ANIT induced cholestasis(mL/h)A

Bile flow

2Y = 0.018X + 1.25
r = 0.93

101020304050 (mg/h)

Bile acids(mg/h)

BCholesterol

0.5

Y = 10.72X + 42.7 (× 10-3)
r = 0.9301020304050 (mg/h)

Bile acids(mg/h)

CPhospholipids

2010Y = 0.525X + 0.28
r = 0.97

01020304050 (mg/h)

Bile acids

Fig. 1. Correlations between biliary bile acid secretion and bile flow (A), biliary bile acid secretion andbiliary cholesterol secretion (B) and biliary bile acid secretion and biliary phospholipid secretion (C) incontrols and ANIT treated rats. Broken lines in (A) are given under the assumption that the bile aciddependent bile flow is not changed by ANIT (see text).

is assumed that bile acid dependent bile flow is
not influenced by ANIT. Based on this assumption, dots representing the bile acid secretionin the region less than 10 mg/h are considered to
be on the regression lines shown by brokenlines having the same slope with the solid line.
Fukumoto et al. (1980) and Lock et al. (1982)
reported that the bile acid independent bile flow
was inhibited in ANIT treated rats from a study
measuring the biliary clearance of erythritol.
This conclusion was consistent with the present

data (Fig. 1A). Furthermore, the bile flow in
ANIT treated rats markedly increased on day 4,
but this increase was due to the bile acid dependent bile flow, because the plots for day 4 werecompletely matched with the regression line between biliary bile acid secretion and bile flow
(Fig. 1A). On the other hand, the cholesteroland phospholipid secretions were linearly related to the bile acid secretion closing the origin(Figs. 1B and C), and the molar ratio of cholesterol and phospholipid in the bile was 1:22.
65

K. Uchida et al.
Table 7. Changes in serum and liver lipid levels after _-naphthylisothiocyanate (ANIT) administration in rats

0Body weight(g) 328 ± 3.5
Serum total cholesterol
67 ± 1.7(mg/dL)

1Days after ANIT administration
24311 ± 3.3*
147 ± 10.1*

302 ± 4.9*204 ± 20.0*

309 ± 4.9*170 ± 6.9*610304 ± 4.2*
84 ± 3.3*309 ± 4.9*75 ± 3.5

Phospholipids (mg/dL) 134 ± 1.4305 ± 26.3* 375 ± 46.3* 257 ± 13.8
150 ± 11.3
135 ± 3.771 ± 3.0*
63 ± 4.6*
63 ± 3.5*
63 ± 4.0*
60 ± 2.2*
Triglyceride (mg/dL) 84 ± 5.0
C/P ratio
0.50 ± 0.017 0.48 ± 0.014 0.55 ± 0.019 0.66 ± 0.024* 0.56 ± 0.020 0.56 ± 0.014*
Liver weight
3.7 ± 0.06
4.1 ± 0.06* 4.6 ± 0.07*
4.2 ± 0.09* 4.0 ± 0.08
(g/100g body weight) 3.8 ± 0.10
Total cholesterol (mg/g) 3.3 ± 0.07
3.5 ± 0.16
3.8 ± 0.09* 3.7 ± 0.08*
3.5 ± 0.07* 3.5 ± 0.03*
45.5 ± 1.13 45.0 ± 0.69
Phospholipids (mg/g) 46.9 ± 0.83 49.1 ± 0.67 46.1 ± 1.17 45.3 ± 0.79(mg/g) 3.7 ± 0.18
4.7 ± 0.56
2.9 ± 0.28* 4. 3 ± 0.17
3.1 ± 0.16* 3.0 ± 0.16*
Triglyceride
Mean ± SE in 5 rats.
C/P, cholesterol/phospholipids.
* Statistically significant compared with day 0 (P < 0.05).

This value is not consistent with the cholesterol
and phospholipid ratio in the liver membrane ofrats (Daum, 1985).
Biliary bile acid excretion is under the regulation of transporters, the bile salt export pump(bsep) (Gerloff et al., 1998) and multidrug resistance associated protein 2 (mrp2) (Paulusmaet al., 1996), in the canalicular membrane of thehepatocyte. Bsep is concerned with the excretion of amino acid conjugated bile acids andmrp2 with the excretion of bile acid sulfates andglucuronides. Biliary cholesterol and phospholipid secretions are related to multidrug resistance 2 (mdr2) P-glycoprotein. Phospholipid secretion is completely, and the cholesterol secretion is partially, dependent on mdr2 gene expression (Oude Elferink et al., 1996).
Cholestasis induced by lipopolysaccharide(Kubitz et al., 1999; Lee et al., 2000), phalloidin(Rost et al., 1999), ethinylestradiol (Lee et al.,2000), or bile duct ligation (Lee et al., 2000;
Paulusma et al., 2000) is accompanied by changes
in bsep and/or mrp2. ANIT produced cholestasis
and decreased biliary cholesterol and phospholipid secretion in parallel with the bile acid secretion (Figs. 1B and C), but ANIT is reported tohave no effect on mrp2 (Ogawa et al., 2000). Theeffect of ANIT on bsep is not yet known.
`-Muricholic acid in the biliary bile aids increased especially on day 4, the day when thebiliary bile acid secretion markedly increased,with a concomitant decrease in cholic acid.
These changes, however, were normalized on
day 6 or later. Maeyama (1977) also reported
an increase of `-muricholic acid in ANIT treated rats even after the bile flow was recovered.
The increase of `-muricholic acid and a decrease of cholic acid were also found in the serum, urine and feces. In the feces, the increaseof `-muricholic acid was found as the increaseof t-muricholic acid formed from `-muricholicacid by the action of intestinal bacteria.
The increase of `-muricholic acid is also reported in bile duct ligated rats (Kinugasa et al.,
1981; Takita et al., 1988; Fujio et al., 1989), indicating that cholestasis causes an increase in `muricholic acid formation. Takita et al. (1988)
suggested that an alternative metabolic pathway
in bile acid synthesis via 26-hydroxycholesterol,3`-hydroxy-5-cholenoic acid and lithocholicacid contributes to the increase of `-muricholicacid in bile duct ligated rats. `-Muricholic acidformation is known to increase after the feedingof cholesterol in rats (Uchida et al., 1977), which
results in an elevation of the liver cholesterollevel. In the case of ANIT treatment (the present experiment) and bile duct ligation (Kinugasaet al., 1981), a minor or no increase in the livercholesterol level was found, though significant
hypercholesterolemia was produced. The mechanism for this increase of `-muricholic acid incholestasis is not yet known.
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ANIT induced cholestasis

Acknowledgments: We sincerely thank Messrs. Haruto
Takase, Yasuharu Nomura, Masumi Kadowaki andMasao Masui of Shionogi Research Laboratories fortheir skillful technical assistance, and also Ms. Yoshiko
Kurosawa of The Cell Science Research Foundation,Mr. Morio Oohori of Shionogi Co. Ltd., and Ms.
Yumiko Uyama of the Division of Medical Biochemistry, Department of Pathophysiological and Therapeutic Science, School of Medicine, Tottori University
for their help in preparing the manuscript.

The serum bile acid concentration markedly
increased after ANIT treatment and the urinary
bile acid excretion increased being delayed
about 1 day more than the increase of the serumbile acids. The enormous amounts of urinary
bile acids on day 2 came, we expect, from thebile acid pool in the enterohepatic circulation.
Fecal bile acid excretion decreased duringcholestasis, but increased after day 5 or later
exceeding the initial level. This increase is areflection of the increase of the bile acid synthesis in the liver. Hepatic cholesterol synthesis
and cholesterol 7_-hydroxylation activities arereported to increase in bile duct ligated rats
(Danielsson, 1973; Cooper and Ockner, 1974;
Adler et al., 1977). When one lobe of the liver
was experimentally ligated, cholesterol synthesis increased only in the ligated lobe (Cooperand Ockner, 1974; Adler et al., 1977). Therefore, cholestasis is considered to increase hepatic cholesterol synthesis, and probably subsequent bile acid synthesis, in rats.
Fecal sterol excretion decreased on days 1–
2 reflecting a decrease in feces dry weight, butmarkedly increased on days 3–4 and then gradually decreased to the initial level. The composition ratios of coprostanol and cholesterol werealmost identical through out the experimental
period, suggesting that the intestinal bacteria
concerned with the sterol transformation were
not changed by ANIT treatment. On the otherhand, the secondary bile acid composition waschanged after ANIT treatment. t-Muricholicacid increased and hyodeoxycholic acid decreased after days 5–6 or later. `-Muricholicacid is transformed to t-muricholic acid andthen to hyodeoxycholic acid by the intestinalbacteria. Therefore, the intestinal bacteria concerned with the bile acid metabolism werechanged in the ANIT treated rats.
The serum cholesterol and phospholipid
levels increased for 4 to 5 days after ANIT
treatment. Since enhancement of cholesterol
absorption is not the reason, the increase may
be a reflection of biliary epithelial cell necrosis(Orsler et al., 1999).

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Received April 26, 2002; accepted May 17, 2002
Corresponding author: Prof. Kazuo Yamada68