The CYP2D gene subfamily: analysis of the molecular basis of the debrisoquine 4-hydroxylase deficiency in DA rats

The DA rat has been proposed as an animal model for the human debrisoquine 4-hydroxylase/bufuralol 1'-hydroxylase genetic deficiency. To determine the mechanism of this deficiency, we isolated and sequenced five cDNAs in the CYP2D gene subfamily including a new IID1 allele and two cDNAs of novel P450s, designated IID3 and IID5. IID3 and IID5 cDNA-deduced amino acid sequences contained 500 and 504 residues with calculated molecular weights of 56,683 and 57,081, respectively. IID5 displayed 20 amino acid differences with the IID1, yet bore only 72% and 76% similarity to IID2 and IID3. Despite an overall nucleotide similarity of 80-98% between the 4 cDNAs, a region of 134 nucleotides of sequence exists that contains only 1 base difference. This region is probably the result of gene conversion events between the P450 IID genes. Although all IID cDNAs were expressed into immunodetectable proteins using the COS cell SV40-based expression system, only IID1 could effectively catalyze the oxidation of the prototype substrate bufuralol. Expression of a cDNA isolated in an earlier study [Gonzalez, F. J., Matsunaga, T., Nagata, K., Meyer, U. A., Nebert, D. W., Pastewka, J., Kozak, C. A., Gillette, J., Gelboin, H. V., & Hardwick, J. P. (1987) DNA 6, 149-161], previously called db1 and now designated IID1v, produced a protein with a drastically reduced activity as compared to cDNA-expressed IID1 despite only four amino acid differences between the two cDNA-deduced protein sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absence of hepatic cytochrome P450bufI causes genetically deficient debrisoquine oxidation in man

The common genetic deficiency of drug oxidation known as debrisoquine/sparteine-type polymorphism was investigated with bufuralol as prototype substrate. In human liver microsomes the 1'-hydroxylation of bufuralol is catalyzed by two functionally distinct P-450 isozymes, the high-affinity/highly stereoselective P450bufI and the low-affinity/nonstereoselective P450bufII. We demonstrate that P450bufI is unique in hydroxylating bufuralol in a cumene hydroperoxide (CuOOH) mediated reaction whereas P450bufII is active only in the classical NADPH- and O2-supported monooxygenation. In microsomes of liver biopsies of in vivo phenotyped poor metabolizers of debrisoquine or sparteine, the CuOOH-mediated activity was drastically reduced. Rabbit antibodies against a rat P-450 isozyme with high bufuralol 1'-hydroxylase activity (P450db1) precipitated exclusively P450bufI-type activity from solubilized microsomes. Western blotting of microsomes with these antibodies revealed a close correlation between the immunoreactive protein and CuOOH-mediated (+)-bufuralol 1'-hydroxylation. No immunoreactive protein was detected in liver microsomes of in vivo phenotyped poor metabolizers. These data provide evidence for a specific deficiency of P450bufI and are consistent with the complete or almost complete absence of this protein in the liver of poor metabolizers.     

High-resolution imaging reveals a limit in spatial resolution of blood flow measurements by microspheres

Density of 15-microm microspheres after left atrial application is the standard measure of regional perfusion. In the heart, substantial differences in microsphere density are seen at spatial resolutions <5 ml, implying perfusion heterogeneity. Microsphere deposition imaging permits a superior evaluation of the distribution pattern. Therefore, fluorescent microspheres (FMS) were applied, FMS deposition in the canine heart was imaged by epifluorescence microscopy in vitro, and the patterns were observed compared with MR images of iron oxide microspheres (IMS) obtained in vivo and in vitro. FMS deposition in myocardial slices revealed the following: 1) a nonrandom distribution, with sequentially applied FMS of different color stacked within the same vessel, 2) general FMS clustering, and 3) rather large areas devoid of FMS (n = 3). This pattern was also seen in reconstructed three-dimensional images (<1 nl resolution) of FMS distribution (n = 4). Surprisingly, the deposition pattern of sequentially applied FMS remained virtually identical over 3 days. Augmenting flow by intracoronary adenosine (>2 microM) enhanced local microsphere density, but did not alter the deposition pattern (n = 3). The nonrandom, temporally stable pattern was quantitatively confirmed by a three-dimensional intermicrosphere distance analysis of sequentially applied FMS. T2-weighted short-axis MR images (2-microl resolution) of IMS revealed similar patterns in vivo and in vitro (n = 6), as seen with FMS. The observed temporally stable microsphere patterns are not consistent with the notion that microsphere deposition is solely governed by blood flow. We propose that at high spatial resolution (<2 microl) structural aspects of the vascular network dominate microsphere distribution, resulting in the organized patterns observed.     

A naturally occurring mutation in the SLC21A6 gene causing impaired membrane localization of the hepatocyte uptake transporter

The organic anion transporter SLC21A6 (also known as OATP2, OATP-C, or LST-1) is involved in the hepatocellular uptake of a variety of endogenous and xenobiotic substances and drugs. We analyzed 81 human liver samples by immunoblotting and found one with a strongly reduced amount of SLC21A6 protein suggesting mutations in the SLC21A6 gene. The SLC21A6 cDNA from this sample contained five base pair changes in one allele; three of the mutations resulted in amino acid substitutions designated SLC21A6-N130D, SLC21A6-P155T, and SLC21A6-L193R. The former two were polymorphisms (SLC21A6*1b and SLC21A6*4), whereas SLC21A6-L193R represents the first naturally occurring mutation identified in one allele of the SLC21A6 gene, which affects protein maturation and organic anion transport. We introduced each of the mutations into the SLC21A6 cDNA and established stably transfected MDCKII cells expressing the respective mutant SLC21A6 protein. Immunofluorescence microscopy and uptake measurements were used to study localization and transport properties of the mutated proteins. Both proteins carrying the polymorphisms were sorted to the lateral membrane like wild-type SLC21A6, but their transport properties for the substrates cholyltaurine and 17beta-glucuronosyl estradiol were altered. Importantly, most of the mutant protein SLC21A6-L193R was retained intracellularly, and this single amino acid exchange abolished transport function.     

Distinction between human cytochrome P450 (CYP) isoforms and identification of new phosphorylation sites by mass spectrometry

In mammals, Cytochrome P450 (CYP) enzymes are bound to membranes of the endoplasmic reticulum and mitochondria, where they are responsible for the oxidative metabolism of many xenobiotics as well as organic endogenous compounds. In humans, 57 isoforms were identified which are classified based on sequence homology. In the present work, we demonstrate the performance of a mass spectrometry-based strategy to simultaneously detect and differentiate distinct human Cytochrome P450 (CYP) isoforms including the highly similar CYP3A4, CYP3A5, CYP3A7, as well as CYP2C8, CYP2C9, CYP2C18, CYP2C19, and CYP4F2, CYP4F3, CYP4F11, CYP4F12. Compared to commonly used immunodetection methods, mass spectrometry overcomes limitations such as low antibody specificity and offers high multiplexing possibilities. Furthermore, CYP phosphorylation, which may affect various biochemical and enzymatic properties of these enzymes, is still poorly analyzed, especially in human tissues. Using titanium dioxide resin combined with tandem mass spectrometry for phosphopeptide enrichment and sequencing, we discovered eight human P450 phosphorylation sites, seven of which were novel. The data from surgical human liver samples establish that the isoforms CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C8, CYP2D6, CYP3A4, CYP3A7, and CYP8B1 are phosphorylated in vivo. These results will aid in further investigation of the functional significance of protein phosphorylation for this important group of enzymes.     

RNA‐Interference Approach to Study Functions of NADPH : Cytochrome P450 Oxidoreductase in Human Hepatocytes

Human NADPH : cytochrome P450 oxidoreductase (POR) is encoded by a single gene on chromosome 7q11.2. This flavoprotein donates electrons derived from NADPH to a variety of acceptor proteins, including squalene monooxygenase, heme oxygenase, cytochrome b₅, and many microsomal cytochromes P450 (CYPs), which are involved in oxidative drug metabolism, steroidogenesis, and other functions. Numerous aspects related to cellular POR expression have not been systematically investigated. Interestingly, POR expression is lower compared to CYPs and may thus be limiting for monooxygenase activities, but conversely, POR knock‐out in mice resulted in compensatory upregulation of CYPs. POR may also influence intracellular cholesterol and lipid homeostasis. To systematically investigate such effects, we developed specific POR gene silencing in cell lines and primary human hepatocytes by RNA interference using small interfering RNAs (siRNAs). In HepG2 cells, POR mRNA could be reduced by 95% over 4 days accompanied by reduced protein content and activity. In primary human hepatocytes, POR mRNA knock‐down was less effective and more variable. Analysis of CYPs indicated induction of CYP3A4 but not CYP1A2 or CYP2D6. These results demonstrate that POR can be efficiently and almost completely silenced in HepG2 cells and, at least partially, in primary human hepatocytes. This will allow systematic studies of various consequences of POR variability in human cells.