-,OYICOLOGY AND APPI.IED PHARMACOLOGY 59, 467-475 (1981)

1,3-Butanediol Pretreatment on the Cholestasis Induced in Rats by Manganese-Bilirubin Combination, Taurolithocholic

Acid, or cr-Naphthylisothiocyanate

EVE DE LAMIRANDE AND GABRIEL L. PLAA

I .3-Butanediol Pretreatment on the Cholestasis Induced in Rats by Manganese-Bilirubin Combination, Taurolithocholic Acid. or cu-Naphthylisothiocyanate. DE LAMIRANDE.

E., AND PI.AA. G. L. (1981). To.rico/. Appl. Pl~trrn~rcol. 59, 467-475. Haloalkane-induced hepatonecrogenesis can be potentiated by the prior administration of ketones (acetone, chlordecone) or ketogenic substances (1,3-butanediol, alloxan). We therefore investigated the possibility that the ketosis induced in rats by 1.3.butanediol (BD) would potentiate choles- tasis induced by manganese-bilirubin (Mn-BR) combination, taurolithocholic acid (TLC), and a-naphthylisothiocyanate (ANIT). In the Mn-BR studies. the depression of bile flow was more severe with 15 or 20 mgikg bilirubin and the threshold dose for manganese was also affected. Furthermore. the protection usually afforded by sulfobromophthalein (BSP) or phenoldibromophthalein disulfonate (DBSP) was lessened in the BD-pretreated group. Potentiation of cholestasis also occurred with TLC. and this was evident in the longer duration of the effect in the pretreated rats. Hyperbilirubinemia was more severe in BD- pretreated rats 74 hr after ANIT (50-200 mgikg) but not 14 or 18 hr after treatment. Two mechanisms, increased metabolism and increased cell susceptibility, are proposed to explain these effects. It is suggested that ketogenic substances can enhance cholestatic. as well as necrogenic. liver injury.

Many studies have been conducted con- and chlordecone (Kepone) (Hewitt et (I/.. cerning the potentiation of haloalkane- 1979; Curtis et (il., 1979). Alcohols that are induced hepatotoxicity. Microsomal enzyme metabolized to ketones, such as isopropanol inducers like phenobarbital (Garner and (Traiger and Plaa. 1971, 1972). 2-butanol McLean, 1969; Scholler, 1970) and 3-methyl- (Traiger and Bruckner, 1976), and 1,3- cholanthrene (Lavigne and Marchand, 1974) butanediol (Hewitt and Plaa, 1979), also can potentiate the hepatotoxic response to potentiate haloalkane toxicity. Furthermore, haloalkanes: increased synthesis of reactive the diabetic state produced by alloxan or metabolites is thought to be responsible for streptozotocin also leads to a potentiated this potentiation. The potentiating agents response (Hanasono et rrl., 1975). It has seem to be structurally quite diverse. been proposed that the generation of However, several of them have a common abnormal quantities of ketonic substances structural component. a cdrbonyl moiety: within the liver increases the sensitivity of acetone (Traiger and Plaa, 1972), 2-butanone laboratory animals to the hepatotoxic (Traiger and Bruckner, 1976). 2-hexanone effects of haloalkane solvents (Hewitt et al., and 2,5-hexanedione (Hewitt et rrl., 1980a). 1979, 1980a,b).

467 0041-008X/8 l/090467-09$02,00/O Copyright ( 1981 by Acadrmlc Prc\\. Inc. 411 right\ of rrproduction I” any form r~~vsrved

468 DE LAMIRANDE AND PLAA

All the previously mentioned studies concern potentiation of the known necrotic properties of haloalkanes. However, it has recently been shown that low doses of car- bon tetrachloride (Ccl,) administered to rats pretreated with chlordecone cause severe cholestasis in addition to necrosis (Curtis ei 111.. 1979). This result is particularly sur-

prising since high doses of Ccl,, given alone, do not exhibit marked cholestatic effects. This finding suggests that the pre- treatments mentioned above might possibly increase the sensitivity of laboratory ani- mals, not only to necrotic but also to cho- lestatic lesions. We therefore investigated the possibility that the ketotic state in- duced by 1,3-butanediol pretreatment would potentiate the cholestatic responses ob- served in two different models of experi- mental cholestasis: manganese-bilirubin (Mn-BR) and taurolithocholic acid (TLC).

Each of these models of cholestasis has its own characteristics. The Mn-BR model was first introduced by Witzleben et ul. ( 1968) and further developed by de Lamirande and Plaa (1978, 1979a,b). It was noted that the combination of low and noncholestatic doses of manganese and bilirubin produces a rapid and reversible reduction in bile flow if the substances are injected in the proper sequence and during a certain time interval: manganese seems to act through an inter- mediate, possibly the canalicular mem- brane. In the TLC model, the cholestatic response is rapid, reversible and dose re- lated (Priestly et (11.. 1971); intracanalicu- lar precipitation (Javitt and Emerman, 1968) or canalicular membrane alteration (Kakis and Yousef, 1978) appears to be responsible for the cholestasis.

After considering the results obtained with the two models, we decided to perform preliminary studies with a third model of cholestasis, cu-naphthylisothiocyanate (ANIT). The object was to see if the poten- tiation with the other two readily controlled models might also be evident in the more complicated ANIT system. With ANIT the

cholestasis and hyperbilirubinemia are dose dependent, but appear after a latent period of many hours; the effect is probably caused by a toxic degradation product and the lesion is reversible only after a number of days (Eliakim et al., 1959: Plaa and Priestly, 1976).

We chose 1.3-butanedioi (BD) as the pre- treatment since it is relatively nontoxic in rats (LDSO = 30 g/kg, p.0.) and is me- tabolized to ketone bodies (P-hydroxybu- tyrate and acetoacetate) within the liver of rats after oral administration (Mehlman et ul., 1975). Furthermore, BD has been considered for use as a synthetic source of dietary calories for humans and animals (Miller and Dymsza, 1967: Mehlman et ul., 1975: Tobin et crl., 1975). Hewitt et ul. (1979, 1980b) observed that BD pretreat- ment could potentiate Ccl, toxicity in rats. It is therefore important to inquire whether BD will increase the frequency or the severity of hepatotoxic responses (necrosis and/or cholestasis) due to drugs or chemicals.

METHODS

Animuls. Male Sprague-Dawley rats (Canadian Breeding Farm Ltd.) weighing 170 to 300 g were maintained on Charles River Rat Chow and water ud lihirrrm until pretreatment began. BD (10% solu- tion in water) was then substituted for the tap water, for 7 days, in pretreated animals. From the amount of water consumed. it was estimated that the rats in- gested about 10 g/kg/day of BD. Control rats still re- ceived tap water. Production of ketosis by BD pre- treatment was confirmed by urinalysis with Ames Co. Labstix.

On Day 8, the animals were anesthetized with sodium pentobarbital (60 mgikg, i.p.) and catheters were placed in the common bile duct (PE-IO tubing) and in a femoral vein (PE-50 tubing). Anesthesia was maintained throughout the biliary secretion studies. Body temperature was monitored via a rectal probe (YSI Thermoregulator) and maintained at 37°C by means of an infrared lamp to eliminate temperature- dependent changes in bile flow (Roberts rr ul., 1967). Bile was collected over 15 or 30.min periods and meas- ured volumetrically.

7r~tmrrrt.s. Each treatment described below was administered to BD-pretreated and to nonpretreated rats.

Monohydrated manganese sulfate tMnS0, H,O)

1,3-BUTANEDIOL ON EXPERIMENTAL CHOLESTASIS 469

was dissolved in 0.9% NaCl and injected intravenously (7.5, 15, or 30 mg/kg and 2.25, 4.5, or 9.0 mg Mnikg, respectively) 15 min after beginning the bile secretion study. Freshly prepared aqueous solutions of bilirubin (0.52 g NaCl, 0.52 Na&03, and 0.5 g bilirubin per 100 ml) were injected intravenously (15 to 40 mg/kg) alone or 15 min after manganese administration. For the pro- tection studies, sulfobromophthalein (BSP; Dade Laboratories; 15 mg/kg) or dibromosulphthalein (DBSP; Societi d’etudes et de recherches biologiques, Paris; 6.25 or 12.5 mgikg) in 0.9% NaCl was injected intravenously 5 min after manganese administration. Another group received the vehicles of all three sub- stances.

Sodium taurohthocholate was dissolved in a vehicle containing 10% albumin, 5% dextrose, and 0.45% NaCl (Javitt. 1966) and injected (15, 20, or 25 mgfkg and 29.6, 39.5, or 49.4 PmoUkg, respectively) intra- venously. Another group received an equivalent volume of the vehicle.

ANIT was dissolved in corn oil and injected (50- 200 mg/kg, i.p.) on Day 7 (14, 18, or 24 hr before the cannulations). Another group received corn oil alone. After the bile flow studies the animals were decapi- tated and blood was collected. Serum bilirubin con- centration (total and conjugated) was determined by Nosslin’s modification (1960) of the method of Jen- drassik and Grof (1938).

Sfuristics. Data were submitted to statistical analysis using either Student’s t test or an analysis of variance, followed by the Student-Newman-Keuls test (Sokal and Rohlf, 1969). The 0.05 level of proba- bility was used as the criterion of significance.

RESULTS

Mn -BR Studies

We observed that 1,3-butanediol pre- treatment alone had no effect on bile flow. Manganese (4.5 to 9.0 mg/kg) or bilirubin ( 15 to 40 mg/kg) given alone did not influence bile flow either in BD-pretreated or nor- mal rats.

In all nonpretreated rats combination of the two treatments, Mn(4.5 mg/kg) plus bilirubin (BR), produced a fall in bile how. The magnitude of the decrease depended upon the dose of BR and the maximal ef- fect was always observed 60 min after BR injection.

BD pretreatment produced some modifi- cation of this response. With 15 mg/kg BR

A Mn + BR (iSmg/kg)

4 t kl 8R

MntBR (20mg/kg)

Mn * BR (25mglkg)

Mn BR

30 60 90 120 150 TIME (mm)

FIG. I. Effects of 1,3-butanediol (BD) pretreatment on cholestasis induced by manganese-bilirubin com- bination. Manganese (4.5 mg Mnikg) was injected 15 min before bihrubin (BR) at (A) 15 mg/kg, (B) 20 mgikg, and (C) 25 mgikg. Each point represents the mean ? SE for 6 to 10 rats. (-.-) BD alone: (-) Mn-BR; (- - -) BD + (Mn-BR). (*) Significantly dif- ferent from initial 0- to 15-mitt value (0- to 15-min period) in the same experiment. ( $) Bile flow fall (‘%) significantly different between BD-pretreated and normal rats.

(Fig. lA), bile flow decreased in the same manner in all animals for the initial 60-min period, but as the nonpretreated group rapidly recovered, the BD-pretreated group worsened. The maximal fall in bile flow

470 DE LAMIRANDE AND PLAA

TABLE I

EFFECT OF 1 ,3-BUTANEDIOL ON BSP AND DBSP PROTECTIVE EFFECT IN MANGANESE-BIURUIHN-INDLKED CHOLESTASIS”

Bile Bow decrease (9)?

Treatment” Without BD

Mn (4.5 mgikg) + BSP (15 mgikg) + BR (25 mgikg) 1.1 -c 3.2 (6) Mn (4.5 mgikg) + DBSP (6.25 mgikg) + BR (25 mgikg) 12.2 r 8.4 (9) Mn (4.5 mgikg) + DBSP (12.5 mgikg) + BR (25 mgikg) 19.0 i_ 7.5 (10)

With BD

24.0 5 6.4” (5) 52.6 t 10.1* (6) 34.5 i 9.1 (5)

’ Each value represents the mean t SE, and the number of rats in each group is indicated in parentheses. ’ Injection times of manganese (Mn). BSP or DBSP, and bilirubin (BR) were 15,20, and 30 min. respectively,

after beginning biliary secretion study. All treatments were administered to 1.3-butanediol (BD)-pretreated rats and to normal rats.

r Bile flow was always measured 60 min after bilirubin injection. * Significantly different from nonpretreated group (p < 0.05).

(52.2 2 3.6%,p < 0.05) wasreached90min after BR injection and cholestasis was still present 30 min later. With 20 mg/kg BR (Fig. lB), bile flow decreased more rapidly in BD-pretreated than in normal rats; 30 min after BR injection, bile flow decreased 55.7 * 5.2 and 19.3 * 9.4%, respectively (p < 0.005). Cholestasis was still more severe in BD-pretreated rats 60 min after BR in- jection. The Mn(4.5 mg/kg)-BR(25 mg/kg) combination produced similar bile flow curves (onset, severity, and recovery) in BD-pretreated and in normal rats (Fig. 10.

de Lamirande and Plaa (1978) observed that while the size of the manganese load does not influence the severity of the Mn- BR cholestasis, a threshold dosage (2.25 mg/ kg) for manganese does exist. BD pretreat- ment also modified the response to the threshold dose for Mn. In the BD-pre- treated rats, 2.25 mg/kg of Mn was suf- ficient to induce a 52.7 r 9.8% fall in bile flow 60 min after BR (25 mg/kg) injection. In the nonpretreated group this treatment schedule effected only a 25.2 2 7.5% fall in bile flow; the difference between the BD- pretreated and nonpretreated groups was statistically significant (p < 0.05).

Intravenous administration of BSP af- fords protection against Mn-BR cholesta- sis (Witzleben and Boyce, 1975a,b; de

Lamirande and Plaa, 1978, 1979a,b). This protective effect was lessened in BD- pretreated rats: 15 mg/kg BSP did not totally prevent Mn(4.5 mg/kg)-BR(25 mg/kg) cho- lestasis as it did in normal rats (Table 1). This could be the result of an accelerated BSP conjugation, since BSP-glutathione (BSP-GSH) affords no protection against Mn-BR cholestasis (de Lamirante and Plaa, 1978). We therefore replaced BSP by one of its analogues, DBSP; this substance is excreted in bile without biotransforma- tion but in all other respects resembles BSP (Klaassen and Plaa, 1968). As shown in Table 1, the dosage of DBSP had to be in- creased from 6.25 to 12.5 mg/kg to abolish the statistically significant difference ob- served between BD-pretreated and normal rats. However, cholestasis was still present (bile flow fall, 34.5 i 9.1%) in BD-pre- treated rats. Thus, BSP and DBSP ex- hibited diminished protective effects in BD- pretreated rats.

TLC Studies

Table 2 shows that BD pretreatment potentiated the cholestasis observed I5 and 4.5 min after TLC injection with all three TLC dosages used. The times for recovery were also much longer in BD-pretreated

1,3-BUTANEDIOL ON EXPERIMENTAL CHOLESTASIS 471

TABLE 2

E~FEC~SOF 1,3-BUTANEDIOLPRETREATMENTON TAUROL.ITHOCHOLIC ACID-INDUCED CHOLESTASIS"

Time after TLC

,Clll"b TLC &se (mgiksl

Bile Row decrease (%I”

WIthout BD With BD

I? 15 35.4 f 3.6(11) 54.3 i- 4.4’ (14, 20 52.0 + 4.2 (91 69.2 t 4.8’ (61 25 67.3 2 1.9 161 75.? z 2.6’ (61

45 IS 8.2 f 3.7 37.2 z 5.8* 20 31.2 z 3.5 46.8 f 5.3* 25 36.8 t_ 6.0 63 5 f_ 9.0’

105 IS 9.4 z 3.4 24.1 t 6.2* 20 20.9 z 4. I 20.1 r 2.2 25 21.2 2 5.2 51.6 2 9.7;

” Each value represents Ihe mean 2 SE. and the number of rats in each group I\ indicated in parentheses.

’ Bile Row was measured IS. 45. and 105 m m aher TLC injectmn. * Significantly ddTerent from nonpretreated group (p < 0.051

rats that received 15 or 25 mg/kg of TLC (Table 2). Figure 2 depicts the bile tlow curves obtained after the injection of 25 mg/kg TLC. Cholestasis was more severe in BD-pretreated animals 15 to 105 min after treatment.

A NIT Studies

With two dosages tested (150 and 200 mg/kg), bile flow recorded 24 hr after ANIT treatment was very low and this parameter could not be used to differentiate normal and BD-pretreated rats (Table 3). The bile was colorless in both cases. However, BD- pretreated rats exhibited a more severe hyperbilirubinemia than did normal rats 24 hr after ANIT treatment (Table 3). Both total and conjugated bilirubin concentra- tions were increased, but the proportion of unconjugated bilintbin remained the same in all groups. At 14 and 18 hr after ANIT treatment the BD-pretreated and normal rats could not be differentiated on the basis of bile flow and hyperbilirubinemia (Table 3). Other groups of rats were treated with varying dosages (50-200 mg/kg) of ANIT and total plasma bilirubin concen- trations measured 24 hr later. The BD-

pretreated rats again exhibited a more severe hyperbilirubinemia (Table 4).

DISCUSSION

The results presented in this study show that rats pretreated with 1,3-butanediol, a ketogenic substance, are more responsive to the cholestatic effect of Mn-BR com- bination and TLC.

Manganese given alone in large doses causes cholestasis in rats (Witzleben et al., 1968). Furthermore, bilirubin alone can cause cholestasis in monkeys (Gartner el nf., 1971). However, we observed that the bile flow of BD-pretreated rats was unaffected when manganese (2.5 to 9.0 mg/kg) or bilirubin (15 to 40 mg/kg) was given alone.

Combination of the two treatments (man- ganese and bilirubin) results in cholesta- sis. The intensity and duration of the cholestasis depend upon the dose of biliru- bin administered (de Lamirande and Plaa, 1978, 1979b). In the present experiments the cholestasis was more severe and/or lasted longer with the two lower dosages of bilirubin in BD-pretreated rats. Further-

FIG. 2. Effect of 1.3.butanediol (BD) pretreatment on taurolithocholic acid-induced cholestasis. Tauro- lithocholic acid (TLC), 25 mg/kg, was injected at zero time. Each point represents the mean 4 SE for six rats. (6--) BD vehicle: (-.-) TLC: (---) BD + TLC. (*) Significantly different from the initial value in the same experiment. ($ ) Bile flow fall (%;) significantly different between BD-pretreated and normal rats.

472 DE LAMIRANDE AND PLAA

TABLE 3

EFFECT OF I,%BUTANEDIOL PRETREATMENT ON ANIT-INDUCED HYPERBILIRUBINEMIA

AND DIMINUTION OF BILE FLOW”

Treatment Bile flow

(Fl/min/lOO g)

Total plasma Conjugated plasma bilirubin bilirubin

(mg/IOO ml) (mg/lOO ml)

Vehicle BD + vehicle ANIT (200 mgikg), 14 hr BD + ANIT (ZOO mg/kg), 14 hr ANlT (200 mglkg). 18 hr BD + ANIT (200 mg/kg), 18 hr ANIT (200 mgikg), 24 hr BD + ANIT (200 mg/kg), 24 hr ANIT (150 mgikg), 24 hr BD + ANIT (150 mgikg). 24 hr

7.78 t 0.65 (5) 0.094 i 0.008 (5) 0.028 c 0.004 (5) 7.87 2 0.32 (5) 0.051 k 0.010 (51 0.028 ‘- 0.007 (61 8.09 k 0.74 (4) 0.39 + 0.05” (41 0.17 i 0.03* (4) 6.14 2 0.89 (5) 0.35 + 0.10* (6) 0.18 -t 0.07* (6) 3.83 f 1.62* (6) 1.18 f 0.41* (6) 0.80 i 0.?7* (6) 3.00 2 0.91* (6) 1.09 -t 0.30* (6) 0.62 I? 0.18” (6) 0.65 2 0.23* (4) 1.93 rf- 0.24* (6) 1.15 5 0.16’ (6, 1.17 ? 0.63* (6) 3.23 t 0.33** (9, 2.35 ? 0.36”* (61 1.17 -c 0.52” (5) 1.30 2 0.19” (4) 0.90 2 0.15; (41 1.99 t 0.81* (9) 2.32 +- 0.20** (1 I) 1.66 z 0.14?” (10)

n Each value represents the mean t SE, and the number of results in each experiment is indicated In parentheses. The parameters were measured 14. 18, or 24 hr after treatment with ANIT.

* Sigificantly different from corresponding vehicle or BD + vehicle value. ** Significantly different from BD + vehicle value and from nonpretreated ANIT value.

more, the threshold dosage of manganese appeared to be lower in BD-pretreated rats. The protection offered by BSP and DBSP was also lessened in BD-pretreated animals. Thus, BD appears to potentiate the cholesta- tic effect of Mn-BR combination: The

TABLE 4

EFFECT OF I,3-BUTANEDIOL PRETREATMENT ON

THE HYPERBILIRUBINEMIC RESPONSE TO VARIOUS

DOSAGES OF ANIT AT 24 hr”

Dose (m&g)

200 150 100 75 50

Total plasma bilirubin (mg/lOO ml)

ANIT BD + ANIT*

2.52 -t 0.28 3.02 + 0.28 1.95 3 0.33 2.91 f_ 0.39 1.96 5 0.29 2.74 2 0.18 1.28 ? 0.17 1.84 ? 0.37 0.95 i- 0.27 1.66 i 0.45

1* Each value is the mean 2 SE of six rats. The values for the rats treated with the vehicle or BD + vehicle were 0.12 2 0.01 and 0.10 + 0.01 mgilO0 ml, re- spectively.

* An analysis of variance indicates that the 1.3. butanediol pretreatment groups are significantly different (p < 0.05) from the nonpretreated groups.

diminution in bile flow is more severe, the threshold dosage of manganese is altered, and the protective effects of BSP and DBSP are lessened.

The taurolithocholic acid (TLC) choles- tatic model is characterized by the rapidity, the reversibility, and the dose dependence of the effect. With the three dosages of TLC used, the decrease in bile flow was more pronounced and lasted longer in BD-pre- treated rats. In this regard, the effect of BD on TLC-induced cholestasis resembles the effect previously observed with the Mn-BR model.

In the case of ANIT our studies in- dicate that the situation might be more com- plex. When bile flow was measured, no in- dication of BD potentiation was evident. Yet, the hyperbilirubinemic response to ANIT was significantly enhanced in BD- pretreated rats at 24 hr. ANIT exerts an effect on the hepatocyte and on the bile ductular cells; it also affects bilirubin syn- thesis and disposition (Plaa and Priestly. 1976). It is not clear that the increased hyperbilirubinemia observed with BD pre- treatment actually reflects the potentiation

I ,3-BUTANEDIOL ON EXPERIMENTAL CHOLESTASIS 473

of ANIT cholestasis. To clarify this situa- tion a number of additional experiments would be required. Furthermore, recovery from ANIT-induced cholestasis needs to be investigated in BD-treated animals. How- ever, the enhanced hyperbilirubinemia ob- served certainly is encouraging and in- dicates that a more intensive investigation of the ANIT model is warranted.

There is some indication in the literature that ketonic or ketogenic substances appear to increase the cholestatic potential of other chemicals. Plaa et crl. (197.5) observed that, after a pretreatment with isopropanol or its metabolite, acetone. the hyperbilirubinemic response to a high dose of ANIT (300 mg/kg, p.o.) was significantly enhanced. Isopro- panol pretreatment was also capable of ele- vating plasma bilirubin concentrations after CCI, exposure (Traiger and Plaa, 1971). Re- cently Curtis rt al. (1979) have shown that, after pretreatment with chlordecone. a ketone, low dosages of Ccl, caused necro- sis as expected, but also exhibited im- portant cholestatic properties.’ These last two experiments are particularly interest- ing. since hyperbilirubinemia and choles- tasis are not prominent features of Ccl, hepatotoxicity in rats.

Previous reports (Hewitt and Plaa, 1979; Hewitt ct cl/., 1980b) show that BD pre- treatment potentiates the Ccl,-induced hepatonecrogenic response. The data pre- sented in this study extend the potentiating property to two quite different models of

’ We performed an experiment with isopropanol

(2.5 ml/kg. IX hr before Ccl,) and Ccl, (0.1 ml/kg)

and measured bile flow to see if cholestasis be-

came evident during the potentiation. The bile flow

was compared to that observed in a group treated

with a large dose of CCI, (I.0 ml/kg) given alone.

The values recorded (~llmin/lOO g) were: isopropanol.

II.24 + 0.72: Ccl, (0.1 ml/kg), 9.73 +- 0.61: Ccl,

( I .O ml/kg). 9.23 2 0.88: isopropanol i- Ccl, (0. I ml/

kg). 4.32 i- 0.73 (/I < 0.05 vs all other values). Thus,

isopropanol-treated rats did exhibit diminished bile

flow when challenged with the small dose of Ccl,, yet

no change in bile flow was observed when the large

dose of Ccl, was given alone.

cholestasis: Mn-BR and TLC. It is of in- terest to see if the two mechanisms formu- lated (Cot.5 rt al., 1974: Hewitt et al., 1979, 1980b; Cianflone et uf., 1980) to ex- plain the potentiation of haloalkane hepa- totoxicity can be used to explain our results with the cholestatic models. The first hy- pothesis proposes that the potentiation is consecutive to an enhancement of bio- transformation leading to the production of toxis metabolites. The second hypothesis proposes that the potentiation is consecu- tive to an increased susceptibility of the subcellular organelles of the hepatocyte.

Both hypotheses can be used to explain the potentiation of Mn-BR cholestasis seen after BD pretreatment. It appears that Mn- BR cholestasis proceeds via an intermediate (de Lamirande and Plaa, 1978, 1979a,b). Furthermore, the cholestasis depends upon the dose of bilirubin used. Potentiation by BD could be due to an increased amount of bilirubin available for the bilirubin-inter- mediate reaction. This could explain why potentiation was not observed with the highest dose of bilirubin, since the maxi- mal effect was already attained even with- out pretreatment. The second explanation would be that an increase in cell sus- ceptibility to manganese (facilitated forma- tion of the intermediate) occurs or that the subsequent reaction of the intermediate with bilirubin is facilitated. This hypothesis is supported by the observed modification of the Mn threshold dosage needed to ini- tiate the response.

In the TLC model it is difficult to invoke increased biotransformation to explain the observed potentiation since TLC is ex- creted unchanged in bile. Acceleration of TLC uptake and/or transport to the site of action is conceivable. However, increased cell susceptibility appears to be a more at- tractive mechanism. Kakis and Yousef (1978) proposed that TLC-induced choles- tasis is caused by an alteration of canalicular membrane structural integrity; incorpora- tion of bile acids and changes in the choles-

474 DE LAMIRANDE AND PLAA

terol:phospholipid ratio would hinder nor- mal membrane activity. BD pretreatment possibly affects canalicular membranes and thus facilitates the action of TLC and the membranes.

The ANIT model is quite different from the Mn-BR or TLC models. ANIT is bio- transformed to a toxic metabolite through the MFO system before it exerts its cholestatic properties. Hyperbilirubinemia is marked 24 hr after ANIT administration but still increases to reach a maximum on Day 5 after the treatment. This response is modi- fied by altering MFO enzymic activity (Plaa and Priestly, 1976). Phenobarbital acceler- ates and increases ANIT effects (Roberts and Plaa, 1965). In the present study the BD-induced increase in MFO enzymic ac- tivity could be responsible for the poten- tiation of hyperbilirubinemia observed at 24 hr. However, the lack of different responses at 14 and 18 hr argues against this hypothesis, although it cannot be totally rejected. On the other hand, the data ap- pear to be consistent with the increased cell susceptibility hypothesis as well. The ANIT metabolite may not be formed more rapidly or in greater amounts, but once formed, its reaction with the more sus- ceptible hepatic cell would provoke greater effects.

In all three models, the BD effect can be explained by increased metabolism (uptake, transport, or biotransformation) of the in- jected substances or by increased cellular (especially membranes) susceptibility to these substances. Since these two mecha- nisms have been invoked to explain poten- tiation of haloalkane-induced hepatotox- icity after pretreatment with ketonic or ketogenic substances, we suggest that a common cellular alteration could be the first event for two quite different hepatotoxic manifestations, necrosis and cholestasis.

We can also wonder whether the poten- tiation of cholestasis observed after BD pretreatment bears some relationship to situations likely to occur in humans. Tobin et N/. (1975) did not note ketonemia

in human subjects fed with BD-enriched bread (5% of the total daily caloric intake: 15 g BD/subject/day: 0.2 g/kg/day). How- ever, little is known about the relationships among ketonemia, intrahepatic ketosis, and potentiation of cholestasis. Further- more, if BD is used for parenteral nutri- tion, the dose administered would probably be higher and simultaneous to various treatments, possibly including drugs that can induce cholestasis. It appears that this possibility warrants further investigation.

ACKNOWLEDGMENTS

This work was supported by a grant and a student- ship from the Medical Research Council of Canada. The expert technical assistance of Miss The&e Vaillancourt is gratefully acknowledged,

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