ABSTRACT
CYP2D6 is a highly polymorphic human gene responsible for a large variability in the disposition of more than 100 drugs to which humans may be exposed. Animal models are inadequate for preclinical pharmacological evaluation of CYP2D6 substrates because of marked species differences in CYP2D isoforms. To overcome this issue, a transgenic mouse line expressing the human CYP2D6 gene was generated. The complete wild-type CYP2D6 gene, including its regulatory sequence, was microinjected into a fertilized FVB/N mouse egg, and the resultant offspring were genotyped by both polymerase chain reaction and Southern blotting. CYP2D6-specific protein expression was detected in the liver, intestine, and kidney from only the CYP2D6 humanized mice. Pharmacokinetic analysis revealed that debrisoquine (DEB) clearance was markedly higher (94.1 +/- 22.3 l/h/kg), and its half-life significantly reduced (6.9 +/- 1.6 h), in CYP2D6 humanized mice compared with wild-type animals (15.2 +/- 0.9 l/h/kg and 16.5 +/- 4.5 h, respectively). Mutations in hepatic nuclear factor 4alpha (HNF4alpha), a hepatic transcription factor known to regulate in vitro expression of the CYP2D6 gene, could affect the disposition of CYP2D6 drug substrates. To determine whether the HNF4alpha gene modulates in vivo pharmacokinetics of CYP2D6 substrates, a mouse line carrying both the CYP2D6 gene and the HNF4alpha conditional mutation was generated and phenotyped using DEB. After deletion of HNF4alpha, DEB 4-hydroxylase activity in CYP2D6 humanized mice decreased more than 50%. The data presented in this study show that only CYP2D6 humanized mice but not wild-type mice display significant DEB 4-hydroxylase activity and that HNF4alpha regulates CYP2D6 activity in vivo. The CYP2D6 humanized mice represent an attractive model for future preclinical studies on the pharmacology, toxicology, and physiology of CYP2D6-mediated metabolism.
Subject(s)
Cytochrome P-450 CYP2D6/metabolism , DNA-Binding Proteins , Debrisoquin/pharmacokinetics , Mice, Transgenic/metabolism , Phosphoproteins/metabolism , Transcription Factors/metabolism , Adrenergic Agents/pharmacokinetics , Alleles , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Cytochrome P-450 CYP2D6/genetics , Gene Deletion , Gene Dosage , Gene Transfer Techniques , Hepatocyte Nuclear Factor 4 , Humans , Male , Mice , Mice, Knockout/metabolism , Phosphoproteins/genetics , Transcription Factors/geneticsABSTRACT
The sequence and organization of the CYP1A cluster on human chromosome 15 was determined. A human genomic clone from a BAC library, containing both CYP1A1 and CYP1A2 genes, was isolated and sequenced. The results of Southern blot analysis using human genomic DNA were compatible with the structure of the BAC clone. The CYP1A1 and CYP1A2 genes are separated by a 23 kb segment that contains no other open reading frames. The CYP1A1 and CYP1A2 genes are in opposite orientation, revealing that the 5' flanking region is in common between the two genes. Analysis of the sequence obtained revealed the presence of xenobiotic response elements (XREs) previously reported for CYP1A1 and CYP1A2 and several additional consensus sequences for putative XREs. The presence of all the XREs upstream of both genes suggest that some of the regulatory elements known to control CYP1A1 gene expression, could also control CYP1A2 gene expression.
Subject(s)
Chromosomes, Human, Pair 15/genetics , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A2/genetics , Gene Expression Regulation , Gene Order , Multigene Family , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP1A2/biosynthesis , Genetic Markers , Humans , Molecular Sequence Data , Restriction MappingABSTRACT
A yellow-eyed mutant was discovered in a strain of Heliothis virescens, the tobacco budworm, that already exhibited a mutation for yellow scale, y. We investigated the inheritance of these visible mutations as candidate markers for transgenesis. Yellow eye was controlled by a single, recessive, autosomal factor, the same type of inheritance previously known for y. Presence of the recombinant mutants with yellow scales and wild type eyes in test crosses indicated independent segregation of genes for these traits. The recombinant class with wild type scales and yellow eyes was completely absent and there was a corresponding increase of the double mutant parental class having yellow scales and yellow eyes. These results indicated that a single factor for yellow eye also controlled yellow scales independently of y. This gene was named yes, for yellow eye and scale. We hypothesize that yes controls both eye and scale color through a deficiency in transport of pigment precursors in both the ommochrome and melanin pathways. The unlinked gene y likely controls an enzyme affecting the melanin pathway only. Both y and yes segregated independently of AceIn, acetylcholinesterase insensitivity, and sodium channel hscp, which are genes related to insecticide resistance.
