The role of bile salt composition in liver pathology of mdr2 (−/−) mice: differences between males and females

Journal of Hepatology 1991; 26: 138-145 Printed in Denmark . All rights reserved Munksgaard Copenhagen

Copyrighf 0 European Association for the Study of the Liver 1997

Journal of Hepatology ISSN 0168-8278

The role of bile salt composition in liver pathology of mdr2 (-/-) mice: differences between males and females

Carin M J van Nieuwerk’,2, Albert K. Green’, Roelof Ottenhoff’, Michael van Wijland’, . .

Marius A. van den Bergh Weerman 2, Guido N. J. Tytgati, Johan J. A. OfferhaLts and

Ronald P J. Oude Elferink’

Departments of ‘Gastrointestinal and Liver Diseases and, ‘Pathology, Academic Medical Center, Amsterdam, The Netherlands

Background/Aims: The mouse mdr2 gene encodes a P-glycoprotein expressed in the canalicular membrane of the hepatocyte. Mice in which this gene has been inactivated (mdr2 -/-) show a defect in biiary phospholipid and cholesterol secretion and develop non-suppurative cholangitis. We hypothesized that secretion of bile salts without lipids initiates this liver disease. Methods: To delineate the pathologic process, mdr2 (-/-) mice were fed different bile salt-supplemented diets for 22 weeks after weaning. Aspects of liver pathology including eosinophilic bodies, portal inflammation, ductular proliferation, mitotic activity and fibrosis were semiquantitatively scored. Results: It was observed that liver pathology was more severe in female than in male mice when fed a purified control diet. This correlated with a more hydrophobic bile salt composition of female vs. male bile. When increasing amounts of cholate were added to the diet

D UE TO THEIR detergent properties, bile salts are cytotoxic compounds. Many studies have dem-

onstrated that incubation of cells with micellar concen- trations of bile salts leads to rapid cell death (l-3). For different types of bile salts a clear correlation was observed between the degree of cytotoxicity and hydrophobicity. The presence of phosphatidylcholine and cholesterol in bile inactivates the cytotoxicity of bile salts by the formation of mixed micelles (2,4,5). Hence it was hypothesized that one of the main functions of

Received 29 March; revised 27 June; accepted I July 1996

Correspondence: C. M. J. van Nieuwkerk, MD, Dept. of Gastrointestinal and Liver Diseases, Academic Medical Center, C2, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Fax. (20) 6917033. Tel. (20) 5664301.

(0.01% and O.l%), the secretion of taurocholate increased and this was accompanied by a more severe liver pathology. At the high dose of cholate (O.l%), the bile salt compositions of male and female mice became similar, as did the severity of the histological score. Addition of cholate to the diet did not induce liver pathology in (+/+) mice. Addition of ursodeoxycholate to the diet (0.5%) led to a near complete re- placement of biliary bile salts by tauroursodeoxycholate and this reduced pathology and dissipated the difference between males and females. Conclusions: These observations support our hypothesis that liver pathology in the mdr2 (-/-) mouse is caused by bile salts and depends on the hydrophobicity c.q. cytotoxicity of biliary bile salts.

Key words: Bile constituents; Bile salt hydrophobicity; Gender difference; mdr2 knockout mice.

biliary lipid secretion is to reduce the cytotoxic action of the high concentration of bile salts. Indeed, biliary lipid defective mdr2 (-/--) knockout mice develop liver disease (6). The liver pathology in these animals was characterized by a picture of inflammatory non- suppurative cholangitis, of which bile duct proliferation and portal inflammation were the hallmarks (7). On the basis of the studies mentioned above, it was hypothesized that this pathology was induced by toxic- ity of bile salts without lipids in the biliary tree. The endogenous bile salt pool of mice is very hydrophilic, i.e. it consists of bile salts with a relatively high critical micellar concentration (CMC). About 70% of the murine biliary bile salts consists of tauromuricholate, the remainder being taurocholate (8,9). To test our hypothesis, mdr2 (-/-) knockout mice were fed cholate or

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Bile salt hydrophobicity and liver pathology in mdr2 (-/-) mice

ursodeoxycholate, which rendered the biliary bile salts more hydrophobic or hydrophilic, respectively. In accordance with our hypothesis, we observed that cholate-fed mice developed liver pathology more rapid- ly, while in ursodeoxycholate-fed mice the progressive pathology was halted (10).

Further studies suggested that females developed more severe liver disease than male mdr2 (-/-) knockout mice. In the present report we investigated whether this phenomenon could be related to the cytotoxicity of different types of bile salts. Mice were fed a diet containing 0.01% or 0.1% cholate or 0.5% ursodeoxycholate. Progression of liver disease was monitored by measure- ment of liver enzymes in serum and by liver histology. The severity of the pathological process was different between male and female (-/--) knockout mice and could be related to the hydrophobicity of the bile salt pool. These data support our hypothesis that liver pathology in mdr2 (-/-) knockout mice is caused by secretion of bile salts without lipids which are cytotoxic.

Materials and Methods Previously the generation of homozygous mdr2 (-/-) mice, which lack a functional mdr2 gene, has been described (6). These animals had the 129/OLA strain as genetic background. To improve the poor breeding ef- ficiency of this strain, mdr2 (-/-) mice were serially crossbred with the FVB strain until a 94% FVB background was obtained. These (-/-) mice develop similar liver abnormalities to those with a 129/OLA genetic background (10). All mice were bred in our own col- ony; the applied procedures were in accordance with the institutional guidelines of the University of Amsterdam for experimental animals and approved by the local committee. Immediately after weaning, mdr2 (-/-) mice and wild type (+/+) mice were divided into three different diet groups with at least four male and four female (-i-) mice and three male and three female (+/+) mice in each diet group: purified control diet containing 20% (w/w) casein (K4068.02; Hope Farms, The Netherlands), and the same diet supplemented with either 0.5% (w/w) ursodeoxycholate or 0.1% (w/w) cholate. Because of high mortality in the (-I-) mice on 0.1% cholate diet, we later added an- other group consisting of at least four male and four female (-/-) and three male and three female (+/+) mice which were fed 0.01% (w/w) cholate. All mice had ad libitum access to water. After 22 weeks on the diets the animals were anesthetized by i.p. injection of 1 ml/kg Hypnorm (fentanyl/fluanisone) and 10 mg/kg di- azepam. The abdomen was opened and the gallbladder was cannulated after distal ligation of the common bile

duct. Bile was collected for a period of 2 h, after which the liver was resected for further analysis.

Histochemistry Tissue samples were fixed by immersion in a mixture of ethanol, acetic acid and formalin (8: 1:2 [v/v] (11). Five-micrometer-thick serial paraffin sections were stained according to standard histochemical procedures, with hematoxylin and eosin (H&E), periodic-acid Schiff (PAS), periodic-acid Schiff after diastase digestion (PAS-D), Gomorri’s reticulin stain, Perl’s iron stain and Van Giesson’s elastic stain.

Light microscopy Two independent investigators judged the liver pathology in a blinded manner. The variation in the scoring by the separate investigators was less than the biological variation between mice in one experimental group. Liver histology was semi-quantatively scored for five histo- pathological parameters in four grades (O-3) of severity (7): mitotic activity, portal inflammation, ductular proliferation, Councilman bodies and fibrosis. This scoring system is the same as previously described (10). Mitotic activity (mitotic figures per lobule at a magnification of 400x) was: absent (0) less than three (l), three to five (2), or more than five (3). The degree of inflammation in the portal triads was graded as follows: absent (0), no inflammatory cells present; (l), only scattered inflammatory cells present in the mesenchyma of the portal triads; (2) increasing inflammatory infiltrate associated with cholangiolitis, polymorphonuclear leukocytes in- vading bile ducts; and (3) destructive cholangiolitis with presence of fibrosis. Ductular proliferation per high power field at magnification of 250~ was graded as follows: absent (0) no proliferation present; (1) no more than one portal tract involved; (2), one to five portal tracts involved; and (3), all portal tracts involved. The number of acidophilic (‘Councilman’) bodies in each lobule at a magnification of 400X was graded as follows: absent (0) less than three (1) three to ten (2), more than ten (3). The grade of fibrosis was scored as follows: (0), no fibrosis present; (l), expansion of the portal triad by increase of fibrotic tissue; (2) sporadic presence of porto-portal septate fibrosis in one or two lobules; (3), septate fibrosis surrounding the majority of lobules. Adding the scores for these various parameters, gave a maximum total histology score of 15.

Bile and serum analysis Activities of serum liver enzymes were determined by routine clinical tests on a Hitachi analyzer according to the instructions of the manufacturer. Total choline content of phosphatidylcholine and sphingomyelin in

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bile was determined enzymatically with phospholipase D and choline oxidase (12). Cholesterol was assayed enzymatically with cholesterol oxidase (13). Total bile salt concentration was measured spectrophotometr- ically with 3a-hydroxysteroid dehydrogenase (14). Bile salt species from selected bile samples were analyzed by HPLC as described by Ruben et al. (15) modified as follows: samples were diluted 1:200 in eluent (methanol 60%, 3 mM K2HP04, pH 3.75; 40%). Twenty micro- liters of this sample were injected on the HPLC column (Cl 8 Chromspher 5 ,um, Chrompack with a flow of 800 pl/min). Peaks were detected using an UV detector at 205 nm.

All bile samples were also investigated by light microscopy to see whether stones or sludge were present.

Statistics All values represent means? standard deviation of three or more animals in each group of (-/-) knockout mice and (+/+) wild type mice. Differences between the groups were analyzed by Wilcoxon’s two sample test. Significance was assumed at ~~0.05.

Results Bile formation and bile salt composition in male vs. female mice We fed mdr2 (+/+) and (-/-) mice different bile salt supplemented diets for a period of 22 weeks after weaning. The effect on bile formation in male and female mice is shown in Table 1. As described before, mdr2 (-/-) mice have a negligible amount of phospholipid in their bile and only secrete substantial amounts of cholesterol when fed a high cholate (0.1%) diet. Bile flow was also significantly higher in (-/-) vs. (+/+) mice. Female mice from both the mdr2 (+/+) and the (-/--) strain had a significantly higher bile salt secretion than male mice. Upon cholate feeding bile salt secretion increased and was no longer different between females and males. In male mdr2 (--I-) mice on control diet the glutathione (GSH) secretion in bile was significantly higher than in females. GSH secretion in males declined drastically upon cholate feeding and became identical to that in female mice.

In agreement with previous reports (8,9), HPLC analysis of the various bile samples revealed only two major bile salt species, taurocholate and tauromuri-

TABLE 1

Bile formation in male and female mdr2 (-/-) mice on various diets

Bile flow Bile salt PL Chol. GSH ALP1 jd min-’ . 100 g jd . min-’ . 100 g 1 min-’ . 100 g

(-/-) mice Control diet

Male (8) 9.7kO.5 2262 107 0.08~0.09 0 lOk4.3 4232269 Female (4) 10.9k3.6 369+97* 0.03kO.06 0 2.7?0.6** 539-t 149

0.01% cholate

Male (6) 10.6k2.9 3582 105” 0.0320.05 0.23t0.33 6.7k3.6 5215403 Female (5) 8.8kl.O 424k 130 0.0620.04 0.03~0.05 5.922.9 3182100

0.1% cholate

Male (6) 14.4t2.6a 517?81a 1.42?1.17” 4.3k2.0 1.9t1.8” 25802770” Female (4) 16.6k5.9 4992350 0.9320.89 2.023.1 1.622.7 42OOk2830”

0.5% UDCA

Male (3) 13.8-e7 438283 0.05-cO.08 0 7.31 kO.83 12082470 Female (4) 11.423.5 624k283 0.30t0.23 0.18kO.35 9.1Ok1.44 16215809

(+/+) mice Control diet

Male (4) 6.022.4 179576 Ilk4 l.lkO.7 24.026.0 151256 Female (4) 5.5kl.O 381260 32-c8** 1.7-cO.8 18.1k3.1 234239

0.01% cholate

Male (3) 10.4kl.l 454215 35&l 7.421.7 44.3211.7 212531 Female (4) 5.OkO.6 411282 3524 2.250.4 25.053.5 311257

0. I % cholate

Male (6) 8.0k1.3 308k68 33.129.5 7.052.4 31.1k5.2 283486 Female (4) 6.6k1.4 3962252 43.4220 6.Ok3.8 31.0k6.1 2882193

0.5% UDCA

Male (4) 9.7k3.3 5472 148 28.729.5 6.5k2.8 34.2217 5892239 Female (4) 8.721.7 886+ 184* 81.6?29.6* 7.722.1 50.258.7 7872541

* JKo.05 ** p<O.Ol female vs. male. a ~~0.05 experimental vs. control diet.

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fied control diet was significantly higher than in male mice. In the 0.01% cholate-supplemented (-i-) group the serum ALP was only 2-2.5 times higher in both genders compared to the control, diet group, but there was no significant difference any more. However, 0.1% cholate feeding induced a dramatic increase in serum ALP levels in (-/--) in both genders. ALAT levels demonstrated a similar picture; on control diet female mice had significantly higher levels, while in the bile- salt-supplemented groups no significant difference was observed. The serum bilirubin was not elevated and showed no differences between male and female (-/-) mice. On a 0.01% cholate-containing diet serum bilirubin varied strongly; in some female mice a marked elevation was observed, while in others normal levels were measured. Male (-/-) mice on this diet had normal serum bilirubin levels. In contrast, serum bilirubin in the 0.1% cholate-containing diet group was severely elevated in both male and female (-/-) mice, sug- gesting that cholestasis had set in. UDCA feeding also

TC concentration of bile salt pool

100

80

60

40

20

0

control diet 0.01% CA 0.1% CA

m Male mdr2 -I- = Female mdr2 -I-

Fig. I. Taurocholate concentration (TC) in bile of female and male mdd (-/-) mice on different diets. On the y-axis, percentages of the bile salt pool taken by TC are given. TheJigure gives only the taurocholate content of bile; the remainder of the biliary bile salts consisted of tauromuricholate.

TABLE 2

Serum level of alkaline phosphatase (ALP), alanine aminotransferase (ALAT) and bilirubin in male and female mdr2 (-/-) and (+/+) mice after 22 weeks on various diets

cholate, with only traces of taurochenodeoxycholate. Female mice on control diet also had a more hydrophobic composition of biliary bile salts. Figure 1 shows that female (-/-) bile contains an almost twofold higher concentration of taurocholate than male (-I-) bile (3424 vs 18+2%), the remainder being tauromuricholate. A significant difference between males and females was also observed in the mar2 (-, t) strain: in male mice on control diet the TC concentration was 122 1% and in female mice 47 * 3% (not shown). Tauromuricholate was efficiently replaced by taurocholate after feeding 0.1% cholate-supplemented diet. Addition of 0.01% cholate to the diet did not lead to significant change in the bile salt composition, although the total bile salt secretion increased significantly in comparison with the control diet. When mdr2

(-/-) mice were fed with ursodeoxycholate-supplemented diet (0.5%), the biliary bile salts were replaced by tauroursodeoxycholate to an equal extent in female (n=3) and male (n=3) animals (7823 and 75?6%, respectively), the remainder being tauromuricholate. A similar picture was observed in mdr2 (+/+) mice.

n ALP ALAT Bilirubin (IU/l) (IU/l) WI)

(-/-) mice Control diet

Male Female

0.01% cholate Male Female


0.5% UDCA Male Female

90240 112~55 202?46* 202?52

251 4t1

4 4

3 5

178?6 215274 574*309 271?59

321 69?56

3 22605475 4802 309 151261 3 36985 1783 26026 223296

4 454-+ 147 250243 9*3 4 269257 2402204 14219

(-I-/+) mice Control diet

Male Female



0.5% UDCA Male Female

4 4423 5729 221 4 6228” 45?19 2*1

2 3226 76251 3-tl 4 4927 59+15 221

4 61222 35-c9 221 4 8126 34’-2 121 Hepatic damage in mdr2 (-/-) and (+/+) mice after

bile salt feeding

Table 2 shows the serum levels of alkaline phosphatase (ALP), alanine aminotransferase (ALAT) and bilirubin after 22 weeks on the experimental diet. The serum ALP level in female (-/-) mice after 22 weeks on puri-

4 4627 32-c2 251 4 5525 3026 121

* p<O.Ol, a p<O.Ol; this was probably not a real high value because in the other three experimentai diet groups there was no increase in ALP compared with male (+/+) mice on control diet.

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C. M. J. van Nieuwkerk et al.

Mean total histological score

control diet 0.01% CA 0.1% CA 0.5% UDCA

m Male mdr.2 -I- = Female mdr2 -I-

Fig. 2. Mean total histological scores plus range in male and female m&2 (-/-) mice on different diets. These are the mean total scores for each of the 5 parameters after 22 weeks on the different diets. Each parameter can score at maximum 5 points; maximum mean total score is thus 1.5. P-values (not shown) in the m&2 (-/-) mice on purified control diet and in the mice on 0.01% cholate-containing diet are 0.02 and CO.05 respectively. In the mdr2 (+/+) mice the mean histological scores plus range in male and female mice after 22 weeks diet as follows (not shown): purtfied control diet: male mice 0.66; range O-2 vs 0.33; range O-I in male mice. 0.01% Cholate-containing diet. male mice 0.5; range O-I vs 0.75; range O-I in female mice. 0.1% Cholate-containing diet: male mice 2; range 2 vs I; range 1 in female mice. 0.5% UDCA-containing diet: male mice 0; vs 0.75 range O-I in female mice.

caused an elevation in ALP and ALAT levels, which were not different between male and female mice. For comparison, the values of these markers in mdr2 (+/

+) mice on the different experimental diets are also given.

Liver histology in male and female (-/--) mice

One (male) out of nine (-/--) mice died on a 0.01% cholate-containing diet during the course of the study, and four out of eight mice on a 0.1% diet, all male mice: three after 6 weeks and one after 22 weeks. The mean total histological score for male and female mdr2

(-I-) mice on the various diets is given in Fig. 2. Fe- male (-I-) mice on a purified control diet showed significantly higher histopathology scores than male (-/-) mice (mean total histology score 7.5; range 6- 10 vs 4.8; range 4-6, p=O.O2). Especially portal inflammation and ductular proliferation contributed to this higher score and were mainly responsible for the difference. A representative picture of the histology in

female mdr2 (-I-) mice on control diet is given in Fig. 3A and B. Both male and female (-/-) mice on a 0.01% cholate-containing diet showed more prominent portal inflammation and ductular proliferation than animals on control diet (Fig. 3C and D). The overall histological score in females on 0.01% cholate was still significantly higher than for male mice (mean histological score 10.8; range 8-13 vs. 6.3; range 67, ~~0.05). However, (-/-) mice on a 0.1% cholate-containing diet demonstrated an even worse microscopic picture, consisting of extreme portal inflammation, ductular proliferation and also severe fibrosis, and this picture was independent of gender (mean histological score 12; range 12 in both groups). In addition, the overall pathological picture decreased on UDCA-supplemented diet and the gender difference completely disappeared (mean histological score in males 4; range 4 vs. 4; range 2-6 in female (-/-) mice). A representative picture of the histology UDCA-containing diet is given by in Fig. 3E. Im- portantly, when looking solely at the mitotic index and Councilman bodies, it turned out that the number of mitotic figures and Councilman bodies was not significantly different in the various diet groups among male and female (-/ -) mice.

In (+/+) mice, liver histology was minimally affected by 0.1% CA; the mean histological score in female mice was 0.33 (range Crl) vs. 0.66 (range O-2) in male mice.

Discussion The liver pathology in mdr2 (-/-) mice is characterized by a gradually progressive picture that mainly affects the portal triads. The main features are ductular proliferation and portal inflammation with relatively little hepatic damage and only weak fibrosis. The nature of the bile salt species in bile of the mdr2 (-/-) mouse clearly determines the course and outcome of this pathological process. The majority of the endogenous murine bile salt pool consists of tauromuricholate, which is a relatively hydrophilic bile salt. We have already shown that re- placement of the bile salt pool by taurocholate, as occurs on a diet containing 0.1% cholate, gives a more severe pathological picture and is almost lethal to the animals. Under those conditions extreme proliferation of bile duct epithelium is observed, accompanied by heavy inflammation of the portal fields and striking fibrosis (biliary cirrhosis). After 22 weeks on this diet animals are without exception cholestatic, as demonstrated by a high serum bilirubin content, and they also have high serum levels of aminotransferase and alkaline phosphatase activities (10).

In the present study we have further delineated this liver disease. The main conclusion from these experi-

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Bile salt hydro~hobicity and liver pcrthology in mdd i-/-j mice

ments must be that the initiation and prolongation of the pathological process is triggered by only minor changes in the bile salt composition of the secreted bile. Several observations support this conclusion.

Firstly, female m&2 (-/-) mice developed a more severe histological picture than their male counterparts when fed a purified control diet. This correlated with a relatively small difference in bile salt composition: in female mice 34% of the secreted bile salts consisted of tauro~holate, while this was 18% in male mice, the remainder being tauromuricholate in both sexes. That

Fig. 3. (A/B) Microscopy of the liver of u mdd knockout mouse after 22 weeks on purified control diet; female mice (A) show more ductulur proliferation and portal injlam- ~nution thun male mice (3). original magn~~~ution: X400. (CID). Microscopy of the liver af a mdr2 knockout mouse after 22 weeks on 0.01% CA-containing diet; female mice (C) show more pronounced portal injlammation and ductulur proltferation than the male mice (D). Original magni- ~~ation: X400. (E). ~i~ros~o~y of the liver of a mdr2 knockout mouse after 22 weeks on 0.5% UDCA-containing diet;female mice show the same degree ofpathology us mule mice (not shown). Original mugncjication: ~400.

this difference in endogenous bile salt composition was the cause of the difference in pathology was demonstrated by the fact that the difference in pathology could be annihilated by substituting the bile salt pool either with tauroursodeoxycholate or with taurocholate. In the first case the histology improved and became equal in both genders, while in the latter case the pathology worsened but again became equal in males and females. The reason for the difference in bile salt composition and bile salt output in male vs. female mice is as yet unexplained. It is, however, independent

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C. M. J. van Nieuwkerk et al.

of the mdr2 (-/-) phenotype because the same differences were observed in wild type mice. Total bile salt synthesis and/or cholesterol synthesis may be higher in female mice. In addition, expression of the separate enzymes in the biosynthetic pathways of cholate vs. muricholate may be under hormonal control and thus be expressed to a different extent in male and female mice.

Secondly, feeding male mice a diet which contained a very low amount of cholate (0.01%) did not significantly change the bile salt composition, but only increased the total bile salt output by about 50%. Never- theless, this led to a significant increase in the mean histological score as well the serum levels of alkaline phosphatase and alanine aminotransferase. The lack of change in the bile salt composition on a low cholate- diet, accompanied by an increased total bile salt output, is unexplained; apparently feeding low amounts of cholate increases not only the output of taurocholate but also that of tauromuricholate. A possible explanation might be that the presence of cholate in the in- testine increases the (re)absorption of cholesterol, thereby increasing precursor availability for bile salt synthesis which, in the mouse predominantly produces muricholate.

The data show that male mdr2 (-/-) mice on purified diet have such a low proportion of taurocholate in their bile that the pathology is very mild; no damage to the liver parenchyma is observed and very little portal inflammation and ductular proliferation. This corre- lates with only a minor elevation of alkaline phosphatase and alanine aminotransferase in the serum, an- other argument that damage to hepatocytes is virtually negligible under these conditions. It suggests that the bile duct epithelial cells are the most sensitive target of lipid-free bile, to which they respond with a proliferat- ive reaction. Only when bile becomes more cytotoxic, with a higher proportion of taurocholate, does the liver parenchyma also become involved, and the reaction of bile duct epithelial cells becomes more pronounced. In this context it should also be noted that male mdr2 (-/-) mice on a UDCA-containing diet had a significant increase in both ALP and ALAT This increase in serum enzymes on a UDCA-containing diet was not observed in (+/+) mice. The high rate of TUDC secretion that occurs on this diet could be harmful in mdr2 (-/-) mice. In contrast, histological observation of bile duct proliferation and portal inflammation decreased on a UDCA-diet compared to control diet. This observation also supports the notion that injury to the parenchymal cells and the bile duct epithelial cells leads to separate pathological entities.

Analysis of the biliary glutathione output in mice on various diets revealed a clear correlation with the sever-

ity of the liver disease. In (-/--) mice on a high cholate diet, GSH was barely detected in bile (1.9 and 1.6 nmol - min- 1 - 100 g in male and female mice resp.). However, in male mice on control diet, which have only very mild liver pathology, the GSH output was 5-fold higher, which was still about 40% of that in (+/+) mice on a control diet. In female (-/-) mice on a control diet, the GSH output was almost four-fold lower than in their male counterparts. This clearly suggests that the decrease in biliary GSH output in mdr2 (-/-) mice is a secondary consequence of liver disease. It is not likely that this is due to increased breakdown of GSH in the biliary tree, since gamma-glutamyl- transferase activity is extremely low in murine liver. A possible explanation might be that there is down-regulation of the transporter responsible for GSH secretion during disease.

In human pathology, inflammatory non-suppurative destruction of bile ducts is a feature of various cholestatic liver diseases. The two most important entities in which this morphology predominates are primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). The etiology and pathophysiologic mechanisms of these two disorders are not completely understood, but immunologic, cytotoxic and infectious noxes may be important factors. There is no correlation between the bile salt pool composition and severity of these cholestatic diseases. However, PSC is predominantly found in males (16) and PBC in females (17); the reason for this gender difference is also unknown. The present data make comparison with the human situ- ation difficult; human bile contains cholate-, chenode- oxycholate- and deoxycholate-conjugates, each of which is more cytotoxic than tauromuricholate. We have shown here that relatively low amounts of taurocholate in bile of mdr2 (-/-) mice initiate the pathological process of bile duct proliferation and portal inflammation. Hence, as we have stated previously, the prediction would be that deficiency of biliary lipid secretion in man would lead to severe liver pathology, if not acute liver failure.

In conclusion, small changes in the bile salt composition in mdr2 (-/-) mice were found to influence the extent of liver pathology dramatically. This is in line with our previously postulated hypothesis that liver pathology in mdr2 (-/-) mice is directly related to cytotoxicity of the secreted bile salts in the absence of biliary lipids. Biliary lipids apparently serve an important function in the cytoprotection against bile salts.

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