Effects of fructose-induced hypertriglyceridemia on hepatorenal toxicity of acetaminophen in rats

Exp Toxic Patho11995; 47: 509-516 Gustav Fischer Verlag Jena

l)Safety Research Laboratories, Yamanouchi Pharmaceutical CO., LTD., Tokyo, Japan Department of Veterinary Pathology, Faculty of Agriculture, The University of Tokyo, Tokyo, Japan

2)

Effects of fructose-induced hypertriglyceridemia on hepatorenal toxicity of acetaminophen in rats KATSUHIKO ISHIDA!), TAKANORI HANADAl), TOSHIHARU SAKAI l) and KUNIO D0I 2 ) With 4 figures and 4 tables Received: February 20, 1995; Revised: March 30, 1995; Accepted: April 12, 1995 Address for correspondence: KATSUHIKO ISHIDA, Safety Research Laboratories, Yamanouchi Pharmaceutical CO., LTD., 1-1-8, Azusawa, Itabashi-Ku, Tokyo 174, Japan. Kay words: Fructose-induced hypertriglyceridemia; Hypertriglyceridemia; Hepatorenal toxicity; Acetaminophen, toxicity; Hepatotoxicity; Nephrotoxicity.

Summary The mode of hepatorenal toxICIty of acetaminophen (AAP) was compared between fructose-induced hypertriglyceridemic and normal rats. The hypertriglyceridemic and normal rats received a single dose of AAP (0, 750 and 900 mg/kg ip) at week 5 of fructose-treatment. At 24 hrs after AAP-dosing, they were sacrificed and examined blood biochemically and histopathologically. Hepatotoxicity as indicated by an increase in plasma AL T and AST activities and centrilobular necrosis of hepatocytes was more severe in the normal rats than in the hypertriglyceridemic ones. In contrast, nephrotoxicity as indicated by an increase in plasma urea nitrogen content and necrosis of epithelial cells in the proximal straight tubules was more severe in the hypertriglyceridemic rats than in normal ones. Thus, in the fructose-induced hypertriglyceridemic rats, as compared with normal ones, hepatotoxicity of AAP became apparently less severe, whereas nephrotoxicity of AAP became significantly more severe.

Introduction Risk assessment of new Iy developing drugs for human is generally discussed based on the results of toxicological studies using healthy animals. On the other hand, it is easy to presume that the toxicity of drugs may be modified in the diseased animals because of modified drug metabolism, detoxification and pharmacokinetics. This is important because drugs are administered to the diseased human. However, little is known about the modification of drug toxicity in the diseased animal models. This study was undertaken to compare the mode ofhepatorenal toxicity of acetaminophen (AAP) between fructose-induced hypertriglyceridemic and normal rats.

Overdose of AAP is well known to induce necrosis of centrilobular hepatocytes (BLACK 1984; BoYD and BERECZKY 1966; DAVIDSON and EASTHAM 1966; HINSON et al. 1977; MITCHELL et al. 1973; PRESCOTT et al. 1971; PROUDFOOT and WRIGHT 1970) and of uriniferous tubule epithelial cells (BOYER and ROUPF 1971; KLEINMANN et al. 1980; COBDEN et al. 1982; DAVENPORT and FINN 1988; McMURTRY et al. 1978; NEWTON et al. 1985) both in human and experimental animals. In addition, it is said that fructose induces hypertriglyceridemia in rats through an overproduction of hepatic VLDL triglyceride and an impaired rate of removal ofVLDL triglyceride (IWATA et al. 1990; MAZUR et al. 1992; YOSHINO et al. 1992).

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 stainless steel wire bottomed cage in an animal room under controlled conditions (temperature: 23 ± 3 °C, humidity: 55 %, lighting: 13hr (8:00-21 :00), 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. Treatment: The rats were divided into 6 groups (Gl-G6) of 10 (fig. 1). Rats of GI-G3 and G4-G6 were given tap water or 25 % fructose (Dai-ichikogyo Seiyaku Co. Ltd., Tokyo) in their drinking water ad libitum for 5 weeks, respectively. Fructose and food consumptions were recorded every 3 days, and body weight every 7 days. At week 3 of fructose-treatment, plasma samples were separated from heparinized blood collected from each rat by retroorbital sinus puncture, and stored at -20°C until used for the examination of the effects of fructose on blood biochemical parameters. Exp Toxic Pathol 47 (1995) 6

509

Period on

test Group

I 24hrs

5Wks

I

1- 3 ~

t

~

t

4 - 6 Fig. 1. Experimental Protocol. Rats were given tap water (G I-G3) ':il or 25 % fructose (G4-G6) • in drinking water ad libitum for 5 weeks. At week 5, they were treated with vehicle (G I and G4) or AAP at a concentration of 750 (G2 and G5) and 900 (G3 and G6) mg/kg (... ). At 24 hr after dosing of AAP, They were sacrificed (i).

After completion of the 5 weeks treatment, rats of G2, G3, G5 and G6 were given a single ip dose (750 mg/kg for G2 and G5 , and 900 mg/kg for G3 and G6) of AAP (Wako Pure Chemical Industries Inc., Tokyo) as a warmed (40°C) suspension (35 mg/ml) in 1 % carboxymethyl-cellulose (CMC) solution. Rats of Gland G4 were given CMC alone in the same way. At 24 hr after AAP-administration, all rats were killed by exsanguination from the abdominal aorta under ether anesthesia, and plasma samples were stored at -20 °C until used. The liver and kidney were weighed and then fixed in 10 % neutral buffered formalin. Blood biochemistry: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (UN),

triglyceride, total cholesterol, glucose, total protein and albumin in plasma were measured using an automatic analyzer (Model Hitachi 736, Hitachi Co. Ltd., Tokyo). Urinalysis: At week 4 of fructose-treatment, rats were individually placed in stainless metabolism cages with free access to water (GI-G3) or 25 % fructose solution (G4-G6). Urine samples were collected overnight under the fasting condition. They were analyzed for pH, protein, glucose, ketones, urobilinogen and occult blood semiquantitatively (BM test, Yamanouchi Pharmaceutical Co. Ltd. , Tokyo) in order to examine the effect of fructose-treatment on urinary parameters. Histopathology: The formalin-fixed liver and kidney were embedded in paraffin, sectioned at 5 11m, and stained with hematoxylin-eosin (HE) or by periodic acid-Schiff method (PAS) for microscopic examinations. The severity of hepatic injury was graded and scored as follows; 1) no lesion, 2) hepatocyte vacuolation in a few centrilobular areas, 3) hepatocyte necrosis in a few centrilobular areas, 4) hepatocyte necrosis in almost all centrilobular areas, and 5) hepatocyte necrosis in both centrilobular and midzonal areas . The severity of renal injury was also graded and scored as follows; 1) no lesion, 2) single cell necrosis in the proximal straight tubules in the medullary ray, 3) necrosis of epithelial cells in the proximal straight tubules in the medullary ray, 4) necrosis of epithelial cells in some proximal straight tubules in both the medullary ray and the outer stripe of the outer medulla, and 5) necrosis of epithelial cells in almost all proximal straight tubules in both the medullary ray and the outer stripe of the outer medulla.

Table 1. Effect of fructose-treatment on body weight, fructose and food consumptions and plasma biochemistry in SD rats a).

Group • Body Weight (g) - week 0 - week 5 • Fructose consumption b) - (g/animal/day) • Food consumption b) - (g/animal/day) • Blood BiochemistryC) (mg/dl) - Triglyceride (lUll) -AST (IU/l) -ALT (mg/dl) - Cholesterol (g/dl) - Albumin (mg/dl) - Glucose

non-treatment 1-3

fructose-treatment

347.8 ± 1.9 534.3 ± 5.0

348.3 ± 2.4 549.1 ± 6.3

N.D.

10.1 ± 0.1

d)

25.4 ± 0.2 205.5 ± 76.8 ± 42.5 ± 71.4 ± 2.2 ± 160.3 ±

4-6

10.4 2.5 1.5 2.4 0.0 3.3

17.6 ± 0.2 e) 391.4 ± 29.4 e) 71.0 ± 3.6 41.3 ± 1.8 79.8 ± 3.5 2.2 ± 0.0 163.6 ± 5.9

a) Rats were given tap water or 25 % fructose in drinking water ad libitum for 5 weeks. Values are means ± SE of 30 rats b) Fructose and food consumption were measured throughout the experiment. c) Blood biochemistry was measured at week 3 of treatment. d) N.D.: not determined. e) Significantly different from non-treated rats (p < 0.01)

510

Exp Toxic Pathol 47 (1995) 6

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

140

80 60

~

........

""

::z:

Effects of fructose-treatment

=> c;Q

Body weight gain, and fructose and food consumptions were shown in table 1. In the fructose-treated rats, fructose consumtion was 10.1 ± 0.1 g/animal/day, and fructose-treatment brought about no changes in body weight gain throughout the experiment. On the other hand, food consumption was about 30 % lower in fructose-treated rats (17.6 ± 0.2 g/animal/day) than in nontreated ones (25.4 ± 0.2 g/animal/day). All blood biochemical parameters except for plasma triglyceride showed no significant changes. Plasma triglyceride levels were significantly higher in fructosetreated rats than in non-treated ones (table 1). As to urinalysis, there were no abnormal findings in any parameters.

Acute toxicity of AAP Mortality: Death occurred to 3 rats of G6. Blood biochemical findings: As shown in table 2, there were no differences in any parameters between G 1 and G4, except for plasma triglyceride level which was significantly higher in G4 (table 1). G2 and G3 showed significant increases in ALT and AST activities, and the values were higher in G2. The increases in these enzyme

•

I

-0

•

40 20 0

treatment

~.,.

100 ,§

o non-

0

120

• fructosetreatment

0

O~SO·OO-O 0 10

100

1000

10000

ALT ( lUl l) Fig. 2. Relationship between plasma ALP activity and UN content in non-hypertriglyceridemic and fructose-induced hypertriglyceridemic rats intoxicated with AAP. activities were also observed in G5 and G6, but the values were significantly lower than those in G2 and G3. As to the changes in UN contents, as compared to Gland G4, G2 and G3 showed no significant changes while G5 and G6 showed significant increases. In fig. 2, the relationship between ALT activity and UN content was shown. Organ weight: As shown in table 3, there were no differences in the liver weight among all groups, and in the kidney weight among Gl , G2, G3 and G4. However, G5 and G6 showed significant increases in the kidney weight in a dose-dependent manner. Histopathology: Hepatocytes filled with PAS-positive granules, i.e. glycogen granules, were observed all

Table 2. Effect of fructose-treatment on ALT, AST and urea nitrogen after dosing with acetaminophen in SD rats a).

ALT (IU/l)

acetaminophen (mg/kg)

Group

non-treatment

0 750 900

1 2 3

59.3 ± 13.5 1136.6 ± 523.9 c) 270.3 ± 69.3 c)

4 5 6

43.4 ± 5.1 150.3 ± 48.2 b)d) 118.5 ± 9.9 b)d)

0 750

108.9 ± 18.7 2450.5 ± 866.4 c) 913.3 ± 168.2 c)

4 5

106.1 ± 23.2 470.5 ± 73.9 c)d) 721.2 ± 117.6 c)

15.8 ± 1.0 22.9 ± 5.1 34.1 ± 13.1

4

AST (IU/l)

900

1 2 3

UN (mg/d1)

0 750 900

2 3

fructose-treatment Group

6

5 6

13.3 ± 70.6 ± 100.8 ±

1.1 9.8 c)e) 2.9 c)d)

a) Rats were given tap water or 25 % fructose in drinking water ad libitum for 5 weeks, received a single ip injection of AAP at week 5 and were killed 24 hr later. Values are means ± SE. b) P < 0.05, c) p < 0.01: Significantly different from treatment-matched and vehicle-dosed rats. d) p < 0.05, e) p < 0.01: Significantly different from non-treated and dose-matched rats.

Exp Toxic Pathol 47 (1995) 6

511

512

Exp Toxic Patho147 (1995) 6

Table 3. Effect of fructose-treatment on liver and kidney weights after dosing with acetaminophen in SD rats a). acetaminophen (mg/kg) Group

non-treatment

Group

fructose-treatment

Liver weight (g)

0 750 900

2 3

18.8 ± 0.8 16.9 ± 0.5 18.4 ± 0.6

4 5 6

21.4 ± 1.1 19.6±0.7 20.2 ± 0.6

Liver weight (%/g B.W.)

0 750 900

1 2 3

3.5 ± 0.1 3.2 ± 0.1 3.4 ± 0.1

4 5 6

3.9±0.2 3.6 ± 0.1 3.6 ± 0.1

Kidney weight (g)

0 750 900

1 2 3

3.1 ± 0.1 3.1 ± 0.2 3.5 ±0.2

4 5 6

3.1 ± 0.1 4.0 ± 0.2 b)d) 4.7 ± 0.2 c)e)

Kidney weight (%/g B.W.)

0 750 900

2 3

0.58 ± 0.02 0.59 ±0.03 0.66 ± 0.05

4 5 6

0.56 ± 0.02 0.74 ± 0.03 b)d) 0.83 ± 0.02 c)d)

a) Rats were given tap water or 25 % fructose in drinking water ad libitum for 5 weeks, received a single ip injection of AAP at week 5 and were killed 24 hr later. Values are means ± SE. b) P < 0.05, c) p < 0.01 : Significantly different from treatment-matched and vehicle-dosed rats. d) p < 0.05, e) p < 0.01 : Significantly different from non-treated and dose-matched rats.

over the hepatic lobules in G4 while they were found only in the centrilobular area in G 1 (fig. 3a and b). Except this, there was no difference in hepatic pictures between G 1 and G4. The liver of G2 and G3 showed prominent lesions which were characterized by necrosis of hepatocytes in the centrilobular areas (fig. 3c). In the lesions, sinusoidal congestion and accumulation of neutrophils were simultaneously observed. In contrast, the liver of G5 and G6 showed less severe lesions which were characterized by vacuolation of hepatocytes in a few centrilobular areas (fig. 3d, table 4). The kidney of Gland G4 showed no changes. In the kidney of G2, single cell necrosis was sporadically seen in the proximal straight tubules of the medullary ray and such cells frequently desquamated from the epithelium into the lumen" (fig. 4a). The kidney of G3 showed focal epithelial cell necrosis in the above-mentioned portion of the nephron (fig. 4b).

~ ~

In contrast, the kidney of G5 and G6 showed prominent lesions which were characterized by marked epithelial cell necrosis in the proximal straight tubules in the medullary ray and the outer stripe of the outer medulla (fig. 4c). In addition, hyaline casts within distal nephrons in both cortex and medulla and epithelial cell necrosis in the thick ascending limbs in the inner stripe of the outer medulla were also observed.

Discussion Alteration of developmental mode of AAP-toxicity has also been reported in streptozotocin-induced diabetic rats and spontaneous hyperlipidemic rats (PRICE and JOLLOW 1982; PRICE and JOLLOW 1986; TUNTATERDTUM et al. 1993). Their diseased conditions are however too complex to clarify the major factor which causes an alteration in the mode of AAP-toxicity in these animal models. In

Fig. 3. Liver. a. Non-hypertriglyceridemic rat of G 1. PAS x 20. b. Fructose-induced hypertriglyceridemic rat of G4. Hepatocytes filled with PAS-positive granules are seen all over the hepatic lobules. PAS x 20. c. Non-hypertriglyceridemic rat of G3 (AAP, 900 mg/kg). Necrosis of hepatocytes is seen in the centrilobular area. HE x 100. d. Fructose-induced hypertriglyceridemic rat of G6 (AAP, 900 mg/kg). Vacuolation of hepatocytes is seen in the centrilobular area. HE x 100. Exp Toxic Patho1 47 (1995) 6

513

Table 4. Effect of fructose-treatment on liver and kidney injury after dosing of acetaminophen in SD rats a ).

acetaminophen (mg/kg)

0

Group Liver injury score 1 score 2 score 3 score 4 score 5 mean of score Kidney injury score 1 score 2 score 3 score 4 score 5 mean of score

lOb)

0 0 0 0 1.0

non-treatment 750 900

fructose-treatment 0 750

900

2

3

4

5

6

2 0 2 4 2 3.4 d)

0 3 1 0 3.3

d)

10 0 0 0 0 1.0

2 4 2 2 0 2.4 d)

2 5 0 0 0 1.7 c)e)

4 5 0 0 2.4 d)

0 4 5 1 0 2.7 d)

10 0 0 0 0 1.0

0 0 1 7 2 4.1

0 0 0 0 7 5.0 d)e)

lOb)

0 0 0 0 1.0

6

d)e)

Rats were given tap water or 25 % fructose in drinking water ad libitum for 5 weeks, received a single ip injection of AAP at week 5 and were killed 24 hr later. b) Number of animals which were scored by the criteria given in Material and methods. c) p < 0.05, d) P < 0.01: Significantly different from treatment-matched and vehicle-dosed rats. e) p < 0.01: Significantly different from non-treated and dose-matched rats. a)

contrast, fructose-induced hypertriglyceridemic rats used in the present study showed only an increase in plasma triglyceride level, and so they are considered to be of great benefit for the examination of the effects of hypertriglyceridemia on the mode of toxicity of various chemicals. In the fructose-induced hypertriglyceridemic rats, as compared with non-hypertriglyceridemic ones, hepatic toxicity became apparently less severe, whereas renal toxicity became significantly more severe judging from the results of blood biochemical and histopathological examinations. As the AAP dosage increases, the glucuronidation and sulfation pathways, i.e. major pathways of AAP (JOLLOw et al. 1974), become saturated and more AAP metabolism is diverted to that through cytochrome P-450, i.e. a minor metabolic pathway of AAP (DAHLIN et al. 1984; HARBISON et al. 1988). At sufficiently high doses, glutathione (GSH) becomes depleted, leaving N-acetyl-p-benzoquinone imine (NAPQI), i.e. an electrophilic metabolic intermediate, free to bind to possibly critical cellular proteins, and this causes hepatic necrosis (DAHLIN et al. 1984; MITCHELL 1973). On the other hand, nephrotoxicity of AAP results from the cytochrome P-450-independent N-deacetylation of AAP to p-aminopheno1 (NEWTON et al. 1985). Therefore the cause of alteration in liepato514

Exp Toxic Patho147 (1995) 6

renal tOXICIty of AAP in the fructose-induced hypertriglyceridemic rats is expected as follows: 1) alteration in the distribution of AAP, 2) alteration in the metabolism, conjugation or excretion of AAP, and 3) alteration in the sensitivity of tissues or cells against the metabolic toxicant. In mice, AAP bioactivation occurs to greater extent in the liver than in other tissues, making liver the critical organ for AAP toxicity. If hepatic metabolism is inhibited, then renal and pulmonary toxicity becomes critical (JEFFERY 1991). Like this, the enhancement of AAPnephrotoxicity in the fructose-induced hypertriglyceridemic rats may be brought about by an inhibition of hepatic AAP metabolism. The streptozotocin-induced diabetic rats show an apparent resistance to AAP-hepatotoxicity (PRICE and JOLLOW 1982; PRICE and JOLLOW 1986). Obese Zucker rats developing a spontaneous hypertriglyceridemia (BRAY 1977) are also resistant to AAP-hepatotoxicity in comparison with lean littermates growing normally (TUNTATERDTUM et al. 1993). This increased resistance is believed to be associated with an enhancement in the capacity of conversion of AAP into nontoxic metabolites, an enhancement of hepatic levels of glutathione (GSH) (PRICE and JOLLOW 1982; PRICE and JOLLOW 1986; CHAUDHARY et al. 1993), or differences in activities of

,'

..

....

•• .....

,..

"

:

......; , ......... ,. ::.

~

......

,

' 1! ("

" (

JPh. ,;.

hepatic cytochrome PA50 isozymes (JEFFERY et al. 1991; TUNTATERDTUM et al. 1993). Similar mechanisms may be involved in the development of resistance of fructoseinduced hypertriglyceridernic rats against AAP-hepatotoxicity. Further detailed studies are now in progress to elucidate the mechanism by which the hepatorenal toxicity of AAP is altered in the fructose-induced hypertriglyceridernic rats.

References 1. BLACK M: Acetaminophen hepatotoxicity. Annu Rev Med 1984; 35: 577-593. 2. BOYER TD, ROUFF SC: Acetaminophen-induced hepatic necrosis and renal failure. J Am Med Assoc 1971; 218: 440-441.

'~. ~.

'"

Fig. 4. Kidney. a. Non-hypertriglyceridemic rat of G2 (AAP, 750 mg/kg). Desquamation of necrotic epithelial cells is seen in the proximal straight tubules in the medullary rays (arrow head). HE x 80. b. Non-hypertriglyceridemic rat of G3 (AAP, 900 mg/kg). Focal necrosis of epithelial cells in the proximal straight tubules in the medullary rays. HE x 80. c. Fructose-induced hypertriglyceridemic rat of G5 (AAP, 750 mg/kg). Massive necrosis of epithelial cells in the proximal straight tubules in the medullary rays and the outer stripe of the outer medulla. HE x 40.

3. BOYD EM, BERECZKY GM: Liver necrosis from paracetamol. Br J Pharmacol Chemother 1966; 25: 606-614. 4. BRAY GA: The Zucker-fatty rat: a review. Federation Proc 1977; 36: 148-153. 5. CHAUDHARY IP, TUNTATERDTUM S, McNAMURA PJ, et al.: Effect of genetic obesity and phenobarbital treatment on the hepatic conjugation pathways. J Pharmacol Exp Ther 1993;265: 1333-1338. 6. COBDEN I, RECORD CO, WARD MK, KERR DNS: Paracetamol-induced acute renal failure in the absence of fulminant liver damage. Brit Med J 1982; 284: 21-22. 7. DAVENPORT A, FINN R: Paracetamol (acetaminophen) poisoning resulting in acute renal failure without hepatic coma. Nephron 1988; 50: 55-56. 8. DAVIDSON DG, EASTHAM WN: Acute liver necrosis following overdose of paracetamol. Br Med J 1966; 2: 497-499. 9. DAHLIN DC, MrwA GT, Lu A YH, NELSON SD: N-acetylp-benzoquinone imine: A cytochrome P-450-mediated Exp Toxic Pathol 47 (1995) 6

515

10.

11.

12.

13.

14.

15.

16.

17.

oxidation product of acetaminophen. Proc Natl Acad Sci USA 1984; 81: 1327-1331. HARVISON PJ, GUENGERICH FP, RASHED MS, NELSON SD: Cytochrome P-450 isozyme selectivity in the oxidation of acetaminophen. Chem Res Toxicol 1988; 1: 47-52. HINSON JA, NELSON SD, MITCHELL JR: Studies on the microsomal formation of arylating metabolites of acetaminophen and phenacetin. Mol PharmacoI1977; 13: 625-633. IWATA K, INAYAMA T, KATO T: Effects of Spirulia platensis on plasma lipoprotein lipase activity in fructoseinduced hyperlipidemic rats. J Nutr Sci Vitaminol 1990; 36: 165-171. JEFFERY EH, ARNDT K, HASCHED WM: The rule of cytochrome P-450 2EI in bioactivation of acetaminophen in diabetic and acetone-treated mice. Adv Exp Med BioI 199I; 283: 249-251. JEFFERY EH: Biochemical Basis of toxicity. In HASCHEK WM, ROUSERAUX CG (eds.): Handbook oftoxicologic pathology. Academic Press INC 1991; Chap 5 pp.49-70. JOLLOW DJ, THORGEIRSSON SS, POTIER WZ, et al.: Acetaminophen-induced hepatic necrosis. 6. Metabolic disposition of toxic and non toxic doses of acetaminophen. Pharmacology 1974; 12: 251-271. KLEINMANN JG, BREITENFfELD RV, ROTH DA: Acute renal failure associated with acetaminophen ingestion: Report of a case and review of the literature. Clin Nephrol 1980; 14: 201-205. MAZUR A, GUEUX E, FELGINES C, et al.: Effect of dietary fermentable fiber on fatty acid synthesis and triglyceride secretion in rats fed fructose-based diet: Studies with sugar-beet fiber. Proc Society Exp BioI Med 1992; 199: 345-350.

18. McMuRTRY RJ, SNODGRASS WR, MITCHELL JR: Renal necrosis, glutathione depletion and covalent binding after acetaminophen. Toxicol Appl Pharmacol 1978; 46: 87-100. 19. MITCHELL JR, JOLLOW DJ, PATIER WZ, et al.: Acetaminophen-induced hepatic necrosis. 4. Protective role of glutathione. J Pharmacol Exp Ther 1973; 187: 211-217. 20. NEWTON JF, Kuo CH, DESHONE GM, 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. 21. PRESCOTI LF, WRIGHT N, ROSCOE P, BROWN SS: Plasma-paracetamol half-life and hepatic necrosis in patients with paracetamol overdose. Lancet 1971; 1: 519-522. 22. PRICE VF, JOLLOW DJ: Increased resistance of diabetic rats to acetaminophen-induced hepatotoxicity. J Pharmacol Exp Ther 1982; 220: 504-513. 23. PRINCE VF, JOLLOW DJ: Strain differences in susceptibility of normal and diabetic rats to acetaminophen hepatotoxicity. Biochem Pharmacoll986; 35: 687-695. 24. PROUDFOOT AT, WRIGHT N: Acute paracetamol poisoning. Brit Med J 1970; 557-558. 25. TUNTATERDTUM S, CHAUDHARY IP, ClBULL M, et al.: Acetaminophen hepatotoxicity: Influence of phenobarbital and J3-naphthoflavone treatment in obese and lean Zucker rats. Toxicol Appl Pharmacol 1993; 123: 219-225. 26. YOSHINO G, MATSUSHITA M, IWAI M, et al.: Effect of mild diabetes and dietary fructose on very-low-density lipoprotein triglyceride turnover in rats. Metabolism 1992; 41 : 236-240.

Experimental and Toxicologic Pathology Publisher: Gustav Fischer Verlag l ena GmbH, Postfach 100537, D-07705 lena; Telefon (0 36 41) 62 63; Telefax (0 36 41) 62 6500. Managing editor: Prof. Dr. Dr. F. Bolck, Institute of Pathology, Friedrich Schiller University, Ziegelmuhlenweg I, D-07743 lena. Type setting: Satz Repro Grafik GmbH Leipzig, KreuzstraBe 20, D-041 03 Leipzig. Printing, binding: Druckerei zu Altenburg GmbH, GutenbergstraBe 1, D-04600 Altenburg. Advertising sales: Gustav Fischer Verlag lena GmbH, Anzeigenverwaltung, Frau A. Schutz, Postfach 100537, D-07705 Jena; Telefon (0 36 41) 62 64 30, Telefax (0 36 41) 62 65 00. The price list from October 1st, 1994 is effective at present. Distribution and subscription agency: Gustav Fischer Verlag Jena GmbH, Zeitschriftenvertrieb, Frau B. Dressler, Postfach 100537, D-07705 lena; Telefon (0 36 41) 62 64 44, Telefax (0 36 41) 62 65 00. For the USA and Canada only: VCH Publishers. Inc., Distribution Center, 303 N.W. 12th Avenue, Deerfield Beach, FL 33442-1788; Telefon (305) 4 28 55 66, Telefax (3 05) 4 288201. Terms of delivery (1995): I volume consisting of 6 issues. Subscription rate (1995): DM 648.- lOS 4795.- I sFr 622.50 (plus postage); single issue price: DM 125.- lOS 925.- I sFr 120.- (plus postage). Preference-Price for members of Gesellschaft fur Toxikologische Pathologie DM 298.- I OS 2205.- I sFr 286.- (plus postage). We accept the following credit cards: VisalEurocard/Mastercardl American Express (please specify Card No. and Expiry Date). Subscription information: Please, send your order to your scientific bookshop, to your hitherto dealer or directly to our publishing house. If not terminated the SUbscription will be effective until recalled. If no discontinuance is given until October, 31 st the delivery of the journal will be continued. Banking connections: Postgiroamt Leipzig. Konto-Nr. 149249-903. BLZ 860 10090; Deutsche Bank, Konto-Nr. 3 907 656, BLZ 82070000; Commerzbank AG, Filiale lena, Konto-Nr. 2581 122, BLZ 820400 00. Copyright: The articles published in this journal are protected by copyright. All rights are reserved. No part of the journal may be reproduced in any form without the written permission of the publisher. Printed in Germany © Gustav Fischer Verlag lena GmbH 1995