ATP-dependent transport of glutathione and glucuronate conjugates from hepatocytes into bile is mediated by a distinct member of the ATP-binding cassette superfamily. We have cloned and sequenced the canalicular isoform of the multidrug resistance protein from rat liver, and termed it cMrp. This membrane glycoprotein is composed of 1541 amino acids with an identity of 47.8% with the human multidrug resistance protein (MRP) and of 41.9% with the yeast cadmium factor (YCF1). The carboxyl-terminal 130 amino acids of the human hepatocyte canalicular isoform of MRP (cMRP) were 80.2% identical with rat cMrp. cMrp was not expressed in the liver of two mutant rat strains, the Eisai hyperbilirubinemic rat and the GY/TR- mutant, which are deficient in the ATP-dependent transport of conjugates across the canalicular membrane. Immunoblotting using an antibody raised against the carboxyl terminus of cMrp detected the glycoprotein of about 190 kDa only in the canalicular membrane from normal liver. Double immunofluorescence and confocal laser scanning microscopy localized cMrp exclusively to the canalicular membrane domain of hepatocytes and demonstrated its loss in the hyperbilirubinemic mutant rat. The results identify cMrp as a canalicular transport protein with a novel sequence and with a function similar to the one of the MRP.
ABSTRACT:Recently, a chimeric mouse line in which the liver could be replaced by more than 80% with human hepatocytes was established in Japan. Because the chimeric mouse produces human albumin (hAlb), replacement by human hepatocytes could be estimated by the hAlb concentration in the blood of chimeric mice. In this study, we investigated human major cytochrome P450 (P450) in the livers of chimeric mice by mRNA, protein, and enzyme activity using real-time polymerase chain reaction, Western blot analysis, and high-performance liquid chromatography, respectively. Chimeric mice with humanized liver generated using hepatocytes from a Japanese and white donor were used. Human P450 mRNAs were expressed in the liver of chimeric mice, and major human P450 proteins such as CYP1A2, CYP2C9, and CYP3A4 were detected. The expression of P450 mRNA and protein was correlated with the hAlb concentration in the blood. The enzyme activities such as diclofenac 4-hydroxylase activity, dexamethasone 6-hydroxylase activity, and coumarin 7-hydroxylase activity, activities that are specific to human P450 but not to murine P450, were increased in a hAlb concentration-dependent manner. The chimeric mice with nearly 90% replacement by human hepatocytes demonstrated almost the same protein contents of human P450s and drug-metabolizing enzyme activity as those of the donor. It was confirmed that genomic DNA from the livers of the chimeric mice and that from the liver of the donor exhibited the same genotype. In conclusion, the chimeric mice exhibited a similarly efficient capacity of drug metabolism as humans, suggesting that they could be a useful animal model for drug development.
These results suggest that the microglia/macrophages recruited to the crushed site are the possible cellular sources of the ETs, which caused reciprocal activation of astrocytes. Blocking the ETB receptors by BQ-788 rescued RGCs, most likely by attenuating neuroinflammatory events.
Recent studies have suggested that vitamin D activities involve vitamin D receptor (VDR)-dependent and VDR-independent effects of 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3) and 25-hydroxyvitamin D 3 (25(OH)D 3) and ligand-independent effects of the VDR. Here, we describe a novel in vivo system using genetically modified rats deficient in the Cyp27b1 or Vdr genes. Type II rickets model rats with a mutant Vdr (R270L), which recognizes 1,25(OH) 2 D 3 with an affinity equivalent to that for 25(OH)D 3 , were also generated. Although Cyp27b1-knockout (KO), Vdr-Ko, and Vdr (R270L) rats each showed rickets symptoms, including abnormal bone formation, they were significantly different from each other. Administration of 25(OH)D 3 reversed rickets symptoms in Cyp27b1-KO and Vdr (R270L) rats. Interestingly, 1,25(OH) 2 D 3 was synthesized in Cyp27b1-KO rats, probably by Cyp27a1. In contrast, the effects of 25(OH)D 3 on Vdr (R270L) rats strongly suggested a direct action of 25(OH)D 3 via VDR-genomic pathways. These results convincingly suggest the usefulness of our in vivo system. The active form of vitamin D 3 , 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3), plays important roles in osteogenesis, calcium homeostasis, cellular differentiation, and immune responses 1. 1,25(OH) 2 D 3 is generated by two hydroxylation steps from vitamin D 3 : C-25 hydroxylation by hepatic CYP2R1 and CYP27A1 and subsequent 1α-hydroxylation by renal 1α-hydroxylase (CYP27B1) 2. The vitamin D receptor (VDR) mediates the genomic action of active vitamin D 3. Binding of active vitamin D 3 to the VDR triggers its heterodimerization to the retinoid X receptor and subsequent translocation to the nucleus. This translocation results in regulating target gene expression by formation of the VDR complex on vitamin D-responsive elements in the promoter regions of target genes, such as osteocalcin and osteopontin in bones and the calcium channels and calbindins in intestines 3. CYP24A1, one of the well-known vitamin D target genes, is involved in inactivating 1,25(OH) 2 D 3 through sequential metabolism that starts with C-24 or C-23 hydroxylation of 1,25(OH) 2 D 3 4. A variety of vitamin D derivatives have been developed as drugs for rickets, osteoporosis, psoriasis, secondary hyperparathyroidism, and chronic kidney disease. Because all of these compounds show high affinity for the VDR, these pharmacological actions are considered to be VDR mediated. However, as with 1,25(OH) 2 D 3 , they might also have non-VDR-mediated actions. Thus, pharmacological action studies of vitamin D derivatives are essential for future drug discovery.
Drug development of a potential analgesic agent 5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidine was withdrawn because of its limited hepatotoxic effects in humans that could not be predicted from regulatory animal or in vitro studies. In vivo formation of glutathione conjugates and covalent binding of a model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine were investigated in the present study after intravenous administration to chimeric mice with a human or rat liver because of an interesting capability of human cytochrome P450 1A2 in forming a covalently bound metabolite in vitro. Rapid distribution and elimination of radiolabeled 5-n-butyl-pyrazolo[1,5-a]pyrimidine in plasma or liver fractions were seen in chimeric mice after intravenous administration. However, similar covalent binding in liver was detected over 0.17-24 h after intravenous administration. Radio-LC analyses revealed that the chimeric mice with humanized liver preferentially gave the 3-hydroxylated metabolite and its glutathione conjugate in the plasma and liver. On the contrary, chimeric mice with a rat liver had some rat-specific metabolites in vivo. Analyses by electrophoresis with accelerator mass spectrometry of in vivo radiolabeled liver proteins in chimeric mice revealed that bioactivated 5-n-butyl-pyrazolo[1,5-a]pyrimidine bound nonspecifically to a variety of microsomal proteins including human P450 1A2 as well as cytosolic proteins in the livers from chimeric mice with humanized liver. These results suggest that the hepatotoxic model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine was activated by human liver microsomal P450 1A2 to reactive intermediate(s) in vivo in humanized chimeric mice and could relatively nonspecifically bind to biomolecules such as P450 1A2 and other proteins.
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