Influence of agents affecting monooxygenase activity on taurolithocholic acid-induced cholestasis

Toxicology Letters, 63 (1992) 243-252 0 1992 Elsevier Science Publishers B.V. All rights reserved 03784274/92/$5.00

243

TOXLET 02804

Influence of agents affecting monooxygenase on taurolithocholic acid-induced cholestasis

Lena Dahlstriim-King’, D$artement

activity

Johanne Couture and Gabriel L. Plaa

de pharmacologic, Facultt+ de mddecine, Universitk de Montreal, Montreal, Quebec (Canada)

(Received 26 May 1992) (Accepted 25 August 1992) Key words: Cholestasis; Taurolithocholate;

Monooxygenase activity; Cytochrome P-450; Bile flow

SUMMARY In rats, pretreatment with certain ketones results in enhanced taurolithocholic acid (TLCA)-induced reduction in bile flow, whereas pretreatment with inhibitors of protein synthesis diminishes the effect on bile flow of cholestatic regimens. In the present study, the possible role of cytochrome P-450 in the ketone potentiation phenomenon was investigated. Male rats were pretreated with inducers or inhibitors of hepatic cytochrome P-450 and the impact of these pretreatments on TLCA-induced cholestasis assessed. Phenobarbital, 3-methylcholanthrene, chlordecone or mirex were used as inducers, and SKF 525-A, piperonyl butoxide, or cobaltous chloride as inhibitors of monooxygenase activity. Phenobarbital and 3-methylcholanthrene pretreatment enhanced TLCA-induced reduction of bile flow, while mirex and chlordecone were without effect. The three inhibitors of monooxygenase activity did not diminish TLCA-induced cholestasis. Instead, piperonyl butoxide and cobaltous chloride appeared to enhance the action of TLCA. Consequently, an increase in cytochrome P-450 (or specific isozymes) as a common denominator in the potentiation phenomenon appears unlikely. While hepatic proteins may play an important role in the potentiation of TLCAinduced cholestasis following pretreatment with ketones, the pattern of potentiation after pretreatment of rats with different inducers or inhibitors of cytochrome P-450 does not appear to implicate this family of proteins.

Correspondence to: Dr. Gabriel L. Plaa, Departement de pharmacologic, Faculte de mtdecine, Universite de Montreal, Montreal, Quebec, Canada H3C 357. ‘Present address: Parke-Davis Research Institute, Division of Warner-Lambert Speakman Drive, Mississauga, Ontario, Canada L5K lB4.

Canada

Inc., 2270

244

INTRODUCTION

Pretreatment of rats with ketones or ketogenic compounds has only a minimal effect on hepatic integrity or function; yet such pretreatments markedly alter the effect of several hepatotoxicants. The hepatonecrogenic properties of haloalkanes are potentiated [ 11; the cholestatic potentials of chloroform and carbon tetrachloride are unmasked [2-4]; and the cholestatic effects of two experimental regimens, a manganese-bilirubin (MnBR) combination and taurolithocholic acid (TLCA), are enhanced [3,5-71. Enhanced monooxygenase activity has been shown to be partly responsible for the potentiation of the necrotic effect of haloalkanes [8-121. Increased susceptibility of hepatocellular organelles, however, also seems to be implicated [13]. Pretreatment of rats with inhibitors of protein synthesis leads to a reduced response with several cholestatic regimens [14,15]. In contrast, ketones enhance the effects of these same cholestatic regimens. Since ketones can induce hepatic monooxygenases, the aim of the present study was to address the issue of the possible involvement of cytochrome P-450 in ketone potentiation of cholestasis. Animals were pretreated with inducers or inhibitors known to affect monooxygenase activity; the impact of these pretreatments on TLCA-induced cholestasis was then assessed. MATERIALS AND METHODS

Chemicals Chlordecone (99+% pure) was obtained from Applied Science Laboratory Inc. (State College, PA, USA) and mirex (98+% pure) from Chem Service (West Chester, PA, USA). Bovine serum albumin, cobaltous chloride, 3-methylcholanthrene (practical grade), piperonyl butoxide (90% pure), sodium taurolithocholate, urethane, and phenobarbital were purchased from Sigma Chemical Co. (St. Louis, MO, USA). SKF 525-A was a gift from SmithKline Beecham Pharmaceuticals (King of Prussia, PA, USA). Animals Sprague-Dawley rats from Charles-River Canada, Inc. (St-Constant, Quebec, Canada) were used in all experiments. The animals weighed between 200-300 g and were acclimated to the animal quarters (temperature 22 & 4°C and humidity 30-70%) with alternating 12-h light and dark cycles for at least 4 days prior to the start of the experiments. The rats were housed in wire-mesh bottom, stainless steel cages and had free access to pelleted rat food (Agway Pro-Lab No. 4020) and water. Experimental protocol Pretreatments. Phenobarbital (PB), 3-methylcholanthrene (3-MC), chlordecone or mirex were used as inducers and SKF 525-A, piperonyl butoxide (Pbut) or cobaltous chloride as inhibitors of monooxygenase activity. 3_Methylcholanthrene, chlordecone, mirex, and piperonyl butoxide were diluted in Mazola corn oil; pheno-

245

barbital sodium, SKF 525-A, and cobalt were dissolved in 0.9% NaCl. Compounds administered by gavage were given in a volume of 10 ml/kg; when the route of administration was i.p., the volume was 4 ml/kg, and S.C.injections were given in a volume of 1 ml/kg. PB (344 mmol/kg, i.p.) was administered daily for either 1, 3, or 5 days; 3-MC (74 mmol/kg, i.p.) was administered daily for 4 days; chlordecone (102 mmol/kg, p.o.) and mirex (92 mmol/kg, p.o.) were administered for 1 day. The TLCA cholestatic challenge (20 or 30 pmollkg, i.v.) was injected 18 h after the last dose of the inducer. Pbut (4.06 mmol/kg, i.p.) and SKF 525-A (103 mmol/kg, i.p.) were administered 1 h before the TLCA challenge. Cobalt (250 mmol/kg, s.c.) was given 24 h before the TLCA challenge. Measurement of bile Jrow. After the different pretreatments, the animals were anesthetized with urethane (1 g/kg, i.p.) and a femoral vein cannulated with polyethylene tubing PE-50, the common bile duct with PE-10, and the trachea with PE-240. Body temperature was monitored with a rectal thermoprobe (YSI Thermoregulator, Yellow Springs Instrument Co., Yellow Springs, OH, USA) and maintained at 37°C with a thermostatically controlled infrared lamp. TLCA was dissolved in 0.45% NaCl and 10% albumin to a final concentration of 10 PmoVml. TLCA was injected over 1 min into the femoral vein (24 ml/kg body weight depending on the dose) and the cannula flushed with a small amount of 0.9% NaCl. The bile was collected at 15- or 30-min intervals for 3 h. The volume was determined gravimetrically assuming a density of 1.O. Bile flows were calculated as a percentage change of the basal bile flow (calculated from the bile collected in a 15-min period prior to the injection of the cholestatic regimen). Statistical methods When appropriate, data were submitted to analysis of variance (ANOVA); comparisons of significant differences between the means were done using the StudentNeuman-Keuls procedure [16]. An CIof 0.05 was used as the level of significance. RESULTS

Effect of inducers on TLCA-induced reduction in bile flow The temporal effect of phenobarbital pretreatment on TLCA-induced bile flow reduction is illustrated in Figure 1. Phenobarbital pretreatment generally resulted in a more severely depressed bile flow; this was more evident with the smaller dose of TLCA. The effect, however, was statistically significant only following the 5-day administration. Figure 2 depicts the effects of 3_methylcholanthrene, chlordecone, and mirex pretreatment on the reduced bile flow produced by the same dosages of TLCA. 3-Methylcholanthrene enhanced the cholestatic effect of both doses of TLCA, while mirex and chlordecone had no effect on TLCA-induced bile flow reduction.

246

30 pmol TLCAJkg

20 pmof TLGNkg

PB 344 pmot/kg ip 18 h prior 0

+

SALINE

+

~E~B~JTAL

-20 -40

-60 -80 -100

PB 344 pmol/kg ip 3 days prior

PB 344 .wnoVkg ip 5 days prior

* ~~0.05 vs vehicle

-60 -80 -100

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0

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0

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*

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90

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TtME AFTER TLGA (min)

pretreatment on TLCA-induced reduction in bileflow.Each curve sents the mean t SE of six animals. ‘PsO.05 vs. vehicle-treated control.

Fig. 1. Effect of phenobarbital

repre-

Effect of inhibitors ON TLCA-induced reduction in bi~e~ow Figure 3 shows the effect of piperonyl butoxide, SKF 525-A and cobaltous chloride on TLCA-induced cholestasis. Piperonyl butoxide enhanced the effect of the larger dose of TLCA. SKF 525-A pretreatment, however, did not affect the response to TLCA. Pretreating the animals with cobalt enhanced the action of TLCA at 20 flrnoI/ kg but only at the two last collection periods.

20 pmol TLCA/kg

30 pmol TLWkg MX 92 pmolkg PO 18 h prior

+

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CD 102 pmol/kg po 18 h prior

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0

+

CORNOIL 344EMYLCHOLANiVRENE

p
-100 0

30

60

90

120

0

30

60

90

120

TIME AFTER Tl_CA (min) Fig. 2. Effect of mirex, chlordecone, and 3-methyicholanthrene

pretreatment on TLCA-induced reduction in bile flow. Each curve represents the mean + SE of six animals. ‘IQO.05 vs. vehicle-treated control.

Effect of the deferent pretreo~e~t~ on liver weight and basal bile~ow Table I summarizes the effects of the different pretreatments on liver weight, liverto-body weight ratio, as well as basal bile flow. With increasing duration of pheaobarbital pretreatment, there was an increase in liver weight and the liver-to-body weight

248

20 pmol

30 pmol

TLCAikg Pbut 4.06

pmol/kg

TLCAfkg

ip 1 h prior

0

-o-

CORNOIL

-

PIPERONYL BUTOXIDE

~~0.05 vs vehicle -20

-80

SKF 525-A 103 pmoi/kg ip 1 h prior -d-+

act

SALINE SKF525.A

-100

Co 250 pmol/kg SC24 h prior -

SALINE

+

COBALT

p.zO.05 vs vehicle

-8O-1907

0

1 30

.

, 6a

.

I 90

1 120

0

30

60

90

120

TIME AFTER TLCA (min) Fig. 3. Effect of piperonyl butoxide, SKF 525-A, and cobaltous chloride pretreatment on TWA-induced reduction in bile flow. Each curve represents the mean + SE of six animals. ‘PcO.05 VS. respective vehicletreated control.

ratio. The effect on basal bile flow was, however, maximal after a single dose of phenobarbital and did not augment with the duration of pretreatment. 3-Meth-

249

TABLE I EFFECT OF PRETREATMENT WITH INDUCERS AND INHIBITORS OF MONOOXYGENASE ACTIVITY ON LIVER WEIGHT AND BILE FLOW” Group

Liver weight (g)

Liver weight/ body weight

Basal bile flow @l/min/lOO g)

(%)

Controls PB18h PB 3 days PB 5 days

Controls Mirex

10.67 10.43 (-2.2) 11.86b (11.1) 13.17b (23.4)

7.50 8.04b (7.2)

Controls Chlordecone

8.00 7.32 (-8.5)

Phenobarbital pretreatment 3.67 3.99b (8.7) 4.34b (18.2) 4.40b (19.9) Mirex pretreatment 3.26 3.50b (7.4) Chlordecone pretreatment 3.28 3.14 (-4.4)

5.57 8.02b (58.2) 7.50b (47.9) 7.34b (44.8)

6.19 8.46b (36.7)

5.81 6.60 (13.6)

Controls 3-MC

3-Methylcholanthrene pretreatment 3.34 8.48 10.45b 4.22b (23.2) (20.8)

6.05 6.34 (4.6)

Controls Pbut

Piperonyl butoxide pretreatment 3.64 10.32 3.76 10.60 (3.3) (2.7)

5.61 8.82b (57.2)

Controls SKF 525-A

Controls Cobalt

7.85 7.62 (-2.9)

SKF 525-A pretreatment 3.38 3.34 (-1.2)

Cobaltous chloride pretreatment 3.60 9.63 3.47 8.71b (-9.5) (-3.6)

6.34 6.38 (1.1)

5.56 7.62b (37.0)

’ The values are the mean for the control group and a combined mean from groups receiving 20 or 30 pmol TLCAkg; % change from control is given in the parentheses. “Significantly different (P
250

ylcholanthrene caused an increase in liver weight without affecting basal bile flow. Increased liver weight and basal bile flow were observed after pretreatment with mirex, but not with chlordecone. There was a significant body weight loss over 24 h in animals pretreated with cobaltous chloride. The reduction affected liver weight as well, and the liver-to-body weight ratio remained unaltered. Pretreatment with both cobalt and piperonyl butoxide significantly increased basal bile flow. SKF 525-A pretreatment, on the other hand, did not affect basal bile flow. DISCUSSION

Methyl isobutyl ketone (MIBK) and methyl n-butyl ketone (MnBK) enhance the cholestatic response to TLCA or to a combination of manganese and bilirubin [5-71. In addition, they have been shown to be inducers of cytochrome P-450 in the rat [17-191. The pattern of induction of cytochrome P-450 by MIBK resembles that of MnBK [ 181 and phenobarbital [ 193. The purpose of the present study, therefore, was to assess whether the mechanism whereby ketones potentiate TLCA-induced cholestasis somehow involves hepatic cytochrome P-450 isozymes. The underlying working hypothesis was that the reduction in bile flow following TLCA is mediated in part by interaction with the enzymes. The effects of the different pretreatments employed in this study have been well characterized. Phenobarbital is an inducer of cytochrome P450IIB in the rat, while 3-methylcholanthrene induces P450IA [20]. Chlordecone and mirex induce catalytically different forms of cytochrome P-450 in the rat [21], and in the mouse there are indications they induce different isozymes of P-450 [22]. Administration of a single dose of chlordecone or mirex increases cytochrome P-450 activity in rats and mice [9,11,23]. Piperonyl butoxide and SKF 525-A inhibit monooxygenase activity by forming complexes with cytochrome P-450, while cobalt is a more generally acting inhibitor altering cellular heme metabolism [24]. If specific isozymes of cytochrome P-450 were involved in the potentiation of TLCA-induced cholestasis, one would expect that inducers of monooxygenase activity should enhance or exert little effect on TLCA-induced cholestasis itself, whereas inhibitors should diminish or prevent the reduction in bile flow following TLCA administration. In the present study, only phenobarbital and 3-methylcholanthrene enhanced the effect of TLCA; mirex and chlordecone pretreatment did not alter the response. Overall, the results obtained with these inducers are generally consistent with what we could expect. The results obtained with the inhibitors, however, are contrary to our expectations. The three inhibitors of cytochrome P-450 (piperonyl butoxide, SKF 525-A, and cobalt) did not reduce TLCA-induced cholestasis. Actually, two of the pretreatments appeared to enhance the action of TLCA. Consequently, overall, the evidence against cytochrome P-450 playing an important role in the TLCA potentiation phenomenon appears stronger than the evidence favoring a role for the enzyme(s).

251

The results of this study also show that pretreatment with structurally diverse chemicals can enhance TLCA-induced cholestasis. All compounds that acted as potentiators in these experiments were associated with an increase in liver weight and, except for 3_methylcholanthrene, an increase in basal bile flow. The effect on liver mass and bile flow occurred, as evidenced by a lack of effect on plasma alanine aminotransferase activity and bilirubin concentrations (data not shown), with little or no impact on liver function and hepatic integrity. In previous studies, pretreatment of rats with ketones enhancing the effect of cholestatic regimens also resulted in increased liver weight and bile flow [5-71. However, while phenobarbital administered for 3 days increased liver weight and bile flow, it did not enhance the response to TLCA. As well, a single dose of phenobarbital given for 18 h increased basal bile flow without affecting TLCA. Thus, increased liver mass and bile flow may be contributing factors in the potentiation phenomenon, but these characteristics alone do not account for the potentiation. We believe that hepatic proteins are involved in the potentiation of TLCA-induced cholestasis associated with ketone pretreatment, because (i) unaltered protein synthesis has been shown to be important in bile acid-induced cholestasis [14,15] and, furthermore, (ii) TLCA-induced cholestasis in MIBK-pretreated rats is less affected by inhibition of protein synthesis than it is in nonpretreated rats [25]. The pattern of response after pretreatment of rats with different inducers or inhibitors of cytochrome P-450, however, indicate that this family of proteins is unlikely to play a dominant role. Consequently, other hepatic proteins need to be considered. ACKNOWLEDGEMENTS

This study was supported by the Medical Research Council of Canada. L. Dahlstrom-King was supported by Fonds pour la formation de chercheurs et l’aide a la recherche, Quebec, Canada. REFERENCES Plaa, G.L. (1988) Experimental evaluation of haloalkanes and liver injury. Fundam. Appl. Pharmacol. 10, 5633570. Curtis, L.R., Williams, W.L. and Mehendale, H.M. (1979) Potentiation of the hepatotoxicity of carbon tetrachloride following preexposure to chlordecone (Kepone) in the male rat. Toxicol. Appl. Pharmacol. 51, 283-293. de Lamirande, E. and Plaa, G.L. (1981) 1,3-Butanediol pretreatment on the cholestasis induced in rats by manganese-bilirubin combination, taurolithocholic acid, or oz-naphthylisothiocyanate. Toxicol. Appl. Pharmacol. 59, 467475. Hewitt, L.A., Ayotte, P. and Plaa, G.L. (1986) Modifications in rat hepatobiliary function following treatment with acetone, 2-butanone, 2-hexanone, mirex or chlordecone and subsequently exposed to chloroform. Toxicol. Appl. Pharmacol. 83,465-473. Plaa, G.L. and Ayotte, P. (1985) Taurolithocholate-induced intrahepatic cholestasis: potentiation by methyl isobutyl ketone and methyl n-butyl ketone in rats. Toxicol. Appl. Pharmacol. 80,228-234. Vezina, M. and Plaa, G.L. (1987) Potentiation by methyl isobutyl ketone of the cholestasis induced in

rats by a manganese-bilirubin

combination

or manganese

alone. Toxicol.

Appl.

Pharmacol.

91. 477-

483. 7 Vezina, M. and Plaa, G.L. (1988) Methyl isobutyl induced

in rats by a manganese-bilirubin

ketone metabohtes

combination

or manganese

and potentiation

of the cholestasis

alone. Toxicol.

Appl. Pharmacol.

92.419427. 8 Sipes, LG., Stripp, B., Krishna. somal activity by pretreatment

G.. Maling. H.M. and Gillette, J.R. (1973) Enhanced hepatic microof rats with acetone or isopropanol. Proc. Sot. Exp. Biol. Med. 142,

237-240. 9 Cianflone,

D.J., Hewitt, W.R., Villeneuve,

alterations

of chloroform

D.C. and Plaa. G.L. (1980) Role of biotransformation

hepatotoxicity

produced

by Kepone

and mirex. Toxicol.

in the

Appl. Pharmacol.

53, 140-149. 10 Branchflower,

R.V. and Pohl, L.R. (1981) Investigation

form-induced

hepatotoxicity

and nephrotoxicity

ofthe

mechanism

by methyl n-butyl

of the potentiation

ketone.

Toxicol.

of chloro-

Appl. Pharmacol.

61.407413. 11 Hewitt,

L.A., Hewitt,

and rats treated 12 Hewitt.

L.A.,

acetone-,

W.R. and Plaa. G.L. (1983) Fractional

with chlordecone Valiquette,

2-butanone-,

01. Pharmacol. 13 Hewitt,

in mice

C. and Plaa.

G.L.

(1987) The role of biotransformation-detoxification

in

and 2-hexanone-potentiated

hepatic

Appl. Toxicol.

localization 3, 489495.

chloroform-induced

hepatotoxicity.

Can. J. Physi-

65,2313-2318.

L.A., Palmason,

C., Masson,

ganelles in the mechanisms 14 Yousef,

of “CHCI,

or mirex. Fundam.

I.M., Tuchweber,

cycloheximide,

S. and Plaa. G.L.

of ketone-potentiated B. and Weber,

an inhibitor

of protein

(1990) Evidence

chloroform-induced

A. (1983) Prevention

synthesis.

Principles

of or-

Liver IO, 3548.

of lithocholate-induced

cholestasis

by

Life Sci. 33. 103-l IO.

15 Dahlstrom-King, L. and Plaa, G.L. (1989) Effect of inhibition induced by taurolithocholate. lithocholate and a manganese-bilirubin Pharmacol. 38,254332549. 16 Winer. B.J. (1971) Statistical

for the involvement

hepatotoxicity.

in Experimental

of protein synthesis on cholestasis combination in the rat. Biochem.

Design. 2nd Edn., McGraw-Hill

Book Compa-

ny, Montreal. 17 Vtzina,

M., du Souich,

P., Jutras,

on cytochrome

P-450 oxidases.

Pharmacologist

M., Kobusch,

A.B., du Souich,

P., Greselin.

ketones 18 Vtzina,

form-induced

hepatotoxicity

L. and Plaa, G.L.

by methyl isobutyl

(1988) Inductive

properties

of aliphatic

mono-

30. A75. E. and Plaa, G.L. (1990) Potentiation

ketone and two metabolites.

of chloro-

Can. J. Physiol.

Pharma-

col. 68. 105551061. 19 Kobusch, A.B.. Plaa, G.L. and du Souich, P. (1989) Effects of acetone hepatic mixed-function oxidase. Biochem. Pharmacol. 38, 3461-3467. 20 Ioannides, chemical 21 Kaminsky,

C. and Parke, toxicity

D.V. (1987) The cytochromes

and carcinogenesis.

Biochem.

L.S., Piper, L.J., MacMartin,

cytochrome 22 Lewandowski.

M., Levi. P. and Hodgson,

Toxicol.

potentiation

24 Testa, B. and Jenner. P. (1981) Inhibitors Metab. Rev. 12, 1-l 17. 25 Dahlstriim-King. taurolithocholic

M.J. (1978) Induction

E. (1989) Induction

involved

in

of hepatic microsomal

43, 3277338.

of cytochrome relationships

of chloroform-induced of cytochrome

on

36.4197~4207.

Appl. Pharmacol.

and chlordecone. J. Biochem. Toxicol. 4, 1955199. 23 Hewitt, L.A., Caillt, G. and Plaa, G.L. (1986) Temporal detoxication, and chlordecone Pharmacol. 64,4777482.

rr-butyl ketone

P448 - a unique family of enzymes

Pharmacol.

D.N. and Fasco,

P-450 by mirex and Kepone.

and methyl

P-450 isozymes

between

biotransformation,

hepatotoxicity.

P-450s and their mechanism

L. and Plaa. G.L. (1987) Cycloheximide in methyl acid-induced cholestasis. Toxicologist 17, 58.

isobutyl

by mirex

ketone

Can.

J. Physiol.

of action. potentiation

Drug of