Mutant animals with hyperbilirubinemia have been widely used as animal models for human diseases. Among these mutant animals, Eisai hyperbilirubinem-.
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Comparison of the Disposition Behavior of Organic anions in an Animal Model for Wilson's Disease (Long-Evans Cinnamon rats) with that in Normal Long-Evans Agouti rats Shirou ITAGAKI1, Mitsuru SUGAWARA2, Michiya KOBAYASHI2, Katsumi MIYAZAKI2, Takeshi HIRANO1 and Ken ISEKI1,* 1Department
of Clinical Pharmaceutics and Therapeutics, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan 2Department of Pharmacy, Hokkaido University Hospital, Sapporo, Japan Full text of this paper is available at http://www.jssx.org
Summary: Long-Evans Cinnamon (LEC) rats have an abnormality similar to that observed in Wilson's disease in humans and are therefore a good animal model for the study of Wilson's disease. LEC rats develop hereditary hepatitis and severe jaundice. Mutant animals with hyperbilirubinemia have been widely used as animal models for human diseases. Among these mutant animals, Eisai hyperbilirubinemic rats (EHBR) have defective biliary excretion of organic anions. Thus, biliary excretion of sulfobromophthalein (BSP) and urinary excretion of phenolsulfonphthalein (PSP) in LEC rats were compared with those in Long-Evans Agouti (LEA) rats. In LEC rats, the excretion of BSP, a multidrug Abcc2) substrate, was signiˆcantly decreased compared to that in resistance-associated protein 2 (Mrp2 W LEA rats. It has been reported that the transport function for organic anions on the kidney is maintained in EHBR. However, the urinary excretion of PSP is impaired in LEC rats. It is possible that organic anion transporters responsible for the urinary excretion of PSP in LEA rats and EHBR are impaired in LEC rats. It is important to elucidate the relationship between organic anion secretion and Wilson's disease.
Key words: Long-Evans Cinnamon rats; transporter; urinary excretion; organic anion; Wilson's disease bilirubinemic rats (EHBR) have defective biliary excretion of organic anions and bile acid glucuronide and sulfate.9,10) Sulfobromophthalein (BSP) and phenolsulfonphthalein (PSP) are widely used clinically as drugs for testing liver and renal function, respectively.11,12) In this study, biliary excretion of BSP and urinary excretion of PSP in LEC rats were compared with those in Long-Evans Agouti (LEA) rats. We found distinct diŠerences between the disposition behavior of organic anions in LEC rats and that in LEA rats.
Introduction Mutant animals have been widely used as animal models for the study of human diseases. Long-Evans Cinnamon (LEC) rats develop hepatitis and severe jaundice spontaneously and liver cancer appears in long-surviving rats after recovery from jaundice.1–7) Therefore, LEC rats provide a pertinent model for basic and clinical studies of hepatitis and liver cancer. LEC rats also have an abnormally high copper accumulation in the liver and show biochemical features that are very similar to those found in Wilson's disease.8) LEC rats are therefore a good animal model for the study of Wilson's disease in humans. Mutant animals with hyperbilirubinemia have been widely used as animal models for the study of human diseases. Among these mutant animals, Eisai hyper-
Materials and Methods Chemicals: Phenolsulfonphthalein and sulfobromophthalein were purchased from Wako Pure Chemical (Osaka, Japan). All other reagents were of the highest grade available and used without further puriˆcation.
Received; October 29, 2003, Accepted; January 13, 2004 *To whom correspondence should be addressed : Ken ISEKI, Ph.D., Department of Clinical Pharmaceutics & Therapeutics, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12-jo, Nishi-6-chome, Kita-ku, Sapporo 060-0812, Japan. Tel. & Fax. ＋81-11-706-3770, E-mail: ken-i＠pharm.hokudai.ac.jp
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Fig. 1. Time proˆles of the plasma concentration and the cumulative biliary excretion of BSP in LEA and LEC rats. The time proˆles of plasma concentrations (A) and cumulative biliary excretions (B) were determined after i.v. injection of BSP (45 mmol W kg) in LEA rats (open symbols) and LEC rats (closed symbols). Each value represents the mean with S.D. of three (LEA rats) to four (LEC rats) determinations. ***Pº0.001, signiˆcantly diŠerent from values in LEA rats.
Animals: Male LEC and LEA rats, aged 6 to 9 weeks (200¿350 g in weight), were obtained from the Center for Experimental Plants and Animals of Hokkaido University (Sapporo, Japan). Male LEA rats were used as controls. The experimental protocols were reviewed and approved by the Hokkaido University Animal Care Committee in accordance with the ``Guide for the Care and Use of Laboratory Animals'' as adopted by the National Institutes of Health. In vivo study: The procedure for the in vivo study has been described previously.13) Three to four male LEA rats and LEC rats were used in all experiments. The rats were anesthetized with sodium pentobarbital kg weight, i.p.). The common bile duct of each (40 mg W rat was cannulated with a blunted 24-gauge hypodermic needle shaft to collect bile specimens. A substrate solution (45 mmol W kg BSP or 2.2 mmol W kg PSP) was injected through the femoral vein. Blood was collected at 1, 15, 30, 45 and 60 min after injection. Plasma was prepared by centrifugation (850×g for 15 min) of blood samples. Methanol, corresponding to a double volume of plasma, was added to each plasma specimen. After centrifugation of the mixture (12,000×g for 15 min), the concentration of substrate in the supernatant was measured. Bile specimens were collected at 0¿15, 15¿30, 30¿45 and 45¿60 min after injection. The whole contents of the bladder were withdrawn with a syringe at 60 min after injection. Analytical procedures: Substrates were determined using an HPLC system equipped with a Hitachi L-6000 VIS detector.14) The column pump and L-4200H UV W was a Hitachi ODS Gel #3053 (4 mm i.d.×250 mm). C and 0.7 Column temperature and ‰ow rate were 559 mL W min, respectively. In the assay for PSP, a mobile phase containing 20z acetonitrile and 50 mM H3 PO4
Table 1.
Kinetic parameters of BSP after i.v. administration to rats
mL) AUC (mmol min W Xbile0–1h (mmol W kg) CLbile (mL W min W kg)
LEA rats
LEC rats
3.14±0.42 18.8±1.40 6.02±0.37
5.92±0.32*** 11.6±0.90*** 1.97±0.25***
kg) was injected through the femoral vein. Blood and BSP (45 mmol W bile specimens were collected at the speciˆed times. Xbile0–1h represents the cumulative amounts excreted into bile over a period of 60 min. The kinetic parameters were calculated from the values shown in Fig. 1. Each value represents the mean with S.D. of three (LEA rats) to four (LEC rats) determinations. ***Pº0.001, signiˆcantly diŠerent from values in LEA rats.
with pH adjusted to 3.0 by NaOH was used. In the assay for BSP, a mobile phase containing 25z acetonitrile and 100 mM H3 PO4 with pH adjusted to 7.4 by NaOH was used. The wavelengths of the detectors for PSP and BSP were 432 nm and 279 nm, respectively. The lower limits of quantitation for PSP and BSP were 50 pmol W mL and 100 pmol W mL, respectively. Data analysis: The area under the plasma concentration-time curve (AUC) was estimated by the trapezoidal rule using the plasma data from 0 to 60 min. The clearance values of bile and urine (CLbile and CLurine) were determined by dividing the amounts excreted into bile and urine, respectively, from 0 to 60 min by the AUC from 0 to 60 min. Statistical analysis was carried out using Student's t-test, and a value of Pº0.05 was considered signiˆcant. Results Biliary excretion of BSP in LEC rats: Fig. 1 shows the time proˆles of the plasma concentration and the cumulative amount of BSP excreted into bile in LEC
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Fig. 2. Time proˆles of the plasma concentration and the urinary excretion of PSP in LEA and LEC rats. PSP was injected through the femoral kg). The time proˆles of plasma concentrations (A) and the ratio of the excreted amount of PSP over a period of 60 min in a urine vein (2.2 mmol W sample to the percent of the injected amount (B) were determined in LEA rats (open symbols) and LEC rats (closed symbols). Each value represents the mean with S.D. of three determinations. *Pº0.05, signiˆcantly diŠerent from values in LEA rats.
Table 2.
Kinetic parameters of PSP after i.v. administration to rats LEA rats
mL) AUC (nmol min W Xurine0–1h (nmol W kg) CLurine (mL W min W kg)
588±99.6 367±67.4 0.63±0.10
LEC rats 401±33.7* 2.01±3.48* 0.10±0.01**
kg) was injected through the femoral vein. Blood and PSP (2.2 mmol W urine specimens were collected at the speciˆed times. Xurine0–1h represents the cumulative amounts excreted into urine over a period of 60 min. The kinetic parameters were calculated from the values shown in Fig. 2. Each value represents the mean with S.D. of three determinations. *Pº0.05, **Pº0.01, signiˆcantly diŠerent from values in LEA rats.
rats. The plasma concentration of BSP was signiˆcantly increased in LEC rats (Fig. 1A). Furthermore, the amount of biliary excretion of BSP in LEC rats was signiˆcantly less than that in LEA rats (Fig. 1B). Based on these results, the calculated CLbile value of BSP in LEC rats was signiˆcantly lower than that in LEA rats (Table 1). Urinary excretion of PSP in LEC rats: The amount of urinary excretion of PSP over a period of 60 min after intravenous injection was determined. The plasma concentration of PSP was signiˆcantly decreased in LEC rats (Fig. 2A). Furthermore, the amount of urinary excretion of PSP in LEC rats was markedly lower than that in LEA rats (Fig. 2B). Based on these results, the calculated CLurine value of PSP in LEC rats was signiˆcantly lower than that in LEA rats (Table 2). Discussion The LEC rat is a mutant strain established at the Center for Experimental Plants and Animals of Hokkaido
University. LEC rats are recognized to be a unique and useful animal model of human Wilson's disease.8) Wilson's disease is an autosomal recessive disorder of copper metabolism characterized by hepatic cirrhosis and neuronal degeneration. Wilson's disease gene is expressed as a single 7.5-kb transcript in the normal rat liver, and the LEC rat is deˆcient in this expression.15) Male LEC rats suŠer from ‰uminant hepatitis at around 5 months of age. It has been reported that the levels of serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities and total blood bilirubin in LEC rats before the onset of hepatitis and those in age-matched LEA rats were not signiˆcantly diŠerent and that the values were within the normal range.16) It is well known that the biliary excretion of many xenobiotics is mediated by a primary active organic anion transporter located on the bile canalicular membrane, which has been deˆned as multidrug resistance-associated protein 2 (Mrp2 W Abcc2), and it has been shown that hereditary dysfunction of Mrp2 is responsible for hyperbilirubinemia.17) Taking of these ˆndings into consideration, it is possible that organic anion secretion in LEC rats is impaired. In the present study, biliary excretion and urinary excretion of organic anions in LEC rats were compared with those in LEA rats. BSP is widely used clinically as a drug for testing liver function.11) We performed kinetic analysis of the biliary excretion of BSP. Signiˆcantly decreased excretion of BSP was observed in LEC rats. Biliary excretion of an Mrp2 substrate in EHBR was signiˆcantly less than that in SD rats.9,10) It has been reported that BSP is a substrate for Mrp2.18) These results suggest that the activity level of Mrp2 may be decreased in LEC rats and that
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LEC rats have characteristics similar to those of EHBR. PSP is widely used clinically as a drug for testing renal function.10) The urinary excretion of PSP is impaired in LEC rats. Secretion of organic anions in renal proximal tubules involves the uptake of organic anions across basolateral membranes into cells and exit into the lumen across brush-border membranes.19) Recently, we have found that rat organic anion transporter 3 (rOat3 W Slc22a8) is involved in the renal uptake of PSP on the basolateral membrane of the proximal tubules.14) On the other hand, the transporters responsible for the transport of PSP in the brush-border (apical) membrane are not fully understood. In rat renal brush-border membranes, a potential-sensitive transport system, an anion exchanger and primary active transporters are involved in the transport of organic anions. Recently, we reported that PSP is a substrate for Mrp2.13) However, it has been reported that the transport function for organic anions on the kidney was maintained in EHBR and that the contribution of Mrp2 to urinary excretion was minor.20,21) It is possible that organic anion transporters responsible for the urinary excretion of PSP in LEA rats are impaired in LEC rats. Deˆcient expression of the Wilson's disease gene may aŠect the function of these organic anion transporters. Previous studies have revealed that the concentration of albumin in LEC rats is lower than that in LEA rats.22) Most amphipathic organic anions tightly bind to albumin in the circulation. The increased unbound fractions of BSP and PSP due to the low albumin level would aŠect secretion of these compounds. In summary, LEC rats have not only impaired canalicular transport of organic anions but also impaired urinary excretion of organic anions. Molecular analysis is needed to clarify these transport systems. It is important to elucidate the relationship between organic anion secretion and Wilson's disease. Results of molecular analysis might lead to an increase in the therapeutic index. References 1)
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