Cholestasis as a liver protective factor in paracetamol acute overdose

Gen. Pharmac. Vol. 26. No. 7, pp. 1619-1624, 1995

0306-3623(95)00061-5

Pergamon

Copyright © 1995 ElsevierScienceInc. Printed in Great Britain. All rights reserved. 0306-3623/95 $9.50 + 0.00

Cholestasis as a Liver Protective Factor in Paracetamol Acute Overdose C R I S T I N A A C E V E D O , 1 L A U R A B E N G O C H E A , ~ D A N I E L M. T C H E R C A N S K Y , 1 G R A C I E L A O U V I l q A , 2 J U A N C. P E R A Z Z O , 2 NI~STOR L A G O , 2 A B R A H A M L E M B E R G 2 a n d M O D E S T O C. R U B I O 1'3. 1Cdtedra de Farmacologia y 2Catedra de Fisiopatolog:a, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956, Buenos Aires (1113), Argentina [Fa~" 541-962-5341] 3ININFA, C O N I C E T (Received 11 November 1994)

Abstract-1. The effect of paracetamol overdoses on its disposition was investigated in cholestatic rats. 2. Paracetamol plasma concentration and hepatic accumulation decrease about 70-80% in cholestatic rats. 3. Cholestatic rats intoxicated with paracetamol showed less hepatic damage as concluded from biochemical and histological findings. These data are correlated with liver and plasmatic paracetamol. 4. These results indicate a decrease in paracetamol toxicity related to stagnant bile. Key Words: Cholestasis, paracetamol, liver

INTRODUCTION

Paracetamol (APAP) is a widely used analgesic and antipyretic agent, which is considered safe when taken at usually therapeutic doses for limited periods of time. This drug is metabolized by the liver primarily to paracetamol glucuronide (5507o) and paracetamol sulphate (33070) (Cumming et al., 1967). The hepatotoxic effect of APAP is due to the fact that a small fraction of it undergoes cytochrome P450 mediated metabolism (Mitchell et al., 1973; Potter et al., 1973) to form N-acetyl-p-benzoquinone imine (NAPQI), a highly reactive intermediate (Hinson et al., 1981). Following a therapeutic dose this metabolite is detoxified by conjugation with glutathione and then excreted in urine (Jollow et al., 1974) or bile (Hinson et al., 1982) as cysteine and mercapturic acid conjugated. At higher doses, however, hepatic stores of glutathione may be depleted and the chemically reactive metabolite can initiate a number of primary effects, including covalent binding to tissue macromolecules, causing cellular necrosis (Potter et al., 1973; Jollow et al., 1973).

*To whom all correspondence should be addressed. GP Z6:7-L

As the liver is the main site of drug metabolism in the body, it is not surprising, therefore, that the liver disease can produce significant effects upon drug disposition. In spite of extensive studies, the mechanisms determining changes in the pharmacokinetics of drugs in liver disease are still poorly understood, and, therefore, drug handling is highly variable and difficult to predict. In general, oxidative drug metabolism is impaired in liver disease and the degree o f impairment of oxidization differs between drugs. On the other hand, drug conjugation appears to be less affected by liver disease (Secor and Schenker, 1987; Wilkinson, 1986). Moreover, in the case of conjugation by glucuronidation, there is no evidence of impairment of the metabolism of temazepam (Ghabrial et al., 1986), lorazepam (Krauss et al., 1978), furosemide (Villeneuve et al., 1986), oxacepam (Shull et al., 1976). However, there is impairment in cirrhosis, and in other liver disease such as acute viral hepatitis on the elimination of chloramphenicol (Narang et al., 1981) and naproxen (Williams et al., 1984). Glucuronidation represents a major pathway by which the body inactivates and eliminates a wide variety of lipid-soluble endogenous and exogenous compounds (Dutton, 1966). This metabolic route is catalyzed by a family of enzymes, the UDP-glucuronyl 1619

1620

Cristina Acevedo et al.

transferases (UDPGTs), which are located in endoplasmic reticulum of cells from a number of tissues and are encoded by a multigene family (Mulder, 1992). These proteins are responsible for the glucoronidation of hundreds o f xenobiotics, clinically used drugs and many endogenous substances as bilirrubin and steroid hormones. Probably because of its location (membranebound enzymes), its activity is modified under various conditions: age, diet, ethnicity, drug therapy and disease states. Previous reports showed that during cholestasis, the activity of hepatic microsomal UDPGTs is variable, depending on the drug utilized as substrate (Ouvifia et al., 1993). An increment of APAP glucuronidation was observed in vitro in cholestatic rats (Ouvifia et al., 1994). As this effect was described in hepatic microsomal membrane, it could indicate a permissive effect of cholestasis on the glucuronidation of this drug. In consideration to these results, the present study was undertaken to investigate possible relationships between the above described in vitro data and an in vivo model of APAP overdose during experimental cholestasis.

MATERIAL A N D METHODS

Animals and animal treatments Male Wistar rats (initial body weight 200-250 g) were used in all experiments. They had free access to a standard commercial diet (Purina Chow) and to water ad libitum. Animals were divided in four groups of 4 rats each one, as follows: group I: cholestatic rats injected with tylose 1070;group II: cholestatic rats injected with APAP; group III: control rats injected with APAP; group IV: control rats injected with tylose 1070. Sham operated rats were used as controls. Animals treated with APAP were injected intraperitoneally (i.p.) with a single dose of 2 g/kg body weight, in a 1070 tylose (carboxymethylcellulose) solution. Extrahepatic cholestasis was performed under slight ether anesthesia, by surgical ligation and secondary section of the common bile duct proximal to bifurcation. A P A P or its vehicle were given after 7 days of the surgical procedure to induce cholestasis; after 24 h the animals were anesthetized with ether and blood samples were collected from the abdominal aorta. The portal vein was perfused with 20 ml of ice-cold saline and the animals were killed by exsanguination. Livers were removed, weighed and stored on ice. Plasma were processed immediately for biochemical tests and an aliquot of them and livers were kept frozen until the time o f analysis.

Experimental protocol In each group of rats the following studies were performed: liver histology, biochemical determinations and quantification of APAP in plasma and liver tissue. Liver histology. Livers were removed and pieces of them were fixed in 1007oneutral-buffered formaline. After fixation, rat livers were routinely dehydrated and flat embedded in paraplast for optical microscopy. Sections were trimmed and cut to include all lobes of each liver, wherever possible. Sections were stained with hematoxylin and eosin, PAS and Masson trichromic. Hepatocellular necrosis was graded for each section on a scale of 0 to 4, according to the following criteria (Douidar et al., 1985): (0) no necrosis; (1) small focal areas involving less than 10070 of cross-sectional area; (2) several focal areas with at least one large hemorrhagic area, involving 10-30°70 of cross-sectional area; (3) two or more large areas of hemorrhagic necrosis, involving 30-6007o of cross-sectional area and (4) massive hemorrhagic necrosis with multiple confluent large areas involving 60070 of cross-sectional area. Biochemical determinations. The activities of alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and gamma-glutamil transpeptidase (GGT) in serum, as well as the concentration of total (TB) and direct (DB) bilirubin were assayed using commercial kits of Boehringer Mannheim (Argentine).

Quantification o f A P A P in plasma and liver tissue. The concentration in plasma and liver tissue were determined by means of high performance liquid chromatography (HPLC) according to Gotelli et al., 1977. Livers were homogenized in a glass-teflon homogenizer with perchloric acid 0.4 M. APAP was determined by extracting aliquots of tissue homogenate supernatant and plasma with ethyl acetate. Organic phase was evaporated. The dried extracted was reconstituted in 150 ~tl of methanol before injection onto a reverse phase C18 column; the separation was achieved at an effluent flow-rate of I ml/min at 40°C and UV detection was at 254 nm. Mobil phase consist of acetonitrile-phosphate buffer pH = 4.4 (15:85). Acetanilide was used as internal standard.

Chemicals APAP (4-acetamidophenol, 9807o) were purchased from Aldrich Chemical Company (Milwaukee, WI, USA). HPLC grade methanol, acetontfile and ethyl acetate were from J. T. Baker (Phillipsburg, N J, USA). All other chemicals used were of analytical reagent grade.

Cholestatic liver disease and APAP

1621

Statistics Results are expressed as means _+ standard error of the mean (SEM). Data were statistically analyzed by means of one-factor analysis of variance (ANOVA). For comparison between two groups, Student's t-test was used. A difference was judged significant if P < 0.05. RESULTS

Liver pathology The hepatic lesion incidence is shown in Fig. 1. Group I showed classical patterns of cholestatic liver: bile duct duplication, stasis and biliary thrombus in bile ducts (grade 0). Group II showed centrilobular focal necrosis in just a few areas in three animals (grade 1), and the two remaining animals showed cholestasis features. In this group it was noticed that apoptotic p h e n o m e n a was observed in all the animals (Fig. 1 a,b). Significantly less necrosis was observed in this group when it was compared to group III. In addition, changes related to cholestasis were also observed. Group III showed widespread confluent necrosis area (grade 3), as well as cellular mobilization and centrilobular fatty changes were observed (Fig. lc). Group IV showed normal liver.

Quantitation of APAP in plasma and liver tissue Plasma A P A P concentration over 24 hr after treatment with 2 g/kg i.p. of A P A P in groups II and III are shown in Fig. 2. As it can be seen plasma A P A P levels from group II were lower than group III (86.2 _+ 3.9°7o of decrease). On the other hand, liver A P A P accumulation in the same conditions are indicated in Fig. 3. In accordance with the former results, A P A P concentration in liver cholestatic rats showed a decrease of 66.7 _+ 6.2070 when compared to control group.

Biochemical tests Results obtained in serum biochemical tests are shown in Table 1 and 2. Biochemical marker enzymes of cholestasis, such as G G T and AP, and bilirubin are increased in cholestatic rats (group I and II) compared to controls (group IV) (Table 1) and the following increments were observed (expressed in fold over control values): TB: 45 and 50-fold, DB" 30 and 30-fold, GGT: 5 and 6-fold and AP: 5.5 and 4.5-fold, groups I and II, respectively. Serum pseudocholinesterase activity remained unchanged (data not shown). As a measure of hepatotoxicity, LDH, ALT and AST, were measured (Table 2). Paracetamol effect (group III) can be appreciated by the serum increment of cytosolic enzymes: LDH is elevated 2-fold, ALT 17-fold and A S T 6-fold over control values (group IV) as shown in Table 2. On the other hand, L D H and ALT showed a decrease in para-

Fig. 1. Histopathological findings in rat liver 24 hr after administered APAP in both, with or without experimental cholestasis. (a) Group II: low-power view shows focal necrotic areas (grade 1, arrowheads), scattered apoptotic cell (arrow) and cholestasis adjacent to the central vein (hematoxylineosin). (b) Group II: high-power view showing two apoptotic cells adjacent to normal hepatocytes (hematoxylin-eosin, x 128). (c) Group III: low-power view shows extensive areas of hemorrhagic necrosis (grade 3). There are no inflammatory cells surrounding the necrotic area (hematoxylin-eosin).

cetamol-treated cholestatic rats (group II) to about 50% and 30°/o, respectively, related to its control (group III).

DISCUSSION Paracetamol is an intermediate clearance drug; its clearance depends both on metabolism and blood sup-

1622

Cristina Acevedo et al. 1200-

1000

.-g

0

800

-

600

-~

400

200

0

_

L

Control

Cholestasis

Fig. 2. Plasma concentrations of APAP 24 hr after treatment with 2 g/kg i.p. of APAP in control (group III) and cholestatic (group II) rats. Each bar and vertical line represents the mean and SEM of the experiments. *P < 0.05 compared with the control.

ply to the liver. Extensive pharmacokinetic studies suggested that APAP might be a useful test drug to study glucuronidation (Prescott, 1980). Lipid composition of the microsomal membrane might play a role in some pathways of APAP metabolism and changes of the lipidic composition were reported in cholestasis (Bengochea et al., 1987). Despite these alterations the synthesis of UDP-glucuronic acid and of glucuronides are retained in acutely injured liver. Because APAP is metabolized by the liver, and this tissue is the major site of injury after overdose, it could be speculated that patients with liver disease may be at risk when using the drug. In theory, hepatotoxicity could develop if high

15

12

~

9

~

6

/] Control

Cholestasis

Fig. 3. Hepatic accumulation of APAP 24 hr after treatment with 2 g/kg i.p. of APAP in control and cholestatic rats. Each bar and vertical line represents the mean and SEM of the experiments. *P < 0.05 compared with the control.

blood levels of APAP occur because of delayed metabolism or if there is increased production of toxic metabolite. A variety of mechanisms have been put forward to account for the persistence of glucuronidation in liver diseas~ These include a) activation of latent glucuronyltransferase enzymes, b) the presence of extrahepatic glucuronidation which would be unaltered in liver disease (Hoyumpa et al., 1989), c) location of UDPglucuronyltransferase in parts of the liver lobule less affected by the liver disease, d) the reduction of enterohepatic recycling of the glucuronide, which might tend to increase the clearance of the parent drug and thereby offset the effect of decreased glucuronide formation (Ghabrial et al., 1986). Our biochemical, histological, serum and tissue APAP parameters show that during cholestasis APAPinduced toxicity is attenuated. The present study showed lower APAP concentrations in both liver and plasma from cholestatic rats; it could be due to a limited uptake in the parenchymal cells or higher capacity in the conjugation mechanism, as consequence of bile accumulation. Cholestatic phenomena observed in group I are also present in group II, but a minor degree of necrosis was evident in group II when it is compared to group III (grade 1 vs grade 3). It is well known that apoptotic phenomena is present when, in death cells, inflammatory mechanisms are involved. The presence of these cells in group II constitutes evidence for a different path of cellular injury absent or masked in group III. A possible explanation for cellular apoptosis might be related to the presence of cytokines such as tumoral necrosis factor and interleukines, since it has been reported that the inductive capacity of these molecules related to the expression and/or up regulation of adhesion molecules already described in other liver pathologies accompanies apoptotic phenomena (Ohlinger et al., 1993). Previous reports (Bengochea et al., 1985; 1987) demonstrated that in vitro APAP glucuronidation is increased in cholestasis, probably due to physicochemical changes of the membrane in which UDPGTs are located. On the other hand we might consider a possible role of P-glycoprotein. It is an energy dependent plasma membrane drug-efflux pump, capable of reducing the intracellular concentration of a variaty of hydrophobic xenobiotics. It is located on the bile canalicular domain of the hepatocyte, and it is encoded by mdrl, a member of multidrug resistent gene family (Endicott and Ling, 1989). Schrenk et al. (1993) demonstrated that obstructive cholestasis results in a pronounced increase in MDR gene expression in rat and monkey liver. These authors showed that the induction of mdrl a and

Cholestatic liver disease and A P A P

1623

Table 1. Biochemical cholestatic parameters (TB, DB, GGT and AP) in serum of cholestatic rats and their respective controls Paracetamol g/kg

Group I

-

II III IV

2 2 --

Treatment

n

cholestasis cholestasis ---

5 5 4 5

TB mg/dl 9 10 1 0.2

± ± ± +

DB mg/dl

2* 1" 0.2* 0.1

3 3 1 0.1

± ± ± ±

GGT IU/I

1" 1" 0.2* 0.02

5 6 1 1

± ± ± ±

AP IU/I

1" 1" 0.3 0.1

659 567 216 122

+ ± ± ±

9* 4* 9* 8

TB: total bilirubin; DB: direct bilirubin; GGT; gamma-glutamil transpeptidase; AP: alkaline phosphatase. Values are means ± SEM of n experiments. *p < 0.05 vs group IV.

Table 2. Cytolitic hepatic parameters (LDH, AST and ALT) in serum of cholestatic rats and their respective controls Group I II IIl IV

Paracetamol g/kg

Treatment

n

-2 2 -

cholestasis cholestasis -

5 5 4 5

LDH IU/1 344 243 502 220

+ + + +

36t 29* 27t 28

ALT IU/I 63 48 153 9

+ + + +

3t 4 *t 9 ~f 3

AST IU/1 295 285 290 47

± ± ± +

17i" 13"~ 91" 6

LDH: lactic dehydrogenase; ALT: alanine aminotransferase; AST: aspartate aminotransferase. Values are mean _+ SEM of n experiments. *P < 0.05 vs group III; t p < 0.05 vs group IV.

b in c h o l e s t a s i s is c o r r e l a t e d w i t h t h e d e g r e e o f c h o l e s t a sis as reflected in p l a s m a b i l i r u b i n levels, s u g g e s t i n g t h a t bile c o n s t i t u e n t s - still to be i d e n t i f i e d - m a y b e e n d o g enous transcriptional regulators of the mdrl genes and s u b s t r a t e s f o r t h e P - g l y c o p r o t e i n efflux p u m p . It is p o s sible t h a t A P A P b e c o m e s a s u b s t r a t e for P-glycoprotein d u r i n g c h o l e s t a s i s , p r o v i d i n g a m e c h a n i s m o f safety, c a p a b l e o f d i m i n i s h i n g A P A P t o x i c i t y in c h o l e s t a t i c livers. The evidence available suggests there may be multip l e m e c h a n i s m s i n t e r r e l a t e d in t h e p r o t e c t i v e effect o f c h o l e s t a s i s o n A P A P i n d u c e d cell d a m a g e . F u r t h e r s t u d i e s a r e n e c e s s a r y to d e t e r m i n e if prese n t d a t a a l s o s u g g e s t t h a t p a t i e n t s w i t h c h o l e s t a t i c liver d i s e a s e m a y t o l e r a t e larger d o s e s o f A P A P t h a n n o r mal individuals.

Acknowledgments-This work was supported by a grant 079/91 o f the University of Buenos Aires. REFERENCES

Bengochea L., Ouvifia G. a n d Lemberg A. (1985) Liver microsomal bilirubin UDP-glucuronyl transferase disturbances in bile duct ligated rats. Biochem. Biophys. Res. Comm. 130, 163-167. Bengochea L., Ouvifia G., Sozzani P. and Lemberg A. (1987) Protein and lipid distribution in rat liver microsomal membranes after bile duct ligation. Biochem. Biophys. Res. Comm. 144, 980-985. C u m m i n g A., King M. L. a n d Martin B. K. (1967) A kinetic study o f drug elimination. T h e excretion o f paracetamol and its metabolites in man. Br. J. Clin. Pharmacol. 29, 150-157. Douidar S. M., Boor P J. and A h m e d A. E. (1985) Potentiation o f the hepatotoxic effect o f acetaminophen by prior administration of salicylate. J. Pharmacol. Exp. Ther. 233, 242-248. Dutton G. J. (1966) The biosynthesis of glucuronides. In Gluc-

uronic Acid, Free and Combined (Edited by Dutton G. J.), pp. 187-199. Academic Press, New York. Endicott J. a n d Ling V. (1989) The biochemistry of P-glycoprotein mediated multidrug resistence. Ann. Rev. Biochem. 58, 137-171. Ghabrial H., D e s m o n d P. V., Watson K. J. R., Gilbers A. J., H o r m a n P. J. et al. (1986). T h e effects of age and chronic liver disease on the elimination of temazepam. Eur. J. Clin. Pharmacol. 30, 93-97. Gotelli G. R., Kabra P. M. and Marton L. J. (1977) Determination of acetaminophen and phenacetin in plasma by high pressure liquid chromatography. Clin. Chem. 23, 957. Hinson J. A., Pohi L. R., M o n k s T. J. and Gillette J. R. (1981) Acetaminophen-induced hepatotoxicity. Life ScL 29, 107116. Hinson J. A., Monks T. J., H o n g M., Highet R. J. and Pohl L. R. (1982) 3-(glutathion-S-yl) acetaminophen: a biliary metabolite o f acetaminophen. Drug Metab. Disp. 10, 47-50. H o y u m p a A., Brown M., J o h n s o n R., Troxell R. and Snowdy P. (1989) Effects of liver disease on the disposition of ciramadol in h u m a n s . J. Clin. Pharmacol. 29, 217-224. Jollow D. J., Mitchell J. R., Potter W. Z., Davis D. C., Gillette J. R. and Brodie B. B. (1973) Acetaminophen-induced hepatic necrosis. II. Role of covalent binding in vivo. J. Pharmacol. Exp. Ther. 187, 195-202. Jollow D. J., Thorgeirsson S. S., Potter W. Z., H a s h i m o t o M. and Mitchell J. R. (1974) Acetaminophen induced hepatic necrosis. VI Metabolic disposition of toxic and non-toxic doses of acetaminophen. Pharmacology 12, 251-271. Krauss J. W., D e s m o n d P. V., Marshall J. P., J o h n s o n R. E, Schenker S. et al. (1978) Effect of age a n d liver disease on disposition o f lorazepam. Clin. Pharmacol. Ther. 24, 411-419. Mitchell J. R., JoUow D. J., Potter W. Z., Davis D. C., Gillette J. R. and Brodie B. B. (1973) Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism. J. Pharmacol. Exp. Ther. 187, 185-194. Mulder G. (1992) Glucuronidation and its role in regulation o f biological activity of drugs. Ann. Rev. Pharmacol. Toxicol. 32, 25-49. Narang A. P. S., Datta D. V., Nath N. and Mathur V. S. (1981) Pharmacokinetic study of chloramphenicol in patients with liver disease. Eur. J. Clin. Pharmacol. 20. 479-483. Ohlinger W., Dinges H. P., Zatloukal K., Mair S., Gollowitsch E a n d Denk H. (1993) Immunohistochemical detection of

1624

Cristina Acevedo et al.

tumor necrosis factor, other cytokines and adhesion molecules in human livers with alcoholic hepatitis. Virh. Arch. A. Pathol. Anat. Histopatol. 423, 169-176. Ouvifia G., Pavese A., Lemberg A. and Bengochea L. (1993) Aumento de la capacidad de conjugaci6n del acetaminofen en colestasis experimental. Acta Gastroenter. Latinoamer. 23, 71-74. Ouvifia G., Lemberg A. and Bengochea L. (1994) Changes in liver drug glucuronidation during cholestasis are not predictable. Arch. Int. Physiol. Biochem. et Biophys. 102, 121-123. Potter W. Z., Davis D. C., Mitchell J. R., Jollow D. J., Gillette J. R. and Brodie B. B. (1973) Acetaminophen induced hepatic necrosis. III Cytochrome P-450 mediated covalent binding in vitro. J. Pharmacol. Exp. Ther. 187, 203-210. Prescott L. E (1980) Kinetics and metabolism of paracetamol and phenacetin. Br. J. Clin. Pharmacol. 10, 291S-298S. Schrenk D., Gant T., Preisegger K., Silverman J., Marino P. and Thorgeirsson S. (1993) Induction of multidrug resis-

tance gene expression during cholestasis in rats and nonhuman primates. Hepatology 17, 854-860. Secor J. W. and Schenker S. (1987). Drug metabolism in patients with liver disease. In Advances in InternaIMedicine (Edited by Stollerman), vol. 32, pp. 379-406. Year Book Medical Publishers, Chicago. Shull H. J., Wilkinson G. R., Johnson R. and Schenker S. 0976) Normal dispersion of oxacepam in acute viral hepatitis and cirrhosis. Annals Int. Med. 84, 420-425. Villeneuve J. P., Veerbeeck R. K., Wilkinson (3. R. and Branch R. A. (1986) Furosemide kinetics and dynamics in patients with cirrhosis. Clin. Pharm. Ther. 40, 14-20. Wilkinson G. R. (1986) Influence of hepatic disease on pharmacokinetics. In Applied Pharmacokinetics" Principles o f Therapeutic Drug Monitoring (Edited by Evans et aL), 2nd ed, pp 116-138. Applied Therapeutics, Spokane. Williams R. L., Upton R. A., Cello J. P., Jones R. M., Blitstein M. et al. (1984) Naproxen disposition in patients with alcoholic cirrhosis. Eur. J. Clin. Pharmacol. 27, 291-296.