The effect of α-naphthylisothiocyanate on bile secretion prior to and during the onset of cholestasis in the rat

Toxicology Letters, 10 (1982) 427-435 Elsevier Biomedical Press

427

THE EFFECT OF a-NAPHTHYLISOTHIOCYANATE ON BILE SECRETION PRIOR TO AND DURING THE ONSET OF CHOLESTASIS IN THE RAT*

SIMON LOCK**, JOCELYN LAVIGNE** and GABRIEL L. PLAA Dkpartement de Pharmacologic, Facultk de MPdecine et FacultP des Etudes Supkieures, Universitt! de Montrt!al, Montrt?al, Quebec (Canada H3C 357) (Received October lst, 1981) (Accepted October 8th, 1981)

SUMMARY The effect of cu-naphthylisothiocyanate (ANIT) on bile flow, erythritol clearance, bile acid excretion and bilirubin excretion was studied in rats. The median time-to-effect (Et50) for the appearance of cholestasis was about 15 h. ANIT failed to exert a gradual effect on bile flow or erythritol clearance before the onset of cholestasis. However, 3 h before complete cessation of bile flow, a rapid decline in bile flow and bile acid excretion was observed. Bile acid-independent flow was markedly reduced. However, the bile acid-dependent component was also affected.

INTRODUCTION

ANIT, when administered acutely, is a useful model substance for studying the pathogenesis of chemically induced intrahepatic cholestasis [l]. The onset of hyperbilirubinemia appears to occur between 12 and 15 h after treatment. Bilirubin uptake and excretory mechanisms are also adversely affected by ANIT; this effect increases with the time following ANIT treatment. In rats, complete cessation of bile flow occurs by 24 h after oral administration of ANIT [2]. Recently, Fukumoto et al. [3] demonstrated that at 18 h the decrease in bile flow was about 30%; they showed that the bile acid-independent component was depressed at this time.

*Supported by the Medical Research Council of Canada. **Present addresses: (S.L.) Biology Division, Oak Ridge National Laboratories, Oak Ridge, TN (U.S.A.); (J.L.) Departement de Sante Communautaire, Hopital Saint-Luc, Montreal, Quebec (Canada). Abbreviations: ANIT, ol-naphthylisothiocyanate; effect.

CMC, carboxymethyl cellulose; ET50, median time-to-

037%4274/82/000&OCOO/$O2.75 0 Elsevier Biomedical Press

428

These observations imply that toxic effects commence within a short time after administration of ANIT. Since the hepatocyte is one site of ANIT toxicity [I, 31, the purpose of the present study was to see whether early changes in canalicular bile formation could be detected prior to the onset of cholestatis. The temporal effects of ANIT on total bile flow, canalicular bile flow, bile acid excretion and bilirubin excretion have been investigated. MATERIALS

AND METHODS

materials ANIT (Eastman Kodak Rochester, NY), either suspended in IS% CMC or dissolved in corn oil; [14C]erythritol (specific activity 1.8 mCifmmo1, 14.5 &i/mg; (Amersh~/Searle Corp. ~lington Heights, IL) dissolved in 0.9~0 NaCI to give a concentration of 10 &i/ml; hydroxysteroid dehydrogenase (Worthington Biochemicals Corp. Freehold, NJ); bile salts and NAD+ (Sigma Chemical Co. St. Louis, MO); and the Monitor-Jendrassik Bilirubin Kit (American Monitor Corp. Indianapolis, IN). Animals Male Sprague-Dawley rats (200-280 g) (Bio-Breeding Laboratories Ltd Ottawa, Ontario) were housed in stainless steel wire cages and maintained on Purina Rat Chow and water ad lib. determination of the median time-to-effect (Et.50)for ANIT-induced cholesfasis Rats were randomly divided into 6 groups of 10 animals each and given ANIT (300 mg/kg) by gavage. 5 h after treatment food and water were restored and allowed ad lib. for the remainder of the experiment. Time intervals from 12 to 36.6 h were selected for the determination of the presence of cholestasis; each time point was multiplied by 1.25 to yield the next time of sampling. The presence or absence of cholestasis was determined by an indirect fluorescein method 141. In this procedure fluorescein (20 mg/kg) is injected i.v.; 15 min later the animal is anesthetized (ether); the viscera are exposed and visualized under ultraviolet light. In control rats, intense fluorescence is seen in the bile duct and duodenum, whereas during cholestasis, no fluorescence is observed. The percentage of each group exhibiting cholestasis was calculated and the Et50 determined graphically. Bile flow and bile acid excretion during continuous bile drainage Rats were lightly anesthetized with ether and the bile duct was cannulated with PE-10 tubing. The cannula (90 cm long) was passed under the skin and exteriorized at the back of the head. ANIT (300 mg/kg) or the vehicle was administered by

429

gavage. The animals were placed in individual cages and continuous bile drainage was m~ntained for the entire experiment; water was available ad lib. to prevent dehydration. Bile was collected in tubes contained in ice buckets outside the cage. Bile volumes were measured at hourly intervals for a total of 14 h. Aliquots were taken for measurement of biliary bilirubin [5] and total bile acid 161. Bile flow and bile acid excretion during discontinuous bile drainage Rats were treated with ANIT (300 mg/kg, p.o.) or the vehicle. 30 min before bile collection (4, 8, 12 or 16 h after treatment), the animals were anesthetized (ethyl carbamate-phenobarbital mixture). The trachea was cannulated as were the femoral artery, femoral vein and bile duct. The renal pedicles were ligated prior to suturing the abdominal incision. Body temperature was monitored and maintained at 36-37°C to prevent decreases in bile flow due to hypothermia 171. [14C]Er~hritol was injected (10 &i/kg, iv.) and at the end of a 15min equilibration period, which coincided with the prescribed times (4, 8, 12 or 16 h), bile samples were collected every 15 min for 1 h. Bile volume was measured. Blood samples were taken at 15 and 45 min after the beginning of bile collection. Plasma and bile samples were counted in a Packard Model 3324 scintillation spectrometer and erythritol clearance was determined. Bile was also analyzed for bilirubin and total bile acids (see above). Bile fLow durkg development of cholestasis Rats (215-240 g) were administered ANIT (300 mglkg) by gavage. At 16 h each animal was anesthetized, the trachea and femoral vein were ~annulated and a bile fistula was prepared using PE-10 tubing. Body temperature was monitored and maintained at 36-37°C. Bile was collected over 30-min intervals until cholestasis was complete. The bile volume was recorded and aliquots were analyzed for total bile acids. RESULTS

Fig. 1 depicts the time-to-effect for the appearance of cholestasis in rats treated with ANIT. Within an 8-h time period (12-20 h after ANIT) the number of animals exhibiting cholestasis rose from about 20% to 80%. Thus the onset was relatively rapid once the latency period (about 10 h) was surpassed. An excellent linear correlation (r =0.995) was obtained, and the Et50 was calculated to be 15.3 h. On the basis of these results we expected that ANIT should exhibit a slow gradual effect on bile flow and on the bile constituents during the latency period before the onset of cholestasis. The data presented in Table I, however, show that ANIT exerted no effect on bile flow when biliary drainage was maintained throughout a 14-h observation period. Furthermore, when bile acid excretion was monitored it diminished (from about 1.5 to 0.2 pmol/min/kg) in a similar manner in both control and treated groups. Bilirubin excretion was also unaffected.

430

0

5

IO

15

20

25

33

35

HOURS

Fig.

1. Percentage

of rats exhibiting

cholestasis

at varying

times after the oral administration

of ANIT

(300 mg/kg).

TABLE

I

EFFECT

OF ANIT ON BILE FLOW,

RATS OVER A 14-h PERIOD

BILE ACID EXCRETION

FOLLOWING

AND BILIRUBIN

EXCRETION

IN

BILE DUCT CANNULATIONa

Time

Bile flow

Bile acid excretion

Bilirubin

after

&l/min/kg)

bmol/min/kg)

bg/min/kg)

excretion

ANIT (h) Vehicle

ANIT

Vehicle

ANIT

Vehicle

ANIT

1

44+-IO

35f

11

1.88 + 0.70

1.18kO.33

2.53 f 0.41

2.29 * 0.55

2

51*

13

46f

11

1.54f 0.23

1.45 + 0.24

3.87 f 0.65

3.18 + 0.42

3

42+

9

45*

12

1.16?

1.35 * 0.33

4.18 + 0.77

4.12 + 0.62

4

46+

9

46& 12

1.15 t 0.17

1.59 f 0.34

5.55 * 0.94

4.63 + 0.83

5

51f

1

41 * 10

1 .Ol f 0.23

1.21 + 0.25

5.17 * 0.77

4.48 f 0.82

6

49*

3

41+

9

0.61 f 0.15

0.76 + 0.13

4.74 + 0.30

4.50 f 0.53

7

46+

3

37+

8

0.34 zlz0.07

0.42 +- 0.07

4.25 + 0.40

3.80 + 0.34

8

46+

3

37+

9

0.26 + 0.04

0.32 & 0.05

3.75 f 0.27

3.50 * 0.50

0.22

9

45f

3

36+

8

0.23 f 0.03

0.30 + 0.04

4.06 * 0.55

3.65 * 0.47

10

48+

4

34+

8

0.23 ? 0.06

0.17 * 0.01

4.55 + 0.47

3.21 + 0.56

11

45*

5

35*

9

0.18 f 0.04

0.16 + 0.02

4.22 z? 0.55

3.3 1 + 0.46

12

48*

4

33+

8

0.18 f 0.05

0.14 + 0.02

4.42 f 0.55

2.88 + 0.52

I3

47+

4

34*

6

0.17 f 0.05

0.16 + 0.04

4.03 f 0.35

3.10 + 0.40

14

47+

4

35*

5

0.16 + 0.04

0.13 * 0.01

4.22 f 0.38

3.45 f 0.68

aVehicleor ANIT (300 mg/kg, treatment.

Analyses

p.o.) was administered

were performed

to rats whose bile ducts were cannulated

on samples obtained

during

continuous

bile drainage.

prior to

Each value is

the mean + SE of 4 rats.

Table II depicts the results obtained when the bile duct was cannulated at varying times after treatment. Thus the biliary drainage was discontinuous and an intact enterohepatic circulation was maintained up to the moment of cannulation. There

47 + 8 55 rt 6 62+6 64&5

61+ 8 68+_ 8 71 * 10 682 5

57+ 1 48rt 11 58+ 16 651 9 72~ 58t 67& 73+

8 12 17 8

1.29 1.23 1.16 1.06

1.26 1.23 1.15 1.12

1.43 + 0.22 1s7 * 0.45 1.71 +0.09 1.73 * 0.12

1.02r~O.12 1.13 + 0.08 1.11 kO.39 0.93 zt 0.14

ANIT

2.OOzrO.13 3.06 rt 0.60 3.96 f- 0.66 1.99 3- 0.30

Vehicle

Bilirubin excretion (&nin/kg)

2.23 2.54 3.30 3.22

rfi0.5 1 f 0.58 i 0.61 + 0.29

ANIT

AND BILIRLJBIN EXCRETION

IN RATS AT

aVehicle or ANIT (300 mg/kg, p.o.) was administered to rats. At the times indicated the rats were anesthetized and the bile duct cannulated; bile was collected for 1 h. Each value is the mean Z!Z SE of 3-5 rats.

4 8 12 16

ANIT

Vehicle

Vehicle

ANIT

ANIT

Vehicle

Vehicle

BiIe acid excretion &mol/min/kg)

Erythritol biIe:plasma ratio

Erythritol clearance (&mm/kg)

Time Bile flow after &I/min/kg) ANIT (h)

BILE ACID EXCRETION

CLEARANCE,

EFFECT OF ANIT ON BILE FLOW, ERYTHRITOL VARYING TlMES AFTER T~ATMENTa

TABLE II

t

432

TABLE

III

EFFECT

OF ANIT

ON BILE FLOW

AND

BILE ACID

EXCRETION

IN RATS

16-23

h AFTER

TREATMENTa Time after ANIT

Rat

1

Rat 2

Rat 3

Rat 4

Rat 5

Rat 6

(h)

At’

Bb

A

B

A

A

A

A

16.0-16.5

14

1.57

40

0.60

86

1.49

53

1.06

91

1.46

75

2.19

16.5-17.0

54

1.05

37

0.48

83

1.33

53

0.92

94

1.42

80

2.11

B

B

B

B

17.0-17.5

46

0.67

34

0.39

89

1.33

45

0.67

91

1.37

77

2.08

17.5-18.0

45

0.48

31

0.30

88

1.18

39

0.51

80

1.12

71

1.72

18.0-18.5

42

0.35

31

0.26

82

1.07

33

0.35

65

0.82

62

1.56

18.5-19.0

31

0.20

25

0.18

74

0.99

35

0.26

52

0.50

57

1.61

0.12

67

0.83

26

0.17

34

0.23

45

1.03

-

60

-

-

38

0.74

19.0-19.5

4

-

22

19.5-20.0

0

-

0

20.0-20.5 20.5-21

.O

0

-

0

47

0.52

33

0.53

36

0.34

28

0.40

21 .O-21.5

38

0.19

25

-

21.5-22.0

24

0.12

20

-

22.0-22.5

0

-

17

0.13

22.5-23.0

14

0.09

23.0-23.5

0

aANIT

(300 m&kg,

cannulated.

p.o.) was administered

Bile was collected

bA, bile flow bl/min/kg);

continuously

to rats. At 16 h each rat was anesthetized until cholestasis

B, bile acid excretion

-

and the bile duct

was evident.

&mol/min/kg).

IOO-

G

90.

<

80-

’

60.

m

,o_.’ 0

“““‘.“““““I”’ 01 03

0.5 BILE

Fig. 2. Relationship (300 mg/kg).

a7

0.9

ACID

EXCRETION

II

13 (pm&

15 /mln/

17

between bile flow and bile acid excretion

Data points were obtained

19

21

kg)

16-23 h after oral administration

of ANIT

from Table III.

were no significant differences in bile flow between ANIT-treated and vehicletreated animals. Erythritol clearance was also unaffected by ANIT. At the early times bile acid and bilirubin excretion were unaffected. However, at 16 h animals treated with ANIT excreted significantly less bile acid and more bilirubin than did the control animals (P < 0.05, t-test).

433

In the last experiment, in which the bile duct was cannulated 16 h after treatment with ANIT, cholestasis was present in 6 of the 12 rats at the time of cannulation. The remaining 6 rats were maintained under anesthesia and bile flow was monitored until cholestasis was clearly established (Table III). In all cases bile ‘flow and bile acid excretion decreased with time. The mean time for cessation of bile flow was 20.5 + 0.64 h. The bile flow rates were plotted against the bile acid excretion rates (Fig. 2). A linear relationship with a good correlation (r = 0.85; n = 50) was obtained. DISCUSSION

The Et50 for ANIT-induced cholestasis in the present study is virtually identical to the value determined in mice [4]. It also agrees well with the onset of hyperbilirubinemia in rats (between 12 and 15 h) reported previously [2] and the diminution in bile flow (18 h) noted by Fukumoto et al. [3]. These observations indicate that there is a latency period before the acute hepatotoxic results of ANIT manifest themselves. One possible explanation for a latency period would be that ANIT perturbs the bile secretory mechanism, but that the effect occurs gradually. Both bile aciddependent and bile acid-independent components of bile flow have been characterized [8], and an interaction may exist between these two components [8-l 11. One can expect that ANIT might modify these components. The data (Tables I and II) show that ANIT does not have a gradual effect on total bile flow, either where biliary drainage is continuous or with an intact enterohepatic circulation. Erythritol clearance, which is a measure of canalicular bile flow [S], was also unaffected, indicating the absence of an early effect of ANIT on canalicular bile flow. The absence of an early gradual ANIT effect on bile flow when biliary drainage was continuous (Table I) was not unexpected, since bile duct cannulation prior to ANIT treatment can prevent the full development of cholestasis and hyperbilirubinemia [12]. However, the lack of an early effect on flow in those rats with an intact enterohepatic circulation (Table II) was unexpected, since the Et50 for cholestasis was found to be about 15 h. When the bile acid excretion data are examined, no early effect of ANIT was evident. The constant diminution in bile acid excretion over the 14-h period (Table I) is a normal finding since the enterohepatic circulation had been interrupted. When the enterohepatic circulation remained intact (Table II), a significant difference in bile acid excretion was only observed at 16 h after ANIT, a time when cholestasis became evident in about 50% of the animals. Fukumoto et al. [3] also reported decreases in bile acid excretion 15 and 18 h after ANIT administration. We interpret these data to indicate that ANIT-induced cholestasis is not due to a gradual modification of bile formation. However, after the latency period, cholestasis occurs relatively rapidly. Fig. 1 shows that in a period of less than 8 h the

434

number of rats exhibiting cholestasis increased from 20 to 8OVo. The bile flow and bile acid excretion data obtained in animals 16 to 23 h after ANIT treatment (Table III) also support such a conclusion. In 5 of 6 rats, once bile flow began to diminish, a rapid decline in bile flow and bile acid excretion was observed during the next 3 h. At 16-23 h after ANIT administration, it is clear that ANIT did perturb bile formation, when one compares bile flows at different bile acid concentrations. The equation for the linear regression of the ANIT data depicted in Fig. 2 was calculated to be: (Y = 24.9 + 33.4 X; r = 0.85, P < 0.05). A similar calculation for the vehicle-treated rats (Tables I and II) yielded the following equation (Y = 45.1 + 4.6 X; r = 0.51, P < 0.05). A comparison of these equations indicates that the Yintercept was smaller in the ANIT-treated animals (45.1 vs. 24.9 Fl/min/kg). Assuming that the Y-intercept is a measure of the bile acid-independent fraction, then ANIT decreased this component markedly. When the slopes of the regression lines are compared, it is evident that in ANIT-treated rats this parameter was much larger (33.4 vs. 4.6), indicating that following ANIT treatment the bile flow was much more dependent upon bile acid excretion. Therefore, after ANIT treatment, bile acid-independent flow is reduced, and the bile acid-dependent component becomes the major participant in bile formation. ANIT treatment markedly decreases bile acid excretion 16-21 h later (Table III). Comparing these excretion rates with those obtained for the first 5 h in vehicletreated animals, whose bile ducts were also cannulated (Table I), this effect is evident. In the ANIT group (Table III, 20.5-21 h) the rates ranged from 0 to 0.40 pmol/min/kg, whereas in the vehicle-treated group (Table I, 5 h) the mean value was 1.01 k 0.23 ~mol/min/kg. In both experiments, we are comparing bile acid excretion for the first 5 h immediately after cannulation of the bile duct. We conclude, therefore, that ANIT affects the bile acid-dependent component as well as the bile acid-independent component. These conclusions are consistent with those expressed by Fukumoto et al. [3] who observed a diminution in bile acid-independent bile flow after ANIT. Their results did not permit a definitive conclusion regarding the bile acid-dependent component, but they did show that at 18 h; ANIT-treated rats no longer responded (increased bile flow) to taurocholic acid injection. At 18 h their rats no longer responded to secretin injection, but did so at 15 h. A number of investigators have attempted to study the effects of ANIT and other cholestatic chemicals on the enzymic activity of hepatic plasma membranes [I]. Mg2+ -ATPase, Na+ -K+ -ATPase and 5 ‘-nucleotidase activities are reported to be depressed 1 to 3 days after ANIT administration [13]. Since these studies were performed after cessation of bile flow, it cannot be assumed that the changes caused the diminution in bile flow and were not the result of cholestasis. Our results indicate that the question should be reinvestigated, particularly during the 15-to 24h period after ANIT treatment. Furthermore, canalicular-enriched plasma membranes should be employed for such studies.

435 REFERENCES 1 G.L. Plaa and B.C. Priestly, Intrahepatic cholestasis induced by drugs and chemicals, Pharmacol. Rev., 28 (1976) 207-273. 2 N. Indacochea-Redmond and G.L. Plaa, Functional effects of cY-naphthylisothiocyanate in various species, Toxicol. Appl. Pharmacol., 19 (1971) 71-80. 3 Y. Fukumoto, K. Okita, T. Kodama, K. Noda, T. Harada, M. Mizuta and T. Takemoto, Studies of ol-naphthylisothiocyanate-induced hepatic disturbance, Hepato-Gastroenterol., 27 (1980) 457-464. 4 G.L. Plaa and B.A. Becker, Demonstration of bile stasis in the mouse by a direct and indirect method, J. Appl. Physiol., 20 (1965) 534-537. 5 B. Nosslin, The direct diazo reaction of bile pigments in serum, Stand. J. Clin. Lab. Invest., 12 (1960) Suppl. 49, 1-176. 6 P. Talalay, Enzymic analysis of steroid hormones, Meth. Biochem. Anal., 8 (1960) 119-143. 7 R.J. Roberts, C.D. Klaassen and G.L. Plaa, Maximum biliary excretion of bilirubin and sulfobromophthalein during anesthesia-induced alteration of rectal temperature, Proc. Sot. Exp. Biol. Med., 125 (1967) 313-316. 8 J.L. Boyer, New concepts of mechanisms of hepatocyte bile formation, Physiol. Rev., 60 (1980) 303-326. 9 C.D. Klaassen, Bile flow and composition during bile acid depletion and administration, Can. J. Physiol. Pharmacol., 52 (1974) 334-348. 10 C. Balabaud, K.A. Kron and J.J. Gumucio, The assessment of the bile salt non-dependent fraction of canalicular bile water in the rat, J. Lab. Chn. Med., 89 (1977) 393-399. 11 S. Lock, H.P. Witschi and G.L. Plaa, The effect of cycloheximide on bile flow in rats, Proc. Sot. Exp. Biol. Med., 161 (1979) 546-553. 12 R.J. Roberts and G.L. Plaa, The effect of bile duct ligation, bile duct cannulation, and hypothermia on a-naphthylisothiocyanate-induced hyperbilirubinemia and cholestasis in rats, Gastroenterology, 50 (1966) 768-774. 13 P.J. Hertzog, P.S. Bhathal, P.R. Dorling and R.N. Le Page, ol-Naphthylisothiocyanate-induced cholestasis in the rat: Studies of liver plasma membrane enzymes, Pathology, 7 (1975) 13-23.