Interindividual variation of drug effects in humans can be attributed to many factors. Among the factors, the rate of drug metabolism has been regarded as the most important one. A genetic defect of enzymes involved in drug metabolism, particularly cytochrome P450 (CYP), has been believed to be one of the important causal factors of adverse drug reactions. There are multiple gene mutations for CYP causing the poor metabolizer phenotype. The occurrence of genetic polymorphism has been seen in genes for CYP1A1, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A5. Among them, the molecular mechanisms of genetic polymorphisms of CYP2D6 (debrisoquine/sparteine type) and CYP2A6 (coumarin type) in Japanese have been the focus of investigations. Only 20 - 30% of the Japanese population that shows the CYP2D6 poor metabolizer phenotype can be diagnosed by gene analysis. By other means, we found two new mutations, CYP2D6/J9 and CYP2D6/C8, in Japanese. With regard to CYP2A6, we discovered SM-12502, a new probe drug in addition to coumarin, that is currently under development; this drug is mainly metabolized by CYP2A6. Using this drug as a probe, we found poor metabolizers and analyzed the genes for CYP2A6. We found a new mutation (CYP2A6 whole deletion) responsible for the poor metabolizer phenotype. These results should contribute to the selection of an effective drug prescription and a reduced incidence of adverse effects.
Cytochromes P450 (CYP) are a superfamily of hemoproteins that metabolize various foreign compounds. The human CYP2C subfamily is one of the subfamilies of the CYP2 family and it consists of four members of CYP isoforms, CYP2C8, CYP2C9, CYP2C18 and CYP2C19. A well-characterized genetic polymorphism occurs in CYP2C19, which is associated with the 4'-hydroxylation of S-mephenytoin. There are two phenotypes, extensive metabolizers and poor metabolizers (PM) of mephenytoin. The frequency of PM in the Japanese population is 20%, while only 3% of Caucasians are PM of mephenytoin. Two defective alleles, designated as CYP2C19∗2 and CYP2C19∗3, have been described, and the latter mutation has been detected only in Oriental populations. Recently, an allelic variant of CYP2C9 that causes substitution of Leu359 for Ile359 has been shown to be associated with the decreased metabolic clearance of various therapeutic agents including warfarin, tolbutamide and phenytoin. The frequency of this variant allele in the Japanese population is 2%, while those of the Caucasians are 6-9%. Although the role of CYP2C18 in the drug metabolism remains obscure, we have recently found that defective alleles of CYP2C19∗3 and CYP2C18m1 are completely linked, suggesting that PM of CYP2C19 with CYP2C19∗13 alleles is a PM of CYP2C18 and vice versa.
Arachidonic acid is metabolized to biologically active substances by three major enzyme systems including cyclooxygenases, lipoxygenases and cytochrome P450s. The third pathway, P450-dependent pathway, includes allylic oxidation, ω-hydroxylation, and epoxidation of arachidonic acid. Of these metabolites, the physiological role of 20-hydroxyeicosatetraenoic acid (20-HETE) produced by CYP4A isoforms has been extensively studied. 20-HETE affects ion transport, constricts blood vessels and participates in tubuloglomerular feed back. Increased production of 20-HETE is a major factor in elevating blood pressure in spontaneously hypertensive rats (SHR). We have found that CYP4A2 level in SHR is much higher than that of normotensive rat. Recently, factors of endothelial origin other than nitric oxide and prostaglandins were reported. Inhibitors of P450-dependent arachidonic acid metabolism greatly reduce the vasodilator effect and this factor is speculated to be an epoxide of arachidonic acid. We have isolated CYP2C23 from rat kidney and have found that it produces arachidonic acid epoxides. We have investigated changes in the CYP2C23 levels in physiological and pathophysiological conditions. Multiple pathways of arachidonic acid metabolism by P450 have been reported and the diverse properties of these metabolites and the wide distribution of the P450 system make them prime candidates for participation in regulatory mechanisms of the circulation and transporting epithelia.
Nitric oxide (NO) reacts with iron, superoxide, thiols and oxygen. Although NO reversibly interacts with the heme-iron of P450, the pathophysiological role of this interaction remains to be elucidated. We found that hepatic levels of P450 markedly decreased in endotoxemic rats, particularly when the rate of NO generation was increased. To determine the possible role of NO in the modulation of the structure and function of P450, changes in the levels and activities of P450 isozymes were determined in liver microsomes from normal and endotoxemic rats. Electron spin resonance analysis revealed that incubation of microsomes with the NO donor NOC-7 rapidly generated NO-P450 adducts. Microsomal levels of NO-P450 adducts increased and peaked at 10 min after incubation and decreased thereafter; it disappeared completely within 60 min. In contrast, microsomal levels of the low-spin ferric form and CO-differential spectrally detectable P450 rapidly decreased during the initial 10 min; the signal intensity for P450 recovered thereafter. Western blot analysis using specific antibodies against CYP3A2 and CYP2C11 isozymes revealed no detectable degradation of these isoforms. Effect of NO on the catalytic activity of the enzymes was also determined by using testosterone as the substrate. The hydroxylation activity in microsomes rapidly decreased during the initial 10 min and disappeared slowly thereafter. These results suggested that NO might form dissociable complexes with the heme moiety of P450 and irreversibly inactivate them. The mechanism for P450 inactivation by NO and the role of NO-P450 interaction in the pathogenesis of liver injury in endotoxemia are discussed.
Cytochrome P450c17 (P450c17) catalyzes 17α-hydroxylation and 17, 20-lyase reactions. This enzyme plays a key role in determination of the balance between glucocorticoids and steroid sex hormones. In this review, we discuss recent progress in the studies of both transcriptional regulation of CYP17 encoding P450c17 and enzymatic regulation of P450c17. Several transcription factors involved in cAMP-dependent transcription of the gene have been isolated and identified to be members of the atypical homeodomain “TALE” superfamily containing Pbx, Meis, Pknox and TGIF families. The studies of enzymatic regulation of P450c17 suggest that cytochrome b5 (b5), a heme protein, may switch the reaction of P450c17 by enhancing the 17, 20-lyase activity to increase the level of plasma C19 steroids. The importance of b5 in the synthesis of C19 steroids has also been shown in a clinical study reporting that the external genitalia was abnormal in a patient having a defect in b5. Therefore, this enhancement by b5 on the lyase activity of P450c17 may beessential to normal sexual differentiation in humans and also important in control of an optimal balance between sex steroid hormones and glucocorticoids. In addition, the age-dependent expression of P450c17 in immature rat livers is also discussed.
Aromatase (estrogen synthetase) catalyzes a key step in estrogen biosynthesis and plays an important role in reproductive processes in the ovary and placenta. In this context, aromatase is an enzyme producing estrogen as an endocrine female sex hormone. Recently, it has reported that aromatase is also present in various extra-gonadal tissues and is tissue-specifically regulated by various factors. This tissue-specific regulation of human aromatase gene is realized by alternative utilization of multiple exon 1's; exons 1a, 1b, 1c, 1d, 1e, and 1f that are specific for expression in the placenta, skin fibroblasts/fetal liver, ovary, ovary/prostate/testis, placenta, and brain, respectively. Each of the tissue-specific exon 1's is flanked by a unique promoter containing basic and regulatory elements. The facts that (i) aromatase is distributed in various gonadal and extra-gonadal tissues and (ii) regulated tissue-specifically by various factors, (iii) estrogen participates in specific physiological functions of various tissues, and (iv) estrogen receptor is also distributed in various tissues strongly indicate that estrogen locally produced by aromatase acts in various tissues as a multi-functional paracrine or autocrine hormone. This idea was discussed in connection with the Kd value for the estrogen receptor and serum concentration of estrogen.
In the present review, we described the procedures of production of polyclonal and monoclonal antibodies and single chain Fv molecule (scFv). Among several animal species, the rabbit is the best animal for polyclonal antibody production and the mouse is the best animal for monoclonal antibody production. In this review, we discussed problems that might be encountered when trying to produce antibodies. Polyclonal antibodies are easily produced in rabbits by immunizing them with gluthatione-S-transferase fusion proteins. However, it is difficult to eliminate nonspecifically reacting antibodies, even after the antibodies were purified from sera by an antigen column. Monoclonal antibody production is a time-consuming process, but if successful, will produce a very useful reagent due to no limitation of supply and constant quality. We described monoclonal antibody production by means of gluthatione-S-transferase fusion protein. scFv is a portion of the antibody and is constructed by PCR of VH and VL regions of the antibody. We recommend that scFv should be constructed from a hybridoma that secretes monoclonal antibody, although some researchers have claimed that scFv can be constructed from the spleen of immunized mice. The expression of scFv is a promising approach to analyze the function of one of the subtypes, when the original monoclonal antibody can block the function of the protein.