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Cholemic transgenic mice: A novel animal model to investigate the effects of bile acids
Journal of Pharmacological and Toxicological Methods 50 (2004) 231 – 235 www.elsevier.com/locate/jpharmtox
Brief communication
Cholemic transgenic mice: A novel animal model to investigate the effects of bile acids Predrag Ljubuncica, Ibrahim Yousef b, Arieh Bomzona,* a
Department of Pharmacology, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P.O. Box 9649, Haifa 31096, Israel b Department of Pharmacology, University of Montreal, C.P. 6128, Surccursale A, Montreal, Quebec, Canada H3C 3J7 Received 3 March 2004; accepted 21 June 2004
Abstract Introduction: Laboratory investigations into cholestatic liver disease and the effects of cholemia on organ function are long-standing subjects of scientific enquiry. A widely-used strategy to investigate these topics relies on animal-based research using experimental animal models. Targeted inactivation of the spgp gene, the gene responsible for expressing the bile salt export pump (BSEP) in the hepatocyte canalicular membrane impairs the canalicular secretion of bile salts resulting in systemic cholemia. The results of in vitro experiments have established bile acids as pro-oxidants and the collection of unambiguous in vivo data on the pro-oxidant activity of bile acids in the existing models of cholemia cannot be done. Therefore, we decided to use these genetically modified mice to determine whether this model of cholemia has evidence of extrahepatic or systemic oxidative stress, one of the features of cholestatic liver disease. Methods: The extent of lipid peroxidation in livers, kidneys, hearts and brains harvested from cholemic homozygous (spgp / ) mice using the thiobarbituric acid reactive substances (TBARS) assay. The data were compared to equivalent data collected from heterozygous (spgp +/ ) and control mice. Results: We found (1) substantial increases in malondialdehyde (MDA) levels in the brains and hearts; (2) a moderate increase in MDA levels in the kidneys; and (3) no change in MDA levels in the livers of the homozygous cholemic mice compared to the control and heterozygous mice. Discussion: The transgenic mouse model of cholemia has an intact enterohepatic circulation and is uncomplicated by the adverse consequences of hepatotoxins or biliary surgery. Hepatocellular injury, as well as plasma and tissue accumulation of bilirubin and other liver-derived compounds are also negligible. Although this preliminary study could not establish a causal relationship between cholemia and oxidative stress, we believe this model is worthy of further investigation to study the impact of short-term and long-term cholemia on diverse physiological and biochemical functions such as trying to establish a causal role for bile acids in the development of oxidative stress in cholestatic liver disease. D 2004 Elsevier Inc. All rights reserved. Keywords: Bile acids; Oxidative stress; Lipid peroxidation; Malondialdehyde; Methods
1. Introduction Laboratory investigations into cholestatic liver disease and the effects of cholemia on organ function are longstanding subjects of scientific enquiry. A widely-used strategy to investigate these topics relies on animal-based research using experimental animal models. These models
* Corresponding author. Tel.: +1 972 4 8295259; fax: +1 972 4 8524978. E-mail address: [email protected] (A. Bomzon). 1056-8719/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2004.06.002
fall into one of several categories: surgical-induced models such as bile duct ligation; toxic injury of liver using hepatotoxins such as carbon tetrachloride or thioacetamide or hormones such as ethinyl estradiol; or sepsis-induced cholestasis (Bomzon & Blendis, 1990; Lee & Boyer, 2000). Many human genes have a mouse ortholog and the production of genetically modified animals has substantially increased our capabilities to investigate the roles of endogenous substances in the pathogenesis and course of disease (Bedell, Largaespada, Jenkins, & Copeland, 1997; Vaitukaitis, 1998). The canalicular secretion of bile salts
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from the liver into bile is a key process in the enterohepatic circulation of bile acids. The bile salt export pump (BSEP) is a canalicular-specific ATP-binding cassette transporter. It was originally called the sister of P-glycoprotein (spgp) because of its homology to the family of P-glycoproteins that conferred multidrug resistance in a large number of cell types (Childs, Yeh, Hui, & Ling, 1998). It was subsequently renamed as BSEP to emphasize its dependence on ATP (Gerloff et al., 1998). Targeted inactivation of the spgp gene in mice causes cholemia due to impaired canalicular secretion of bile salts (Wang et al., 2002). This mouse model has elevated plasma concentrations of bile salts and an intact enterohepatic circulation; therefore, there is negligible hepatocellular damage and the consequences of biliary surgery, hepatotoxins, sepsis and the plasma and tissue accumulation of bilirubin and other liver or biliary metabolites have no impact. Bile acids are known pro-oxidants (Ljubuncic, Fuhrman, Oiknine, Aviram, & Bomzon, 1996; Sokol, Devereaux, Khandwala, & O’Brien, 1993; Sokol, WinklhoferRoob, Devereaux, & McKim, 1995). This characteristic has been established from the results of in vitro experiments. Collecting unambiguous in vivo data on the prooxidant activity of bile acids in the existing models of cholemia cannot be done. Therefore, we decided to use these genetically modified mice to determine whether this model of cholemia has evidence of extrahepatic or systemic oxidative stress, one of the features of cholestatic liver disease.
used for lipid peroxidation determination by the thiobarbituric acid reactive substances (TBARS) assay (see next section for specific details). Protein contents of the individual tissue homogenates were determined by the method described by Lowry, Rosebrough, Farr, and Randall (1951). 2.2. Assessment of the extent of lipid peroxidation
2. Methods
The assessment of the extent of lipid peroxidation relied on individual determinations of the thiobarbituric acid reactive substances (TBARS) content in hepatic, kidney, heart and brain homogenates prepared from the three groups of experimental mice (Draper & Hadley, 1990). Malondialdehyde (MDA) is an end product of peroxidative decomposition of polyeonic fatty acids in the lipid peroxidation process and its accumulation in tissues is indicative of the extent of lipid peroxidation (Draper & Hadley, 1990). In an earlier publication, we proved bile acids did not interfere with the TBARS assay (Ljubuncic et al., 1996). TBARS reagent (1 ml) was added to a 0.5 ml aliquot of tissue homogenate and heated for 20 min at 1008C. The antioxidant, butylated hydroxytoluene, was added before heating of samples. After cooling on ice, the samples were centrifuged at 840g for 15 min and the absorbance of the supernatant was read at 532 nm. Sample blanks for each sample were prepared and assessed in the same way to correct for A532 contribution due to the sample. The results were expressed as MDA equivalents (expressed in nmol MDA) using 1,1,3,3-tetraethoxypropane as the standard. Duplicate determinations were made and the average of the two measurements was used in the subsequent statistical analysis.
2.1. Preparation of tissue homogenates
2.3. Statistical analysis of the data
Tissue homogenates were prepared from four normal (wild-type) (sgpg +/+), eight heterozygous (sgpg +/ ), and 10 homozygous (spgp / ) mice. The sgpg / mice were prepared using transgenesis in embryonic stem cells (see previously published paper, Wang et al., 2002). Briefly, animal transgenesis involved inactivating the spgp gene in two different embryonic stem cell lines and subsequent implantation of the stem cells into pseudo-pregnant mice. Chimeric progeny were then mated with wild C57Black/6J mice to produce hemizygous mice (sgpg +/ ), which in turn were mated to produce the homozygous mice (sgpg / ). The impact of this transgenesis on bile acid regulation of hepatic function was studied in heterozygous and homozygous mice in Canada (Wang et al., 2002). Upon completion of these investigations, the livers, kidneys, heart and brains were harvested and couriered to Israel on dry ice. Each tissue was cut into small pieces by fine scissors and then homogenized in ice-cold 100 mM sodium phosphate buffer, pH 7.4 at 4 8C with a Potter–Elvehjem glass homogenizer. Separate aliquots of each homogenate were
The data are expressed as meansFstandard error of the mean. The data were analyzed by one-way analysis of variance using a Dunnett’s post-test. Pb0.05 (two-tailed) was considered significant.
3. Results Fig. 1 describes the accumulation of TBARS or MDA equivalents in the liver (A), kidney (B), brain (C) and heart (D) in the three groups of mice. Focusing on individual tissues, the amount of TBARS or MDA equivalents in the livers of the three groups of mice were not different from each other (Fig. 1A). Levels of TBARS or MDA equivalents tended to be higher in the kidneys of the heterozygous and homozygous mice than in the control mice but the differences were not statistically significant (Fig. 1B). However, the levels of TBARS or MDA equivalents in brain and heart homogenates prepared from the homozygous mice were two to three times higher than
P. Ljubuncic et al. / Journal of Pharmacological and Toxicological Methods 50 (2004) 231–235
233
Fig. 1. The tissue concentrations of malondialdehyde (MDA) in livers (A), kidneys (B), brains (C) and hearts (D) harvested from four control (black), eight heterozygous (spgp +/ ) (dark grey) and 10 homozygous (spgp / ) (light grey) mice. *( pb0.05) and **( pb0.01) represents the significance of the difference from control.
levels measured in the identical tissues of control and heterozygous mice (Fig. 1C and D).
4. Discussion Bile acids, and in particular hydrophobic types, are prooxidants. The basis of this assertion comes from experimental reports on the pro-oxidant effects of bile acids in cultured hepatocytes (Sokol et al., 1993, 1995) and macrophages (Ljubuncic et al., 1996). For several years, we have tried to implicate bile acids as one of the causal or contributory factors in the development of oxidative stress in non-hepatic tissues in cholestatic liver disease (Bomzon, Holt, & Moore, 1997; Ljubuncic, Tanne, & Bomzon, 2000). The existing models of cholemia have hampered our efforts to verify this hypothesis. They were created to emphasize one or more of the specific features of cholestatic liver disease and different methods of induction impose limitations on the research value of the model. For example, bile duct-ligated rats have severe hepatocellular injury with gross disruption of hepatic architecture and exhibit signs of nephropathy (Bomzon, Jacob, & Better, 1996), cardiopathy (Lee & Bomzon, 1990) and the hepatopulmonary syndrome (Fallon, Abrams, McGrath, Hou, & Luo, 1997). They are also susceptible to sepsis (Kennedy, Parks, Clements, & Rowlands, 1995; Parks, Clements, Smye, Pope, Rowlands, & Diamond, 1996; Reynolds, Murchan, Leonard, Clarke, Keane, & Tanner, 1996; Wen Ding, Andersson, Norgren, Stenram, & Bengmark, 1992) and are oxidatively stressed (Kra¨henbu¨hl, Talos, Lauterburg, & Reichen, 1995; Ljubuncic et al., 2000). They are also moribund after 6 weeks (Bomzon & Blendis, 1990). Because the enterohepatic circulation is surgically disrupted, liver or biliary metabolites, including bile acids, accumulate in the plasma, liver and extrahepatic tissues. As a result, this
model precludes the opportunity to ascertain a causal contribution of bile acids for some of these extrahepatic sequelae because their accumulation occurs together with that of bilirubin and other compounds. In the other models, a different set of issues arise. Spontaneous resolution of the hepatocellular damage occurs if administration of the hepatotoxin stops (Bomzon & Blendis, 1990). Therefore, data can only be collected while the animal is intoxicated. Similarly, data collected in the septic models makes it difficult to distinguish between the effects of sepsis and those of liver or biliary metabolites on extrahepatic organ function. The development of the spgp / mouse model of cholemia creates a new opportunity to consolidate this idea because it is free from all these limitations. Moreover, it is unique because one of the specific bile acid transporters in canalicular membrane of the hepatocyte has been inactivated resulting in an untainted or pure cholemia of longer duration that cannot be obtained in the other models. Systemic oxidative stress is one of the features of cholestatic liver disease encompassing the kidneys, heart and brain (Bomzon et al., 1997; Ljubuncic et al., 2000; Orellana, Rodrigo, Thielemann, & Guajardo, 2000; Panozzo, Basso, Balint, Zaninotto, Bonvicini, & Plebani, 1995). We found increased extents of lipid peroxidation in the brains and hearts of the homozygous transgenic mice thereby providing the evidence of systemic oxidative stress in these animals. Wang et al. (2002) reported four-fold increases in plasma bile acid levels in the cholemic spgp / mice when compared to plasma levels in the wild spgp +/+ type mice. In the absence of information of other potential contributors to the development of systemic oxidative stress, it is tempting to equate association with causality in this mouse model of cholemia. Therefore, we think it is not unreasonable to assume bile acids through their pro-oxidant activity could account for the oxidative processes detected in the heart and brains of these
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mice. In order to support or even refute the hypothesis, future investigations should be directed towards establishing the levels and types of bile acids in the tissues of these mice and their pro-oxidant potential. We also reported that the extent of lipid peroxidation was greater in the kidneys of these mice than control or heterozygous mice, the increase did not quite reach a level of statistical significance. The organs came from mice used in an unrelated project undertaken in Canada, thereby limiting the number and types of tissues that could be studied. Therefore, it is possible a type II error exists that could be overcome by increasing the group sample sizes to ensure that the data are unambiguous. The TBARS assay is a well-validated system having a considerable amount of historical control data (Esterbauer & Cheeseman, 1990; Halliwell & Chirico, 1993) and is extensively used by many investigators to establish the presence of an oxidative process in tissues. This assay is a target of criticism where its critics claim the measurement of F 2-isoprostanes (prostaglandins formed by free radical-mediated lipid peroxidation levels) is a more reliable method to detect lipid peroxidation in biological fluids such as plasma and urine (De Zwart, Meerman, Commandeur, & Vermeulen, 1999; Morrow et al., 1999). Therefore, it is possible that the use of this method would bestow statistical significance to the kidney data. For these reasons, additional validation studies involving more animals are needed to establish this transgenic mice model of cholemia as a valuable addition to the existing models. In summary, this transgenic mouse model of cholemia has an intact enterohepatic circulation and is uncomplicated by the adverse consequences of hepatotoxins or biliary surgery. Hepatocellular injury, as well as plasma and tissue accumulation of bilirubin and other liver-derived compounds are also negligible. As a result, we believe this model is worthy of further investigation to study the impact of short-term and long-term cholemia on diverse physiological and biochemical functions such as trying to establish a causal role for bile acids in the development of oxidative stress in cholestatic liver disease. Acknowledgements This research was supported by the Center for Absorption in Science of the Ministry of Immigrant Absorption and the Committee for Planning and Budgeting of the Council for Higher Education under the framework of the KAMEA Program. References Bedell, M. A., Largaespada, D. A., Jenkins, N. A., & Copeland, N. G. (1997). Mouse models of human disease: Part II. Recent progress and future directions. Genes and Development, 11, 11 – 43.
Bomzon, A., & Blendis, L. M. (1990). Animal models of liver disease. In A. Bomzon, & L. M. Blendis (Eds.), Cardiovascular complications of liver disease (pp. 9 – 28). Boca Raton7 CRC Press. Bomzon, A., Holt, S., & Moore, K. (1997). Bile acids, oxidative stress, and renal function in biliary obstruction. Seminars in Nephrology, 17, 549 – 562. Bomzon, A., Jacob, G., & Better, O. S. (1996). Jaundice and the kidney. In M. Epstein (Ed.), The kidney in liver disease (pp. 423 – 446). Philadelphia7 Hanley & Belfus. Childs, S., Yeh, R. L., Hui, D., & Ling, V. (1998). Taxol resistance mediated by transfection of the liver-specific sister gene of Pglycoprotein. Cancer Research, 58, 4160 – 4167. De Zwart, L. L., Meerman, J. H. N., Commandeur, J. N. M., & Vermeulen, N. P. E. (1999). Biomarkers of free radical damage applications in experimental animals and in humans. Free Radical Biology & Medicine, 26, 202 – 226. Draper, H. H., & Hadley, M. (1990). Malondialdehyde determination as an index of lipid peroxidation. Methods in Enzymology, 186, 421 – 431. Esterbauer, H., & Cheeseman, K. H. (1990). Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods in Enzymology, 186, 407 – 421. Fallon, M. B., Abrams, G. A., McGrath, J. W., Hou, Z. Y., & Luo, B. (1997). Common bile duct ligation in the rat: a model of intrapulmonary vasodilatation and hepatopulmonary syndrome. American Journal of Physiology: Gastrointestinal and Liver Physiology, 272, G779 – G784. Gerloff, T., Stieger, B., Hagenbuch, B., Madon, J., Landmann, L., Roth, J., Hofmann, A. F., & Meier, P. J. (1998). The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. Journal of Biological Chemistry, 273, 10046 – 10050. Halliwell, B., & Chirico, S. (1993). Lipid peroxidation: its mechanism, measurement, and significance. American Journal of Clinical Nutrition, 57, 715S – 725S. Kennedy, J. A., Parks, R. W., Clements, W. D. B., & Rowlands, B. J. (1995). Failure of macrophage activation in experimental obstructive jaundice: association with bacterial translocation. British Journal of Surgery, 82, 1433 – 1434. Kra¨henbu¨hl, S., Talos, C., Lauterburg, B. H., & Reichen, J. (1995). Reduced antioxidative capacity in liver mitochondria from bile duct ligated rats. Hepatology, 22, 607 – 612. Lee, J., & Boyer, J. L. (2000). Molecular alterations in hepatocyte transport mechanisms in acquired cholestatic liver disorders. Seminars in Liver Disease, 20, 373 – 384. Lee, S. S., & Bomzon, A. (1990). The heart in liver disease. In A. Bomzon, & L. M. Blendis (Eds.), Cardiovascular complications of liver disease (pp. 81 – 102). Boca Raton7 CRC Press. Ljubuncic, P., Fuhrman, B., Oiknine, J., Aviram, M., & Bomzon, A. (1996). Effect of deoxycholic acid and ursodeoxycholic acid on lipid peroxidation in cultured macrophages. Gut, 39, 475 – 478. Ljubuncic, P., Tanne, Z., & Bomzon, A. (2000). Evidence of a systemic phenomenon for oxidative stress in cholestatic liver disease. Gut, 47, 710 – 716. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265 – 275. Morrow, J. D., Chen, Y., Brame, C. J., Yang, J., Sanchez, S. C., Xu, J., Zackert, W. E., & Roberts, L. J. (1999). The isoprostanes: unique prostaglandin-like products of free-radical-initiated lipid peroxidation. Drug Metabolism Reviews, 31, 117 – 139. Orellana, M., Rodrigo, R., Thielemann, L., & Guajardo, V. (2000). Bile duct ligation and oxidative stress in the rat: effects in liver and kidney. Comparative Biochemistry and Physiology. C, Comparative Pharmacology Toxicology and Endocrinology, 126, 105 – 111. Panozzo, M. P., Basso, D., Balint, L., Zaninotto, M., Bonvicini, P., & Plebani, M. (1995). Renal functional alterations in extrahepatic cholestasis: can oxidative stress be involved? European Surgical Research, 27, 332 – 339.
P. Ljubuncic et al. / Journal of Pharmacological and Toxicological Methods 50 (2004) 231–235 Parks, R. W., Clements, W. D. B., Smye, M. G., Pope, C., Rowlands, B. J., & Diamond, T. (1996). Intestinal barrier dysfunction in clinical and experimental obstructive jaundice and its reversal by internal biliary drainage. British Journal of Surgery, 83, 1345 – 1349. Reynolds, J. V., Murchan, P., Leonard, N., Clarke, P., Keane, F. B. V., & Tanner, W. A. (1996). Gut barrier failure in experimental obstructive jaundice. Journal of Surgical Research, 62, 11 – 16. Sokol, R. J., Devereaux, M., Khandwala, R., & O’Brien, K. (1993). Evidence for involvement of oxygen free radicals in bile acid toxicity to isolated rat hepatocytes. Hepatology, 17, 869 – 881. Sokol, R. J., Winklhofer-Roob, B. M., Devereaux, M. W., & McKim, J. M. (1995). Generation of hydroperoxides in isolated rat hepatocytes and hepatic mitochondria exposed to hydrophobic bile acids. Gastroenterology, 109, 1249 – 1256.
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Vaitukaitis, J. L. (1998). Animal models of human disease for the 21st century. Laboratory Animal Science, 48, 562 – 564. Wang, M., Salem, M., Yousef, I. M., Tuchweber, B., Lam, P., Childs, S. J., Helgason, C. D., Ackerley, C., Phillips, J. P., & Ling, V. (2002). Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis. Proceedings of the National Academy of Sciences of the United States of America, 98, 2011 – 2016. Wen Ding, J., Andersson, R., Norgren, L., Stenram, U., & Bengmark, S. (1992). The influence of biliary obstruction and sepsis on reticuloendothelial function in rats. European Journal of Surgery, 158, 157 – 164.
Brief communication
Cholemic transgenic mice: A novel animal model to investigate the effects of bile acids Predrag Ljubuncica, Ibrahim Yousef b, Arieh Bomzona,* a
Department of Pharmacology, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P.O. Box 9649, Haifa 31096, Israel b Department of Pharmacology, University of Montreal, C.P. 6128, Surccursale A, Montreal, Quebec, Canada H3C 3J7 Received 3 March 2004; accepted 21 June 2004
Abstract Introduction: Laboratory investigations into cholestatic liver disease and the effects of cholemia on organ function are long-standing subjects of scientific enquiry. A widely-used strategy to investigate these topics relies on animal-based research using experimental animal models. Targeted inactivation of the spgp gene, the gene responsible for expressing the bile salt export pump (BSEP) in the hepatocyte canalicular membrane impairs the canalicular secretion of bile salts resulting in systemic cholemia. The results of in vitro experiments have established bile acids as pro-oxidants and the collection of unambiguous in vivo data on the pro-oxidant activity of bile acids in the existing models of cholemia cannot be done. Therefore, we decided to use these genetically modified mice to determine whether this model of cholemia has evidence of extrahepatic or systemic oxidative stress, one of the features of cholestatic liver disease. Methods: The extent of lipid peroxidation in livers, kidneys, hearts and brains harvested from cholemic homozygous (spgp / ) mice using the thiobarbituric acid reactive substances (TBARS) assay. The data were compared to equivalent data collected from heterozygous (spgp +/ ) and control mice. Results: We found (1) substantial increases in malondialdehyde (MDA) levels in the brains and hearts; (2) a moderate increase in MDA levels in the kidneys; and (3) no change in MDA levels in the livers of the homozygous cholemic mice compared to the control and heterozygous mice. Discussion: The transgenic mouse model of cholemia has an intact enterohepatic circulation and is uncomplicated by the adverse consequences of hepatotoxins or biliary surgery. Hepatocellular injury, as well as plasma and tissue accumulation of bilirubin and other liver-derived compounds are also negligible. Although this preliminary study could not establish a causal relationship between cholemia and oxidative stress, we believe this model is worthy of further investigation to study the impact of short-term and long-term cholemia on diverse physiological and biochemical functions such as trying to establish a causal role for bile acids in the development of oxidative stress in cholestatic liver disease. D 2004 Elsevier Inc. All rights reserved. Keywords: Bile acids; Oxidative stress; Lipid peroxidation; Malondialdehyde; Methods
1. Introduction Laboratory investigations into cholestatic liver disease and the effects of cholemia on organ function are longstanding subjects of scientific enquiry. A widely-used strategy to investigate these topics relies on animal-based research using experimental animal models. These models
* Corresponding author. Tel.: +1 972 4 8295259; fax: +1 972 4 8524978. E-mail address: [email protected] (A. Bomzon). 1056-8719/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2004.06.002
fall into one of several categories: surgical-induced models such as bile duct ligation; toxic injury of liver using hepatotoxins such as carbon tetrachloride or thioacetamide or hormones such as ethinyl estradiol; or sepsis-induced cholestasis (Bomzon & Blendis, 1990; Lee & Boyer, 2000). Many human genes have a mouse ortholog and the production of genetically modified animals has substantially increased our capabilities to investigate the roles of endogenous substances in the pathogenesis and course of disease (Bedell, Largaespada, Jenkins, & Copeland, 1997; Vaitukaitis, 1998). The canalicular secretion of bile salts
232
P. Ljubuncic et al. / Journal of Pharmacological and Toxicological Methods 50 (2004) 231–235
from the liver into bile is a key process in the enterohepatic circulation of bile acids. The bile salt export pump (BSEP) is a canalicular-specific ATP-binding cassette transporter. It was originally called the sister of P-glycoprotein (spgp) because of its homology to the family of P-glycoproteins that conferred multidrug resistance in a large number of cell types (Childs, Yeh, Hui, & Ling, 1998). It was subsequently renamed as BSEP to emphasize its dependence on ATP (Gerloff et al., 1998). Targeted inactivation of the spgp gene in mice causes cholemia due to impaired canalicular secretion of bile salts (Wang et al., 2002). This mouse model has elevated plasma concentrations of bile salts and an intact enterohepatic circulation; therefore, there is negligible hepatocellular damage and the consequences of biliary surgery, hepatotoxins, sepsis and the plasma and tissue accumulation of bilirubin and other liver or biliary metabolites have no impact. Bile acids are known pro-oxidants (Ljubuncic, Fuhrman, Oiknine, Aviram, & Bomzon, 1996; Sokol, Devereaux, Khandwala, & O’Brien, 1993; Sokol, WinklhoferRoob, Devereaux, & McKim, 1995). This characteristic has been established from the results of in vitro experiments. Collecting unambiguous in vivo data on the prooxidant activity of bile acids in the existing models of cholemia cannot be done. Therefore, we decided to use these genetically modified mice to determine whether this model of cholemia has evidence of extrahepatic or systemic oxidative stress, one of the features of cholestatic liver disease.
used for lipid peroxidation determination by the thiobarbituric acid reactive substances (TBARS) assay (see next section for specific details). Protein contents of the individual tissue homogenates were determined by the method described by Lowry, Rosebrough, Farr, and Randall (1951). 2.2. Assessment of the extent of lipid peroxidation
2. Methods
The assessment of the extent of lipid peroxidation relied on individual determinations of the thiobarbituric acid reactive substances (TBARS) content in hepatic, kidney, heart and brain homogenates prepared from the three groups of experimental mice (Draper & Hadley, 1990). Malondialdehyde (MDA) is an end product of peroxidative decomposition of polyeonic fatty acids in the lipid peroxidation process and its accumulation in tissues is indicative of the extent of lipid peroxidation (Draper & Hadley, 1990). In an earlier publication, we proved bile acids did not interfere with the TBARS assay (Ljubuncic et al., 1996). TBARS reagent (1 ml) was added to a 0.5 ml aliquot of tissue homogenate and heated for 20 min at 1008C. The antioxidant, butylated hydroxytoluene, was added before heating of samples. After cooling on ice, the samples were centrifuged at 840g for 15 min and the absorbance of the supernatant was read at 532 nm. Sample blanks for each sample were prepared and assessed in the same way to correct for A532 contribution due to the sample. The results were expressed as MDA equivalents (expressed in nmol MDA) using 1,1,3,3-tetraethoxypropane as the standard. Duplicate determinations were made and the average of the two measurements was used in the subsequent statistical analysis.
2.1. Preparation of tissue homogenates
2.3. Statistical analysis of the data
Tissue homogenates were prepared from four normal (wild-type) (sgpg +/+), eight heterozygous (sgpg +/ ), and 10 homozygous (spgp / ) mice. The sgpg / mice were prepared using transgenesis in embryonic stem cells (see previously published paper, Wang et al., 2002). Briefly, animal transgenesis involved inactivating the spgp gene in two different embryonic stem cell lines and subsequent implantation of the stem cells into pseudo-pregnant mice. Chimeric progeny were then mated with wild C57Black/6J mice to produce hemizygous mice (sgpg +/ ), which in turn were mated to produce the homozygous mice (sgpg / ). The impact of this transgenesis on bile acid regulation of hepatic function was studied in heterozygous and homozygous mice in Canada (Wang et al., 2002). Upon completion of these investigations, the livers, kidneys, heart and brains were harvested and couriered to Israel on dry ice. Each tissue was cut into small pieces by fine scissors and then homogenized in ice-cold 100 mM sodium phosphate buffer, pH 7.4 at 4 8C with a Potter–Elvehjem glass homogenizer. Separate aliquots of each homogenate were
The data are expressed as meansFstandard error of the mean. The data were analyzed by one-way analysis of variance using a Dunnett’s post-test. Pb0.05 (two-tailed) was considered significant.
3. Results Fig. 1 describes the accumulation of TBARS or MDA equivalents in the liver (A), kidney (B), brain (C) and heart (D) in the three groups of mice. Focusing on individual tissues, the amount of TBARS or MDA equivalents in the livers of the three groups of mice were not different from each other (Fig. 1A). Levels of TBARS or MDA equivalents tended to be higher in the kidneys of the heterozygous and homozygous mice than in the control mice but the differences were not statistically significant (Fig. 1B). However, the levels of TBARS or MDA equivalents in brain and heart homogenates prepared from the homozygous mice were two to three times higher than
P. Ljubuncic et al. / Journal of Pharmacological and Toxicological Methods 50 (2004) 231–235
233
Fig. 1. The tissue concentrations of malondialdehyde (MDA) in livers (A), kidneys (B), brains (C) and hearts (D) harvested from four control (black), eight heterozygous (spgp +/ ) (dark grey) and 10 homozygous (spgp / ) (light grey) mice. *( pb0.05) and **( pb0.01) represents the significance of the difference from control.
levels measured in the identical tissues of control and heterozygous mice (Fig. 1C and D).
4. Discussion Bile acids, and in particular hydrophobic types, are prooxidants. The basis of this assertion comes from experimental reports on the pro-oxidant effects of bile acids in cultured hepatocytes (Sokol et al., 1993, 1995) and macrophages (Ljubuncic et al., 1996). For several years, we have tried to implicate bile acids as one of the causal or contributory factors in the development of oxidative stress in non-hepatic tissues in cholestatic liver disease (Bomzon, Holt, & Moore, 1997; Ljubuncic, Tanne, & Bomzon, 2000). The existing models of cholemia have hampered our efforts to verify this hypothesis. They were created to emphasize one or more of the specific features of cholestatic liver disease and different methods of induction impose limitations on the research value of the model. For example, bile duct-ligated rats have severe hepatocellular injury with gross disruption of hepatic architecture and exhibit signs of nephropathy (Bomzon, Jacob, & Better, 1996), cardiopathy (Lee & Bomzon, 1990) and the hepatopulmonary syndrome (Fallon, Abrams, McGrath, Hou, & Luo, 1997). They are also susceptible to sepsis (Kennedy, Parks, Clements, & Rowlands, 1995; Parks, Clements, Smye, Pope, Rowlands, & Diamond, 1996; Reynolds, Murchan, Leonard, Clarke, Keane, & Tanner, 1996; Wen Ding, Andersson, Norgren, Stenram, & Bengmark, 1992) and are oxidatively stressed (Kra¨henbu¨hl, Talos, Lauterburg, & Reichen, 1995; Ljubuncic et al., 2000). They are also moribund after 6 weeks (Bomzon & Blendis, 1990). Because the enterohepatic circulation is surgically disrupted, liver or biliary metabolites, including bile acids, accumulate in the plasma, liver and extrahepatic tissues. As a result, this
model precludes the opportunity to ascertain a causal contribution of bile acids for some of these extrahepatic sequelae because their accumulation occurs together with that of bilirubin and other compounds. In the other models, a different set of issues arise. Spontaneous resolution of the hepatocellular damage occurs if administration of the hepatotoxin stops (Bomzon & Blendis, 1990). Therefore, data can only be collected while the animal is intoxicated. Similarly, data collected in the septic models makes it difficult to distinguish between the effects of sepsis and those of liver or biliary metabolites on extrahepatic organ function. The development of the spgp / mouse model of cholemia creates a new opportunity to consolidate this idea because it is free from all these limitations. Moreover, it is unique because one of the specific bile acid transporters in canalicular membrane of the hepatocyte has been inactivated resulting in an untainted or pure cholemia of longer duration that cannot be obtained in the other models. Systemic oxidative stress is one of the features of cholestatic liver disease encompassing the kidneys, heart and brain (Bomzon et al., 1997; Ljubuncic et al., 2000; Orellana, Rodrigo, Thielemann, & Guajardo, 2000; Panozzo, Basso, Balint, Zaninotto, Bonvicini, & Plebani, 1995). We found increased extents of lipid peroxidation in the brains and hearts of the homozygous transgenic mice thereby providing the evidence of systemic oxidative stress in these animals. Wang et al. (2002) reported four-fold increases in plasma bile acid levels in the cholemic spgp / mice when compared to plasma levels in the wild spgp +/+ type mice. In the absence of information of other potential contributors to the development of systemic oxidative stress, it is tempting to equate association with causality in this mouse model of cholemia. Therefore, we think it is not unreasonable to assume bile acids through their pro-oxidant activity could account for the oxidative processes detected in the heart and brains of these
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mice. In order to support or even refute the hypothesis, future investigations should be directed towards establishing the levels and types of bile acids in the tissues of these mice and their pro-oxidant potential. We also reported that the extent of lipid peroxidation was greater in the kidneys of these mice than control or heterozygous mice, the increase did not quite reach a level of statistical significance. The organs came from mice used in an unrelated project undertaken in Canada, thereby limiting the number and types of tissues that could be studied. Therefore, it is possible a type II error exists that could be overcome by increasing the group sample sizes to ensure that the data are unambiguous. The TBARS assay is a well-validated system having a considerable amount of historical control data (Esterbauer & Cheeseman, 1990; Halliwell & Chirico, 1993) and is extensively used by many investigators to establish the presence of an oxidative process in tissues. This assay is a target of criticism where its critics claim the measurement of F 2-isoprostanes (prostaglandins formed by free radical-mediated lipid peroxidation levels) is a more reliable method to detect lipid peroxidation in biological fluids such as plasma and urine (De Zwart, Meerman, Commandeur, & Vermeulen, 1999; Morrow et al., 1999). Therefore, it is possible that the use of this method would bestow statistical significance to the kidney data. For these reasons, additional validation studies involving more animals are needed to establish this transgenic mice model of cholemia as a valuable addition to the existing models. In summary, this transgenic mouse model of cholemia has an intact enterohepatic circulation and is uncomplicated by the adverse consequences of hepatotoxins or biliary surgery. Hepatocellular injury, as well as plasma and tissue accumulation of bilirubin and other liver-derived compounds are also negligible. As a result, we believe this model is worthy of further investigation to study the impact of short-term and long-term cholemia on diverse physiological and biochemical functions such as trying to establish a causal role for bile acids in the development of oxidative stress in cholestatic liver disease. Acknowledgements This research was supported by the Center for Absorption in Science of the Ministry of Immigrant Absorption and the Committee for Planning and Budgeting of the Council for Higher Education under the framework of the KAMEA Program. References Bedell, M. A., Largaespada, D. A., Jenkins, N. A., & Copeland, N. G. (1997). Mouse models of human disease: Part II. Recent progress and future directions. Genes and Development, 11, 11 – 43.
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