Yonago Acta medica 2002;45:59–68_-Naphthylisothiocyanate (ANIT) Induced Cholestasis in Rats
Kiyohisa Uchida*†, Yoshio Ogura*, Nobuo Yamaga* and Kazuo Yamada**Division of Medical Biochemistry, Department of Pathophysiological and Therapeutic Science,
School of Medicine, Tottori University, Yonago 683-8503 and †The Cell Science Research
Foundation, Osaka 541-0045 Japan
In order to distinguish the disorder of bile acid and cholesterol metabolism in _-naphthylisothiocyanate (ANIT)-induced cholestasis, we examined changes in bile acid levels andcompositions in bile, serum, feces and urine, as well as cholesterol levels in bile, serum,
liver and feces in Wistar male rats (10–13 weeks) after a single oral administration of
100 mg/kg of ANIT. The bile flow and the biliary secretions of cholesterol, phospholipids
and bile acids markedly decreased on days 1 and 2 but increased over the normal values
on day 4 and then returned to the normal ranges. The fecal excretion of bile acids
decreased after the treatment and remained low by day 4 but markedly increased
thereafter. The urinary excretion of bile acids changed almost in parallel with serum
bile acid level, increasing to 37 mg/day on day 2, 28 mg/day on days 3–4 but to a trace
on days 5–6. The urinary bile acids on day 2 mainly consisted of cholic acid while thoseon days 3–4 and biliary bile acids on day 4 were mostly `-muricholic acid. The serum
cholesterol level markedly increased maximally on day 2 and decreased thereafter. The
fecal excretion of sterols, cholesterol and coprostanol, decreased on days 1–2 but rather
increased thereafter. These data suggest that the cholestasis induced by ANIT is very
similar to that in bile duct ligated rats for a short period but not to those ligated forlong periods. In addition, the present data suggest that the bile acid independent bileflow is impaired and the daily synthesis of bile acids, especially `-muricholic acid, isincreased in the ANIT induced cholestasis.
Key words: bile secretion; biliary lipids; fecal bile acids and sterols; _-naphthylisothiocyanate;
serum lipids; urinary bile acids_-Naphthylisothiocyanate (ANIT) is a compound which causes cholestasis, hepatocellularand biliary epithelial cell necrosis, bile duct obstruction and biliary epithelial cell hyperplasia
in rats (Plaa and Priestly, 1976). The changes inbiliary epithelial cells are reported to be comparable to that induced by bile duct ligation(Kossor et al., 1995).
Previously, we examined the changes in bile
acid metabolism after bile duct ligation in rats
(Kinugasa et al., 1981, Takita et al., 1988) andthose after the release of bile duct ligation(Fujio et al., 1989). A striking change in bile
duct ligated rats was a marked increase in `muricholic acid with a concomitant decrease ofcholic acid. The change was normalized withina week after the release of ligation regardless ofthe duration of ligation, but the changes in bileflow and biliary bile acid secretion were different according to the duration. Release after a 1week ligation showed a temporal rebound increase in both bile flow and biliary bile acid secretion, but release after a 2-week ligation
caused a continuous hypersecretion of the bile
without any rebound increase in biliary bile
acid secretion (Fujio et al., 1989).
In the present experiments, we examined thechanges in bile flow and bile acid metabolism
after ANIT administration until cholestasis wascompletely recovered in rats.

Abbreviations: ANIT, _-naphthylisothiocyanate; bsep, bile salt export pump; GLC, gas-liquid chromatography; mdr2, multidrug resistance 2; mrp2, multidrug resistance associated protein 2

59

K. Uchida et al.

portion of the largest lobe of the liver (lobus sinistra externa) was homogenized with 9 volumes of water. The serum and the liver homogenate were extracted in 9 volumes of ethanol by
refluxing for 20 min at 90–95˚C. Total cholesterol was determined with portions of the serum
and liver lipid extracts as previously reported(Uchida et al., 1965). Phospholipids were determined by the method of Gomori (1942).

Materials and Methods
ANIT obtained from Nakarai Pure-Chemicals(Tokyo, Japan) was dissolved in corn oil at a concentration of 100 mg/mL, and a single dose of
ANIT (100 mg/kg) was administered orally torats. This dosage was selected based on the report of Kossor et al. (1995) who examined theeffect of various doses of ANIT (25–150 mg/kg)
in rats and showed that a dose of 100 mg/kg induced a modest cholestasis at 48 h after treatment.
Control rats received 1 mL/kg of corn oil orally.

Biliary lipid determination

Animal treatments
Wistar strain male rats weighing about 300 g(10–13 weeks old) were kept in an air-conditioned room (25 ± 1˚C, 50–60% humidity)
lighted 12 h a day (8:00–20:00) and maintainedon a commercial stock diet ( Japan CLEA CA-1,
Tokyo, Japan).
In the first experiment, rats were individually caged and daily urine specimens were collected for 10 days after the administration of
ANIT. Daily urine volume, diet intake and water intake were recorded. In the second experiment, rats were individually caged and sacrificed 2, 4, 6, 8 and 10 days after the administrationof ANIT to collect bile, blood and liver specimens. Two-day feces specimens were collected prior to sacrifice as described previously(Uchida et al., 1977). On the day of sacrifice,the rats were cannulated of their bile duct with
PE-10 polyethylene tubings in order to collectthe bile for 30 min under sodium pentobarbital
anesthesia (50 mg/kg, intraperitoneally). During the time of bile collection, the rectal temperature was maintained at 36–37˚C using an electric warm plate. The blood was then withdrawn
by heart puncture and the liver was removed forlipid determination. Bile was collected between 9:00–11:00 to avoid any variation due tothe circadian rhythm.

Serum and liver lipid determination
The serum was separated by centrifugation at
3,000 rpm for 15 min after allowing the blood tostand for at least 30 min at room temperature. A
60

The bile was added to 20 volumes of ethanol,
brought to boil once for several minutes, cooled
down to room temperature, and then filtered.
An aliquot of the filtrate was evaporated todryness under a mild stream of nitrogen and theresidue was hydrolyzed at 120˚C for 6 h in 1.25
M sodium hydroxide solution. Sterols were extracted with diethyl ether, the reaction mixture
was acidified with 2 M hydrochloric acid andthen the bile acids were extracted with diethyl
ether. The bile acids were methylated withfreshly prepared diazomethane and then trifluoroacetylated with trifluoroacetic anhydride.
The bile acid derivatives were quantified by
gas-liquid chromatography (GLC) with a 1%
QF-1 column and cholesterol with a 1% SE-30
column (Uchida et al., 1970, 1977). Biliary phospholipids were determined by the methods ofGomori (1942).

Urinary and serum bile acid determination
The urine was applied to an Amberlite XAD-2
column and bile acids were eluted with methanol according to the method of Makino et al.
(1974). The eluate was evaporated to dryness andthe residue was dissolved in 30 mL of acetoneethanol (9:1) containing 0.01 volume of 2 M hydrochloric acid. The solution was left at room
temperature for 2 to 3 days to complete solvolysis.
The reaction mixture was then evaporated to dryness and the residue was subjected to alkalinehydrolysis. After extraction with diethyl ether, thebile acids were methylated and trifluoroacetylated, and analyzed by GLC as mentioned above.
Serum bile acids were isolated by the use ofan Amberlyst A-26 column (Sandberg et al.,

ANIT induced cholestasis
Table 1. Changes in body weight, diet intake and water intake after _-naphthylisothiocyanate(ANIT) administration in rats

0Days after ANIT administration
2
4
6

1

Body weight(g) 338 ± 3.2 315 ± 3.8* 304 ± 2.3* 312 ± 1.5*
Diet intake(g/day) 22 ± 1.0
5 ± 0.8*
3 ± 1.3* 14 ± 2.7*
Water intake (mL/day) 30 ± 4.5
5 ± 1.6*
18 ± 4.433 ± 2.8
Mean ± SE in 5 rats.
—, not determined.
* Statistically significant compared with day 0 (P < 0.05).

1965) and quantified by GLC after alkaline hydrolysis without any procedure for solvolysis.

318 ± 5.2
22 ± 1.0
34 ± 5.3

810332 ± 4.1
26 ± 1.4
39 ± 3.5342 ± 2.7——

tion. Sterols were extracted with diethyl ether,
the reaction mixture was acidified with 2 Mhydrochloric acid and then bile acids were extracted with diethyl ether. The methylated andtrifluoroacetylated bile acids were quantified by
GLC with a 1% QF-1 column and sterols with a
1% SE-30 column (Uchida et al., 1977; Kinugasa
et al., 1981). The extraction efficiency of sterols
and bile acids from feces was over 92.5%(Kinugasa et al., 1981).

Fecal sterol and bile acid determination
The feces were dried and powdered, according
to the procedures described previously (Uchida
et al., 1977). An aliquot of the pulverized feces
was extracted with 20 volumes of boiling ethanol for 1 h and filtered after cooling down toroom temperature. The extraction procedures
were repeated three times. The combined filtrate was evaporated to dryness under reducedpressure, and the residue was hydrolyzed at
120˚C for 6 h in 1.25 M sodium hydroxide solu-

Statistical analysis
Data are expressed as mean ± SE. The difference
between the means of variables was calculated

Table 2. Changes in urinary bile acids after _-naphthylisothiocyanate (ANIT) administration in rats

0

1

2

Days after ANIT administration
3–4‡
5–6‡

7–8‡

9–10‡

Urine volume (mL/day) 12 ± 1.8
15 ± 1.2*
11 ± 2.0
15 ± 3.3
18 ± 2.2
19 ± 2.1*
20 ± 2.1*
Urinary bile acids
0.01 ± 0.00 2.01 ± 0.56* 37.29 ± 3.74* 28.07 ± 3.10* 0.09 ± 0.01* 0.07 ± 0.01* 0.05 ± 0.01*(mg/day)
Bile acid composition (%)
Lithocholic acid
31 ± 4.7<1<1<1
17 ± 5.0
6 ± 0.8*
11 ± 3.3*
Chenodeoxycholic acid 3 ± 2.2<1
1 ± 0.0
2 ± 0.5<1
ND
ND_-Muricholic acid2 ± 1.7
3 ± 0.4
1 ± 0.1
3 ± 0.2
2 ± 0.6
3 ± 1.0
3 ± 1.2`-Muricholic acid
4 ± 0.6
16 ± 5.8
16 ± 1.1*
46 ± 3.4*
16 ± 2.4*
7 ± 2.0
9 ± 2.5
t-Muricholic acid
8 ± 0.6
3 ± 0.4
3 ± 0.2
3 ± 0.9
7 ± 0.8
4 ± 0.8
7 ± 0.7
Ursodeoxycholic acid 1 ± 0.8<1<1
3 ± 0.6<1
ND
ND
Hyodeoxycholic acid<1
<1
<1
1 ± 0.3
ND
ND
ND
Deoxycholic acid
ND
<1
<1
<1
<1
ND
<1
Cholic acid
47 ± 6.4
55 ± 7.0
62 ± 0.931 ± 2.542 ± 7.6
73 ± 1.963 ± 6.6
Peak 8†ND
15 ± 5.4
9 ± 0.8
7 ± 0.8
3 ± 1.2
4 ± 0.8
4 ± 1.2
Others
3 ± 1.0
6 ± 0.6
6 ± 0.2
4 ± 0.3
13 ± 3.3<1
4 ± 0.7
Mean ± SE in 5 rats.
ND, not detectable.
† Unidentified.
‡ Samples of 2-day urine specimens were combined.
* Statistically significant compared with day 0 (P < 0.05).

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K. Uchida et al.

Table 3. Changes in feces dry weight and fecal sterols and bile acids after _-naphthylisothiocyanate (ANIT) administration in rats0

1–2

Days after ANIT administration
3–4
5–6

7–8

9–10

Feces dry weight (g/day) 4.5 ± 0.39 1.3 ± 0.18* 3.6 ± 0.53
4.6 ± 0.22
5.4 ± 0.27
5.7 ± 0.16
Fecal total sterols (mg/day) 7.49 ± 0.71 1.98 ± 0.27* 13.98 ± 2.58* 12.72 ± 1.22* 11.24 ± 0.42* 9.78 ± 1.06
Sterol composition(%)
Coprostanol
54 ± 5.8
46 ± 7.7
57 ± 9.4
50 ± 7.7
50 ± 6.3
56 ± 7.0
Cholesterol
47 ± 4.5
54 ± 11.0
43 ± 9.2
50 ± 11.0
50 ± 2.7
44 ± 5.1
Fecal bile acids (mg/day) 8.17 ± 0.68 2.56 ± 0.34* 1.61 ± 0.25* 14.73 ± 1.28* 12.98 ± 1.47* 11.26 ± 0.69*
Bile acid composition (%)
Lithocholic acid
10 ± 0.5
7 ± 1.0
26 ± 3.0*
7 ± 1.7
7 ± 1.0
7 ± 0.7`-Muricholic acid
6 ± 0.6
15 ± 5.9
13 ± 1.4*
16 ± 3.2*
10 ± 1.9
8 ± 1.3
18 ± 3.849 ± 5.3*
47 ± 3.3*
46 ± 4.2*
t-Muricholic acid
29 ± 5.2
20 ± 2.0
Hyodeoxycholic acid
22 ± 5.5
22 ± 5.3
7 ± 2.6*
2 ± 0.4*
4 ± 1.2*
6 ± 1.9*
Deoxycholic acid
26 ± 1.4
28 ± 2.6
18 ± 1.2*
17 ± 1.4*
24 ± 1.5
26 ± 0.7
Peak 8† and others
8 ± 0.4
9 ± 1.5
18 ± 1.2*
10 ± 1.3
9 ± 0.8
7 ± 0.6
Mean ± SE in 5 rats.
† Unidentified.
* Statistically significant compared with day 0 (P < 0.05).

by Student’s t-test. Values of P < 0.05 were considered to be statistically significant.

days 1–2 but was restored after days 5–6. The
fecal sterol excretion decreased once on days 1–
2, but markedly increased on days 3–4 and thengradually decreased. The composition ratios ofcoprostanol and cholesterol were almost identical in all the groups. The fecal bile acid excretion decreased after ANIT administration to thelowest level on days 3–4, but in turn markedly
increased on days 5–6 attaining almost twice
the level of the control rats, and gradually declined thereafter. Deoxycholic acid comprised
about 25% in the control rats but decreased ondays 3–4 and 5–6, and was then restored to theinitial level. Instead, `-muricholic acid and tmuricholic acid increased while deoxycholicacid decreased. Hyodeoxycholic acid decreased after days 3–4, and remained low thereafter,
while t-muricholic acid increased and kept
high levels after days 5–6.
Table 4 shows the changes in the bile flow
and biliary lipid secretions after ANIT administration. The bile flow markedly decreased, almost to 1/10 that of the initial level, on days 1
and 2, but increased showing higher levels than
the initial level on days 4 and 10. Biliary bile
acid secretion also decreased on days 1 and 2,
increased on day 4 and was restored to the control level after day 6. The biliary cholesterol

Results
As shown in Table 1, the body weight decreased
and showed the lowest value on day 2 after ANIT
administration and recovered gradually thereafter. Diet and water intake decreased markedly on the first day but diet intake recovered tothe initial level by day 6 and the water intake by
day 4.
Urine volume increased on day 1, returnedto the initial level on day 2, and slightly increased thereafter. The urinary excretion of bile
acids markedly increased on day 2 and probably
on day 3, and then rapidly decreased. The major component in urinary bile acids was cholicacid in the control rats, but `-muricholic acidincreased after ANIT administration and became the major component on days 3–4. Cholicacid rather decreased on days 3–4, but gradually
increased thereafter in accordance with the decrease of `-muricholic acid (Table 2).
The changes in the feces dry weight, andfecal sterol and bile acid excretions are shownin Table 3. The feces dry weight decreased on
62

ANIT induced cholestasis

Table 4. Changes in bile flow and biliary lipid secretions after _-naphthylisothiocyanate (ANIT)
administration in rats

0

1

Days after ANIT administration
2
4

6

10

Bile flow(mL/h) 1.43 ± 0.02 0.12 ± 0.02* 0.12 ± 0.04* 1.83 ± 0.11* 1.23 ± 0.17 1.60 ± 0.05*
Total cholesterol (mg/h) 0.20 ± 0.01 0.05 ± 0.00 0.05 ± 0.00
0.43 ± 0.05* 0.18 ± 0.03 0.19 ± 0.01
Phospholipids (mg/h) 5.03 ± 0.39 0.27 ± 0.07* 0.24 ± 0.06* 20.22 ± 3.74* 6.74 ± 0.90 7.53 ± 0.81*
Bile acids(mg/h) 12.90 ± 0.82 0.37 ± 0.26* 0.52 ± 0.16* 34.49 ± 5.32* 12.61 ± 1.37 16.72 ± 1.49
Mean ± SE in 5 rats.
* Statistically significant compared with day 0 (P < 0.05).

and phospholipid secretions changed almost inparallel with the bile acid secretion.
Striking changes in the biliary bile acid composition were an increase of `-muricholic acid anda concomitant decrease of cholic acid on days 2
and 4. The compositions of the other bile acids
were not changed significantly (Table 5).
As shown in Table 6, the serum bile acidconcentration markedly increased on day 1 andmaximally on day 2 (about 30-fold) but abruptly declined on day 4 almost attaining the control
level. A decrease in cholic acid and an increasein `-muricholic acid were also noted on days 2
and 4. Chenodeoxycholic acid showed highvalues on day 0 and day 4, but the reason forthis large variation was not clear. Such a highvalue for chenodeoxycholic acid was detected
neither in the urine, feces nor bile.
The correlations between biliary bile acidsecretion and bile flow, biliary bile acid secretion and biliary cholesterol secretion, and biliary bile acid secretion and biliary phospholipid
secretion are shown in Fig. 1. As shown in Fig.
1A, a linear regression line was obtained between the bile acid secretion and bile flow in theregion where the bile acid secretion was over 10
mg/h, by which the bile acid dependent bileflow was calculated to be 0.018 mL/mg and thebile acid independent bile flow to be 1.25 mL/h.
However, in the region where the bile acid secretion was less than 10 mg/h, the spots were
under the regression line.
On the contrary, the cholesterol and phospholipid secretions were linearly related to thebile acid secretion in all regions. It was calculated from these relationships that the cholesterol/bile acids ratio in the bile was 0.012 andthe phospholipids/bile acids ratio was 0.27 on a

Table 5. Changes in biliary bile acid composition after _-naphthylisothiocyanate (ANIT) administration in rats
Composition
(%)

0

1

Days after ANIT administration
2
4

Lithocholic acid
1 ± 0.1<1<1
Chenodeoxycholic acid
3 ± 0.8
1 ± 0.4
1 ± 0.2*_-Muricholic acid
3 ± 0.5
2 ± 0.6
2 ± 0.1`-Muricholic acid
24 ± 4.5
25 ± 10.3
48 ± 7.3*
t-Muricholic acid
1 ± 0.1
2 ± 0.2
1 ± 0.1
Hyodeoxycholic acid
2 ± 0.4
4 ± 0.7
1 ± 0.2
Deoxycholic acid
2 ± 0.1
1 ± 0.1*
1 ± 0.1*
Cholic acid
41 ± 0.8
38 ± 9.6
31 ± 6.8
9 ± 1.5
Peak 8†14 ± 2.4
16 ± 2.1
Others
9 ± 0.6
12 ± 0.9*
8 ± 0.7
Mean ± SE in 5 rats.
† Unidentified.
* Statistically significant compared with day 0 (P < 0.05).

63

1 ± 0.0
5 ± 0.9
4 ± 0.542 ± 5.3*<1
1 ± 0.2
1 ± 0.3*
21 ± 1.6*
16 ± 4.3
9 ± 1.4610<1
2 ± 0.2
3 ± 0.427 ± 7.0
1 ± 0.1
1 ± 0.4
3 ± 0.342 ± 4.4
13 ± 3.7
8 ± 1.6<1
3 ± 0.5
3 ± 0.3
22 ± 4.6
1 ± 0.2
2 ± 0.4
4 ± 0.3*45 ± 2.1
13 ± 2.7
8 ± 1.0

K. Uchida et al.
Table 6. Changes in serum bile acids after _-naphthylisothiocyanate (ANIT) administration in rats

0

1

Days after ANIT administration
2
4

6

10

Bile acids(μg/mL)
4.4
64.4
Composition(%)
Lithocholic acid
ND
ND
Chenodeoxycholic acid
16.9
1.4_-Muricholic acidND
0.7`-Muricholic acid—
6.0
Hyodeoxycholic acid
2.1
4.6
Peak 8†1.4
1.8
Cholic acid
72.2
75.7
Deoxycholic acid
2.5
2.1
Others
5.0
7.6
Samples of 5 rats were combined before analysis.
—, included in cholic acid.
ND, not detectable.
† Unidentified.

124.0

6.6

5.2

3.8

ND
4.4
1.4
56.9
0.7
0.5
31.7
0.1
4.2

ND
18.5
5.0
20.5
1.9
ND
45.1
1.7
7.4

ND
6.0
1.5
5.0
2.4
0.9
69.3
4.2
10.8

ND
6.1
1.6
—
8.6
ND
68.2
4.1
11.4

molar basis. Therefore, the molar ratio of cholesterol and phospholipids in the bile was about
1:22.
The changes in the serum and liver lipid
levels are given in Table 7. The serum cholesterol and phospholipid levels significantly increased on day 1 and further increased on day 2,
but gradually decreased thereafter and returned to control levels on day 10. The cholesterol/phospholipids ratio remained almost constant
but rather increased after day 4. The serum triglyceride level slightly decreased after ANIT
administration. The liver weight per 100 g bodyweight rather increased after day 2, reflecting
decreases in body weight. The liver cholesterollevel slightly increased after ANIT treatment
but the phospholipid levels remained unchanged. The liver triglyceride level decreased on
day 2 but only slightly on days 6 and 10.

The biliary secretions of bile acids, cholesterol
and phospholipids changed almost in parallelwith the bile flow. These changes are almost
consistent with previous reports (Maeyama,
1977, Fukumoto et al., 1980, Lock et al., 1982).
In the previous experiment, we examinedchanges in bile flow and biliary bile acid secretion after release of the bile duct ligation in rats
(Fujio et al., 1989). The release after a shortperiod of obstruction restored normal bile flow
but release after a long period of obstruction did
not. When the ligation was released by 1 week,bile flow and biliary bile acid secretion increased once over the control levels and were thenrestored to the normal ranges. When the ligation was released after 2 weeks, the bile flow
markedly increased and remained high thereafter, while the biliary bile acid secretion gradually increased to the normal range. The present
data in the ANIT treated rats coincide with thepattern observed after a short period ligation.
As shown in Fig. 1A, the spots in the region
where the bile acid secretion was less than 10
mg/h were under the regression line. The bile
acid dependent bile flow is provided by the biliary bile acid composition and is increased bynon-micellar bile acids such as oxo-bile acids(Loria et al., 1989). ANIT treatment causedchanges in bile acid composition, an increase of`-muricholic acid with a concomitant decreaseof cholic acid, but showed no remarkable increase of oxo-bile acids (Table 5). Therefore, it

Discussion
The present experiments not only demonstrated
that ANIT caused cholestasis in rats, but also
clarified the changes in cholesterol and bile acidlevels in the bile, serum, liver, urine and feces inrelation to cholestasis. The bile flow was inhibited, less than 10% of the control, for at least
2 days after a single oral administration of 100
mg/kg of ANIT, but showed hypersecretion on
day 4 and then returned to the control level.
64