The nephrotoxicity of p-aminophenol. I. The effect on microsomal cytochromes, glutathione and covalent binding in kidney and liver

Chem.-Biol. Interactions, 27 (1979)235--243

235

© Elsevier/North-Holland Scientific Publishers Ltd.

THE N E P H R O T O X I C I T Y O F p-AMINOPHENOL. I. THE E F F E C T ON MICROSOMAL CYTOCHROMES, G L U T A T H I O N E AND C O V A L E N T BINDING IN KIDNEY AND L I V E R

C.A. CROWE, A.C. YON(], I.C. CALDER, K.N. HAM and J.D. TANGE Departments of Pathology and Organic Chemistry. University of Melbourne, Parkville, Victoria 3052 (Australia)

(Received December 2nd, 1978) (Accepted April 30th, 1979)

SUMMARY p-Aminophenol administration lowered the microsomal c y t o c h r o m e P-450 and bs content and decreased the activity of NADPH c y t o c h r o m e c reductase in kidney, b u t n o t in liver. Kidney GSH was depleted to 29% of the control value at 2 h, and only partly restored (50% o f control) at 24 h. Liver GSH was transiently decreased, the lowest levels (77% of control) occurring at 30 min. The m a x i m u m level of covalently b o u n d radioactivity was at t w o hours when 16.8% of the total radioactivity in kidney, 1.5% in liver and 3.6% in plasma was protein bound. At this time 81% of the total radioactivity in kidney and 95% of that in the liver was present in the soluble fraction.

INTRODUCTION Although phenacetin has been implicated clinically in the production of renal damage [1,2] and its major metabolite, paracetamol, causes acute hepatic necrosis [3], the acute effects of phenacetin on both liver and kidney in experimental animals are relatively minor, p-Aminophenol is one of the few c o m p o u n d s related to these analgesics which has been found to be nephrotoxic in animal models [4]. The toxicity of all three c o m p o u n d s has been attributed to the formation of toxic reactive intermediate metabolites. The toxicity o f the intermediate is reflected by selective damage at the site of action. Thus in the case of paracetamol there is a relationship between depletion of GSH, covalent binding to protein and hepatotoxicity, following acute administration [5,6] and the concentration of cytochrome P-450 and c y t o c h r o m e bs are decreased [7]. Chronic administration of phenacetin significantly changes the levels of some mitochondrial enzymes in rat kidneys [8] b u t has

236 little effect in the liver [9]; and p-aminophenol selectively affects kidney mitochondrial function [10], microsomal glucose-6-phosphatase activity [11], and giuconeogenesis [12]. In a further study of the mechanisms of action of these compounds we have examined the effect of p-aminophenol on microsomal cytochromes, glutathione and covalent binding in the liver and kidney. MATERIALS AND METHODS

Chemicals DNA sodium salt (Type I), NADPH tetrasodium salt (Type III), and 5,5'-dithiobis-(2-nitrobenzoic acid) were purchased from the Sigma Chemical Company. Cytochrome c was either Sigma type III or Calbiochem A grade, and the glucose-6-phosphate (disodium salt) and GSH were both Calbiochem A grade. Teric X-10 was obtained from ICI Australia Ltd. p-[ring-3H]Aminophenol HCI: p-aminophenol HC1 (0.5 g) was subjected to catalytic exchange with tritiated water by the Radiochemical Centre, Amersham, U.K. The crude product was received in two lots, dissolved in ethanol/water (10 ml, 50%). One lot was added to p-aminophenol HC1 (1.0 g) dissolved in ethanol/water (30 ml). The solution was filtered and the solid washed with methanol (2 × 10 ml). The solvent was removed under reduced pressure and the residue dried under vacuum overnight. Sublimation (120 °, 1 mm) yielded p-[ring-3H]aminophenol HC1 (1.165 g) which showed only one radioactive spot by TLC (Silica Gel GF2s4, CHC13 : EtOH 90 : 10). p-[ring-3H]Aminophenol HC1 (10 mg) from above was added to p-aminophenol HC1 (1.0 g) and dissolved in methanol (15 ml). The solvent was removed and the residue dried under vacuum overnight. Sublimation (120 °, 1 ~mm) yielded p-[ring-3H]-amino phenol HC1 (0.994 g, 9 pCi/mg) as a white solid which showed one radioactive spot by thin layer chromatography (TLC). Treatment o f animals and preparation of samples (i) Microsomal studies. Male Sprague--Dawley rats (170--210 g) were given p-aminophenol HC1 (2.1 mmol/kg) by tail vein injection under light ether anaesthesia. Control animals were injected with 0.9% NaC1 or distilled water. The animals were killed by bleeding under ether anaesthesia and the kidneys and livers removed. All subsequent operations were carried out at 0--4°C. Microsomes were prepared from a 1/10 (w/v) homogenate in 1.15% KC1--0.01 M potassium phosphate buffer (pH 7.4). The homogenate was centrifuged at 11 000 × g for 10 min (SorvaU RC-B refrigerated centrifuge) and the supernatant then centrifuged at 100 000 g for 60 min (Beckman L5-40 ultracentrifuge). The pellet was washed once and resuspended in 0.05 M potassium phosphate (pH 7.6), containing 10 -3 M EDTA. (ii) GSH estimation. Kidneys and livers from control and p-aminophenol treated rats were rinsed in 0.1 M potassium phosphate (pH 7.4) and homogenized in 4.5 vol. of 2% sulphosalicylic acid.

237

(iii) Protein binding and subcellular fractionation studies. For protein binding studies male Sprague--Dawley rats (210--260 g) were given p-[3H]aminophenol HC1 (2.1 mmol/kg; 0.23 mC/mmol) by tail vein injection as before and killed at fixed times after injection by bleeding from the jungular vein under ether anaesthesia. Blood was collected into a heparinized beaker and centrifuged for 10 min at 1500 rev./min to obtain the plasma. The kidney and liver were homogenized in 9 vols. of ice-cold 0.25 M sucrose--1.0 mM Tris--0.1 mM EDTA (pH 7.4), in a glass Potter Elvehjem homogenizer with a teflon pestle driven at 1150 rev./min. The homogenate was filtered through 4 layers of nylon gauze and a 1-ml aliquot taken for analysis. For subcellular fractionation studies, the animals were killed 2 h after, administration of p-[3H]aminophenol and the kidney and liver homogenized as described above. The crude nuclear fraction was obtained from the remainder of the homogenate by centrifuging at 600 × g for 10 rain (Sorvall RC2-B refrigerated centrifuge) and washing the pellet once with 2 vols. of homogenizing medium. The combined supernatants were centrifuged at 10 0 0 0 X g for 10 min to pellet the mitochondrial fraction. The mitochondrial pellet was washed once (2 vols. homogenizing medium) and the combined supernatants from the 10 000 × g spin centrifuged at 100 000 × g for 60 min (Beckman L5-40 ultracentrifuge), giving a microsomal pellet and soluble fraction. The pellets were resuspended in 2 vols. of homogenizing medium. Samples were prepared from the kidney and liver of untreated rats for the determination of the distribution of DNA, protein and marker enzymes in subcellular fractions. Measurements (i) Microsomal studies. Microsomal NADPH cytochrome c reductase was assayed by measuring the rate of reduction of cytochrome c at 550 nm [13]. Cytochromes bs and P-450 were measured as described by Manzel [14] on microsomal suspensions that had been stored overnight at 0--4°C. The extinction coefficients used were 171 and 91 cm -1. mM -1, respectively. Protein content was measured as described by Munro and Fleck [15]. (ii) GSH estimation. GSH was measured on an aliquot of profein-free sulphosalicylic acid supernatant [ 16]. (iii) Protein binding and subeellular fractionation studies. DNA [17], protein [15], succinate-cytochrome c reductase [18] and glucose-6phosphatase [19] were measured in homogenates and in subcellular fractions of kidney and liver from untreated rats. For measurement of total radioactivity in samples from rats given p-[3H]aminophenol, an aliquot of each fraction was made 1 N with respect to NaOH and heated at 50°C to digest the sample. Covalent binding to protein was determined by the method of Jollow et al. [20], by precipitation of the protein with trichloroacetic acid, followed by further washes of the protein with trichloroacetic acid and 80% methanol. The pellet was then dissolved in 1 N NaOH by heating at 50°C. A solubilized sample (1 ml) was added to 8 ml

238 o f scintillation c o c k t a i l (4 g 2 , 5 < l i p h e n y l o x a z o l e per litre o f 2 : 1 t o l u e n e / Teric X-10) a n d 0.1 m l o f 10 N HCI a d d e d t o n e u t r a l i z e t h e N a O H . Samples were c o u n t e d in a P a c k a r d Tri-Carb liquid scintillation c o u n t e r using e x t e r n a l s t a n d a r d i z a t i o n f o r q u e n c h c o r r e c t i o n . T h e results, expressed as n a n o m o l e s p - [ 3 H ] a m i n o p h e n o l , were c a l c u l a t e d o n t h e basis o f t h e specific activity o f t h e i n j e c t e d c o m p o u n d . Statistical analysis. T h e d i f f e r e n c e b e t w e e n c o n t r o l a n d t r e a t e d g r o u p s was a n a l y s e d using a 2-tailed S t u d e n t ' s t-test on t h e d i f f e r e n c e b e t w e e n t h e m e a n s o f t h e t w o groups. P-values less t h a n 0 . 0 5 were c o n s i d e r e d t o be significant. RESULTS

(i) Microsomal estimation. The activity o f m i c r o s o m a l N A D P H c y t o o h r o m e c r e d u c t a s e in k i d n e y was decreased 12 h a f t e r a d m i n i s t r a t i o n o f p - a m i n o p h e n o l , b u t was u n c h a n g e d at t h e earlier times studied. T h e c y t o c h r o m e bs c o n t e n t o f k i d n e y m i c r o s o m e s was decreased at all t i m e s m e a s u r e d f r o m 3 0 rain t o 12 h, w h e r e a s c y t o c h r o m e P - 4 5 0 was significantly decreased o n l y at 4 and 12 h (Table I). T h e r e was n o c h a n g e in liver m i c r o s o m a l N A D P H c y t o c h r o m e c r e d u c t a s e , c y t o c h r o m e b5 o r P - 4 5 0 at a n y time. (ii) GSH estimation. G S H in c o n t r o l animals d i d n o t alter w i t h t i m e a f t e r injection, so t h e average o f values f r o m 1 0 animals m e a s u r e d at d i f f e r e n t t i m e s was t a k e n as the c o n t r o l value, p - A m i n o p h e n o l t r e a t m e n t significantly r e d u c e d G S H in k i d n e y a f t e r 3 0 rain a n d t h e levels c o n t i n u e d t o fall, b e i n g 29% o f t h e c o n t r o l value at 2 h (Fig. 1). T h e d e p l e t i o n o f G S H was f o u n d TABLE I THE EFFECT OF p.AMINOPHENOL TREATMENT ON RAT KIDNEY MICROSOMES Measurements were made on kidney microsomal preparations from control and p-aminophenol treated rats as described in Materials and Methods, and are the mean ± S.E.M. of determinations on preparations from 4 animals. * P < 0.05; ** P < 0.01. Time after injection (h) 0.5 1.0 2.0 4.0 12.0

Control Treated Control Treated Control Treated Control Treated Control Treated

Cytochrome b s (nmol/mg protein)

Cytochrome P-450 (nmol/mg protein)

NADPH eytochrome c reductase ( 4 0 D / m i n / mg protein)

0.25 0.10 0.20 0.11 0.17 0.07 0.16 0.03 0.12 0.08

0.14 0.16 0.14 0.11 0.17 0.10 0.17 0.06 0.12 0.05

0.37 0.40 0.53 0.64 0.35 0.41 0.32 0.31 0.42 0.26

± 0.03 ± 0.02* ± 0.01 ± 0.01" ± 0.03 ± 0.02** ± 0.03 ± 0.01"* ± 0.01 ± 0.01"*

± 0.02 ± 0.02 ± 0.03 ± 0.07 ± 0.04 ± 0.02 ± 0.02 ± 0.02** ± 0.01 ± 0.01"*

± 0.03 ± 0.09 ± 0.09 ± 0.14 ± 0.05 ± 0.03 ± 0.01 ± 0.04 ± 0.04 ± 0.02**

239 10

Liver

! I

Kidney

0.5

1.0

1.5

2.0

Timeafterinjection(hi Fig. 1. E f f e c t o f a d m i n i s t r a t i o n of p - a m i n o p h e n o l , 2.1 m m o l / k g , o n G S H i n k i d n e y a n d liver. Values are t h e m e a n -+ S.E.M. o f d e t e r m i n a t i o n s o n p r e p a r a t i o n s f r o m at least 3 animals. M e a s u r e m e n t o f G S H is as d e s c r i b e d in Materials a n d M e t h o d s .

1 0

~

e

r

| 6 E

!

0.5

!

I

I

1.0 1.5 2.0 Dose p-aminophenol (mmol/kg)

|

2.5

Fig. 2. E f f e c t o f increasing d o s e s o f p - a m i n o p h e n o l o n G S H in k i d n e y a n d liver. V a l u e s are t h e m e a n ± S . E ~ . o f d e t e r m i n a t i o n s o n p r e p a r a t i o n s f r o m a t least 3 animals. GSH was m e a s u r e d as d e s c r i b e d in Materials a n d M e t h o d s 2 h a f t e r a d m i n i s t r a t i o n o f p-aminophenol.

240 to be dose dependent (Fig. 2). At 24 h GSH levels had risen again, but were still only 50% of control. There was a reversible depletion of GSH in the liver, with the lowest levels occurring at 30--45 rain (77% and 78%, respectively, of control). The levels fluctuated for the remainder of the time course, but did not drop below 80% of control (Fig. 1). (iii) Protein binding and subcellu.lar fractionation studies. Following administration of ring-labelled p-[3H] aminophenol HC1 r~dioactivity in the plasma reached a peak at 15 rain, fell at 30--60 rain and reached a second peak at 6 h. The fall in total radioactivity in the plasma corresponded with an increase in radioactivity in the liver and kidney, with the peak concentration in the liver occurring at 1 h, ahead of that in the kidney at 2 h. Radioactivity in the liver dropped rapidly between 1 and 4 h and in the kidney between 2 and 4 h; kidney radioactivity then remained at a concentration about 3--4 times that in the liver. A portion of the radioactivity was covalently bound to protein, with the binding in the kidney being very much greater than in liver and plasma. The binding for all three tissues was maximal at 2 h. The level in the liver remained relatively constant, whilst that in the kidney and plasma began to fall with time. Nevertheless, the level in the kidney was still about ten times that in the liver and plasma at 12 h. In kidney homogenates 2 h after admini-

T A B L E II SPECIFIC ACTIVITY OF TOTAL AND PROTEIN BOUND RADIOACTIVITY IN FRACTIONS FROM KIDNEY, LIVER AND PLASMA 2 H AFTER ADMINISTRATION OF p-[ SH]AMINOPHENOL M e a s u r e m e n t s were m a d e o n h o m o g e n a t e s (H), c r u d e n u c l e a r (CN), m i t o c h o n d r i a l (Mit), m i c r o s o m a l (Mic) a n d soluble (Sol) f r a c t i o n s of k i d n e y a n d liver a n d o n p l a s m a as d e s c r i b e d i n Materials a n d M e t h o d s . All values are t h e m e a n ± S . E ~ I . o f d e t e r m i n a t i o n s o n p r e p a r a t i o n s f r o m 4 animals. Fraction

Kidney Kidney Kidney Kidney Kidney Liver Liver Liver Liver Liver

H CN Mit Mic Sol

H CN Mit Mic Sol

Plasma

n m o l p - [ SH ] a m i n o p h e n o l m g / p r o t e i n Total radioactivity

Protein bound radioactivity

% Radioactivity protein bound

87.39 25.16 17.10 23.63 190.80

± 11.48 ± 2.29 ± 1.94 ± 1.11 ± 27.28

14.27 19.82 12.49 13.70 9.02

± ± ± ± ±

1.51 2.23 0.69 1.20 2.14

16.8 78.8 73.0 58.0 4.7

44.99 5.86 7.64 5.78 121.76

± ± ± ± ±

4.12 0.54 0.39 0.30 5.49

0.68 0.52 0.59 0.65 1.16

± ± ± ± ±

0.09 0.02 0.04 0.08 0.18

1.5 8.9 7.7 11.2 1.0

30.52 ±

2.55

1.09 ± 0.10

3.6

241 TABLE III PERCENTAGE DISTRIBUTION OF p-[~H]AMINOPHENOL, PROTEIN, DNA AND ENZYME ACTIVITY IN SUBCELLULAR FRACTIONS OF RAT KIDNEY AND LIVER Measurements were made on the crude nuclear (CN), mitochondrial (Mit), microsomal (Mic) and soluble (Sol) fractions of kidney and liver as described in Materials and Methods. The results are expressed as a percentage of the content of the homogenate, and are the mean ± S.E.M. of determinations on preparations from 4 animals. Percentage distribution of p-[3H]aminophenol was measured 2 h after administration. Protein, DNA and enzyme activities were measured on preparations from non-injected animals. Fraction

Total radioactivity

Protein

DNA

Succinate cytochrome c reductase

Glucosc6-phosphatase

Kidney CN Kidney Mit Kidney Mic Kidney Sol % Recovery

10.0 3.1 2.3 81.1 96.3

± 0.9 ± 0.2 ± 0.2 ± 1.3 ± 1.2

33.6 13.8 10.8 30.6 88.8

± 2.7 ± 0.4 ± 0.6 ± 2.4 ± 4.6

85.8 ± 5.0 0 9.5 ± 2.8 0 95.2 ± 7.1

44.0 ± 5.7 57.1 ± 6.2 10.8 ± 0.5 0 119.1 ± 3.8

17.3 18.8 47.0 12.3 95.5

± ± ± ± ±

3.6 5.0 4.8 2.8 10.6

Liver CN Liver Mit Liver Mic Liver Sol % Recovery

4.8 2.6 1.9 95.3 104.6

± 0.4 ± 0.1 ± 0.1 -+ 1.8 ± 1.9

37.0 12.8 13.5 29.7 93.1

± 2.6 ~ 0.9 ± 1.0 -+ 2.5 ± 3.5

89.1 ± 1.1 0 3.6 ± 2.4 0 92.7 ± 5.7

48.8 ± 5.5 40.5 ± 3.4 7.3 ± 1.5 0 96.6 ± 5.3

11.4 21.2 50.2 7.4 90.1

± ± ± ± ±

2.3 3.2 5.9 2.9 7.8

s t m t i o n 16.8% o f t h e t o t a l r a d i o a c t i v i t y was p r o t e i n b o u n d , c o m p a r e d w i t h 1.5% f o r liver h o m o g e n a t e s a n d 3.6% f o r plasma. I n all subcellular f r a c t i o n s , t o o , t h e p r o p o r t i o n o f p r o t e i n b o u n d r a d i o a c t i v i t y was greater f o r k i d n e y t h a n f o r liver (Table II). T h e h o m o g e n e i t y o f e a c h f r a c t i o n is i n d i c a t e d b y t h e d i s t r i b u t i o n o f D N A a n d e n z y m e activities (Table III). DISCUSSION T h e early fall in m i c r o s o m a l c y t o c h r o m e s bs a n d P - 4 5 0 c o n t e n t a n d c y t o c h r o m e c r e d u c t a s e activity, t h e d e p l e t i o n o f g l u t a t h i o n e a n d t h e b i n d i n g o f r a d i o a c t i v e p - a m i n o p h e n o l in t h e k i d n e y b u t n o t in t h e liver is c o n s i s t e n t w i t h p - a m i n o p h e n o l b e i n g an a c u t e n e p h r o t o x i n . Like o t h e r effects o n m e t a b o l i s m a n d f u n c t i o n o f renal cells [ 1 0 - - 1 2 ] a n d t h e m o r p h o logical d a m a g e [ 2 1 ] , t h e s e c h a n g e s in m i c r o s o m a l e n z y m e s o c c u r r a p i d l y a f t e r a d m i n i s t r a t i o n o f p - a m i n o p h e n o l . T h e s e studies as well as t h o s e o n t h e isolated p e r f u s e d k i d n e y [ 1 2 ] suggests t h a t t h e n e p h r o t o x i c e f f e c t s o f p - a m i n o p h e n o l are n o t d e p e n d e n t o n m e t a b o l i c t r a n s f o r m a t i o n b y t h e liver. T h e p a t t e r n o f n e p h r o t o x i c i t y f r o m p - a m i n o p h e n o l is d i f f e r e n t f r o m t h e h e p a t o t o x i c i t y d u e t o p a r a c e t a m o l [ 7 ] ; in mice changes in m i c r o s o m a l c y t o c h r o m e s bs a n d P - 4 5 0 a m n o t d e t e c t e d u n t i l 6 h a f t e r m o r p h o l o g i c

242

evidence of liver cell necrosis. Hepatotoxicity of paracetamol has been ascribed to the formation of the reactive metabolite N-acetylbenzoquinoneimine, and liver GSH is depleted by about 70% before the onset of covalent binding and necrosis [5]. Other workers [22] have found that isolated hepatocytes can withstand considerable depletion of GSH but if the depletion is too great or conditions for resynthesis are not favoumble cell damage ensues. Similarly with p-aminophenol, depletion of GSH is one of the prerequisites for toxicity, the depletion occurs rapidly after exposure of the kidney to p-aminophenol. As in the liver, covalent binding to protein and GSH depletion are likely to be due to the formation of a reactive intermediate, probably the benzoquinoneimine. The reactive intermediate is formed within, and acts directly on the kidney; its mode of action, as indicated by the speed and range of its toxic effects, appears to differ substantially from that of the toxic metabolite of paracetamol on the liver. ACKNOWLEDGEMENTS

This work was supported by funds from the National Health and Medical Research Council of Australia and the Australian Research Grants Committee. REFERENCES 1 K.G. Koutsaimanis and H.E. de Wardener, Phenacetin nephropathy, with particular reference to the effect of surgery, Br. Med. J., 4 (1970) 131. 2 T. Murray and M. Goldberg, Analgesic abuse and renal disease, Ann. Rev. Med., 26 (1975) 537. 3 L.F. Prescott and N. Wright, The effects of hepatic and renal damage on paracetamol metabolism and excretion following overdosage. A pharmacokinetic study, Br. J. Pharmacol., 49 (1973) 602. 4 C.R. Green, K.N. Ham and J.D. Tange, Kidney lesions induced in rats by p-aminophenol, Br. Med. J., 1 (1969) 162. 5 J.R. Mitchell, D.J. Jollow, W.Z. Potter, J.R. Gillette and B.B. Brodie, Acetaminopheninduced hepatic necrosis. IV. Protective role of glutathione, J. Pharmacol. Exp. Ther., 187 (1973) 211. 6 W.Z. Potter, S.S. Thorgeirsson, D.J. Jollow and J.R. Mitchell, Acetaminopheninduced hepatic necrosis. V. Correlation of hepatic necrosis, covalent binding and glutathione depletion in hamsters, Pharmacology, 12 (1974) 129. 7 S.S. Thorgeirsson, H.A. Sasame, J.R. Mitchell, D.J. Jollow and W.Z. Potter, Biochemical changes after hepatic injury from toxic doses of acetaminophen or furosemide, Pharmacology, 14 (1976) 205. 8 W. Raab, R. Kramer and C. Moerth, An experimental study of acute and chronic effects of phenacetin on the rat kidney, using clinical~hemical and biochemical methods, Enzyme, 21 (1976) 76. 9 W. Raab, R. Kramer and C. Moerth, Phenacetin and the liver. The influence of phenacetin in acute and chronic doses on membrane-bound mitochondrial enzymes in the rat, Enzyme, 21 (1976) 275. 10 C.A. Crowe, I.C. Calder, N.P. Madsen, C.C. Funder, C.R. Green, K.H. Ham and J.D. Tange, An experimental model of analgesic induced renal d a m a g e - some effects ofp-aminophenol on rat kidney mitochondria, Xenobiotica, 7 (1977) 345.

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