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Effects of fructose-induced hypertriglyceridemia on hepatorenal toxicity of acetaminophen in rats: Role of pharmacokinetics and metabolism of acetaminophen
Exp Toxic Patho11997; 49: 207-215 Gustav Fischer Verlag
lSafety Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd. Tokyo 2Department of Veterinary Pathology, Faculty of Agriculture, The University of Tokyo, Japan
Effects of fructose-induced hypertriglyceridemia on hepatorenal toxicity of acetaminophen in rats: Role of pharmacokinetics and metabolism of acetaminophen KATSUHIKO ISHIDA l, MASASHI SAKAZUME l, MASAMI W ATANABE l, NAMI HIRAIl, HISASHI IKEGAMI l, TOSHIHARU SAKAI l, and KUNIO D0l2 With 4 figures and 2 tables Received: November 18, 1995; Accepted: December 22, 1995
Address for correspondence: KATSUHIKO ISHIDA, Safety Research Laboratories, Yamanouchi Pharmaceutical CO., LTD., 1-1-8, Azusawa, Itabashi-ku, Tokyo 174, Japan.
Key words: Hypertriglyceridemia, fructose-induced; Fructose-induced hypertriglyceridemia; Hepatorena1 toxicity, acetaminophen; Acetaminophen, toxicity; Pharmacokinetics; Renal toxicity, acetaminophen.
Summary Fructose-induced hypertriglyceridemic rats become resistant to hepatotoxicity and susceptible to nephrotoxicity of acetaminophen (APAP), as compared with normal ones. The present study was designed to test the hypothesis that alterations in the distribution of APAP and in the intrinsic susceptibility to toxicants are responsible for the alteration in hepatorenal toxicity of APAP in fructose-induced hypertriglyceridemic rats. Following APAP-administration (750 mg/kg, ip), fructose-pretreated rats (25 % fructose in drinking water for 5 weeks) showed nephrotoxicity of APAP more promptly and more severely than normal ones. Renal APAP-concentrations at the early phase (15 and 30 min. after APAP-administration) were significantly greater in fructose-pretreated rats than those in normal ones. Plasma and hepatic APAP concentrations in fructose-pretreated rats were greater than those in normal ones only at the later phase (plasma; 6 hr, liver; 6 and 12 hr after APAPadministration). There were no significant differences in the APAP-induced depletion of hepatic and renal glutathione and in the basal hepatic and renal cytochrome P-450 contents between these rats. Fructose-pretreated rats were also more susceptible to p-aminophenol (PAP), a nephrotoxic metabolite of APAP, than normal rats. Therefore, enhanced susceptibility to APAP-nephrotoxicity in fructose-pretreated rats may be due, at least in part, to increased renal APAP concentration and increased intrinsic susceptibility to the metabolic nephrotoxic ant.
Introduction 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). Recently we reported that fructoseinduced hypertriglyceridemic rats became resistant to APAP-hepatotoxicity but more susceptible to APAPnephrotoxicity (ISHIDA et al. 1995). Thereafter we also clarified that the modification of APAP-toxicity in the hypertriglyceridemic rats connected with enhancement of fructose metabolism and with overproduction of triglyceride in the liver and kidney (ISHIDA et al. 1997). The mechanism of modification of APAP-toxicity in the hypertriglyceridemic rats is still unclear. However, the cause of alteration in hepatorenal toxicity of APAP in the fructoseinduced hypertriglyceridemic rats is expected as follows: 1) alteration in the distribution of APAP, 2) alteration in the metabolism, conjugation or excretion of APAP, and 3) alteration in the intrinsic susceptibility to toxic metabolites. In the present study, first, plasma, hepatic and renal APAP concentrations, hepatic and renal glutathione contents and hepatorenal1esions were evaluated after APAPadministration. Second, the susceptibility to p-aminophenol, a nephrotoxic metabolite of APAP (NEWTON et al. 1985), was compared between fructose-pretreated and normal rats.
Material and methods Animals: Seven-week-old male Sprague-Dawley (SD) rats weighing 240-270 g were obtained from Charles River Japan Inc. (Kanagawa). They were placed in a hanging Exp Toxic Pathol 49 (1997) 3--4
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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 use. Treatment: The rats were divided into 2 groups and were given tap water (non-pretreated group) or 25 % fructose (Dai-ichikogyo Seiyaku Co. Ltd., Tokyo) in their drinking water (fructose-pretreated group) ad libitum for 5 weeks, respectively. - Time-course study: After completion of the 5-weekpretreatment, the rats received a single ip dose (750 mg/kg) of APAP (Wako Pure Chemical Industries Inc., Osaka) as a warmed (40°C) suspension (35 mg/ml) in 1 % carboxymethyl-cellulose (CMC) solution. At 15 and 30 min. and 1,2, 6, 12, 24, 48, 72, 120 and 168 hrs after APAP-administration, four animals in each group (non-pretreated and fructose-pretreated) were killed by exsanguination from the abdominal aorta under ether anesthesia. As APAP-uninjected controls, four animals in each group were sacrificed without APAP-administration. Plasma samples were stored at -20°C until used. The liver and kidney were removed. A portion of each tissue was stored at -80 °C until measurement, and the rests were fixed in 10 % neutral buffered formalin. - Dose-dependent study: Rats received APAP (0.750 and 900 mg/kg ip, n = 10 for each dosage group) as a warmed (40°C) suspension (35 mg/ml) in CMC. In a separate series of experiments, rats received p-aminophenol hydrochloride (PAP, Tokyo Chemical Industry Co. Ltd., Tokyo; 0, 75 and 150 mg/kg ip, n = 5 for each dosage group) dissolved in saline (100 mg/ml). At 24 hr after administration of APAP or PAP, all rats were killed by exsanguination from the abdominal aorta under ether anesthesia. The liver and kidney were weighed and then fixed in 10 % neutral buffered formalin. Histopathology: The formalin-fixed liver and kidney were embedded in paraffin, sectioned at 5 11m, and stained with hematoxylin-eosin (HE) and by periodic acid-Schiff method (PAS) for microscopic examinations. The severity of hepatorenallesions were assessed by assigning a numerical score ranging from 1 (normal) to 5 (severe) as previously reported (ISHIDA et al. 1995). Measurement of plasma, hepatic and renal APAP concentrations: APAP concentrations in plasma, liver and renal cortex were measured by HPLC according to the method of COLIN et al. (1987) with some modifications. Briefly, liver and renal cortex were rinsed in saline, weighed, and homogenized in ice-cold 1.15 % potassium chloride (250 mg/ml). Aliquots of 100 III of either plasma or tissue homogenates were extracted using 5 ml of acetonitrile: isopropanol (50: 50, v/v) with sulfamerazine (40 Ilg/ml) added as an internal standard. Following shaking (30 sec) and centrifugation (500 g for 2 min.), a 600 III aliquot of the top layer was evaporated at 40°C under vacuum. Each residue obtained was dissolved in 300 III of distilled water and 10 III were then injected into the reversed-phase column at ambient temperature. Instrumentation consisted of a model L6000 pump, a model L-4200 detector, model 655A-40 auto sample injector, a model D-2000 integrator (Hitachi Co. Ltd., Tokyo) and a 150 x 4.6 mm, TSK gelS /Jm pKb-lOO deactivated reverse-phase column (To soh Co. Ltd., Tokyo). The mobile phase consisted of a mixture of distilled wateracetonitrile (86: 14). The flow-rate was 0.8 mllmin. and the 208
Exp Toxic Pathol 49 (1997) 3-4
eluate was monitored at 245 nm. Standard curves were prepared using spiked plasma and tissue homogenates from untreated rats. Measurement of hepatic and renal glutathione (GSH): For GSH determination, 50 mg of liver and renal cortex were homogenized in as ml volume of 0.6 N HCI04 - I mM EDTA and centrifuged (500 g for 10 min). The resultant supernatant was used for determination of hepatic and renal GSH contents by the enzymatic cycling method of TIETZE (1969). Measurement of hepatic and renal cytochrome P-450: Frozen liver and the renal cortex in APAP-uninjected rats in each group were homogenized in ice-cold 1.15 % potassium chloride solution. The homogenates were centrifuged at 9,000 g for 20 min. in a refrigerated centrifuge and the obtained suspensions were centrifuged at 105,000 g for 1 hr in an ultracentrifuge to obtain the microsomal pellets. Each microsomal pellet was stored at -80°C for cytochrome P450 and protein determination. Cytochrome P-450 was determined spectrophotometric ally according to the method of JOHANNESEN and DEPIERRE (1978) and protein by the method of LOWRY et al. (1951), respectively. Statistical analysis: All data are expressed as mean ± standard error (SE). Statistical analysis was done using Student's t-test.
Results Time-course study Histopathological findings: In the kidney from APAP-uninjected controls, there was no difference between non-pretreated and fructose-pretreated rats (fig. la and b). At 2 hrs after APAP-administration, non-pretreated rats showed no lesion, while fructose-pretreated ones showed slight dilatation of proximal straight tubules in the medullary ray (fig. lc and d). At 6 hrs, non-pretreated rats also showed slight dilatation of proximal straight tubules, and fructose-pretreated ones showed marked dilatation of the tubules with degeneration of some epithelial cells (fig. Ie and f). At 12-48 hrs, non-pretreated rats showed only single cell necrosis in the proximal straight tubule epithelia, whereas fructose-pretreated ones showed prominent lesions characterized by prominent epithelial cell necrosis in the proximal straight tubules in the medullary ray and in the outer stripe of the outer medulla (fig. Ig and h). From 72 hrs, non-pretreated rats showed no lesion (fig. Ii). In contrast, fructose-pretreated rats showed tubular damage with regenerative response until 168 hrs (fig. 1j). Most of the rats in both groups showed no hepatic lesion. Plasma, hepatic and renal APAP concentrations: Plasma, hepatic and renal APAP concentrations are shown in fig. 2. In plasma and hepatic APAP concentrations during the first 2 hrs after APAP-administration, there was no significant difference between non-pretreated and fructose-pretreated rats. However, plasma and hepatic APAP concentrations were significantly higher in fructose-pretreated rats than those in non-pretreated ones at
~ cld
Fig. I. Renal histopathology. PAS staining. X 160. a: Non-pretreated and APAP-uninjected rat (control). b: Fructose-pretreated and APAP-uninjected rat. No lesion. c: Nonpretreated rat at 2 hrs after APAP-administration. No lesion. d: Fructose-pretreated rat at 2 hrs after APAP-administration. Slight dilatation of proximal straight tubules in the medullary ray. Exp Toxic Pathol49 (1997) 3-4
209
~ .grh e: Non-pretreated rat at 6 hrs after APAP-administration. Slight dilatation of proximal straight tubules in the medullary ray. f: Fructose-pretreated rat at 6 hrs after APAP-administration. Marked dilatation of proximal straight tubules in medullary ray with a few degenerated and desquamated cells. g: Non-pretreated rat at 24 hrs after APAP-administration. Single cell necrosis of epithelial cells in proximal straight tubules. h: Fructose-pretreated rat at 24 hrs after APAP-administration. Extensive necrosis of epithelial cells in proximal straight tubules. 210
Exp Toxic Pathol49 (1997) 3-4
J i: Non-pretreated rat at 120 hrs after APAP-administration. No lesion. j: Fructose-pretreated rat at 120 hrs after APAP-administration. Regeneration of epithelial cells with basophilic change and papillary cluster in proximal straight tubules.
the later phase (plasma; 6 hrs, hepatic; 6 and 12 hrs). In contrast, renal APAP concentrations in fructose-pretreated rats were significantly higher than those in non-pretreated ones at the early phase (15 and 30 min. after APAP-administration). Although there was no significant difference in renal APAP concentrations from I hr on, those in fructose-pretreated rats tended to be higher at 6 and 12 hrs, as compared with those in non-pretreated ones. Hepatic and renal glutathione (GSH) concentration: In hepatic GSH concentrations, there was no significant difference between non-pretreated and fructosepretreated rats at any time points, and they decreased slowly up to 6 hrs after APAP-administration (fig. 3). Renal GSH concentrations in fructose-pretreated and APAP-uninjected rats were approximately 40 % lower than those in non-pretreated ones. However, in renal GSH concentrations during the first 12 hrs after APAP-administration, there was no significant difference between non-pretreated and fructose-pretreated rats. They decreased rapidly up to 1 hr after APAP-administration and then returned to the normal level by 12 hrs. After that, at 24 hrs, they became higher than the normal level, and the values in fructose-pretreated rats were significantly higher than those in non-pretreated ones.
Hepatic and renal cytochrome P-450 contents: As shown in table 1, there was no significant difference between non-pretreated and fructose-pretreated rats.
Dose-dependent study of AP AP and PAP Both APAP- and PAP-nephrotoxicities indicated by an increase in kidney weight and a severity of renal lesions were more prominent in fructose-pretreated rats than those in non-pretreated ones, and they occurred in a dosedependent manner (fig. 4 and table 2).
Discussion Previous studies reported that fructose-induced hypertriglyceridemic rats became resistant to APAP-hepatotoxicity and susceptible to APAP-nephrotoxicity as compared with normal ones. In the present study, fructosepretreated rats developed tubular dilatation from the early phase, and they showed more severe renal lesions as compared with non-pretreated ones. In fructose-pretreated rats, renal APAP-concentrations at the early phase (15 and 30 min. after APAP-administration) were signifiExp Toxic Pathol49 (1997) 3-4
211
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Hepatic Glutathione
., 2000
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Table 1. Effect of fructose-treatment on hepatic and renal cytochrome P-450 in SD rats a). non-treatment Liver (nmol/mg protein) 0.414 ± 0.031 Kidney (nmol/mg protein) 0.089 ± 0.016
fructosetreatment 0.344 ± 0.031 0.108 ± 0.015
a)Rats were given tap water or 25 % fructose in drinking water ad libitum for 5 weeks. Values are means ± SE. N =6.
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Fig. 4. Effects of APAP or PAP on kidney weight in nonpretreated and fructose-pretreated rats. a: significantly different from pretreatment-matched and vehicle-dosed rats (p < 0.05). b: significantly different from non-pretreated and dose-matched rats (p < 0.05). D: non-pretreated rats, . : fructose-pretreated rats.
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Exp Toxic Patho149 (1997) 3-4
cantly higher than those in non-pretreated ones. The results may indicate that an increased renal APAP-concentration at the early phase is responsible for an enhancement of susceptibility to APAP-nephrotoxicity in fructose-pretreated rats. Since APAP was primarily metabolized in the liver (JOLLOW et a1. 1974), a decrease in the capacity of hepatic metabolism of APAP may be a factor of an increase in APAP concentration in the kidney. In mice, APAP bioactivation occurs to greater extent in the liver than in other tissues, making liver the critical organ for APAP toxicity. If hepatic metabolism is inhibited, then renal and pulmonary toxicity becomes critical (JEFFERY 1991). The liver is also the major site of fructose metabolism and triglyceride production (KAZUMI et a1. 1986), In the previous studies, fructose-, sucrose- and glycerol-pretreatments which produce enhanced metabolism and triglyceride overproduction in the liver showed a clear modification
Table 2. Effects of fructose-pretreatment on renal lesions induced by acetaminophen or p-aminophenol in SD rats. Acetaminophen dose (mg/kg)
non-pretreatment
o
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900
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fructose-pretreatment 150
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a)
of APAP-hepatorenal toxicity, while glucose-, olive oiland Triton WR-1339-pretreatments which do not produce enhanced fructose metabolism and triglyceride overproduction in the liver had no effect on the time course mode of APAP-toxicity development (ISHIDA et al. 1997). Therefore, an increased renal APAP-concentration may be due to a decreased capacity of hepatic APAP-metabolism induced by enhanced fructose metabolism and by overproduction of triglyceride in the liver. On the other hand, the kidney as well as the liver are important sites of fructose disposal (BJORKMAN and FELIG 1982). It was reported that APAP exerts a direct action of the kidney, without influence of hepatic damage or liverderived 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). Thus, the possibility remains that an increased renal APAP concentration in the fructose-induced hypertriglyceridemic rats may occur without influence of alteration in hepatic metabolism of APAP. Plasma and hepatic AP AP concentrations were higher in fructose-pretreated rats than those in non-pretreated ones from 6 hrs after APAP-administration. However, fructose-pretreated rats also tended to show somewhat greater renal APAP-concentrations at the later phase. At that time, severe renal lesions had already been observed in fructose-pretreated rats. Middle-aged rats, which are more susceptible to APAP-nephrotoxicity than younger adults, also showed an increase in APAP-concentration in
plasma and tissues during 2-5 hrs after APAP-administration (T ARLOFF et al. 1989). These effects are thought to be brought about by a reduction in the systemic clearance of APAP due to renal failure (T ARLOFF et al. 1989). Therefore, the increased AP AP concentrations in the later phase observed in the present study may also be brought about in the same way. Renal GSH concentrations were lower in fructose-pretreated and APAP-uninjected rats than those in non-pretreated ones. On the other hand, it is reported that APAPadministration induces a significant reduction in renal GSH (McMURTRY et al. 1978; NEWTON et al. 1982). Thus, it was expected that fructose-pretreated rats, which showed a higher concentration of renal APAP in the early phase, showed a more marked reduction in renal GSH. Really, fructose-pretreated rats showed the same GSH-reduction as non-pretreated ones did, though this significance is not clear. On the other hand, at 24 hrs after APAP-administration, renal GSH concentrations were higher than the controllevel. It is reported that renal or hepatic GSH also rose to the level approximately double as high as the control value in 24 hrs following PAP or diethyl maleate, and thereafter returned to the control level (GARTLAND et al. 1990; WIRTH and THORGEIRSSON 1978). This is explained to be physiological responses to acute depletion of GSH and correlates well with dose or severity of lesions in the kidney (GARTLAND et al. 1990). In the present study, renal aSH contents at 24 hrs APAP-administration were also Exp Toxic Pathol49 (1997) 3-4
213
higher and renal lesions were more marked in fructosepretreated rats than those in non-pretreated rats. Fructose-pretreated rats showed susceptibility to PAPnephrotoxicity as well as APAP-nephrotoxicity, as compared with normal ones. Therefore, fructose-pretreated rats may show severe renal lesions even when PAP is formed at an extent equivalent to that formed in normal rats. Obese overfed SD rats are more sensitive to APAPhepatotoxicity and nephrotoxicity than normal SD rats even at an equivalent initial drug exposure (peak plasma concentration) (CORCORAN and WONG et al. 1987). Further more, when equivalent blood APAP concentrations are produced in 3- and 12-month-old rats (by administering 500 mg/kg iv to 12-month-old and 750 mg/kg iv to 3month-old rats, respectively), nephrotoxicity is present only in older animals (TARLOFF et al. 1989). This is considered to be due to a difference in the intrinsic susceptibility to toxicity of metabolites of APAP. Thus, enhanced susceptibility to APAP-nephrotoxicity in fructose-pretreated rats may be due, in part, to an enhancement of intrinsic susceptibility to toxicity of metabolites of APAP. Although little hepatic lesions were observed in the present study, fructose-pretreated rats were confirmed to show greater resistance to APAP-hepatotoxicity (ISHIDA et al. 1995). APAP-hepatotoxicity is caused by the formation ofNAPQI, which is formed by cytochrome P-450 (DAHLIN et al. 1984). NAPQI binds to GSH and then is excreted (SlEGERS et al. 1983). However, there were no significant differences in hepatic APAP concentration at the early phase, hepatic GSH and hepatic cytochrome P-450 values between fructose-pretreated and non-pretreated rats. Therefore, it is unlikely that the reduction in concentrations of APAP or its hepatotoxic metabolites in the liver contributes to an increase in resistance to APAPhepatotoxicity in fructose-pretreated rats. It was previously shown that fructose was particularly efficient in preventing hypoxic and APAP-induced damage of hepatocytes, and that its protective effect was related to its ability to provide glycolytic ATP (ANN UNDI and DE GROOT 1989; MOURELLE et a!. 1991). Although anaerobic ATP production may provide only approximately 20 % of that required by aerobic tissue, ATP produced glycolytically may be primarily used for maintenance of membrane functions and Ca2+uptake by endoplasmic reticulum which facilitate the survival of the cells (JONES and MASON 1978; MCDONALD et al. 1971; WEISS and LAMP 1979). It has been demonstrated that other carbohydrates such as glucose, xylitol and mannitol do not have the same ability to increase ATP levels as fructose does (SOLS et al. 1964). Thus, even though equivalent concentrations of APAP or metabolites are distributed in the liver of both non-pretreated and fructose-pretreated rats, it is possible that the latter show less severe hepatic lesion than the former ones.
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lSafety Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd. Tokyo 2Department of Veterinary Pathology, Faculty of Agriculture, The University of Tokyo, Japan
Effects of fructose-induced hypertriglyceridemia on hepatorenal toxicity of acetaminophen in rats: Role of pharmacokinetics and metabolism of acetaminophen KATSUHIKO ISHIDA l, MASASHI SAKAZUME l, MASAMI W ATANABE l, NAMI HIRAIl, HISASHI IKEGAMI l, TOSHIHARU SAKAI l, and KUNIO D0l2 With 4 figures and 2 tables Received: November 18, 1995; Accepted: December 22, 1995
Address for correspondence: KATSUHIKO ISHIDA, Safety Research Laboratories, Yamanouchi Pharmaceutical CO., LTD., 1-1-8, Azusawa, Itabashi-ku, Tokyo 174, Japan.
Key words: Hypertriglyceridemia, fructose-induced; Fructose-induced hypertriglyceridemia; Hepatorena1 toxicity, acetaminophen; Acetaminophen, toxicity; Pharmacokinetics; Renal toxicity, acetaminophen.
Summary Fructose-induced hypertriglyceridemic rats become resistant to hepatotoxicity and susceptible to nephrotoxicity of acetaminophen (APAP), as compared with normal ones. The present study was designed to test the hypothesis that alterations in the distribution of APAP and in the intrinsic susceptibility to toxicants are responsible for the alteration in hepatorenal toxicity of APAP in fructose-induced hypertriglyceridemic rats. Following APAP-administration (750 mg/kg, ip), fructose-pretreated rats (25 % fructose in drinking water for 5 weeks) showed nephrotoxicity of APAP more promptly and more severely than normal ones. Renal APAP-concentrations at the early phase (15 and 30 min. after APAP-administration) were significantly greater in fructose-pretreated rats than those in normal ones. Plasma and hepatic APAP concentrations in fructose-pretreated rats were greater than those in normal ones only at the later phase (plasma; 6 hr, liver; 6 and 12 hr after APAPadministration). There were no significant differences in the APAP-induced depletion of hepatic and renal glutathione and in the basal hepatic and renal cytochrome P-450 contents between these rats. Fructose-pretreated rats were also more susceptible to p-aminophenol (PAP), a nephrotoxic metabolite of APAP, than normal rats. Therefore, enhanced susceptibility to APAP-nephrotoxicity in fructose-pretreated rats may be due, at least in part, to increased renal APAP concentration and increased intrinsic susceptibility to the metabolic nephrotoxic ant.
Introduction 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). Recently we reported that fructoseinduced hypertriglyceridemic rats became resistant to APAP-hepatotoxicity but more susceptible to APAPnephrotoxicity (ISHIDA et al. 1995). Thereafter we also clarified that the modification of APAP-toxicity in the hypertriglyceridemic rats connected with enhancement of fructose metabolism and with overproduction of triglyceride in the liver and kidney (ISHIDA et al. 1997). The mechanism of modification of APAP-toxicity in the hypertriglyceridemic rats is still unclear. However, the cause of alteration in hepatorenal toxicity of APAP in the fructoseinduced hypertriglyceridemic rats is expected as follows: 1) alteration in the distribution of APAP, 2) alteration in the metabolism, conjugation or excretion of APAP, and 3) alteration in the intrinsic susceptibility to toxic metabolites. In the present study, first, plasma, hepatic and renal APAP concentrations, hepatic and renal glutathione contents and hepatorenal1esions were evaluated after APAPadministration. Second, the susceptibility to p-aminophenol, a nephrotoxic metabolite of APAP (NEWTON et al. 1985), was compared between fructose-pretreated and normal rats.
Material and methods Animals: Seven-week-old male Sprague-Dawley (SD) rats weighing 240-270 g were obtained from Charles River Japan Inc. (Kanagawa). They were placed in a hanging Exp Toxic Pathol 49 (1997) 3--4
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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 use. Treatment: The rats were divided into 2 groups and were given tap water (non-pretreated group) or 25 % fructose (Dai-ichikogyo Seiyaku Co. Ltd., Tokyo) in their drinking water (fructose-pretreated group) ad libitum for 5 weeks, respectively. - Time-course study: After completion of the 5-weekpretreatment, the rats received a single ip dose (750 mg/kg) of APAP (Wako Pure Chemical Industries Inc., Osaka) as a warmed (40°C) suspension (35 mg/ml) in 1 % carboxymethyl-cellulose (CMC) solution. At 15 and 30 min. and 1,2, 6, 12, 24, 48, 72, 120 and 168 hrs after APAP-administration, four animals in each group (non-pretreated and fructose-pretreated) were killed by exsanguination from the abdominal aorta under ether anesthesia. As APAP-uninjected controls, four animals in each group were sacrificed without APAP-administration. Plasma samples were stored at -20°C until used. The liver and kidney were removed. A portion of each tissue was stored at -80 °C until measurement, and the rests were fixed in 10 % neutral buffered formalin. - Dose-dependent study: Rats received APAP (0.750 and 900 mg/kg ip, n = 10 for each dosage group) as a warmed (40°C) suspension (35 mg/ml) in CMC. In a separate series of experiments, rats received p-aminophenol hydrochloride (PAP, Tokyo Chemical Industry Co. Ltd., Tokyo; 0, 75 and 150 mg/kg ip, n = 5 for each dosage group) dissolved in saline (100 mg/ml). At 24 hr after administration of APAP or PAP, all rats were killed by exsanguination from the abdominal aorta under ether anesthesia. The liver and kidney were weighed and then fixed in 10 % neutral buffered formalin. Histopathology: The formalin-fixed liver and kidney were embedded in paraffin, sectioned at 5 11m, and stained with hematoxylin-eosin (HE) and by periodic acid-Schiff method (PAS) for microscopic examinations. The severity of hepatorenallesions were assessed by assigning a numerical score ranging from 1 (normal) to 5 (severe) as previously reported (ISHIDA et al. 1995). Measurement of plasma, hepatic and renal APAP concentrations: APAP concentrations in plasma, liver and renal cortex were measured by HPLC according to the method of COLIN et al. (1987) with some modifications. Briefly, liver and renal cortex were rinsed in saline, weighed, and homogenized in ice-cold 1.15 % potassium chloride (250 mg/ml). Aliquots of 100 III of either plasma or tissue homogenates were extracted using 5 ml of acetonitrile: isopropanol (50: 50, v/v) with sulfamerazine (40 Ilg/ml) added as an internal standard. Following shaking (30 sec) and centrifugation (500 g for 2 min.), a 600 III aliquot of the top layer was evaporated at 40°C under vacuum. Each residue obtained was dissolved in 300 III of distilled water and 10 III were then injected into the reversed-phase column at ambient temperature. Instrumentation consisted of a model L6000 pump, a model L-4200 detector, model 655A-40 auto sample injector, a model D-2000 integrator (Hitachi Co. Ltd., Tokyo) and a 150 x 4.6 mm, TSK gelS /Jm pKb-lOO deactivated reverse-phase column (To soh Co. Ltd., Tokyo). The mobile phase consisted of a mixture of distilled wateracetonitrile (86: 14). The flow-rate was 0.8 mllmin. and the 208
Exp Toxic Pathol 49 (1997) 3-4
eluate was monitored at 245 nm. Standard curves were prepared using spiked plasma and tissue homogenates from untreated rats. Measurement of hepatic and renal glutathione (GSH): For GSH determination, 50 mg of liver and renal cortex were homogenized in as ml volume of 0.6 N HCI04 - I mM EDTA and centrifuged (500 g for 10 min). The resultant supernatant was used for determination of hepatic and renal GSH contents by the enzymatic cycling method of TIETZE (1969). Measurement of hepatic and renal cytochrome P-450: Frozen liver and the renal cortex in APAP-uninjected rats in each group were homogenized in ice-cold 1.15 % potassium chloride solution. The homogenates were centrifuged at 9,000 g for 20 min. in a refrigerated centrifuge and the obtained suspensions were centrifuged at 105,000 g for 1 hr in an ultracentrifuge to obtain the microsomal pellets. Each microsomal pellet was stored at -80°C for cytochrome P450 and protein determination. Cytochrome P-450 was determined spectrophotometric ally according to the method of JOHANNESEN and DEPIERRE (1978) and protein by the method of LOWRY et al. (1951), respectively. Statistical analysis: All data are expressed as mean ± standard error (SE). Statistical analysis was done using Student's t-test.
Results Time-course study Histopathological findings: In the kidney from APAP-uninjected controls, there was no difference between non-pretreated and fructose-pretreated rats (fig. la and b). At 2 hrs after APAP-administration, non-pretreated rats showed no lesion, while fructose-pretreated ones showed slight dilatation of proximal straight tubules in the medullary ray (fig. lc and d). At 6 hrs, non-pretreated rats also showed slight dilatation of proximal straight tubules, and fructose-pretreated ones showed marked dilatation of the tubules with degeneration of some epithelial cells (fig. Ie and f). At 12-48 hrs, non-pretreated rats showed only single cell necrosis in the proximal straight tubule epithelia, whereas fructose-pretreated ones showed prominent lesions characterized by prominent epithelial cell necrosis in the proximal straight tubules in the medullary ray and in the outer stripe of the outer medulla (fig. Ig and h). From 72 hrs, non-pretreated rats showed no lesion (fig. Ii). In contrast, fructose-pretreated rats showed tubular damage with regenerative response until 168 hrs (fig. 1j). Most of the rats in both groups showed no hepatic lesion. Plasma, hepatic and renal APAP concentrations: Plasma, hepatic and renal APAP concentrations are shown in fig. 2. In plasma and hepatic APAP concentrations during the first 2 hrs after APAP-administration, there was no significant difference between non-pretreated and fructose-pretreated rats. However, plasma and hepatic APAP concentrations were significantly higher in fructose-pretreated rats than those in non-pretreated ones at
~ cld
Fig. I. Renal histopathology. PAS staining. X 160. a: Non-pretreated and APAP-uninjected rat (control). b: Fructose-pretreated and APAP-uninjected rat. No lesion. c: Nonpretreated rat at 2 hrs after APAP-administration. No lesion. d: Fructose-pretreated rat at 2 hrs after APAP-administration. Slight dilatation of proximal straight tubules in the medullary ray. Exp Toxic Pathol49 (1997) 3-4
209
~ .grh e: Non-pretreated rat at 6 hrs after APAP-administration. Slight dilatation of proximal straight tubules in the medullary ray. f: Fructose-pretreated rat at 6 hrs after APAP-administration. Marked dilatation of proximal straight tubules in medullary ray with a few degenerated and desquamated cells. g: Non-pretreated rat at 24 hrs after APAP-administration. Single cell necrosis of epithelial cells in proximal straight tubules. h: Fructose-pretreated rat at 24 hrs after APAP-administration. Extensive necrosis of epithelial cells in proximal straight tubules. 210
Exp Toxic Pathol49 (1997) 3-4
J i: Non-pretreated rat at 120 hrs after APAP-administration. No lesion. j: Fructose-pretreated rat at 120 hrs after APAP-administration. Regeneration of epithelial cells with basophilic change and papillary cluster in proximal straight tubules.
the later phase (plasma; 6 hrs, hepatic; 6 and 12 hrs). In contrast, renal APAP concentrations in fructose-pretreated rats were significantly higher than those in non-pretreated ones at the early phase (15 and 30 min. after APAP-administration). Although there was no significant difference in renal APAP concentrations from I hr on, those in fructose-pretreated rats tended to be higher at 6 and 12 hrs, as compared with those in non-pretreated ones. Hepatic and renal glutathione (GSH) concentration: In hepatic GSH concentrations, there was no significant difference between non-pretreated and fructosepretreated rats at any time points, and they decreased slowly up to 6 hrs after APAP-administration (fig. 3). Renal GSH concentrations in fructose-pretreated and APAP-uninjected rats were approximately 40 % lower than those in non-pretreated ones. However, in renal GSH concentrations during the first 12 hrs after APAP-administration, there was no significant difference between non-pretreated and fructose-pretreated rats. They decreased rapidly up to 1 hr after APAP-administration and then returned to the normal level by 12 hrs. After that, at 24 hrs, they became higher than the normal level, and the values in fructose-pretreated rats were significantly higher than those in non-pretreated ones.
Hepatic and renal cytochrome P-450 contents: As shown in table 1, there was no significant difference between non-pretreated and fructose-pretreated rats.
Dose-dependent study of AP AP and PAP Both APAP- and PAP-nephrotoxicities indicated by an increase in kidney weight and a severity of renal lesions were more prominent in fructose-pretreated rats than those in non-pretreated ones, and they occurred in a dosedependent manner (fig. 4 and table 2).
Discussion Previous studies reported that fructose-induced hypertriglyceridemic rats became resistant to APAP-hepatotoxicity and susceptible to APAP-nephrotoxicity as compared with normal ones. In the present study, fructosepretreated rats developed tubular dilatation from the early phase, and they showed more severe renal lesions as compared with non-pretreated ones. In fructose-pretreated rats, renal APAP-concentrations at the early phase (15 and 30 min. after APAP-administration) were signifiExp Toxic Pathol49 (1997) 3-4
211
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Fig. 2. Time course of acetaminophen concentrations in plasma, liver and kidney in non-pretreated and fructose-pretreated rats. Results are expressed as means ± SE, N =4. *; significantly different from non-pretreated rats (p < 0.05). 2500 r---:-:---::-:---=-:---;--:-;-:---,
Hepatic Glutathione
., 2000
75
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150
Table 1. Effect of fructose-treatment on hepatic and renal cytochrome P-450 in SD rats a). non-treatment Liver (nmol/mg protein) 0.414 ± 0.031 Kidney (nmol/mg protein) 0.089 ± 0.016
fructosetreatment 0.344 ± 0.031 0.108 ± 0.015
a)Rats were given tap water or 25 % fructose in drinking water ad libitum for 5 weeks. Values are means ± SE. N =6.
::J
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Fig. 4. Effects of APAP or PAP on kidney weight in nonpretreated and fructose-pretreated rats. a: significantly different from pretreatment-matched and vehicle-dosed rats (p < 0.05). b: significantly different from non-pretreated and dose-matched rats (p < 0.05). D: non-pretreated rats, . : fructose-pretreated rats.
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Fig. 3. Time course of hepatic and renal glutathione concentrations in non-pretreated and fructose-pretreated rats. Results are expressed as means ± SE, N = 4. *; significantly different from non-pretreated rats (p < 0.05). 212
Exp Toxic Patho149 (1997) 3-4
cantly higher than those in non-pretreated ones. The results may indicate that an increased renal APAP-concentration at the early phase is responsible for an enhancement of susceptibility to APAP-nephrotoxicity in fructose-pretreated rats. Since APAP was primarily metabolized in the liver (JOLLOW et a1. 1974), a decrease in the capacity of hepatic metabolism of APAP may be a factor of an increase in APAP concentration in the kidney. In mice, APAP bioactivation occurs to greater extent in the liver than in other tissues, making liver the critical organ for APAP toxicity. If hepatic metabolism is inhibited, then renal and pulmonary toxicity becomes critical (JEFFERY 1991). The liver is also the major site of fructose metabolism and triglyceride production (KAZUMI et a1. 1986), In the previous studies, fructose-, sucrose- and glycerol-pretreatments which produce enhanced metabolism and triglyceride overproduction in the liver showed a clear modification
Table 2. Effects of fructose-pretreatment on renal lesions induced by acetaminophen or p-aminophenol in SD rats. Acetaminophen dose (mg/kg)
non-pretreatment
o
fructose-pretreatment
750
900
o
750
900
number of animals
10
10
10
10
10
7
score I score 2 score 3 score 4 score 5
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1 4 5 0 0
0 4 5 1 0
10 0 0 0 0
0 0 1 7 2
0 0 0 0 7
p- Aminophenol
non-pretreatment
dose (mg/kg)
0
75
fructose-pretreatment 150
0
75
150
------------------------------------------------------------------
number of animals
5
5
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Number of animals which were scored by the criteria given in Materials and Methods.
a)
of APAP-hepatorenal toxicity, while glucose-, olive oiland Triton WR-1339-pretreatments which do not produce enhanced fructose metabolism and triglyceride overproduction in the liver had no effect on the time course mode of APAP-toxicity development (ISHIDA et al. 1997). Therefore, an increased renal APAP-concentration may be due to a decreased capacity of hepatic APAP-metabolism induced by enhanced fructose metabolism and by overproduction of triglyceride in the liver. On the other hand, the kidney as well as the liver are important sites of fructose disposal (BJORKMAN and FELIG 1982). It was reported that APAP exerts a direct action of the kidney, without influence of hepatic damage or liverderived 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). Thus, the possibility remains that an increased renal APAP concentration in the fructose-induced hypertriglyceridemic rats may occur without influence of alteration in hepatic metabolism of APAP. Plasma and hepatic AP AP concentrations were higher in fructose-pretreated rats than those in non-pretreated ones from 6 hrs after APAP-administration. However, fructose-pretreated rats also tended to show somewhat greater renal APAP-concentrations at the later phase. At that time, severe renal lesions had already been observed in fructose-pretreated rats. Middle-aged rats, which are more susceptible to APAP-nephrotoxicity than younger adults, also showed an increase in APAP-concentration in
plasma and tissues during 2-5 hrs after APAP-administration (T ARLOFF et al. 1989). These effects are thought to be brought about by a reduction in the systemic clearance of APAP due to renal failure (T ARLOFF et al. 1989). Therefore, the increased AP AP concentrations in the later phase observed in the present study may also be brought about in the same way. Renal GSH concentrations were lower in fructose-pretreated and APAP-uninjected rats than those in non-pretreated ones. On the other hand, it is reported that APAPadministration induces a significant reduction in renal GSH (McMURTRY et al. 1978; NEWTON et al. 1982). Thus, it was expected that fructose-pretreated rats, which showed a higher concentration of renal APAP in the early phase, showed a more marked reduction in renal GSH. Really, fructose-pretreated rats showed the same GSH-reduction as non-pretreated ones did, though this significance is not clear. On the other hand, at 24 hrs after APAP-administration, renal GSH concentrations were higher than the controllevel. It is reported that renal or hepatic GSH also rose to the level approximately double as high as the control value in 24 hrs following PAP or diethyl maleate, and thereafter returned to the control level (GARTLAND et al. 1990; WIRTH and THORGEIRSSON 1978). This is explained to be physiological responses to acute depletion of GSH and correlates well with dose or severity of lesions in the kidney (GARTLAND et al. 1990). In the present study, renal aSH contents at 24 hrs APAP-administration were also Exp Toxic Pathol49 (1997) 3-4
213
higher and renal lesions were more marked in fructosepretreated rats than those in non-pretreated rats. Fructose-pretreated rats showed susceptibility to PAPnephrotoxicity as well as APAP-nephrotoxicity, as compared with normal ones. Therefore, fructose-pretreated rats may show severe renal lesions even when PAP is formed at an extent equivalent to that formed in normal rats. Obese overfed SD rats are more sensitive to APAPhepatotoxicity and nephrotoxicity than normal SD rats even at an equivalent initial drug exposure (peak plasma concentration) (CORCORAN and WONG et al. 1987). Further more, when equivalent blood APAP concentrations are produced in 3- and 12-month-old rats (by administering 500 mg/kg iv to 12-month-old and 750 mg/kg iv to 3month-old rats, respectively), nephrotoxicity is present only in older animals (TARLOFF et al. 1989). This is considered to be due to a difference in the intrinsic susceptibility to toxicity of metabolites of APAP. Thus, enhanced susceptibility to APAP-nephrotoxicity in fructose-pretreated rats may be due, in part, to an enhancement of intrinsic susceptibility to toxicity of metabolites of APAP. Although little hepatic lesions were observed in the present study, fructose-pretreated rats were confirmed to show greater resistance to APAP-hepatotoxicity (ISHIDA et al. 1995). APAP-hepatotoxicity is caused by the formation ofNAPQI, which is formed by cytochrome P-450 (DAHLIN et al. 1984). NAPQI binds to GSH and then is excreted (SlEGERS et al. 1983). However, there were no significant differences in hepatic APAP concentration at the early phase, hepatic GSH and hepatic cytochrome P-450 values between fructose-pretreated and non-pretreated rats. Therefore, it is unlikely that the reduction in concentrations of APAP or its hepatotoxic metabolites in the liver contributes to an increase in resistance to APAPhepatotoxicity in fructose-pretreated rats. It was previously shown that fructose was particularly efficient in preventing hypoxic and APAP-induced damage of hepatocytes, and that its protective effect was related to its ability to provide glycolytic ATP (ANN UNDI and DE GROOT 1989; MOURELLE et a!. 1991). Although anaerobic ATP production may provide only approximately 20 % of that required by aerobic tissue, ATP produced glycolytically may be primarily used for maintenance of membrane functions and Ca2+uptake by endoplasmic reticulum which facilitate the survival of the cells (JONES and MASON 1978; MCDONALD et al. 1971; WEISS and LAMP 1979). It has been demonstrated that other carbohydrates such as glucose, xylitol and mannitol do not have the same ability to increase ATP levels as fructose does (SOLS et al. 1964). Thus, even though equivalent concentrations of APAP or metabolites are distributed in the liver of both non-pretreated and fructose-pretreated rats, it is possible that the latter show less severe hepatic lesion than the former ones.
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