We characterized the uptake of fractionated [³H]heparin in rat peritoneal macrophages by investigating the influence of inhibitors of transport and by analyzing binding and internalization processes. Several ligands of scavenger receptors inhibited the binding and subsequent internalization. When temperature was lowered from 37°C to 4°C, the internalization was significantly reduced, while the binding was unchanged, as expected for receptor-mediated endocytosis. However, known inhibitors of receptor-mediated and adsorptive endocytosis of polypeptides and phagocytosis did not affect the uptake. These characteristics are similar to those observed in rat Kupffer cells in our previous study and suggest the involvement of scavenger receptors in the uptake of fractionated [³H]heparin. Finally, although the dissociation constant (K_d) in the macrophage was comparable with that in the Kupffer cell, the internalization rate constant (k_{int, app}) was approximately two-fold larger in the former case, suggesting that the functional regulations of scavenger receptors may differ between these cells.

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The HUGO Human Gene Nomenclature Committee recently developed a new system of nomenclature for the human ATP-binding cassette (ABC) family. Until now more than 40 human ABC-transporter genes have been identified and sequenced. There is accumulating evidence that human ABC proteins are involved in transport of drugs, endogenous substances or ions. It has also been reported that mutations of certain ABC genes result in genetic diseases. Based on the arrangement of molecular structure components, i.e. nucleotide binding domains and topologies of transmembrane domains, hitherto reported human ABC proteins are classified into 7 different sub-families (A to G). The new nomenclature scheme was implemented on October 22, 1999, and detailed information is available on the web site at URL: ‹http://www.gene.ucl.ac.uk./users/hester/abc.html ›. (Nomenclature editor, Dr. Hester Mary Wain, University College London, U.K.).

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This article describes sources and mechanisms of formation of α, β-unsaturated aldehydes, their reactivity with respect to glutathione and amino-groups, their toxicity based on interaction with sulfhydryl and amino targets in cells, and modulation of gene expression by the aldehydes. Among the many different aldehydes generated durimg lipid peroxidation, 4-hydroxy-2 (E)-nonenal (4-HNE) is one of the major products and has been shown to have a number of adverse biological effects. All previous studies on biological and toxicological effects of 4-HNE have been carried out using its racemate. Therefore, nothing has been known of which enantiomer of the racemate is more toxic, more reactive with biomacromolecules, or more readily detoxified by glutathione S-transferase (GST) and alcohol dehydrogenase (ADH) than the other. This article also describes the first evidence for the marked enantioselectivity by 4-HNE enantiomers in the irreversible inactivation of the glycolytic enzyme, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, and in their detoxification by rat liver cytosolic GST A4-4 and ADH.

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Multiple forms cytochrome P450 (P450 or CYP) enzymes play important roles in the oxidation of structurally diverse xenobiotics and endobiotics. P450s are not self-sufficient enzymes and require a reductase as an electron carrier to function as monooxygenases. Recently recombinant P450 enzymes from different sources, e.g., microsomes of human lymphoblastoid cells, of yeast, and insect cells infected with baculovirus systems, and Escherichia coli membranes containing P450 and reductase coexpressed, have been widely used for drug metabolism research. However, the marker activities or kinetic parameters of human P450 enzymes reported from different research laboratories and commercial enzymes manufacturers are not always similar. These differences may be a critical factor for understanding roles of human P450 enzymes involved in drug metabolism. Troglitazone, a new oral antidiabetic drug, is reported not to be oxidized by P450 enzymes. However, cDNA-expressed CYP2C8 and CYP3A4 were active in catalyzing formation of a quinonetype metabolite. Intensity of inhibitory effects of specific P450 inhibitors and anti-P450 antibodies on the quinone-type metabolite formation depended on human liver samples and their P450 status. Azelastine, an antiallergy drug, was N-demethylated by CYP2D6, CYP3A4 and CYP1A2 in human liver microsomes biphasically. Human intrinsic clearance was predictable from P450 contents or kinetic parameters using cDNA-expressed P450 enzymes. The results suggest that different P450 enzymes in human liver have major roles in quinone-type metabolite formation from troglitazone and azelastine N-demethylation and that the hepatic contents of these P450 forms determine which P450 enzymes play the major roles in drug metabolism in individual humans.

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Multiple forms of cytochrome P450 (P450 or CYP) exist in several organs of humans and individual P450 isoforms have considerable, but overlapping, substrate specificities. CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 are identified to date to be the major P450 enzymes involved in the oxidation of xenobiotics including drugs, environmental chemicals, and carcinogens and CYP1A1 and CYP1B1 have been shown to be principally expressed in extrahepatic organs. Most of the xenobiotic chemicals which are ingested clinically or incidentally into the body have been shown to be oxidized by multiple forms of P450 in several organs such as liver, intestine, and lung. Roles of these P450 isoforms in the oxidation of xenobiotics have been shown to be determined by the ratio of V_max to K_m of the P450 isoforms and the levels of expression of these P450 forms in different human samples. Here we show our model studies involving the roles of several P450 enzymes in the oxidation of omeprazole, 7-ethoxycoumarin, and chlorzoxazone using kinetic parameters of recombinant P450 enzymes and contents of individual P450 forms in human liver microsomes. Future work which must be addressed to be examined will be briefly discussed.

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Human CYP2D6 is a causative enzyme in debrisoquine/sparteine-type genetic polymorphism, and is involved as a major enzyme in the oxidation of over 60 drugs that contain a nitrogen or sulfur atom in their chemical structures. Rats also have 6 kinds of CYP2D enzymes such as CYP2D1, CYP2D2, CYP2D3, CYP2D4, CYP2D5 and CYP2D18. Propranolol (PL), a typical β-adrenoceptor blocking agent, is a good substrate for human and rat liver microsomal fractions. Human liver microsomes catalyzed aromatic ring hydroxylations forming 4-and 5-hydroxypropranolols (4-and 5-OH-PLs), and side chain N-desisopropylation forming N-desisopropylpropranolol (NDP). Rat liver microsomes catalyzed aromatic ring 7-hydroxylation in addition to the formation of the three metabolites. PL has an asymmetric carbon at the side chain, yielding R(+)-PL and S(-)-PL. Human liver microsomes showed substrate enantioselectivity of R(+)-PL> S (-) -PL for the formation of the three metabolites, whereas rat liver microsomes exhibited different selectivity: R(+)-PL<S(-)-PL for the formation of 4-OH-PL, 5-OH-PL and NDP; R(+)-PL>S(-)-PL for the formation of 7-OH-PL. Possible mechanisms causing such a differential enantioselectivity in the oxidation of PL by CYP2D6 and CYP2D2 were discussed.

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Cytochrome P450s (P450s) are induced by drugs, environmental chemicals, diseases, and genetic factors. Changes in the levels of P450 have a significant effect on the pharmacokinetics or pharmacodynamics of drugs and endogenous substrates. In experimental animals such as a rat, induction of P450 is detected in hepatic microsomes by drug metabolism assay and Western blotting with antibodies. Assay system of multiple P450 isoforms with Western blotting has been established but it is difficult to apply this method to human subjects because human hepatic tissue must be isolated to assay P450 induction. We tried to develop a less-invasive assay for detection of P450 induction. Detection system of P450 mRNA induction in human lymphocytes in peripheral blood was established using by a competitive PCR or a real-time PCR. Induction of CYP2E1 and CYP3A4 by diabetes and rifampicin, respectively, was detected by this method.

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P450s belonging to the CYP3A subfamily constitute major forms in livers and intestines of human and experimental animals. Expressions of CYP3A forms are induced by treatment with many chemicals, but their profiles differ among humans and experimental animals. Molecular mechanisms of CYP3A gene activation by chemicals had been little known until recently. A novel steroid hormone receptor, PXR (pregnane X receptor), has been isolated from mouse, rat, rabbit and human as a signal transduction molecule in CYP3A gene transactivation. Species differences observed on the induction profiles might be explained by the species specific response of their PXRs to chemicals. However, the diverse induction profiles of CYP3A forms are possible to be caused by interaction of PXR with transcription factors other than RXRα. In support with this idea, we have found that rat CYP3A1 gene is transactivated by an orphan nuclear receptor, ARP-1 with PXR in the presence of inducers. To assess the induction potency of various chemicals for human CYP3A forms, further researches are necessary to clarify the transcriptional regulation of CYP3A forms.

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CYP2A6 has been characterized as a coumarin 7-hydroxylase in humans. A large interindividual difference in the activity of coumarin 7-hydroxylation suggested an existence of genetic polymorphism of this enzyme. In fact, CYP2A6^*2 variant allele which has T→A substitution, leading to amino acid change from Leu₁₆₀ to His₁₆₀, has been found in Caucasian population as the most frequent mutation in poor metabolizers (PM) of coumarin. Although several drugs used clinically or under development such as fadrozole, losigamone and methoxyflurane are recognized at present to be good substrates of CYP2A6, no specific substrate of this CYP isoform has been known until we found a drug, SM-12502. In the phase I trial, 3 out of 28 Japanese subjects were classified as PM of the drug. In vitro studies demonstrated that CYP2A6 played a major role on the metabolism of the drug. Genomic analysis revealed that the PM phenotype was caused by the presence of a novel CYP2A6 gene variant which lacks the entire region of open reading frame encoding the enzyme in the PM. Thus, we designated the variant as “deletion-type” allele. We examined the frequency of individuals carrying homozygous deletion by a genotyping method established in our laboratory. Thus, the frequency was estimated to be 3-4% in Japanese. We found another CYP2A6 gene variant whose 60 bp in the 3'-untranslated region was substituted by the corresponding region of the CYP2A7 pseudogene. This variant was designated as “conversion-type” allele. We found that the allele frequency of the conversiontype was comparable to that of wild-type, CYP2A6^*1 allele in Japanese. We also compared the frequency of the CYP2A6^*2 allele as well as the deletion and the conversion alleles between Japanese and Caucasian. Consequently, a marked interracial difference in the frequency of the genetic variants of the CYP2A6 gene was observed. These results give an interesting insight into racial difference in response to drugs and evolution of the CYP2A gene subfamily in humans.

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