Linkage map of Syrian hamster with restriction landmark genomic scanning.

We have constructed the linkage map with precise genetic analysis of the Syrian hamster, Mesocricetus auratus, according to the restriction landmark genomic scanning (RLGS) spot mapping method. Although only 3.2-6.6% of the total RLGS spots between the two strains, ACN and BIO 14.6, showed genetic variance, 572 loci were found to be polymorphic. Out of 569 RLGS loci and 3 other loci, 531 were mapped with the backcross (ACN x BIO 14.6) F1 x BIO 14.6. The cumulative map was 1111.6 cM, indicating that the spots/loci are located throughout the genome at 1.94 cM intervals on average. Thus, RLGS provides us with a rapid tool to construct the genetic map of any species, even if it has less genetic variation.

Biliary excretion of glycyrrhizin in rats: kinetic basis for multiplicity in bile canalicular transport of organic anions.

PURPOSE: To examine the presence of multiplicity for the biliary excretion of xenobiotic conjugates, we studied the disposition of glycyrrhizin (GR), which has glucuronide within its molecular structure and has the ability to inhibit the biliary excretion of liquiritigenin (LG) glucuronides. METHODS: GR was administered intravenously as a bolus to Sprague-Dawley (SD) rats which received an i.v. infusion of inhibitors (dibromosulfophthalein (DBSP) and indocyanine green (ICG)) at their transport maximum rates. Biliary excretion of GR was also examined in Eisai hyperbilirubinemic rats (EHBR), which have a hereditary defect in the canalicular transport system of several organic anions. RESULTS: Infusion of ICG did not affect the biliary excretion of GR, whereas infusion of DBSP reduced it significantly. The plasma concentration of GR was increased by DBSP but not by ICG. In EHBR, the biliary excretion of GR was severely impaired, resulting in an increase in the plasma concentration of GR. CONCLUSIONS: These findings suggest (1) that the biliary excretion of GR is mediated by the system which is shared by DBSP and LG glucuronides but not by ICG and (2) that this system is hereditarily defective in EHBR. Together with our previous findings, the multiplicity for the biliary excretion of organic anions is shown.

A genetic linkage map of the Syrian hamster and localization of cardiomyopathy locus on chromosome 9qa2.1-b1 using RLGS spot-mapping.

The Syrian cardiomyopathic hamster (BIO14.6) has an inherited form of progressive myocardial necrosis and congestive heart failure. Although widely studied as an animal model for human hypertrophic cardiomyopathy, further genetic analysis has been limited by a scarcity of DNA markers. Until now, only six autosomal linkage groups have been described and the number of polymorphic loci was extremely limited. In this study, we applied the restriction landmark genome scanning (RLGS) spot-mapping method to construct a genetic map of the Syrian hamster (Mesocricetus auratus) using 72 back-cross progeny. Although the polymorphic rate is very low (3-7%) between the strains, 531 polymorphic spots/loci were mapped, showing the power of this approach and reasonable applicability to other organisms lacking a well-defined genetic map. Further, the spot markers which flank the cardiomyopathy (cm) locus were cloned to determine the chromosomal location of cm by fluorescent in situ hybridization (FISH) analysis, resulting in the assignment of the locus to the centromeric region of hamster chromosome 9qa2.1-b1. Several candidate genes responsible for hypertrophic cardiomyopathy in humans have been excluded.

A new mutant rat with turning behavior (Tur).

A new rat mutant with abnormal behavior has been found in a closed colony of Slc: SD. The affected rats turned around after their own tails beginning 4-5 weeks after birth, in response to extroceptive stimuli, such as sound or vibration. The behavior was more pronounced in younger animals. On gross observation, no alteration was found in the cerebrum, cerebellum, or anterior, posterior, or lateral semicircular canals. Light microscopic examination revealed no abnormality in the cerebrum or cerebellum. The trait was controlled by an autosomal dominant gene with incomplete penetrance (78.0%) when transferred to the WKY genetic strain background. It was named "turning" with the gene symbol Tur. A congenic strain was established, which was designated WKY/Eis-Tur.

A new mutant rat with hyperbilirubinuria (hyb).

We have discovered mutant rats with hyperbilirubinuria at the laboratories of Eisai Co., Ltd., Gifu, Japan, and established a new inbred mutant strain, which was designated Eisai Hyperbilirubinuria Rat (EHBR/Eis). They show jaundice immediately after birth, and direct or conjugated hyperbilirubinemia throughout their life. The phenotypes are controlled by a single autosomal recessive gene, hyb with full penetrance. Affected homozygous males seem to have full reproductive capacity. On the other hand, the litter size of hyb/hyb females is significantly reduced at second parturition compared with that at first one. In order to maintain the strain efficiently and to produce both affected (hyb/hyb) and normal control (hyb/+) offspring at the same time, we have mainly been mating hyb/hyb males with hyb/+ females in establishing the inbred strain. We also report here the allele distribution of the EHBR/Eis strain.

Biliary and renal excretions of cefpiramide in Eisai hyperbilirubinemic rats.

Eisai hyperbilirubinemic mutant rats (EHBRs) with conjugated hyperbilirubinemia were recently derived from Sprague-Dawley rats (SDRs). The pharmacokinetic characteristics of the beta-lactam antibiotic cefpiramide (CPM), which is mainly excreted into bile, were investigated in 10- and 20-week-old EHBRs and were compared with those in 20-week-old healthy SDRs. The pharmacokinetic parameters of CPM after an intravenous administration of 20 mg/kg of body weight were estimated for each rat by noncompartmental methods. When compared with age-matched healthy SDRs, significant decreases (by approximately 30%) in the systemic clearance of CPM were observed in 20-week-old EHBRs. The biliary clearance of CPM in 20-week-old EHBRs markedly decreased to less than 10% of that in age-matched healthy SDRs, while total urinary recovery of unchanged CPM increased to threefold and renal clearance doubled. However, no significant differences in any of the pharmacokinetic parameters of CPM were observed between the two groups of EHBRs. There were no significant differences among the three groups in the steady-state volume of distribution of CPM. The present study indicates that hyperbilirubinemia induces an increase in the urinary excretion ability of CPM in return for a reduction in the biliary excretion.

Multiple systems for the biliary excretion of organic anions in rats: liquiritigenin conjugates as model compounds.

Liquiritigenin (LG), 2, 3-dihydro-7-hydroxy-2-(4-hydroxyphenyl)-(S)-4H-1-benzopyran-4-1, is metabolized to five kinds of conjugates (glucuronides and sulfates) that are excreted predominantly into the bile (Shimamura et al., 1993). Using LG as a model compound, we studied the multiplicity for the biliary excretion of conjugates in vivo. LG was administered i.v. as a bolus to Sprague-Dawley rats (SD rats) that received i.v. infusions of the inhibitors [glycyrrhizin (GR), dibromosulfophthalein (DBSP) and indocyanine green (ICG)] at their maximal transport rates. The infusion of GR and that of DBSP reduced significantly the biliary excretion of all the conjugates except LG 4',7-O-disulfate (M3), whereas infusion of ICG did not affect the excretion of conjugates. To minimize the effect of the other tissues on the disposition of the conjugates, the apparent biliary excretion clearance (CLbile,app) was calculated. The CLbile,app values of LG 4'-O-glucuronide (M1), LG 7-O-glucuronide (M2), LG 4'-O-glucuronide 7-O-sulfate (M4) and LG 7-O-glucuronide 4'-O-sulfate (M5) were significantly reduced by GR and DBSP infusion, whereas the CLbile, app value of M3 was not affected by these inhibitors. The CLbile, app values of all the conjugates except M2 were not reduced by ICG infusion. In Eisai hyperbilirubinemic rats, which have a hereditary defect in the canalicular transport system for several organic anions, the biliary excretion clearance of M1, M2, M4 and M5 was markedly reduced, whereas that of M3 was comparable with that in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in the pharmacokinetics and protein binding of enprofylline in Eisai hyperbilirubinemic rats.

Mutant rats possessing conjugated hyperbilirubinemia have recently been established from Sprague-Dawley rats (SDRs) and are called Eisai hyperbilirubinemic rats (EHBRs). The effects of hyperbilirubinemia on the disposition, renal handling, and protein binding behavior of enprofylline, which is mainly excreted into the urine by an active tubular secretion mechanism, were investigated in 9- and 19-week-old EHBRs and compared with their age-matched normal SDRs. Enprofylline was administered intravenously at a dose of 2.5 mg/kg, which exhibits linear kinetics. Pharmacokinetic parameters for both total and unbound enprofylline were estimated by model-independent methods. Both systemic clearance and volume of distribution at steady state of enprofylline significantly increased in 19-week-old EHBRs. However, there were no differences in the glomerular filtration rate estimated as inulin clearance and fraction of urinary excretion as unchanged drug between EHBRs and normal SDRs. Significant decreases in both the binding capacity and number of binding sites were observed in 19-week-old EHBRs, but no such changes were observed between 9-week-old EHBRs and SDRs. Hyperbilirubinemia in EHBRs had no effect on the pharmacokinetics and renal handling of unbound enprofylline. These results indicate that the pharmacokinetics of enprofylline, but not renal handling, glomerular filtration, or tubular secretion are modified in EHBRs by changes in protein binding behavior.

Influence of age on the disposition and renal handling of enprofylline in rats.

The effects of ageing on the pharmacokinetics, renal handling and protein binding of enprofylline were investigated in 6-, 13- and 18-month-old male Fischer 344 rats. Concentrations of enprofylline in plasma and urine were determined by HPLC, and pharmacokinetic parameters were estimated by model-independent methods. No significant differences in the volume of distribution, systemic clearance of enprofylline or urinary recovery of unchanged enprofylline (> 85%) were observed among any of the groups of rats. The dissociation constant and free fatty acid concentration in plasma increased with age. Age-dependent decreases in the systemic clearance for unbound drug were observed, and the volume of distribution for unbound drug tended to decrease with age. The ratio of systemic clearance for unbound drug to the glomerular filtration rate (GFR) decreased with ageing. Ageing was associated with decreases in the apparent maximum capacity of transport (Vmax)(223.33, 160.24 and 142.98 micrograms min-1 kg-1 for 6-, 13- and 18-month-old rats, respectively) and in the tubular secretory intrinsic clearance (Vmax/Km) of enprofylline (75.45, 51.03 and 44.13 mL min-1 kg-1, respectively), while a slight change in the Michaelis-Menten constant (Km) was observed. These results indicate that the mechanism responsible for age-related changes in the disposition and renal handling of enprofylline may be responsible for a decrease in the ability of the tubular anion transport system.

[Establishment of hyperbilirubinuria rat mutant--a new animal model for jaundice].

A new animal model for jaundice, a hyperbilirubinemic rat mutant (EHBR, Eizai hyperbilirubinuria rat), was established from Sprague-Dawley rats. Hyperbilirubinemia was inherited as an autosomal recessive trait. The gene manifesting jaundice was named "hyb". Homozygotes developed jaundice immediately after birth, and a high bilirubin concentration was detected in plasma and urine. The plasma bilirubin levels were high in the neonatal period, but they decreased from 6 to 10 weeks old. In male homozygotes, plasma bilirubin levels increased rapidly until about 40 weeks, and decreased thereafter. Female homozygotes showed slightly high plasma bilirubin levels until 56 weeks, then increased rapidly thereafter. At 72 weeks, the plasma bilirubin level of females was comparable to that of males. About 80 percent of the plasma bilirubin was conjugated. Plasma biochemistry demonstrated the increase of total cholesterol and total bile acid in the homozygotes. Histopathologically, the homozygote was characterized by brown pigment in the hepatocytes, and glomerular lesions with mesangial expansion. From these findings it was considered that EHBR might be a useful animal model for studying constitutional conjugated hyperbilirubinemia and bilirubin metabolism.

Kinetic analysis of hepatobiliary transport of organic anions in Eisai hyperbilirubinemic mutant rats.

The hepatobiliary transport of nonmetabolizable organic anions [cefodizime, dibromosulfophthalein, and indocyanine green (ICG)] was kinetically analyzed in Eisai hyperbilirubinemic rats (EHBR; Sprague-Dawley-derived mutant rats with conjugated hyperbilirubinemia). In EHBR, the biliary excretion of these anions was remarkably impaired, whereas the alteration in initial plasma disappearance was minimal. The kinetic analysis of the disposition of these ligands revealed 1) that the transport rate via bile canalicular membrane was severely impaired in EHBR for cefodizime and dibromosulfophthalein and 2) that the intracellular transport rate of ICG was decreased in EHBR, which contributed more than the decrease in the canalicular membrane transport to the net reduction of the excretion rate of ICG in EHBR. The latter finding was also supported by the in vitro results; the binding of ICG to ligandin(s) in EHBR was less extensive compared with that in normal rats, resulting in the higher distribution of ICG to organelle. Because the ligand molecules bound to organelle diffuse within the cells much more slowly than the molecules in the cytosol, the higher distribution of ICG to organelle in EHBR results in the reduction in the intracellular transport rate. These results indicate that the differential impairment mechanisms can be proposed for the excretion of organic anions: One is the impairment in the transport rate across the canalicular membrane, the other is the impairment in the intracellular transport rate.

Developmental aspects of a unique glutathione S-transferase subunit Yx in the liver cytosol from rats with hereditary hyperbilirubinuria. Comparison with rat fetal liver transferase subunit Yfetus.

The unique glutathione S-transferase (GST) subunit Yx, which is undetectable in normal adult rat liver cytosol, was shown to occur in the liver cytosol of rats with hereditary hyperbilirubinuria (EHB). The Yx subunit is a member of the Alpha-class GST subunits, and is immunologically closely related to the Yc subunit. The Yx subunit has an apparent M(r) of 26,400, different from those of Ya (M(r) 25,800), Yb1 and Yb2 (both M(r) 27,200) and Yc (M(r) 28,400). During postnatal development in livers of EHB rats, the Yx subunit concentration in either sex was highest during the first week post partum and declined rapidly with age. Although the concentration of subunit Yx at 8 weeks of age accounted for about 60% in females and 40% in males of that observed in 1-week-old 'neonatal' male EHB rats, concentrations in females thereafter increased gradually to almost the neonatal level and remained at this high level at least up to 37 weeks of age, whereas the concentration in males did not increase again. Thus the post-pubertal Yx subunit concentration was 2-fold higher in females than in males. In contrast, in normal Sprague-Dawley rat liver, the Yfetus subunit, with the same M(r) as the Yx subunit, had the highest concentration in 10-day-old animals, declined rapidly thereafter, and was not detectable in the post-pubertal period. The Yfetus subunit was also immunoreactive with an antibody against GST YcYc. The analysis of GST subunits by reverse-phase h.p.l.c. revealed that the Yx subunit was eluted at a retention time different from other known subunits, but coincided with that of Yfetus. The N-terminal amino acid sequence of the Yx subunit displayed a high degree of sequence similarity to that of the Yfetus subunit. These data suggest that the Yx subunit in EHB rats may be very similar to, if not identical with, the Yfetus subunit.

A new rat mutant with chronic conjugated hyperbilirubinemia and renal glomerular lesions.

A new mutant strain of inbred Sprague Dawley rats with autosomal recessive hyperbilirubinuria, were studied by biochemical, histologic, and ultrastructural methods. The plasma bilirubin concentration in the homozygote was significantly higher than that of the heterozygote, and about 80% of the bilirubin was conjugated. Plasma BSP and ICG clearance were both severely delayed in the homozygote. Plasma BSP elimination kinetics suggested that the pathophysiologic defect was not hepatic uptake or storage but rather in secretion into bile. Histopathology of the liver demonstrated brown pigment in the hepatocytes that appeared to be lipofuscin. The electron microscopic features of the hepatic pigment resembled those of the Dubin-Johnson syndrome. Homozygote histopathology also revealed glomerular lesions with mesangial expansion and proliferation in the kidneys. Immunohistologic studies disclosed mesangial granular deposition of IgG, IgA, and to a lesser degree, IgM and C3. These renal changes resembled those of IgA nephropathy. The spontaneous hyperbilirubinuric rat (EHBR) may be a useful animal model for studying constitutive conjugated hyperbilirubinemia, bilirubin metabolism, cholestasis, and glomerulonephropathy subsequent to hepatic dysfunction.

Biliary excretion of bile acid conjugates in a hyperbilirubinemic mutant Sprague-Dawley rat.

The hepatic transport of bile acid conjugates was studied in the Eisai hyperbilirubinuria rat, a Sprague-Dawley mutant rat with conjugated hyperbilirubinemia. Serum bile acid levels were increased, bile acid-independent bile flow was decreased and biliary glutathione concentrations were markedly decreased in the Eisai hyperbilirubinuria rat. Biliary excretion of sulfobromophthalein was markedly impaired and almost no glutathione conjugate was excreted in the bile of the Eisai hyperbilirubinuria rat. Biliary excretion of lithocholate-3-O-glucuronide and lithocholate-3-sulfate in the Eisai hyperbilirubinuria rat was markedly delayed, whereas that of lithocholate was only slightly delayed. After [14C]chenodeoxycholate infusion (1 mumol/min/100 gm for 60 min), the increases in bile flow and biliary excretion of isotope in the Eisai hyperbilirubinuria rat were not so prominent as those observed in control rats, and the glucuronide of chenodeoxycholate, which constituted about 15% of biliary chenodeoxycholate in control rats, was not observed in the Eisai hyperbilirubinuria rat. Initial uptake of lithocholate and its glucuronide and sulfate by isolated hepatocytes was not impaired in the Eisai hyperbilirubinuria rat; the profiles of cytosolic bile acid binding proteins in Eisai hyperbilirubinuria rat liver were identical to those in control liver. These data indicate that the Eisai hyperbilirubinuria rat has excretory impairment of organic anions, bile acid glucuronide and sulfate and that it has characteristics very similar to those of the hyperbilirubinemic mutant Wistar rats TR- and GY.

Changes in serum and hepatic levels of immunoreactive prolyl hydroxylase in two models of hepatic fibrosis in rats.

Changes in serum and hepatic levels of immunoreactive prolyl hydroxylase (IRPH) as well as cellular localization of the enzyme were studied in 2 models of hepatic fibrosis, which was induced in male rats either by subcutaneous administration of CCl4 (Group A) or by intraperitoneal injection of porcine serum (Group B). Hepatic fibrosis appeared at the 8th week in Group A and at the 12th week in Group B, and liver cirrhosis developed at the 16th week in both models. Although tissue contents of hydroxyproline (HP) and IRPH increased in both models, only HP levels correlated with the degree of fibrosis. Serum IRPH levels and serum asparate aminotransferase (AST) activities increased, showing a significant positive correlation, in group A, whereas both remained in a control range in Group B. Moreover, in another model which received a single intraperitoneal injection of CCl4, serum IRPH showed a marked increase and then a rapid decrease in parallel with the change in serum AST. Immunohistochemical analysis also showed a difference between the two fibrosis models: in group A, IRPH was positive mainly in parenchymal cells in the peripheral zone of the pseudolobulus, while in group B the staining was diffuse. These results indicate that the elevation of serum IRPH is, at least in part, due to the parenchymal cell damage, and that IRPH levels should be carefully evaluated when being used as a parameter to estimate the activity of fibrogenesis in the liver.

Organic anion transport study in mutant rats with autosomal recessive conjugated hyperbilirubinemia.

The EHBR is a mutant rat strain with congenital conjugated hyperbilirubinemia bred from a Sprague-Dawley rat. Transport of conjugated bilirubin, indocyanine green, and tetrabromosulfophtalein from liver to bile is severely impaired in these rats. Serum bilirubin amounts to 6.0 +/- 0.05 mg/dl (n = 4) in adult rats, with 97% conjugates. The bile flow is reduced to about 65% of the control group, whereas total bile acid in 10-min bile samples is similar. Liver histology of 10 week-old rats revealed neither intracellular pigmentation nor architectural abnormalities.

Inhibitory effect of acyclic retinoid (polyprenoic acid) on the secretion of alpha-fetoprotein in CCl4-treated rats.

A study was conducted to examine the inhibitory effect of acyclic retinoid (polyprenoic acid) on the secretion of alpha-fetoprotein (AFP) in rats with chronic liver damage induced by CCl4. Oral administration of the compound brought about a significant reduction of serum AFP levels at the time when liver cirrhosis was formed. Acyclic retinoid also decreased the activities of serum aminotransferases and ornithine carbamyl transferase, while it increased serum albumin levels, demonstrating the reduction of hepatic parenchymal damage. Significant negative correlation was observed between serum AFP and albumin levels. This cytoprotective effect of the retinoid on the parenchymal cell may well be related to the inhibition of the synthesis and/or secretion of AFP. No significant side effect was observed, despite a long-term administration of the compound. The present finding will provide a potential scope for the future use of acyclic retinoid for the treatment of chronic liver damage.

Inhibitory effect of acyclic retinoid (polyprenoic acid) on hepatic fibrosis in CCl4-treated rats.

A study was conducted to examine the inhibitory effect of acyclic retinoid (polyprenoic acid) on the development of hepatic fibrosis induced by CCl4 in rats. Oral administration of the compound brought about a significant reduction in both serum and tissue levels of immunoreactive prolyl hydroxylase, a key enzyme of collagen formation. The result indicated that the rate of collagen synthesis in the liver was decreased which was consistent with histological findings. Acyclic retinoid also decreased both AST and ALT activities in serum, demonstrating the reduction in hepatic parenchymal damage. This cytoprotective effect on parenchymal cells may be related, at least in part, to inhibition of hepatic fibrosis. No significant side effects were observed, despite a long-term administration of the acyclic retinoid. The present findings suggest the potential scope of therapy of hepatic fibrosis by retinoid.