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Enhanced nephrotoxicity of acetaminophen in fructose-induced hypertriglyceridemic rats: Contribution of oxidation and deacetylation of acetaminophen to an enhancement of nephrotoxicity
Exp Toxic Pathol 1997; 49: 313-319 Gustav Fischer Verlag
ISafety Research Laboratorys, Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan 2Department of Veterinary Pathology, Faculty of Agriculture, The Uni versity of Tokyo, Japan
Enhanced nephrotoxicity of acetaminophen in fructose-induced hypertriglyceridemic rats: Contribution of oxidation and deacetylation of acetaminophen to an enhancement of nephrotoxicity KA TSUHIKO ISHIDA 1, HISASHI IKEGAMI 1, and KUNIO D0I 2 With 5 figures Received: March 19, 1996; Revised: July 18, 1996; Accepted: July 28, 1996 Address for correspondence: KATSUHIKO ISHIDA, Safety Research Laboratories, Yamanouchi Pharmaceutical Co., LTD. , 1-1-8, Azusawa, Itabishi-ku, Tokyo 174, Japan. Key words: Acetaminophen; Bis(p-nitrophenyl)phosphate; Fructose; Hepatotoxicity; Hypertriglyceridemia; Nephrotoxicity; Piperonyl butoxide.
Summary
Introduction
Fructose-induced hypertriglyceridemic Sprague-Dawley (SO) rats become resistant to hepatotoxicity and susceptible to nephrotoxicity of acetaminophen (APAP) as compared with normal SO rats. Fischer-344 rats, which are susceptible to APAP nephrotoxicity, have two toxic metabolic pathways involving cytochrome P450-dependent oxidation of APAP to N-acetyl-p-benzoquinone imine (NAPQI) and P450-independent deacetylation of APAP to p-aminophenol (PAP). SO rats, however, have only the former pathway. This study was undertaken to investigate whether alterations in the metabolic pathways of APAP and in the intrinsic susceptibility to toxic metabolites are responsible for an enhancement of APAP nephrotoxicity in the fructose-pretreated SO-rats. In the non-pretreated rats, the inhibition of APAP oxidation by the MFO inhibitor, piperonyl butoxide, and deacetylation by carboxyesterase inhibitor, bis(p-nitrophenyl)phosphate, did not alter APAP-induced renal lesions. In contrast, these inhibitors protected the fructosepretreated rats from APAP-induced renal lesions. Since there were no differences in the severity of gentamicin-, chloroform, and 45 min-ischemia/reperfusion-induced renal lesions between the non-pretreated and the fructose-pretreated rats, it is unlikely that the increased intrinsic susceptibility to chemicals and their metabolites in the fructose-pretreated rats is a major factor in the enhancement of APAP nephrotoxicity. These results indicate that the enhancement of APAP nephrotoxicity in the fructosse-pretreated rats is due, at least in part, to an alteration in metabolic pathways of APAP.
Acetaminophen (APAP), a widely used analgesic and antipyretic, produces hepatic and renal tubular necrosis in humans and animals following overdosage (BOYER and ROUFF 1971 ; MITCHELL et al. 1973; HINSON 1980; NEWTON et al. 1985). Sprague Dawley (SD) rats are resistant to APAP nephrotoxicity as compared with Fischer-344 (F344) rats (T ARLOFF et al. 1989). In F344 rats, APAP-induced nephrotoxicity involves cytochrome P450-dependent metabolism of APAP to N-acetyl-p-benzoquinone imine (NAPQI) and de acetylation of APAP to p-aminophenol (PAP) (McMURTRY et al. 1978; NEWTON et al. 1985; EMEIGH HART et al. 1991; MUGFORD and TARLOFF 1995). In contrast, the pathway of renal bioactivation of APAP in SD rats is similar to that in the liver (cytochrome P450-dependent metabolism to NAPQI) and deacetylation does not importantly contribute to the bioactivation of APAP (MUGFORD and TARLOFF 1995). Recently, we reported that fructose-induced hypertriglyceridemic SD rats become resistant to APAP hepatotoxicity and susceptible to APAP nephrotoxicity (ISHIDA et al. 1995). Thereafter we also clarified that the enhanced susceptibility to APAP nephrotoxicity in fructose-pretreated rats is considered to be due, at least in part, to significantly but mildly increased renal APAP concentration (111-124 % of nonpretreated rats) at the early phase (15 and 30 min after APAP administration) (ISHIDA et al. (1997); ISHIDA et al. (in press)). However, judging from the severity of renal lesions in the fructose pretreated rats, Exp Toxic Pathol 49 (1997) 5
313
an enhancement of APAP nephrotoxicity in the fructosepretreated rats seems to involve other mechanisms. In this study, we investigated the effects of preinhibition of cytochrome P450-dependent oxidation and P450independent deacetylation of APAP by MFO inhibitor, piperonyl butoxide (PB), and by carboxyesterase inhibitor, bis(p-nitrophenyl)phosphate (BNPP), respectively, on APAP-induced renal lesions in the non-pretreated and the fructose-pretreated rats. We also compared the renal lesions induced by gentamicin which is reabsorbed into renal tubular epithelial cells without metabolic bioactivation, those induced by chloroform which is bioactivated in hepatic and renal tissues as well as APAP and those induced by 45 min-ischemiaJreperfusion between the nonpretreated and the fructose-pretreated rats.
Material and methods Animals: Seven-week old male SD rats weighing 220-250 g were obtained from Charles River Japan Inc. (Kanagawa). They were placed in a hanging stainless steel wire-bottomed cage in an animal room under controlled conditions (temperature: 23 ± 3°C, humidity: 55 ± 10 %, lighting: 13 hr (8:00-21:00), and ventilation: 20 times an hour) and fed pelletted diet (CRF-l, Oriental Yeast Co. Ltd., Tokyo) and tap water ad libitum for 7 days until used. Experiment 1: The rats were divided into 6 groups (Group 1-6) of 9-11 and were given tap water (Group 1-3) or 25 % fructose (Dai-ichikogyo Seiyaku Co. Ltd., Tokyo) in their drinking water (Group 4-6) ad libitum for 3 weeks, respectively. After completion of the 3-week-pretreatment, rats fo Group 2 and 5 and Group 3 and 6 were given PB in olive oil (500 mg/kg, ip, Tokyo Chemical Industry Co. Ltd., Tokyo) or BNPP in distilled water (100 mg/kg, ip, Wako Pure Chemical Industries Inc., Osaka), respectively, 1 hr prior to treatment with AP AP in 1 % carboxymethy lcellulose (CMC) solution (750 mg/kg, ip, Wako Pure Chemical Industries Inc., Osaka). The rats of Group 1 and 4 were treated with APAP alone and used as controls. They were killed by bleeding from the abdominal aorta under ether anesthesia at 24 hr after APAP-treatment. The kidneys were weighed and then fixed in 10 % neutral buffered formalin. Experiment 2: The rats were divided into 8 groups (Group 1-8) of 10 and were given tap water (Group 1,3,5 and 7) or 25 % fructose in their drinking water (Group 2, 4, 6 and 8) ad libitum for 3 weeks, respectively. After completion of the 3-week-pretreatment, 5 rats of each group were treated with APAP in CMC (Group 1 and 2, 750 mg/kg, ip), gentamicin sulfate in saline (Group 3 and 4, 400 mg/kg/day for 2 days, sc, Sigma Chemical Co., St. Louis), chloroform in olive oil (Group 5 and 6, 500 mg/kg, ip, Kanto Chemical Co. Inc., Tokyo) and 45-min-ischemialreperfusion under pentobarbital anesthesia (50 mg/kg, ip) (Group 7 and 8), respectively. The other 5 rats of each group were treated with CMC (Group 1 and 2), saline (Group 3 and 4), olive oil (Group 5 and 6) and sham operation (Group 7 and 8) respectively and used as controls. They were killed at 24 hr after each treatment. The kidneys were weighed and then fixed in 10% neutral buffered formalin. Histopathology: The formalin-fixed kidney was embedded in paraffin, sectioned at 5 11m, and stained with hematoxy314
Exp Toxic Pathol 49 (1997) 5
lin-eosin (HE) and by periodic acid-Schiff method (PAS) for microscopic examinations. The severity of renal lesions was assessed by assigning a numerical score ranging from 1 (normal) to 5 (severe) as previously reported (ISHIDA et al. 1995). Statistical analysis: All data are expressed as mean ± standard error (SE). Statistical analysis was done using Student's t-test. In case that variances were significantly inhomogeneous, Welch's method was used.
Results Experiment 1 In the non-pretreated group, the kidney weight of rats given PB and APAP was greater than that of rats given APAP alone (fig. 1). There was no difference in the kidney weight between rats given APAP alone and rats given BNPP and APAP. The kidney weight of the fructose-pretreated rats was significantly greater than that of the non-pretreated rats irrespective of PB- and BNPP-treatment. In the fructose-pretreated rats, the treatment of PB and BNPP did not alter an APAP-induced increase in kidney weight. The non-pretreated rats given APAP alone showed slight renal lesions characterized by single cell necrosis of epithelial cells in the proximal straight tubules (PSTs) of the medullary ray (fig. 2a). Although non-pretreated rats given PB and APAP showed slight dilatation of the PSTs, the severity of epithelial cell damage was similar to that in the non-pretreated rats given APAP-alone (fig. 2b). Treatment ofBNPP did not alter APAP-induced renal lesions in the non-pretreated rats (fig. 2c). The fructose-pretreated rats given APAP alone showed severe renal lesions characterized by extensive epithelial cell necrosis in most of the PSTs (fig. 2d). PB- and
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o Fig. I. Effect ofPB- and BNPP-treatments on kidney weight of non-pretreated and fructose-pretreated rats given APAP. Results are expressed as means ± SE, N = 9-11. c::J:APAPalone, ~ :PB+APAP, _:BNPP+APAP.
Fig. 2. Renal histopathology. PAS. Bar = 100 11m. A: A non-pretreated rat given APAP alone. Single cell necrosis of epithelial cells in the proximal straight tubules (PSTs) in the medullary ray. B: A non-pretreated rat given PB and APAP. Single cell necrosis of epithelial cells in the PSTs with tubular dilatation in the medullary ray. e: A non-pretreated rat given BNPP and APAP. Single cell necrosis of epithelial cells in the PSTs in the medullary ray. Exp Toxic Pathol 49 (1997) 5
315
Fig. 2. Renal histopathology. PAS. Bar = 100 11m. D: A fructose-pretreated rat given APAP alone. Extensive necrosis of epithelial cells in most of the PSTs. E: A fructose-pretreated rat given PB and APAP. Focal necrosis of epithelial cells in the PSTs with tubular dilatation in the medullary ray. F: A fructose-pretreated rat given BNPP and APAP. Single cell necrosis of epithelial cells in the PSTs with tubular dilatation in the medullary ray. 316
Exp Toxic Pathol49 (1997) 5
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Fig. 3. Effect of PB- and BNPP-treatment on APAP-induced renal lesion in non-pretreated and fructose-pretreated rats. The severity is scored by the criteria given in Material and methods.
Fig. 4. Effect of fructose-pretreatment on kidney weight of the rats treated with APAP, gentamicin, chloroform and 45 min-ischemiaJreperfusion. Results are expressed as means ± SE, N =5.
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BNPP-treatment protected the fructose-pretreated rats from APAP-induced renal lesions (fig. 2e and f). These protective effects were greater in BNPP-treatment than PB-treatment (fig. 3). Experiment 2: The kidney weights of APAP-, gentamicin-, chloroform- and 45 min-ischemiaJreperfusiontreated groups were significantly greater than those of each control group except for the non-pretreated and APAP-treated rats and the fructose-pretreated ischemic rats (fig. 4). Pretreatment of fructose, however, did not alter an increase in kidney weight induced by gentamicin, chloroform and ischemia, although it enhanced an APAP-
: non-pretreatment,
induced increase in kidney weight. Pretreatment of fructose potentiated APAP-induced renal lesions (fig. 5).
Discussion Previous studies reported that the fructose-induced hypertriglyceridemic rats become resistant to APAP hepatotoxicity and susceptible to APAP nephrotoxicity as compared with normal rats (ISHIDA et al. 1995) and that renal APAP concentrations in the fructose-pretreated rats were significantly but mildly greater than those in the non-preExp Toxic Pathol 49 (1997) 5
317
treated ones at the early phase (15 and 30 min, after APAP administration) (ISHIDA et al. (1997». Thus an enhanced APAP nephrotoxicity in the fructose-pretreated rats is considered to be due, at least in part, to an increase in renal APAP concentration. However, such significant but mild increase in renal APAP concentration alone seems to be unable to explain the severity of renal lesions in the fructose-pretreated rats. In this study, preinhibition of both cytochrome P4S0mediated oxidation and cytochrome P4S0-independent deacetylation of APAP reduced the degree of enhancement of APAP nephrotoxicity in the fructose-pretreated rats. Therefore, APAP nephrotoxicity in the fructose-pretreated rats is attributable to APAP bioactivation by two metabolic pathways (oxidation an deacetylation). SD rats are more resistant to APAP nephrotoxicity than F344 rats (TARLOFF et al. 1989). In F344 rats, APAP-induced nephrotoxicity involves both cytochrome P4S0-dependent metabolism of APAP to NAPQI and deacetylation of APAP to PAP (McMuRTRY et al. 1978; NEWTON et al. 1985; EMEIGH HART et al. 1991; MUGFORD and TARLOFF 1995). In SD rats, however, deacetylation does not importantly contribute to the bioactivation of APAP (MUGFORD and TARLOFF 1995). Therefore, an enhancement of APAP nephrotoxicity in the fructose-pretreated SD rats is due not only to cytochrome P4S0-mediated oxidation of APAP but also to cytochrome P4S0-independent deacetylation of APAP which is not responsible for APAP nephrotoxicity in normal SD rats. PB- and BNPP-treatment, however, did not improve APAP-induced increase in the kidney weight and tubular dilatation. Since these treatments can not reduce the degree of an increase in renal APAP concentration in the fructose-pretreated rats, the protective effects of these treatments appear to be partial. In the non-pretreated rats, PB-treatment enhanced slightly the degree of an APAP-induced increase in kidney weight and tubular dilatation. In nonnal rats given APAP, renal APAP concentration is lower than hepatic APAP concentration and than renal APAP concentration in the fructose-pretreated rats (ISHIDA et al. (1997». Normal rats given APAP show severe APAP hepatotoxicity and slight nephrotoxicity (ISHIDA et al. 1995). In the nonpretreated rats, 90 % partial hepatectomy enhanced APAP nephrotoxicity slightly (ISHIDA et al. (in press». In mice, an inhibition of hepatic metabolism of APAP also enhances renal and pulmonary toxicity (JEFFERY 1991). Therefore, PB-treatment-induced decrease in capacity of hepatic AP AP metabolism which is greater than that in the other tissues may be responsible for an enhancement of the degree of APAP-induced increase in kidney weight and tubular dilatation. It was, however, reported that APAP exerted a direct action on the kidney without the influence of hepatic damage or liver-derived toxic metabolites of APAP (TRUMPER et al. 1995). In addition, covalent binding of APAP to renal macromolecules was not altered by total hepatectomy (BREEN et al. 1982). In this study, the extent of enhancement of APAP nephrotoxicity indu318
Exp Toxic Patho1 49 (1997) 5
ced by 90 % partial hepatectomy and by PB-treatment in the non-pretreated rats is less severe than that induced by fructose-pretreatment. These data suggest that a decrease in capacity of hepatic APAP metabolism has only minimal effects on an enhancement of APAP nephrotoxicity. Fructose infusion can lower ATP concentration in tissues possessing the appropriate kinases such as the kidney and liver (BURCH et al. 1970; SUMPIO et al. 1984; SHAPIRO et al. 1989). Basal renal glutathione (GSH) concentrations were lower in the fructose-pretreated rats than in the non-pretreated rats (ISHIDA et al. (1997». Thus, fructoe-pretreatment may enhance renal intrinsic susceptibility to renal lesions, such as decreased concentration of ATP and GSH. Gentamicin, a typical nephrotoxic aminoglycoside, is freely filtered at the glomerulus and reabsorbed via active transport process at the proximal tubular brush borders without metabolic bioactivation (TARLOFF and GOLDSTEIN 1994). Chlorofonn induces hepatorenal toxicity by the production of reactive intermediate, phosgene, through cytochrome P4S0-related biotransformation in the liver and kidney as well as APAP (POHL et al. 1977; KLUWE and HOOK 1981). Phosgene binds to GSH and this conjugate is excreted. Thus, pre-administration of the GSH-depleting agent, diethyl maleate, increases the susceptibility to chloroform nephrotoxicity (KLUWE and HOOK 1981). Ischemic renal injury induces a reduction in ATP concentration due to mitochondrial disturbances as well as APAP (ARNOLD et al 1987; MOURELLE et al. 1991; WILSON et al. 1984). In this study, fructose-pretreatment did not alter gentamicin-, chloroform- and 45 min-ischemia! reperfusion-induced renal lesions. It is also reported that fructose infusion did not affect normal renal functions or recovery from a reversible ischemic insult (SHAPIRO et al. 1987; SHAPIRO et al. 1989). Therefore there was no evidence that alteration of renal intrinsic susceptibility to renal lesions was a major factor in the enhancement of APAP nephrotoxicity in the fructose-pretreated rats. However, since indirect assessment is limited, further detailed studies are required to exclude the alteration of renal intrinsic susceptibility as a contributing factor with respect to enhanced nephrotoxicity in the fructose-pretreated rats. We conclude from these results that an enhancement of APAP nephrotoxicity observed in the fructose-pretreated SD rats is attributable to at least the following two factors: 1) an incrase in renal APAP concentration at the early phase (1S and 30 min after APAP dosing) and 2) bioactivation of APAP via two metabolic pathways: cytochrome P4S0-mediated oxidation of APAP to NAPQI and cytochrome P4S0-independent deacetylation of APAP to PAP which is not responsible for nephrotoxicity in normal SD rats.
References 1. ARNOLD PE, VAN PUTIERN VJ, LUMLERTGUL D: Adenine nucleotide metabolism and mitochondrial calcium transport following renal ischemia. Am J Physiol 1986; 250: F 357-363.
2. BOYER TD, ROUFF SL: Acetaminophen-induced hepatic necrosis and renal failure. J Am Med Assoc 1971; 218: 440-441. 3. BREEN K, WANDSCHEER J-C, PEIGNOUX M et al.: In situ formation of the acetaminophen metabolic covalently bound in kidney and lung. Supportive evidence provided by total hepatectomy. Biochem Pharmacol 1982; 31: 115-116. 4. BURCH HB, LOWLEY OH, MEINHARDT L et al.: Effect of fructose, dihydroxyacetone, glycerol, and glucose on metabolites and related compounds in liver and kidney. J BioI Chern 1970; 245: 2092-2102. 5. EMEIGH HART SG, BEIERSCHMITT WP, BARTOLONE JB et al.: Evidence against deacetylation and for cytochrome P450-mediated activation in acetaminopheninduced nephrotoxicity in the CD-l mouse. Toxicol Appl Pharmacol1991; 107: 1-15. 6. ISHIDA K, HANADA T, SAKAI T et al.: Effects of fructose-induced hypertriglyceridemia on hepatorenal toxicity of acetaminophen in rats. Exp Toxicol Pathol 1995;47: 509-516. 7. ISHIDA K, SAKAZUME M, WATANABE M et al.: Effects of fructose-induced hypertriglyceridemia on hepatorenal toxicity of acetaminophen in rats: Role of pharmacokinetics and metabolism of acetaminophen. Exp Toxicol Patho11997; 49: 207-215. 8. ISHIDA K, SAKAZUME M, IKEGAMI H et al.: Enhanced nephrotoxicity of acetaminophen in fructose-induced hypertriglyceridemic rats: Effect of partial hepatectomy. Exp Toxicol Patho11997; 49: 321-327. 9. HINSON JA: Biochemical toxicology of acetaminophen. Rev Biochem Toxicol 1980; 2: 103-130. 10. JEFFEREY EH: Biochemical Basis of toxicity. In: HASCHEK WM, ROUSERAUX CG (Eds.): Handbook oftoxicology pathology. Academic Press, INC. 1991; Chap. 5: pp.49-70. 11. KLUWE WM, HOOK JB: Potentation of acute chloroform nephrotoxicity by the glutathione depletor diethyl maleate and protection by the microsomal enzyme inhibitor piperonyl butoxide. Toxicol Appl Pharmacol 1981;59:457-466. 12. McMURTRY RJ, SNODGRASS WR, MITCHELL JR: Renal necrosis, glutathione depletion, and covalent binding after acetaminophen. Toxicol Appl Pharmacol 1978; 46: 87-100. 13. MITCHELL JR, JOLLOW DJ, PATTER WZ et al.: Acetaminophen-induced hepatic necrosis. 4. Protective
14. 15.
16.
17. 18.
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20.
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22.
23. 24.
role of glutathione. J Pharmaco1 Exp Ther 1973; 187: 211-217. MOURELLE M, BEALES D, McLEAN AEM: Prevention of paracetamol-induced liver injury by fructose. Biochern Pharmacol1991; 41: 1831-1837. MUGFORD CA, TARLOFF JB: Contribution of oxidation and deacetylation to the bioactivation of acetaminophen in vitro in liver and kidney from male and female Sprague-Dawley rats. Drug Metab Dispos 1995; 23: 290-294. NEWTON JF, Kuo CH, DES HONE DM et al. The role of p-aminophenol in acetaminophen-induced nephrotoxicity: Effect of bis(p-nitrophenyl)phosphate on acetaminophen and p-aminophenol nephrotoxicity and metabolism in Fischer 344 rats. Toxicol Appl Pharmacol 1985; 81: 416-430. POHL LR, BHOOSHAN B, WHITAKER NF et al.: Phosgene: A metabolite of chloroform. Biochem Biophys Res Commun 1977; 79: 684-691. SHAPIRO 11, CHAN L, CHEUNG C et al.: Effects of adenosine triphosphate depletion in the isolated perfused rat kidney. Miner Electrolyte Metab 1987; 13: 415-421. SHAPIRO 11, MILLS S, CHEUNG C et al.: Effect of adenosine triphosphate depletion in vivo on renal function in the rat with and without ischemia. Miner Electrolyte Metab 1989; 15: 276-282. SUMPIO BE, CHAUDRY IH, CLEMENS MG et al.: Accelerated functional recovery of isolated rat kidney with ATP-MgCI 2 after warm ischemia. Am J Physiol 1984; 247: R 1047-1053. TARLOFF JB, GOLDSTEIN RS, MORGAN DG et al.: Acetaminophen and p-aminophenol nephrotoxicity in aging male Sprague-Dawley and Fischer 344 rats. Fund Appl Toxicol 1989; 12: 78-91. TARLOFF JB, GOLDSTEIN RS: Biochemical mechanisms of renal toxicity. In: HODGSON E, LEVI PE (Eds.): Introduction to biochemical toxicology 2nd ed. Appleton & Lange. 1994; Chap. 22: pp.519-546. TRUMPER L, MONASTEROLO LA, OCHOA E et al.: Tubular effects of acetaminophen in the isolated perfused rat kidney. Arch Toxicol 1995; 69: 248-252. WILSON DR, ARNOLD PE, BURKE TJ et al.: Mitochondrial calcium accumulation and respiration in ischemic acute renal failure in the rat. Kidney Int 1984; 25: 519-526.
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ISafety Research Laboratorys, Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan 2Department of Veterinary Pathology, Faculty of Agriculture, The Uni versity of Tokyo, Japan
Enhanced nephrotoxicity of acetaminophen in fructose-induced hypertriglyceridemic rats: Contribution of oxidation and deacetylation of acetaminophen to an enhancement of nephrotoxicity KA TSUHIKO ISHIDA 1, HISASHI IKEGAMI 1, and KUNIO D0I 2 With 5 figures Received: March 19, 1996; Revised: July 18, 1996; Accepted: July 28, 1996 Address for correspondence: KATSUHIKO ISHIDA, Safety Research Laboratories, Yamanouchi Pharmaceutical Co., LTD. , 1-1-8, Azusawa, Itabishi-ku, Tokyo 174, Japan. Key words: Acetaminophen; Bis(p-nitrophenyl)phosphate; Fructose; Hepatotoxicity; Hypertriglyceridemia; Nephrotoxicity; Piperonyl butoxide.
Summary
Introduction
Fructose-induced hypertriglyceridemic Sprague-Dawley (SO) rats become resistant to hepatotoxicity and susceptible to nephrotoxicity of acetaminophen (APAP) as compared with normal SO rats. Fischer-344 rats, which are susceptible to APAP nephrotoxicity, have two toxic metabolic pathways involving cytochrome P450-dependent oxidation of APAP to N-acetyl-p-benzoquinone imine (NAPQI) and P450-independent deacetylation of APAP to p-aminophenol (PAP). SO rats, however, have only the former pathway. This study was undertaken to investigate whether alterations in the metabolic pathways of APAP and in the intrinsic susceptibility to toxic metabolites are responsible for an enhancement of APAP nephrotoxicity in the fructose-pretreated SO-rats. In the non-pretreated rats, the inhibition of APAP oxidation by the MFO inhibitor, piperonyl butoxide, and deacetylation by carboxyesterase inhibitor, bis(p-nitrophenyl)phosphate, did not alter APAP-induced renal lesions. In contrast, these inhibitors protected the fructosepretreated rats from APAP-induced renal lesions. Since there were no differences in the severity of gentamicin-, chloroform, and 45 min-ischemia/reperfusion-induced renal lesions between the non-pretreated and the fructose-pretreated rats, it is unlikely that the increased intrinsic susceptibility to chemicals and their metabolites in the fructose-pretreated rats is a major factor in the enhancement of APAP nephrotoxicity. These results indicate that the enhancement of APAP nephrotoxicity in the fructosse-pretreated rats is due, at least in part, to an alteration in metabolic pathways of APAP.
Acetaminophen (APAP), a widely used analgesic and antipyretic, produces hepatic and renal tubular necrosis in humans and animals following overdosage (BOYER and ROUFF 1971 ; MITCHELL et al. 1973; HINSON 1980; NEWTON et al. 1985). Sprague Dawley (SD) rats are resistant to APAP nephrotoxicity as compared with Fischer-344 (F344) rats (T ARLOFF et al. 1989). In F344 rats, APAP-induced nephrotoxicity involves cytochrome P450-dependent metabolism of APAP to N-acetyl-p-benzoquinone imine (NAPQI) and de acetylation of APAP to p-aminophenol (PAP) (McMURTRY et al. 1978; NEWTON et al. 1985; EMEIGH HART et al. 1991; MUGFORD and TARLOFF 1995). In contrast, the pathway of renal bioactivation of APAP in SD rats is similar to that in the liver (cytochrome P450-dependent metabolism to NAPQI) and deacetylation does not importantly contribute to the bioactivation of APAP (MUGFORD and TARLOFF 1995). Recently, we reported that fructose-induced hypertriglyceridemic SD rats become resistant to APAP hepatotoxicity and susceptible to APAP nephrotoxicity (ISHIDA et al. 1995). Thereafter we also clarified that the enhanced susceptibility to APAP nephrotoxicity in fructose-pretreated rats is considered to be due, at least in part, to significantly but mildly increased renal APAP concentration (111-124 % of nonpretreated rats) at the early phase (15 and 30 min after APAP administration) (ISHIDA et al. (1997); ISHIDA et al. (in press)). However, judging from the severity of renal lesions in the fructose pretreated rats, Exp Toxic Pathol 49 (1997) 5
313
an enhancement of APAP nephrotoxicity in the fructosepretreated rats seems to involve other mechanisms. In this study, we investigated the effects of preinhibition of cytochrome P450-dependent oxidation and P450independent deacetylation of APAP by MFO inhibitor, piperonyl butoxide (PB), and by carboxyesterase inhibitor, bis(p-nitrophenyl)phosphate (BNPP), respectively, on APAP-induced renal lesions in the non-pretreated and the fructose-pretreated rats. We also compared the renal lesions induced by gentamicin which is reabsorbed into renal tubular epithelial cells without metabolic bioactivation, those induced by chloroform which is bioactivated in hepatic and renal tissues as well as APAP and those induced by 45 min-ischemiaJreperfusion between the nonpretreated and the fructose-pretreated rats.
Material and methods Animals: Seven-week old male SD rats weighing 220-250 g were obtained from Charles River Japan Inc. (Kanagawa). They were placed in a hanging stainless steel wire-bottomed cage in an animal room under controlled conditions (temperature: 23 ± 3°C, humidity: 55 ± 10 %, lighting: 13 hr (8:00-21:00), and ventilation: 20 times an hour) and fed pelletted diet (CRF-l, Oriental Yeast Co. Ltd., Tokyo) and tap water ad libitum for 7 days until used. Experiment 1: The rats were divided into 6 groups (Group 1-6) of 9-11 and were given tap water (Group 1-3) or 25 % fructose (Dai-ichikogyo Seiyaku Co. Ltd., Tokyo) in their drinking water (Group 4-6) ad libitum for 3 weeks, respectively. After completion of the 3-week-pretreatment, rats fo Group 2 and 5 and Group 3 and 6 were given PB in olive oil (500 mg/kg, ip, Tokyo Chemical Industry Co. Ltd., Tokyo) or BNPP in distilled water (100 mg/kg, ip, Wako Pure Chemical Industries Inc., Osaka), respectively, 1 hr prior to treatment with AP AP in 1 % carboxymethy lcellulose (CMC) solution (750 mg/kg, ip, Wako Pure Chemical Industries Inc., Osaka). The rats of Group 1 and 4 were treated with APAP alone and used as controls. They were killed by bleeding from the abdominal aorta under ether anesthesia at 24 hr after APAP-treatment. The kidneys were weighed and then fixed in 10 % neutral buffered formalin. Experiment 2: The rats were divided into 8 groups (Group 1-8) of 10 and were given tap water (Group 1,3,5 and 7) or 25 % fructose in their drinking water (Group 2, 4, 6 and 8) ad libitum for 3 weeks, respectively. After completion of the 3-week-pretreatment, 5 rats of each group were treated with APAP in CMC (Group 1 and 2, 750 mg/kg, ip), gentamicin sulfate in saline (Group 3 and 4, 400 mg/kg/day for 2 days, sc, Sigma Chemical Co., St. Louis), chloroform in olive oil (Group 5 and 6, 500 mg/kg, ip, Kanto Chemical Co. Inc., Tokyo) and 45-min-ischemialreperfusion under pentobarbital anesthesia (50 mg/kg, ip) (Group 7 and 8), respectively. The other 5 rats of each group were treated with CMC (Group 1 and 2), saline (Group 3 and 4), olive oil (Group 5 and 6) and sham operation (Group 7 and 8) respectively and used as controls. They were killed at 24 hr after each treatment. The kidneys were weighed and then fixed in 10% neutral buffered formalin. Histopathology: The formalin-fixed kidney was embedded in paraffin, sectioned at 5 11m, and stained with hematoxy314
Exp Toxic Pathol 49 (1997) 5
lin-eosin (HE) and by periodic acid-Schiff method (PAS) for microscopic examinations. The severity of renal lesions was assessed by assigning a numerical score ranging from 1 (normal) to 5 (severe) as previously reported (ISHIDA et al. 1995). Statistical analysis: All data are expressed as mean ± standard error (SE). Statistical analysis was done using Student's t-test. In case that variances were significantly inhomogeneous, Welch's method was used.
Results Experiment 1 In the non-pretreated group, the kidney weight of rats given PB and APAP was greater than that of rats given APAP alone (fig. 1). There was no difference in the kidney weight between rats given APAP alone and rats given BNPP and APAP. The kidney weight of the fructose-pretreated rats was significantly greater than that of the non-pretreated rats irrespective of PB- and BNPP-treatment. In the fructose-pretreated rats, the treatment of PB and BNPP did not alter an APAP-induced increase in kidney weight. The non-pretreated rats given APAP alone showed slight renal lesions characterized by single cell necrosis of epithelial cells in the proximal straight tubules (PSTs) of the medullary ray (fig. 2a). Although non-pretreated rats given PB and APAP showed slight dilatation of the PSTs, the severity of epithelial cell damage was similar to that in the non-pretreated rats given APAP-alone (fig. 2b). Treatment ofBNPP did not alter APAP-induced renal lesions in the non-pretreated rats (fig. 2c). The fructose-pretreated rats given APAP alone showed severe renal lesions characterized by extensive epithelial cell necrosis in most of the PSTs (fig. 2d). PB- and
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Fig. 2. Renal histopathology. PAS. Bar = 100 11m. A: A non-pretreated rat given APAP alone. Single cell necrosis of epithelial cells in the proximal straight tubules (PSTs) in the medullary ray. B: A non-pretreated rat given PB and APAP. Single cell necrosis of epithelial cells in the PSTs with tubular dilatation in the medullary ray. e: A non-pretreated rat given BNPP and APAP. Single cell necrosis of epithelial cells in the PSTs in the medullary ray. Exp Toxic Pathol 49 (1997) 5
315
Fig. 2. Renal histopathology. PAS. Bar = 100 11m. D: A fructose-pretreated rat given APAP alone. Extensive necrosis of epithelial cells in most of the PSTs. E: A fructose-pretreated rat given PB and APAP. Focal necrosis of epithelial cells in the PSTs with tubular dilatation in the medullary ray. F: A fructose-pretreated rat given BNPP and APAP. Single cell necrosis of epithelial cells in the PSTs with tubular dilatation in the medullary ray. 316
Exp Toxic Pathol49 (1997) 5
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Fig. 3. Effect of PB- and BNPP-treatment on APAP-induced renal lesion in non-pretreated and fructose-pretreated rats. The severity is scored by the criteria given in Material and methods.
Fig. 4. Effect of fructose-pretreatment on kidney weight of the rats treated with APAP, gentamicin, chloroform and 45 min-ischemiaJreperfusion. Results are expressed as means ± SE, N =5.
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BNPP-treatment protected the fructose-pretreated rats from APAP-induced renal lesions (fig. 2e and f). These protective effects were greater in BNPP-treatment than PB-treatment (fig. 3). Experiment 2: The kidney weights of APAP-, gentamicin-, chloroform- and 45 min-ischemiaJreperfusiontreated groups were significantly greater than those of each control group except for the non-pretreated and APAP-treated rats and the fructose-pretreated ischemic rats (fig. 4). Pretreatment of fructose, however, did not alter an increase in kidney weight induced by gentamicin, chloroform and ischemia, although it enhanced an APAP-
: non-pretreatment,
induced increase in kidney weight. Pretreatment of fructose potentiated APAP-induced renal lesions (fig. 5).
Discussion Previous studies reported that the fructose-induced hypertriglyceridemic rats become resistant to APAP hepatotoxicity and susceptible to APAP nephrotoxicity as compared with normal rats (ISHIDA et al. 1995) and that renal APAP concentrations in the fructose-pretreated rats were significantly but mildly greater than those in the non-preExp Toxic Pathol 49 (1997) 5
317
treated ones at the early phase (15 and 30 min, after APAP administration) (ISHIDA et al. (1997». Thus an enhanced APAP nephrotoxicity in the fructose-pretreated rats is considered to be due, at least in part, to an increase in renal APAP concentration. However, such significant but mild increase in renal APAP concentration alone seems to be unable to explain the severity of renal lesions in the fructose-pretreated rats. In this study, preinhibition of both cytochrome P4S0mediated oxidation and cytochrome P4S0-independent deacetylation of APAP reduced the degree of enhancement of APAP nephrotoxicity in the fructose-pretreated rats. Therefore, APAP nephrotoxicity in the fructose-pretreated rats is attributable to APAP bioactivation by two metabolic pathways (oxidation an deacetylation). SD rats are more resistant to APAP nephrotoxicity than F344 rats (TARLOFF et al. 1989). In F344 rats, APAP-induced nephrotoxicity involves both cytochrome P4S0-dependent metabolism of APAP to NAPQI and deacetylation of APAP to PAP (McMuRTRY et al. 1978; NEWTON et al. 1985; EMEIGH HART et al. 1991; MUGFORD and TARLOFF 1995). In SD rats, however, deacetylation does not importantly contribute to the bioactivation of APAP (MUGFORD and TARLOFF 1995). Therefore, an enhancement of APAP nephrotoxicity in the fructose-pretreated SD rats is due not only to cytochrome P4S0-mediated oxidation of APAP but also to cytochrome P4S0-independent deacetylation of APAP which is not responsible for APAP nephrotoxicity in normal SD rats. PB- and BNPP-treatment, however, did not improve APAP-induced increase in the kidney weight and tubular dilatation. Since these treatments can not reduce the degree of an increase in renal APAP concentration in the fructose-pretreated rats, the protective effects of these treatments appear to be partial. In the non-pretreated rats, PB-treatment enhanced slightly the degree of an APAP-induced increase in kidney weight and tubular dilatation. In nonnal rats given APAP, renal APAP concentration is lower than hepatic APAP concentration and than renal APAP concentration in the fructose-pretreated rats (ISHIDA et al. (1997». Normal rats given APAP show severe APAP hepatotoxicity and slight nephrotoxicity (ISHIDA et al. 1995). In the nonpretreated rats, 90 % partial hepatectomy enhanced APAP nephrotoxicity slightly (ISHIDA et al. (in press». In mice, an inhibition of hepatic metabolism of APAP also enhances renal and pulmonary toxicity (JEFFERY 1991). Therefore, PB-treatment-induced decrease in capacity of hepatic AP AP metabolism which is greater than that in the other tissues may be responsible for an enhancement of the degree of APAP-induced increase in kidney weight and tubular dilatation. It was, however, reported that APAP exerted a direct action on the kidney without the influence of hepatic damage or liver-derived toxic metabolites of APAP (TRUMPER et al. 1995). In addition, covalent binding of APAP to renal macromolecules was not altered by total hepatectomy (BREEN et al. 1982). In this study, the extent of enhancement of APAP nephrotoxicity indu318
Exp Toxic Patho1 49 (1997) 5
ced by 90 % partial hepatectomy and by PB-treatment in the non-pretreated rats is less severe than that induced by fructose-pretreatment. These data suggest that a decrease in capacity of hepatic APAP metabolism has only minimal effects on an enhancement of APAP nephrotoxicity. Fructose infusion can lower ATP concentration in tissues possessing the appropriate kinases such as the kidney and liver (BURCH et al. 1970; SUMPIO et al. 1984; SHAPIRO et al. 1989). Basal renal glutathione (GSH) concentrations were lower in the fructose-pretreated rats than in the non-pretreated rats (ISHIDA et al. (1997». Thus, fructoe-pretreatment may enhance renal intrinsic susceptibility to renal lesions, such as decreased concentration of ATP and GSH. Gentamicin, a typical nephrotoxic aminoglycoside, is freely filtered at the glomerulus and reabsorbed via active transport process at the proximal tubular brush borders without metabolic bioactivation (TARLOFF and GOLDSTEIN 1994). Chlorofonn induces hepatorenal toxicity by the production of reactive intermediate, phosgene, through cytochrome P4S0-related biotransformation in the liver and kidney as well as APAP (POHL et al. 1977; KLUWE and HOOK 1981). Phosgene binds to GSH and this conjugate is excreted. Thus, pre-administration of the GSH-depleting agent, diethyl maleate, increases the susceptibility to chloroform nephrotoxicity (KLUWE and HOOK 1981). Ischemic renal injury induces a reduction in ATP concentration due to mitochondrial disturbances as well as APAP (ARNOLD et al 1987; MOURELLE et al. 1991; WILSON et al. 1984). In this study, fructose-pretreatment did not alter gentamicin-, chloroform- and 45 min-ischemia! reperfusion-induced renal lesions. It is also reported that fructose infusion did not affect normal renal functions or recovery from a reversible ischemic insult (SHAPIRO et al. 1987; SHAPIRO et al. 1989). Therefore there was no evidence that alteration of renal intrinsic susceptibility to renal lesions was a major factor in the enhancement of APAP nephrotoxicity in the fructose-pretreated rats. However, since indirect assessment is limited, further detailed studies are required to exclude the alteration of renal intrinsic susceptibility as a contributing factor with respect to enhanced nephrotoxicity in the fructose-pretreated rats. We conclude from these results that an enhancement of APAP nephrotoxicity observed in the fructose-pretreated SD rats is attributable to at least the following two factors: 1) an incrase in renal APAP concentration at the early phase (1S and 30 min after APAP dosing) and 2) bioactivation of APAP via two metabolic pathways: cytochrome P4S0-mediated oxidation of APAP to NAPQI and cytochrome P4S0-independent deacetylation of APAP to PAP which is not responsible for nephrotoxicity in normal SD rats.
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