- Email: [email protected]
The relationship between diphenylamine structure and NSAIDs-induced hepatocytes injury
Toxicology Letters 186 (2009) 111–114
Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
The relationship between diphenylamine structure and NSAIDs-induced hepatocytes injury Yan Li a,b , Xin-ming Qi a , Xiang Xue a,b , Xiong-fei Wu a,b , Yuan-feng Wu a,b , Min Chen a,b , Guo-zhen Xing a,b , Yang Luan a , Jin Ren a,∗ a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China b Graduate School of the Chinese Academy of Sciences, Shanghai, China
a r t i c l e
i n f o
Article history: Received 10 December 2008 Received in revised form 6 January 2009 Accepted 6 January 2009 Available online 16 January 2009 Keywords: Mitochondria Liver injury NSAIDs Diphenylamine structure
a b s t r a c t Objective: Many nonsteroidal anti-inflammatory drugs (NSAIDs) with diphenylamine structure induce severe hepatotoxicities. We evaluated the role of diphenylamine structure in liver injuries induced by these NSAIDs. Methods: Effects of diphenylamine, diclofenac and tolfenamic acid on mitochondrial permeability transition (MPT) and efflux of calcium in isolated liver mitochondria as well as on cellular ATP content and mitochondrial membrane depolarization in rat primary hepatocyte cultures were examined. Results: Diclofenac and tolfenamic acid induced cyclosporine A (CsA)-sensitive mitochondrial swelling and membrane depolarization in isolated liver mitochondria. Only diclofenac caused the release of calcium in isolated liver mitochondria. Diphenylamine had no effects on isolated liver mitochondria. All three compounds decreased ATP content and induced mitochondrial membrane depolarization. CsA attenuated these effects, suggesting MPT might be involved in the hepatotoxicities caused by diphenylamine, diclofenac and tolfenamic acid. SKF-525A, a general inhibitor of CYP450, markedly inhibited the injury induced by diphenylamine, but not diclofenac or tolfenamic acid. Conclusion: The hepatotoxicities caused by diclofenac and tolfenamic acid may be attributed to the mitochondrial dysfunction induced by these drugs instead of the diphenylamine structure per se. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used drugs in the world, accounting for 3–9% of total prescriptions in various countries, especially in Europe and North America (Agrawal, 1991; Hughes, 1991; Johnson and Day, 1991). With the wide use of NSAIDs, many adverse effects on liver of NSAIDs have been reported. There were 180 reported cases of diclofenac-related liver injury from November 1988 through June 1991 (Banks et al., 1995). And during a period from 1977 to 1992, 384 of the 539 adverse reactions recorded by WHO were associated with tolfenamic acid (Banks et al., 1995; Schattner et al., 2000). Almost all NSAIDs currently available are known to cause reversible elevation of aminotransferases (Zimmerman, 1990; Brass, 1993; Manoukian and Carson, 1996). The types of NSAIDs-related liver injury range from mild cholestasis to severe hepatocellular injury (Zimmerman and Lewis, 1987; Manov et al., 2006).
∗ Corresponding author. E-mail address: cdser [email protected] (J. Ren). 0378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2009.01.005
Based on their chemical structures, NSAIDs can be categorized into four groups: salicylic acids, anthranilic acids, arylacetic acids and arypropionic acids. The cytotoxicity of diclofenac, tolfenamic acid and anthranilic acids has been attributed to diphenylamine contained in their structures (Fig. 1). Some works have been done to investigate the mechanisms for NSAIDs-induced hepatotoxicities. The cytotoxicity study of 18 NSAIDs using rat primary hepatocytes has been performed, and the results showed that diclofenac and tolfenamic acid belonging to the cytotoxic NSAIDs share diphenylamine in their skeleton. Diphenylamine induced the leakage of lactate dehydrogenase (LDH) to the same degree as diclofenac and tolfenamic acid. In addition, the hepatotoxicities induced by NSAIDs with the diphenylamine structure were attributed to mitochondrial permeability transition (MPT) or the uncoupling of mitochondrial respiration (Masubuchi et al., 1998). In the present study, we analyzed the relationship between diphenylamine structure and NSAIDs-induced hepatocytes injury with isolated liver mitochondria and rat primary hepatocytes, hoping to contribute another possible mechanism for NSAIDs-induced hepatotoxicities
112
Y. Li et al. / Toxicology Letters 186 (2009) 111–114 air atmosphere in Ham’s F-12/DMEM (Invitrogen, Carlsbad, CA, USA) (1:1) medium supplemented with 10% fetal bovine serum (PAA Laboratories GmbH, Linz, Austria) with 100 U/mL penicillin, and 70 g/mL streptomycin. 2.8. Cell viability assays and measurement of cellular ATP content and mitochondrial membrane potential Rat primary hepatocytes were treated with diphenylamine, diclofenac or tolfenamic acid, and cell viabilities of the treated cells were determined using the Cell Counting Kit-8 as instructed by the manufacturer (Dojindo Laboratories, Tokyo, Japan). Intracellular ATP levels in the presence or absence of CsA (0.2 M) were determined using CellTiter-GloTM Luminescent Cell Viability Assay kit according to the instructions of the manufacturer (Promega, Madison, WI, USA). Bioluminescence was measured with Novostar (BMG LABTECH, Offenburg, Germany). Mitochondrial
Fig. 1. Structures of diphenylamine and its structurally related compounds diclofenac and tolfenamic acid. 2. Materials and methods 2.1. Chemicals Diphenylamine (Dpa), diclofenac (Dcf) and tolfenamic acid (Tol), arsenazo III, mitochondria specific dye tetramethylrhodamine ethyl ester (TMRE), SKF-525A and cyclosporine A were purchased from Sigma (St. Louis, MO, USA). All other chemicals and solvents were from Sigma and of analytical grade. 2.2. Animals Sprague–Dawley male rats with an average weight of 200 ± 30 g were supplied by Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai, China). Rats were housed at 20 ± 3 ◦ C and 45–65% humidity with a 12-h light–dark cycle. Drinking water and food were provided ad libitum throughout the study. 2.3. Isolation of liver mitochondria Rat liver mitochondrial fraction was prepared as described (Schneider et al., 1950) with modifications. The liver was isolated and placed in the ice-cold medium containing 250 mM sucrose, 10 mM HEPES-KOH, pH 7.4, and 0.5 mM EGTA. The liver was cut to small cubes with scissors in 5 mL of the medium and homogenized 3 times with a Potter homogenizer. The homogenate was diluted to 10 mL per liver and were centrifuged at 900 × g for 5 min at 4 ◦ C. The supernatant was decanted and further centrifuged at 4500 × g for 5 min. The pellet was suspended in 20 mL of the ice-cold isolation medium and centrifuged at 4500 × g for 10 min. The final mitochondrial pellet was suspended in 1 mL of medium containing 250 mM sucrose and 10 mM HEPES-KOH, pH 7.4. The protein concentration was determined by the method of Lowry (Lowry et al., 1951). 2.4. Measurement of mitochondrial swelling The liver mitochondrial preparation (1.0 mg/mL) was incubated with a reaction buffer containing 210 mM mannitol, 70 mM sucrose, 2 M rotenone, and 5 mM HEPES, pH 7.4 at 30 ◦ C in the presence of 20 M CaCl2 . The mitochondria were energized by 5 mM succinate. Mitochondrial swelling was measured by the change in absorbance at 540 nm over 15 min at room temperature with a SpectraPlus384 microplate reader (Molecular Devices, USA). 2.5. Determination of the isolated mitochondrial membrane potential The mitochondrial membrane potential was monitored with TMRE at 100 nM. After 2 min preincubation with succinate, diphenylamine, diclofenac or tolfenamic acid was added and incubated for 15 min in the presence of Ca2+ . The reaction medium was immediately centrifuged (16,000 × g, 30 s) and fluorescence intensity of the supernatant was measured at the 530/590 nm wavelength pair with Novostar (BMG LABTECH, Offenburg, Germany). 2.6. Measurement of mitochondrial Ca2+ efflux Ca2+ efflux from mitochondria was measured as described (Scarpa, 1979). The reaction medium was used as described above. Arsenazo III (50 M) was added. Absorbance of the medium was monitored at the 675/685 nm wavelength pair during preincubation. Dpa, Dcf or Tol was added 1 min after the energization of the mitochondria followed by 20 min incubation at 30 ◦ C. 2.7. Culture of rat hepatocytes Hepatocytes were isolated by two-step collagenase perfusion as described previously (Berry and Friend, 1969; Seglen, 1972; Orrenius et al., 1976) with some modifications. Monolayer hepatocytes were cultured at 37 ◦ C in a 5% CO2 and 95%
Fig. 2. Effects of diphenylamine, diclofenac and tolfenamic acid on isolated liver mitochondria. (A) Effects of 50 M diphenylamine, diclofenac and tolfenamic acid on the swelling of rat liver mitochondria (1 mg/mL) energized by addition of Ca2+ (20 M final concentration). The results are representatives of at least three experiments. (B) Effects of diphenylamine, diclofenac and tolfenamic acid on mitochondrial membrane potential. The reaction medium was the same as described in (A). The loss of mitochondrial membrane potential of diclofenac and tolfenamic acid was inhibited by 1 M CsA. The values shown are mean ± S.D. from measurement. * P < 0.05, ** P < 0.01 vs. control, ## P < 0.01 vs. each drug without CsA. (C) Effects of 500 M diphenylamine on the swelling of rat liver mitochondria.
Y. Li et al. / Toxicology Letters 186 (2009) 111–114 membrane potential of cultured hepatocytes was evaluated as described (Wu et al., 1990). CsA (0.2 M) was used as an inhibitor of MPT where indicated. Fluorescence readings were taken at 530/590 nm wavelength pair with Novostar (BMG LABTECH, Offenburg, Germany). 2.9. Statistical analysis Results were expressed as mean ± S.D. After homogenetic analysis, homogeneous data were analyzed with one-way analysis of variance (one-way ANOVA) and a post hoc test of least significant difference. To determine intergroup differences, heterogeneous data were analyzed using t’-test. A value of P < 0.05 was considered to be statistically significant.
113
tion of growth (IC50 value). After 24 h treatment, the estimated IC50 values for diphenylamine, diclofenac and tolfenamic acid were 183.2 ± 69.58 M, 332.7 ± 31.38 M and 195.8 ± 19.60 M, respectively (Table 1). All the drugs caused decreases in cellular ATP content and mitochondrial membrane potential. The degrees of injury to the rat primary hepatocytes reflected from which, paralleled with decrease of the cellular ATP content. Among the tested drugs, diphenylamine exerted the strongest effect, depleting the approximately 92% the cellular ATP content and 50% the mitochondrial membrane poten-
3. Results 3.1. Effects of diphenylamine, diclofenac and tolfenamic acid on isolated liver mitochondrial function MPT leads to the disruption of the mitochondrial membranes and mitochondrial swelling. As Fig. 2A shown, in isolated liver mitochondria energized with succinate and Ca2+ , tolfenamic acid caused significant mitochondrial swelling and diclofenac caused moderatel mitochondiral swelling. In contrast, diphenylamine failed to cause any mitochondiral swelling. In addition, CsA completely inhibited the swelling induced by both diclofenac and tolfenamic acid (Fig. 2A). Meanwhile, diclofenac and tolfenamic acid decreased mitochondrial membrane potential significantly in the presence of 20 M Ca2+ (Fig. 2B), which aborted by co-incubation with 1 M CsA, and diphenylamine exerted no effect on mitochondrial membrane potential. Ca2+ release from the mitochondria was induced by diclofenac but not diphenylamine or tolfenamic acid. In addition, Ca2+ release caused by diclofenac was blocked by 1 M CsA (Fig. 3). 3.2. Effects of diphenylamine, diclofenac and tolfenamic acid on rat primary hepatocytes Rat primary hepatocytes were exposed to the increasing concentrations of diphenylamine, diclofenac and tolfenamic acid. Cytotoxicity was measured by the concentration for 50% inhibi-
Fig. 3. Effects of diphenylamine, diclofenac and tolfenamic acid on efflux of mitochondrial calcium. Incubation medium was the same medium as in Fig. 2, plus 50 M arsenazo III as an indicator. The results are representatives of at least three experiments. ** P < 0.01 vs. control, ## P < 0.01 significantly different from each drug without CsA. Table 1 IC50 value of diphenylamine, diclofenac and tolfenamic acid on rat primary hepatocytes after 24 h treatment. IC50 (M)a Diphenylamine Diclofenac Tolfenamic acid
183.2 ± 69.58 332.7 ± 31.38 195.8 ± 19.60
a The values shown are mean ± S.D. For measurement, the result are representatives of at least three experiments.
Fig. 4. Effects of diphenylamine, diclofenac and tolfenamic acid on liver mitochondrial function in rat primary hepatocytes. Hepatocytes were cultured with 250 M or 500 M diphenylamine, 400 M or 800 M diclofenac and 200 M or 400 M tolfenamic acid without or with 0.2 M CsA for 24 h. (A) Effects of diphenylamine, diclofenac and tolfenamic acid on ATP depletion. The results are representatives of at least three experiments. (B) Effects of diphenylamine, diclofenac and tolfenamic acid on decrease of mitochondrial transmembrane potential on rat primary hepatocytes. (C) Effects of inhibition of CYP450 on diphenylamine-induced hepatocytes injury. The rat primary hepatocytes were treated with diphenylamine co-incubation with 10 M SKF-525A for 24 hours. The results are representatives of at least three experiments. ** P < 0.01 vs. control, # P < 0.05, ## P < 0.01 vs. each drug without CsA.
114
Y. Li et al. / Toxicology Letters 186 (2009) 111–114
tial with the concentration of 500 M (Fig. 4A and B). It was shown that all the three drugs, diphenylamine, diclofenac and tolfenamic acid, could cause damages to the rat primary hepatocytes and all these can be attenuated when cocultured with 0.2 M CsA (Fig. 4), suggesting the mechanisms involved in the damages caused by the three drugs may relate with MPT. As shown in Fig. 4C, SKF-525A, a general inhibitor of CYP450, markedly reversed the damages caused by diphenylamine.
In conclusion, we have demonstrated that hepatocyte mitochondrial dysfunction induced by NSAIDs cannot be attributed to the diphenylamine core structure. Conflict of interest There are no conflicts of interest. Acknowledgement
4. Discussion In our study, we evaluated hepatotoxicities caused by diphenylamine and structural related NSAIDs, diclofenac and tolfenamic acid. Diphenylamine (250 M and 500 M), diclofenac (400 M and 800 M) and tolfenamic acid (200 M and 400 M) caused a marked decrease in cellular ATP content and loss of mitochondrial membrane potential in rat primary hepatocytes. CsA, a general inhibitor of MPT, markedly attenuated these effects. These data are consistent with findings by others which suggest that these drugs induce MPT in the primary hepatocytes(Masubuchi et al., 1998; Masubuchi et al., 1999). In isolated liver mitochondria, diphenylamine failed to produce any effect on MPT at concentrations as high as 500 M (Fig. 2C) whereas diclofenac and tolfenamic acid induced CsA-sensitive MPT at doses as low as 50 M (Fig. 2A). Our findings indicate that the hepatotoxicities caused by NSAIDs (e.g. diclofenac and tolfenamic acid) are not attributable to the diphenylamine structure. This is in conflict with a previous study which showed that diphenylamine (250–500 M) could induce MPT in isolated mitochondria without substrate for respiration in incubation buffer and activate MPT in hepatocytes at the same concentration (Masubuchi et al., 2000). Respiration substrates and calcium are present in mitochondria at normal physiological state in hepatocytes. So, in our study, succinate and calcium were present in the incubation buffer. Under our experimental conditions, even 500 M diphenylamine was unable to induce mitochondrial swelling (Fig. 2C) in isolated liver mitochondria. These suggest that diphenylamine structure may not play a major role in activation of MPT in hepatotoxicities induced by diclofenac and tolfenamic acid. We examined the influence of SKF-525A, a general CYP450 inhibitor, on the hepatic injury induced by these drugs, and found that SKF-525A could attenuate diphenylamine-induced ATP depletion. Put together, these findings suggest that MPT has a major role in NSAIDs-induced hepatocyte injury; however, diphenylamine structure may not be involved in MPT activation. Diphenylamineinduced MPT in hepatocytes may be related to its metabolite or reactive oxygen species (ROS), which provides the major inducement of MPT. Our unpublished data indicate that diphenylamine induces burst of ROS in hepatocytes, reaching the peak at 2 h, about 1.3 times of control (data not shown). The detailed mechanism of MPT induced by diphenylamine in hepatocytes needs further research.
The project was supported by National Key Technologies R&D Program (2008ZX09305-007). References Agrawal, N., 1991. Risk factors for gastrointestinal ulcers caused by nonsteroidal antiinflammatory drugs (NSAIDs). J. Fam. Pract. 32 (6), 619–624. Banks, A.T., Zimmerman, H.J., et al., 1995. Diclofenac-associated hepatotoxicity: analysis of 180 cases reported to the food and drug administration as adverse reactions. Hepatology 22 (3), 820–827. Berry, M.N., Friend, D.S., 1969. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J. Cell Biol. 43 (3), 506–520. Brass, E.P., 1993. Hepatic toxicity of antirheumatic drugs. Cleve. Clin. J. Med. 60 (6), 466–472. Hughes, G.R., 1991. The problems of using NSAIDs in the elderly. Scand. J. Rheumatol. Suppl. 91, 15–19. Johnson, A.G., Day, R.O., 1991. The problems and pitfalls of NSAID therapy in the elderly (Part I). Drugs Aging 1 (2), 130–143. Lowry, O.H., Rosebrough, N.J., et al., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193 (1), 265–275. Manoukian, A.V., Carson, J.L., 1996. Nonsteroidal anti-inflammatory drug-induced hepatic disorders. Incidence and prevention. Drug Saf. 15 (1), 64–71. Manov, I., Motanis, H., et al., 2006. Hepatotoxicity of anti-inflammatory and analgesic drugs: ultrastructural aspects. Acta Pharmacol. Sin. 27 (3), 259–272. Masubuchi, Y., Saito, H., et al., 1998. Structural requirements for the hepatotoxicity of nonsteroidal anti-inflammatory drugs in isolated rat hepatocytes. J. Pharmacol. Exp. Ther. 287 (1), 208–213. Masubuchi, Y., Yamada, S., et al., 1999. Diphenylamine as an important structure of nonsteroidal anti-inflammatory drugs to uncouple mitochondrial oxidative phosphorylation. Biochem. Pharmacol. 58 (5), 861–865. Masubuchi, Y., Yamada, S., et al., 2000. Possible mechanism of hepatocyte injury induced by diphenylamine and its structurally related nonsteroidal antiinflammatory drugs. J. Pharmacol. Exp. Ther. 292 (3), 982–987. Orrenius, S., Thor, H., et al., 1976. Isolated rat hepatocytes as an experimental tool in the study of cell injury. Effect of anoxia. Forensic Sci. 8 (3), 255–263. Scarpa, A., 1979. Measurements of cation transport with metallochromic indicators. Methods Enzymol. 56, 301–338. Schattner, A., Sokolovskaya, N., et al., 2000. Fatal hepatitis and renal failure during treatment with nimesulide. J. Intern. Med. 247 (1), 153–155. Schneider, W.C., Hogeboom, G.H., et al., 1950. Intracellular distribution of enzymes VII. The distribution of nucleic acids and adenosinetriphosphatase in normal mouse liver and mouse hepatoma. J. Natl. Cancer Inst. 10 (4), 977–982. Seglen, P.O., 1972. Preparation of rat liver cells. I. Effect of Ca2+ on enzymatic dispersion of isolated, perfused liver. Exp. Cell Res. 74 (2), 450–454. Wu, E.Y., Smith, M.T., et al., 1990. Relationships between the mitochondrial transmembrane potential, ATP concentration, and cytotoxicity in isolated rat hepatocytes. Arch. Biochem. Biophys. 282 (2), 358–362. Zimmerman, H.J., 1990. Update of hepatotoxicity due to classes of drugs in common clinical use: non-steroidal drugs, anti-inflammatory drugs, antibiotics, antihypertensives, and cardiac and psychotropic agents. Semin. Liver Dis. 10 (4), 322–338. Zimmerman, H.J., Lewis, J.H., 1987. Drug-induced cholestasis. Med. Toxicol. 2 (2), 112–160.
Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
The relationship between diphenylamine structure and NSAIDs-induced hepatocytes injury Yan Li a,b , Xin-ming Qi a , Xiang Xue a,b , Xiong-fei Wu a,b , Yuan-feng Wu a,b , Min Chen a,b , Guo-zhen Xing a,b , Yang Luan a , Jin Ren a,∗ a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China b Graduate School of the Chinese Academy of Sciences, Shanghai, China
a r t i c l e
i n f o
Article history: Received 10 December 2008 Received in revised form 6 January 2009 Accepted 6 January 2009 Available online 16 January 2009 Keywords: Mitochondria Liver injury NSAIDs Diphenylamine structure
a b s t r a c t Objective: Many nonsteroidal anti-inflammatory drugs (NSAIDs) with diphenylamine structure induce severe hepatotoxicities. We evaluated the role of diphenylamine structure in liver injuries induced by these NSAIDs. Methods: Effects of diphenylamine, diclofenac and tolfenamic acid on mitochondrial permeability transition (MPT) and efflux of calcium in isolated liver mitochondria as well as on cellular ATP content and mitochondrial membrane depolarization in rat primary hepatocyte cultures were examined. Results: Diclofenac and tolfenamic acid induced cyclosporine A (CsA)-sensitive mitochondrial swelling and membrane depolarization in isolated liver mitochondria. Only diclofenac caused the release of calcium in isolated liver mitochondria. Diphenylamine had no effects on isolated liver mitochondria. All three compounds decreased ATP content and induced mitochondrial membrane depolarization. CsA attenuated these effects, suggesting MPT might be involved in the hepatotoxicities caused by diphenylamine, diclofenac and tolfenamic acid. SKF-525A, a general inhibitor of CYP450, markedly inhibited the injury induced by diphenylamine, but not diclofenac or tolfenamic acid. Conclusion: The hepatotoxicities caused by diclofenac and tolfenamic acid may be attributed to the mitochondrial dysfunction induced by these drugs instead of the diphenylamine structure per se. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used drugs in the world, accounting for 3–9% of total prescriptions in various countries, especially in Europe and North America (Agrawal, 1991; Hughes, 1991; Johnson and Day, 1991). With the wide use of NSAIDs, many adverse effects on liver of NSAIDs have been reported. There were 180 reported cases of diclofenac-related liver injury from November 1988 through June 1991 (Banks et al., 1995). And during a period from 1977 to 1992, 384 of the 539 adverse reactions recorded by WHO were associated with tolfenamic acid (Banks et al., 1995; Schattner et al., 2000). Almost all NSAIDs currently available are known to cause reversible elevation of aminotransferases (Zimmerman, 1990; Brass, 1993; Manoukian and Carson, 1996). The types of NSAIDs-related liver injury range from mild cholestasis to severe hepatocellular injury (Zimmerman and Lewis, 1987; Manov et al., 2006).
∗ Corresponding author. E-mail address: cdser [email protected] (J. Ren). 0378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2009.01.005
Based on their chemical structures, NSAIDs can be categorized into four groups: salicylic acids, anthranilic acids, arylacetic acids and arypropionic acids. The cytotoxicity of diclofenac, tolfenamic acid and anthranilic acids has been attributed to diphenylamine contained in their structures (Fig. 1). Some works have been done to investigate the mechanisms for NSAIDs-induced hepatotoxicities. The cytotoxicity study of 18 NSAIDs using rat primary hepatocytes has been performed, and the results showed that diclofenac and tolfenamic acid belonging to the cytotoxic NSAIDs share diphenylamine in their skeleton. Diphenylamine induced the leakage of lactate dehydrogenase (LDH) to the same degree as diclofenac and tolfenamic acid. In addition, the hepatotoxicities induced by NSAIDs with the diphenylamine structure were attributed to mitochondrial permeability transition (MPT) or the uncoupling of mitochondrial respiration (Masubuchi et al., 1998). In the present study, we analyzed the relationship between diphenylamine structure and NSAIDs-induced hepatocytes injury with isolated liver mitochondria and rat primary hepatocytes, hoping to contribute another possible mechanism for NSAIDs-induced hepatotoxicities
112
Y. Li et al. / Toxicology Letters 186 (2009) 111–114 air atmosphere in Ham’s F-12/DMEM (Invitrogen, Carlsbad, CA, USA) (1:1) medium supplemented with 10% fetal bovine serum (PAA Laboratories GmbH, Linz, Austria) with 100 U/mL penicillin, and 70 g/mL streptomycin. 2.8. Cell viability assays and measurement of cellular ATP content and mitochondrial membrane potential Rat primary hepatocytes were treated with diphenylamine, diclofenac or tolfenamic acid, and cell viabilities of the treated cells were determined using the Cell Counting Kit-8 as instructed by the manufacturer (Dojindo Laboratories, Tokyo, Japan). Intracellular ATP levels in the presence or absence of CsA (0.2 M) were determined using CellTiter-GloTM Luminescent Cell Viability Assay kit according to the instructions of the manufacturer (Promega, Madison, WI, USA). Bioluminescence was measured with Novostar (BMG LABTECH, Offenburg, Germany). Mitochondrial
Fig. 1. Structures of diphenylamine and its structurally related compounds diclofenac and tolfenamic acid. 2. Materials and methods 2.1. Chemicals Diphenylamine (Dpa), diclofenac (Dcf) and tolfenamic acid (Tol), arsenazo III, mitochondria specific dye tetramethylrhodamine ethyl ester (TMRE), SKF-525A and cyclosporine A were purchased from Sigma (St. Louis, MO, USA). All other chemicals and solvents were from Sigma and of analytical grade. 2.2. Animals Sprague–Dawley male rats with an average weight of 200 ± 30 g were supplied by Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai, China). Rats were housed at 20 ± 3 ◦ C and 45–65% humidity with a 12-h light–dark cycle. Drinking water and food were provided ad libitum throughout the study. 2.3. Isolation of liver mitochondria Rat liver mitochondrial fraction was prepared as described (Schneider et al., 1950) with modifications. The liver was isolated and placed in the ice-cold medium containing 250 mM sucrose, 10 mM HEPES-KOH, pH 7.4, and 0.5 mM EGTA. The liver was cut to small cubes with scissors in 5 mL of the medium and homogenized 3 times with a Potter homogenizer. The homogenate was diluted to 10 mL per liver and were centrifuged at 900 × g for 5 min at 4 ◦ C. The supernatant was decanted and further centrifuged at 4500 × g for 5 min. The pellet was suspended in 20 mL of the ice-cold isolation medium and centrifuged at 4500 × g for 10 min. The final mitochondrial pellet was suspended in 1 mL of medium containing 250 mM sucrose and 10 mM HEPES-KOH, pH 7.4. The protein concentration was determined by the method of Lowry (Lowry et al., 1951). 2.4. Measurement of mitochondrial swelling The liver mitochondrial preparation (1.0 mg/mL) was incubated with a reaction buffer containing 210 mM mannitol, 70 mM sucrose, 2 M rotenone, and 5 mM HEPES, pH 7.4 at 30 ◦ C in the presence of 20 M CaCl2 . The mitochondria were energized by 5 mM succinate. Mitochondrial swelling was measured by the change in absorbance at 540 nm over 15 min at room temperature with a SpectraPlus384 microplate reader (Molecular Devices, USA). 2.5. Determination of the isolated mitochondrial membrane potential The mitochondrial membrane potential was monitored with TMRE at 100 nM. After 2 min preincubation with succinate, diphenylamine, diclofenac or tolfenamic acid was added and incubated for 15 min in the presence of Ca2+ . The reaction medium was immediately centrifuged (16,000 × g, 30 s) and fluorescence intensity of the supernatant was measured at the 530/590 nm wavelength pair with Novostar (BMG LABTECH, Offenburg, Germany). 2.6. Measurement of mitochondrial Ca2+ efflux Ca2+ efflux from mitochondria was measured as described (Scarpa, 1979). The reaction medium was used as described above. Arsenazo III (50 M) was added. Absorbance of the medium was monitored at the 675/685 nm wavelength pair during preincubation. Dpa, Dcf or Tol was added 1 min after the energization of the mitochondria followed by 20 min incubation at 30 ◦ C. 2.7. Culture of rat hepatocytes Hepatocytes were isolated by two-step collagenase perfusion as described previously (Berry and Friend, 1969; Seglen, 1972; Orrenius et al., 1976) with some modifications. Monolayer hepatocytes were cultured at 37 ◦ C in a 5% CO2 and 95%
Fig. 2. Effects of diphenylamine, diclofenac and tolfenamic acid on isolated liver mitochondria. (A) Effects of 50 M diphenylamine, diclofenac and tolfenamic acid on the swelling of rat liver mitochondria (1 mg/mL) energized by addition of Ca2+ (20 M final concentration). The results are representatives of at least three experiments. (B) Effects of diphenylamine, diclofenac and tolfenamic acid on mitochondrial membrane potential. The reaction medium was the same as described in (A). The loss of mitochondrial membrane potential of diclofenac and tolfenamic acid was inhibited by 1 M CsA. The values shown are mean ± S.D. from measurement. * P < 0.05, ** P < 0.01 vs. control, ## P < 0.01 vs. each drug without CsA. (C) Effects of 500 M diphenylamine on the swelling of rat liver mitochondria.
Y. Li et al. / Toxicology Letters 186 (2009) 111–114 membrane potential of cultured hepatocytes was evaluated as described (Wu et al., 1990). CsA (0.2 M) was used as an inhibitor of MPT where indicated. Fluorescence readings were taken at 530/590 nm wavelength pair with Novostar (BMG LABTECH, Offenburg, Germany). 2.9. Statistical analysis Results were expressed as mean ± S.D. After homogenetic analysis, homogeneous data were analyzed with one-way analysis of variance (one-way ANOVA) and a post hoc test of least significant difference. To determine intergroup differences, heterogeneous data were analyzed using t’-test. A value of P < 0.05 was considered to be statistically significant.
113
tion of growth (IC50 value). After 24 h treatment, the estimated IC50 values for diphenylamine, diclofenac and tolfenamic acid were 183.2 ± 69.58 M, 332.7 ± 31.38 M and 195.8 ± 19.60 M, respectively (Table 1). All the drugs caused decreases in cellular ATP content and mitochondrial membrane potential. The degrees of injury to the rat primary hepatocytes reflected from which, paralleled with decrease of the cellular ATP content. Among the tested drugs, diphenylamine exerted the strongest effect, depleting the approximately 92% the cellular ATP content and 50% the mitochondrial membrane poten-
3. Results 3.1. Effects of diphenylamine, diclofenac and tolfenamic acid on isolated liver mitochondrial function MPT leads to the disruption of the mitochondrial membranes and mitochondrial swelling. As Fig. 2A shown, in isolated liver mitochondria energized with succinate and Ca2+ , tolfenamic acid caused significant mitochondrial swelling and diclofenac caused moderatel mitochondiral swelling. In contrast, diphenylamine failed to cause any mitochondiral swelling. In addition, CsA completely inhibited the swelling induced by both diclofenac and tolfenamic acid (Fig. 2A). Meanwhile, diclofenac and tolfenamic acid decreased mitochondrial membrane potential significantly in the presence of 20 M Ca2+ (Fig. 2B), which aborted by co-incubation with 1 M CsA, and diphenylamine exerted no effect on mitochondrial membrane potential. Ca2+ release from the mitochondria was induced by diclofenac but not diphenylamine or tolfenamic acid. In addition, Ca2+ release caused by diclofenac was blocked by 1 M CsA (Fig. 3). 3.2. Effects of diphenylamine, diclofenac and tolfenamic acid on rat primary hepatocytes Rat primary hepatocytes were exposed to the increasing concentrations of diphenylamine, diclofenac and tolfenamic acid. Cytotoxicity was measured by the concentration for 50% inhibi-
Fig. 3. Effects of diphenylamine, diclofenac and tolfenamic acid on efflux of mitochondrial calcium. Incubation medium was the same medium as in Fig. 2, plus 50 M arsenazo III as an indicator. The results are representatives of at least three experiments. ** P < 0.01 vs. control, ## P < 0.01 significantly different from each drug without CsA. Table 1 IC50 value of diphenylamine, diclofenac and tolfenamic acid on rat primary hepatocytes after 24 h treatment. IC50 (M)a Diphenylamine Diclofenac Tolfenamic acid
183.2 ± 69.58 332.7 ± 31.38 195.8 ± 19.60
a The values shown are mean ± S.D. For measurement, the result are representatives of at least three experiments.
Fig. 4. Effects of diphenylamine, diclofenac and tolfenamic acid on liver mitochondrial function in rat primary hepatocytes. Hepatocytes were cultured with 250 M or 500 M diphenylamine, 400 M or 800 M diclofenac and 200 M or 400 M tolfenamic acid without or with 0.2 M CsA for 24 h. (A) Effects of diphenylamine, diclofenac and tolfenamic acid on ATP depletion. The results are representatives of at least three experiments. (B) Effects of diphenylamine, diclofenac and tolfenamic acid on decrease of mitochondrial transmembrane potential on rat primary hepatocytes. (C) Effects of inhibition of CYP450 on diphenylamine-induced hepatocytes injury. The rat primary hepatocytes were treated with diphenylamine co-incubation with 10 M SKF-525A for 24 hours. The results are representatives of at least three experiments. ** P < 0.01 vs. control, # P < 0.05, ## P < 0.01 vs. each drug without CsA.
114
Y. Li et al. / Toxicology Letters 186 (2009) 111–114
tial with the concentration of 500 M (Fig. 4A and B). It was shown that all the three drugs, diphenylamine, diclofenac and tolfenamic acid, could cause damages to the rat primary hepatocytes and all these can be attenuated when cocultured with 0.2 M CsA (Fig. 4), suggesting the mechanisms involved in the damages caused by the three drugs may relate with MPT. As shown in Fig. 4C, SKF-525A, a general inhibitor of CYP450, markedly reversed the damages caused by diphenylamine.
In conclusion, we have demonstrated that hepatocyte mitochondrial dysfunction induced by NSAIDs cannot be attributed to the diphenylamine core structure. Conflict of interest There are no conflicts of interest. Acknowledgement
4. Discussion In our study, we evaluated hepatotoxicities caused by diphenylamine and structural related NSAIDs, diclofenac and tolfenamic acid. Diphenylamine (250 M and 500 M), diclofenac (400 M and 800 M) and tolfenamic acid (200 M and 400 M) caused a marked decrease in cellular ATP content and loss of mitochondrial membrane potential in rat primary hepatocytes. CsA, a general inhibitor of MPT, markedly attenuated these effects. These data are consistent with findings by others which suggest that these drugs induce MPT in the primary hepatocytes(Masubuchi et al., 1998; Masubuchi et al., 1999). In isolated liver mitochondria, diphenylamine failed to produce any effect on MPT at concentrations as high as 500 M (Fig. 2C) whereas diclofenac and tolfenamic acid induced CsA-sensitive MPT at doses as low as 50 M (Fig. 2A). Our findings indicate that the hepatotoxicities caused by NSAIDs (e.g. diclofenac and tolfenamic acid) are not attributable to the diphenylamine structure. This is in conflict with a previous study which showed that diphenylamine (250–500 M) could induce MPT in isolated mitochondria without substrate for respiration in incubation buffer and activate MPT in hepatocytes at the same concentration (Masubuchi et al., 2000). Respiration substrates and calcium are present in mitochondria at normal physiological state in hepatocytes. So, in our study, succinate and calcium were present in the incubation buffer. Under our experimental conditions, even 500 M diphenylamine was unable to induce mitochondrial swelling (Fig. 2C) in isolated liver mitochondria. These suggest that diphenylamine structure may not play a major role in activation of MPT in hepatotoxicities induced by diclofenac and tolfenamic acid. We examined the influence of SKF-525A, a general CYP450 inhibitor, on the hepatic injury induced by these drugs, and found that SKF-525A could attenuate diphenylamine-induced ATP depletion. Put together, these findings suggest that MPT has a major role in NSAIDs-induced hepatocyte injury; however, diphenylamine structure may not be involved in MPT activation. Diphenylamineinduced MPT in hepatocytes may be related to its metabolite or reactive oxygen species (ROS), which provides the major inducement of MPT. Our unpublished data indicate that diphenylamine induces burst of ROS in hepatocytes, reaching the peak at 2 h, about 1.3 times of control (data not shown). The detailed mechanism of MPT induced by diphenylamine in hepatocytes needs further research.
The project was supported by National Key Technologies R&D Program (2008ZX09305-007). References Agrawal, N., 1991. Risk factors for gastrointestinal ulcers caused by nonsteroidal antiinflammatory drugs (NSAIDs). J. Fam. Pract. 32 (6), 619–624. Banks, A.T., Zimmerman, H.J., et al., 1995. Diclofenac-associated hepatotoxicity: analysis of 180 cases reported to the food and drug administration as adverse reactions. Hepatology 22 (3), 820–827. Berry, M.N., Friend, D.S., 1969. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J. Cell Biol. 43 (3), 506–520. Brass, E.P., 1993. Hepatic toxicity of antirheumatic drugs. Cleve. Clin. J. Med. 60 (6), 466–472. Hughes, G.R., 1991. The problems of using NSAIDs in the elderly. Scand. J. Rheumatol. Suppl. 91, 15–19. Johnson, A.G., Day, R.O., 1991. The problems and pitfalls of NSAID therapy in the elderly (Part I). Drugs Aging 1 (2), 130–143. Lowry, O.H., Rosebrough, N.J., et al., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193 (1), 265–275. Manoukian, A.V., Carson, J.L., 1996. Nonsteroidal anti-inflammatory drug-induced hepatic disorders. Incidence and prevention. Drug Saf. 15 (1), 64–71. Manov, I., Motanis, H., et al., 2006. Hepatotoxicity of anti-inflammatory and analgesic drugs: ultrastructural aspects. Acta Pharmacol. Sin. 27 (3), 259–272. Masubuchi, Y., Saito, H., et al., 1998. Structural requirements for the hepatotoxicity of nonsteroidal anti-inflammatory drugs in isolated rat hepatocytes. J. Pharmacol. Exp. Ther. 287 (1), 208–213. Masubuchi, Y., Yamada, S., et al., 1999. Diphenylamine as an important structure of nonsteroidal anti-inflammatory drugs to uncouple mitochondrial oxidative phosphorylation. Biochem. Pharmacol. 58 (5), 861–865. Masubuchi, Y., Yamada, S., et al., 2000. Possible mechanism of hepatocyte injury induced by diphenylamine and its structurally related nonsteroidal antiinflammatory drugs. J. Pharmacol. Exp. Ther. 292 (3), 982–987. Orrenius, S., Thor, H., et al., 1976. Isolated rat hepatocytes as an experimental tool in the study of cell injury. Effect of anoxia. Forensic Sci. 8 (3), 255–263. Scarpa, A., 1979. Measurements of cation transport with metallochromic indicators. Methods Enzymol. 56, 301–338. Schattner, A., Sokolovskaya, N., et al., 2000. Fatal hepatitis and renal failure during treatment with nimesulide. J. Intern. Med. 247 (1), 153–155. Schneider, W.C., Hogeboom, G.H., et al., 1950. Intracellular distribution of enzymes VII. The distribution of nucleic acids and adenosinetriphosphatase in normal mouse liver and mouse hepatoma. J. Natl. Cancer Inst. 10 (4), 977–982. Seglen, P.O., 1972. Preparation of rat liver cells. I. Effect of Ca2+ on enzymatic dispersion of isolated, perfused liver. Exp. Cell Res. 74 (2), 450–454. Wu, E.Y., Smith, M.T., et al., 1990. Relationships between the mitochondrial transmembrane potential, ATP concentration, and cytotoxicity in isolated rat hepatocytes. Arch. Biochem. Biophys. 282 (2), 358–362. Zimmerman, H.J., 1990. Update of hepatotoxicity due to classes of drugs in common clinical use: non-steroidal drugs, anti-inflammatory drugs, antibiotics, antihypertensives, and cardiac and psychotropic agents. Semin. Liver Dis. 10 (4), 322–338. Zimmerman, H.J., Lewis, J.H., 1987. Drug-induced cholestasis. Med. Toxicol. 2 (2), 112–160.