. Concentration-dependent effect of acetaminophen on lipid peroxidation.

DISCUSSION

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Y. Tsujimoto et al. The present study was supported partly by a Grant-in-Aid from a Suzuki Memorial Grant of Nihon University School of Dentistry at Matsudo (1993), to Y. T.

References

FIGURE 7. Structures of NSAIDs.

SUMMARY The amount of •OH generated in the Cu21/H2O2 reaction system was decreased by the addition of acetaminophen, sodium salicylate, EDTA (chelating agent) and EPC-K1 (•OH scavenger) in a concentration-dependent manner, in an ESR spin-trapping experiment. The ID50 values of each sample were 3.731024 M for acetaminophen, 8.531024 M for sodium salicylate, 4.131026 M for EPC-K1 and 1.831025 M for EDTA. Cu21/H2O2-dependent lipid peroxidation of the human erythrocyte membrane was inhibited by acetaminophen, EDTA and EPC-K1 in a concentration-dependent manner; however, sodium salicylate enhanced lipid peroxidation. It was shown that acetaminophen had •OH elimination and lipid peroxidation inhibitory abilities, but sodium salicylate had only •OH elimination ability.

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