Biomedical subjects

M Kitada

Publications and source records attributed to M Kitada.

At least 37 records · Page 2Linked to original sources

PubMed1993

Involvement of CYP2D6 in oxidative metabolism of cinnarizine and flunarizine in human liver microsomes.

Oxidative metabolism of cinnarizine (CZ) and its fluorine derivative flunarizine (FZ), both of which are selective calcium entry blockers, was examined in human liver microsomes. The ring-hydroxylations and the N-desalkylations constituted primary metabolic pathways in microsomal metabolism of CZ and FZ. Among these pathways, the ring-hydroxylase (p-hydroxylation) activities at the cinnamyl moiety of both drugs were highly correlated with debrisoquine 4-hydroxylase activity and CYP2D6 content. Quinidine, a selective inhibitor of CYP2D6, suppressed the ring-hydroxylase activities of CZ and FZ. These results suggest that CYP2D6 is involved in the ring-hydroxylation of the cinnamyl moiety of both CZ and FZ in human liver microsomes.

Biotransformation↗

PubMed1993

Purification and characterization of two forms of hepatic microsomal cytochrome P450 from untreated cynomolgus monkeys.

Two forms of cytochrome P450, referred to as P450 CMLb and P450 CMLc, were purified and characterized from hepatic microsomes of untreated cynomolgus monkeys (Macaca irus). The final preparations were apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The minimum molecular weights of P450 CMLb and P450 CMLc estimated from the mobilities on the gel were 48 and 50 kDa, respectively. The N-terminal amino acid sequences of P450 CMLb and P450 CMLc (first 13 residues) were identical, respectively, with those of P450 FI isolated from baboons and P450 MK-2, which has been characterized as a P450 3A enzyme in cynomolgus monkeys. P450 CMLb showed coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation activities. This P450 enzyme reacted with anti-P450 2A6 antibody and acts as a coumarin 7-hydroxylase in hepatic microsomes. On the basis of these results P450 CMLb was classified into the 2A subfamily. P450 CMLb is expressed constitutively as one of the minor forms of P450 in liver microsomes of untreated cynomolgus monkeys and is inducible by phenobarbital. P450 CMLc reacted with anti-P450 3A4 antibody and hydroxylated testosterone at the 6 beta-position. The testosterone 6 beta-hydroxylation activities in hepatic microsomes of cynomolgus monkeys, common squirrel monkeys, and humans were strongly inhibited by the anti-P450 CMLc antibody. These results demonstrate that this P450 enzyme is in the P450 3A subfamily.

Amino Acid Sequence↗

PubMed1993

Effects of bunazosin, a selective alpha 1-blocking agent, and propranolol used alone and in combination on canine ventricular refractoriness and its dispersion during myocardial ischemia.

The individual and combined effects of bunazosin, a selective alpha 1-adrenergic blocking agent, and propranolol on ventricular refractoriness and its dispersion were assessed in 33 chloralose-anesthetized, sympathectomized, and vagotomized dogs 2-3 h after occlusion of the obtuse marginal branches of the circumflex artery. The refractory period was measured in eight sites of the ischemic zone, two sites of the border zone, and two sites of the normal zone with S1-S2 extrastimulus methods. In group 1 dogs (n = 9), coronary artery ligation significantly shortened refractoriness in the ischemic zone (p = 0.023-0.001 in each site). Intravenous (i.v.) administration of a low dose of bunazosin at 0.1 mg/kg significantly blunted the shortening of refractoriness in the ischemic zone (p = 0.026-0.002 in each site), although the values did not reach those observed in the nonischemic zones (both the border and normal zones), where refractoriness remained unchanged. In group 2 dogs (n = 11), a higher dose of i.v. bunazosin, 0.5 mg/kg, significantly blunted the shortening of the refractory period within the ischemic zone (from 149 +/- 14 to 175 +/- 8 ms; mean +/- SD, p < 0.001) and reached the levels of the nonischemic zones (border zone 175 +/- 15 ms, normal zone 170 +/- 14 ms), resulting in a dispersion reduction in refractoriness between the ischemic and nonischemic zones. This dispersion tended to increase again with i.v. administration of 0.2 mg/kg propranolol (ischemic vs. border zone p = 0.034; ischemic vs. normal zone p = 0.089).(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Antagonists↗

PubMed1993

Immunohistochemical and immunoelectron microscopic study of cytochrome P-450 of human fetal livers (P-450HFLa): implications for an onco-feto-placental enzyme.

Cytochrome P-450 of human fetal livers (P-450HFLa) was demonstrated by the avidin-biotin immunoperoxidase technique in tissue samples as follows: human fetal organs, adult livers, human and cynomolgus placenta, and gynecologic organs which were obtained from 40 patients with gynecologic malignancies and 32 patients with benign diseases. P-450HFLa was clearly localized in the cytoplasm and membranes of the hepatocytes, and the fact was confirmed by an immunoelectron microscopic examination. In addition, a semiquantitative assay of staining intensity demonstrated that this enzyme tended to decrease with advancing age. These findings suggest that hepatic P-450HFLa synthesis is inversely proportional to age, and that this enzyme is one of the differentiation antigens. P-450HFLa was also detected immunohistochemically in other fetal organs. The present study thus confirms that P-450HFLa is not specific to the liver and is ubiquitous even in the fetus. Marked positive staining for P-450HFLa was demonstrated in villous syncytiotrophoblasts. In contrast, no positive staining was found in the cynomolgus-monkey placenta, unlike the case for many other placental antigens. These findings lead to the tentative conclusion that P-450HFLa is a feto-placental enzyme peculiar to humans. P-450HFLa was demonstrated to occur very frequently in gynecologic malignancies. The mean positivity rate for all gynecologic malignancies was 85%, while the rate was below 25% for benign gynecologic diseases, indicating that P-450HFLa is one of the onco-feto-placental enzymes. The present study thus suggests that this enzyme could be a promising new tumor marker for gynecologic malignancies.

Animals↗

PubMed1993

Studies on cytochrome P450 responsible for oxidative metabolism of imipramine in human liver microsomes.

The activity of imipramine 2-hydroxylase highly correlated with that of desipramine 2-hydroxylase but not with that of desipramine N-demethylase. The correlation was also found between N-demethylation and 2-hydroxylation when imipramine was used as a substrate, whereas no correlation was observed between them when desipramine was used in place of imipramine. Both activities of desipramine and imipramine 2-hydroxylase were markedly inhibited by quinidine but not by quinine. Although the activity of imipramine N-demethylase was slightly inhibited by both quinidine and quinine, the activity of desipramine N-demethylase was unaffected under the same conditions. The activity of imipramine N-demethylase was roughly correlated with the amounts of P450 3A4 immunochemically determined and the activities of testosterone 6 beta-hydroxylase in human liver microsomes. The P450 3A4 catalyzed imipramine N-demethylation much more efficiently than 2-hydroxylation in a reconstituted system, whereas neither N-demethylation nor 2-hydroxylation of desipramine was catalyzed by P450 3A4. The activity of imipramine N-demethylase was inhibited, to various extents, by anti-P450 3A4 antibodies in human liver microsomes. Taking together these and other results, it is suggested that P450 3A4, other than P450 2Cmp, also partly contributes to N-demethylation of imipramine, depending on human liver microsomes.

Cytochrome P-450 Enzyme Inhibitors↗

PubMed1993

Metabolic activation of aflatoxin B1 by human placental microsomes.

The metabolic activation of aflatoxin B1 by human placental microsomes was studied. Aflatoxin B1 showed relatively high mutagenic activity in Ames test when incubated with human placental microsomes. Addition of alpha-naphthoflavone or aminoglutethimide, known inhibitors of cytochrome P450 1A and P450 19, respectively, into the test system partially inhibited the mutagen-producing activity. It was suggested that the activation of aflatoxin B1 in human placental microsomes is mediated by at least these two forms of cytochrome P450.

Aflatoxin B1↗

PubMed1993

Species differences of testosterone 16-hydroxylases in liver microsomes of guinea pig, rat and dog.

1. In hepatic microsomes, remarkable species differences in the activity of testosterone 16-hydroxylase was observed in guinea pig, dog, and rat. The activity of testosterone 16 beta-hydroxylase was higher than that of 16 alpha-hydroxylase in guinea pig, whereas 16 alpha-hydroxylated testosterone was predominant as the metabolite in dog and rat. 2. Since P4502B isoenzyme has been shown to be a catalyst for testosterone 16-hydroxylations, we compared the catalytic properties of the P4502B subfamily (P450GP-1, P450b and P450PBD-2) purified from liver microsomes of guinea pig, dog, and rat, respectively. P450GP-1, P450b and P450PBD-2 showed different stereoselectivities for hydroxylation of testosterone at the 16-position. 3. P450GP-1, P450b and P450PBD-2 together comprised 47, < 0.1 and 23% of total P450 in liver microsomes of untreated guinea pig, rat and dog, respectively, indicating that the amounts of the P4502B isoenzyme in untreated animals were clearly different in these three animal species. Both 16 alpha- and 16 beta-hydroxylations of testosterone in liver microsomes of phenobarbital-treated guinea pig, rat and dog were inhibited by anti-P450GP-1, anti-P450b and anti-P450PBD-2 antibodies, respectively. 4. These and other results indicate that the species difference observed in testosterone 16-hydroxylation may be, in part, due to differences in the amounts of P450 of the P4502B subfamily, and their stereoselectivities for 16-hydroxylation.

Animals↗

PubMed1993

[Resection of posterior mediastinal cyst under thoracoscopy].

A 52-year-old woman with left posterior mediastinal cyst underwent surgical removal using thoracoscopy. Under general anesthesia by use of one lung ventilation, thoracoscopy was introduced through a trocar at the mid-axillary line of the 7th intercostal space. Grasping forceps were introduced through a incision in the anterior-axillary line of 4th and 5th intercostal space. The lung was held with grasping forceps. The lung was fixed to the region of anterior-mediastinum with the weight of forceps. Then posterior-mediastinum was able to be observed well by the operator. The thin cyst-wall was biopsied and excised as much as possible. Residual cyst-wall was burned by electric knife. A pathological diagnosis probed that the tumor was bronchogenic cyst without malignancy. Post operative pain markedly reduced. The patient rapidly recovered and was discharged on the 6th postoperative day. Thoracoscopic resection of mediastinum cyst may be a useful approach.

Female↗

PubMed1993

Characterization of human liver microsomal cytochrome P450 involved in the reductive metabolism of zonisamide.

Zonisamide (1,2-benzisoxazole-3-methanesulfonamide) was metabolized to 2-sulfamoylacetylphenol (SMAP) in human liver microsomes under anaerobic conditions. The formation of SMAP was remarkably inhibited by cimetidine, n-octylamine, ketoconazole, and carbon monoxide, indicating that a cytochrome P450 is involved in the metabolism of zonisamide to SMAP in human liver microsomes. The SMAP-producing activity did not correlate with the spectrally determined amount of cytochrome P450. In contrast, the SMAP-producing activity from zonisamide correlated closely with the activity of testosterone 6 beta-hydroxylase (r2 = 0.96) and correlated slightly but significantly with the activity of imipramine 2-hydroxylase (r2 = 0.28), but not with those of aniline hydroxylase (r2 = 0.09) or benzphetamine N-demethylase (r2 = 0.20). In addition, immunoquantitation of cytochrome P450 enzymes in 21 human liver microsomal samples revealed that SMAP formation correlated closely with the amount of P450 3A enzyme and correlated moderately well with that of P450 2D6 but not with that of P450 2C enzyme in human liver microsomes. P450 3A4 exhibited SMAP-producing activity in a reconstituted monooxygenase system. The metabolism of zonisamide to SMAP was almost completely inhibited by anti-P450 3A4 antibody but not by anti-P450 2C9 or anti-P450 2D6 antibodies, suggesting that the amount of P450 3A enzyme may be a major factor influencing the level of metabolism of zonisamide to SMAP in human liver microsomes.

Anticonvulsants↗

PubMed1993

[Evaluation of colorectal hepatic metastases with combined modality therapy].

We treated synchronous hepatic metastases in colorectal cancers with combined modality therapy including hepatic resection, selective intra-arterial infusion chemotherapy, and thermocoagulation therapy using a microwave tissue coagulator and compared the value of such therapies. Synchronous hepatic metastases were noted in 563 patients with colorectal cancer who had undergone surgery in our department over the past ten years. Forty-four patients were studied. Six patients underwent hepatic resection only (group A). Another six were subjected to hepatic resection and the residual lesions were treated with selective intra-arterial infusions and thermocoagulation therapy (group B). Fourteen patients were treated with either selective or subselective intra-arterial infusions (group C). The remaining 18 patients were treated in other ways (group D). Cumulative survival, estimated by the Kaplan-Meier method, was compared. One- and three-year cumulative survival rates of patients were 100% and 74% in group A, 100% and 67% in group B, 62% and 0% in group C, and 38% and 5% in group D, respectively. H1 patients accounted for 83% in group A, 33% in group B, 7% in group C, and 22% in group D. The high rate of survival in group A was because of the greater number of H1 patients. One- and three-year survival rated among H2-H3 patients were 100% and 54% in group B, 62% and 0% in group C, and 35% and 14% in group D, respectively. Survival of H2-H3 patients was higher in group B than in group C or D.

Antineoplastic Agents↗

PubMed1993

[Liver metastases from breast cancer: survival and an attempt at cauterization and thermocoagulation therapy combined with selective intra-arterial infusion chemotherapy].

During the past thirteen years, 599 patients with primary breast cancer underwent surgery in our hospital. Nine of them died of liver metastases and the median survival time after detection of liver metastases was five months (range 1-32 months). Four are still alive with liver metastases. Based upon these results, we attempted intraoperative, ultrasound-guided cauterization and thermocoagulation for three patients to improve their prognosis and postoperatively treated them with hepatic arterial infusion chemotherapy. During surgery, a monopolar electrode was inserted with an ultrasonic guide. One round of cauterization lasted for 10 seconds at 100 W, and was repeated until the tissue around the tumor became hyperechoic. The antitumor effect of the therapy was assessed by histological examination of resected liver tissues. Tumor coagulation and necrosis were observed to be consistent with the cauterized area. On CT scanning, treated areas were shown to be of low density and homogeneous. Since this therapeutic approach acts directly on unresectable tumors and is safe, it seems to provide a useful means for intraoperative regional treatment of tumors.

Breast Neoplasms↗

PubMed1993

[Evaluation of subselective intraarterial infusion chemotherapy in far advanced gastric and colorectal cancer].

We investigated the usefulness of subselective intraarterial infusion chemotherapy in far advanced gastric and colorectal cancer. Among gastric and colorectal cancer cases undergoing surgery at our hospital for the past ten years, patients with and without palliative resection were studied. Forty gastric cancer patients and 29 colorectal cancer patients were treated by subselective intraarterial infusion chemotherapy. In gastric cancer cases without resection, a significantly longer survival was noted in patients undergoing this therapy with over 5 g of 5-FU than those treated with conventional chemotherapy. In gastric cancer patients with palliative resection and colorectal cancer patients, survival time was longer by intraarterial infusion with over 5 g of 5-FU, but not significant. Partial response was obtained in 5 cases of gastric cancer and 3 cases of colorectal cancer, and clinical symptoms were improved in 44% of the patients. The main complications of this therapy were myelosuppression and occlusion of the catheter, but none of them were serious. For the improved quality of life of cancer patients, subselective intraarterial infusion chemotherapy is considered a safe and useful form of treatment.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1992

Molecular cloning of monkey liver cytochrome P-450 cDNAs: similarity of the primary sequences to human cytochromes P-450.

Three cDNAs coding for monkey cytochrome P-450 (P450) 2C, 2E and 3A (MKmp13, MKj1 and MKnf2, respectively) were isolated from a lambda gt11 cDNA library of a liver from a 3-methylcholanthrene (3MC)-treated crab-eating monkey, using cDNA fragments for human P450 2C, 2E and 3A as respective probes. MKmp13 and MKnf2 were 1901 and 2032 bp long, containing entire coding regions for polypeptides of 490 and 503 residues, respectively. The deduced N-terminal amino acid sequences of MKmp13 and MKnf2 were identical with those of P450-MK1 and P450-MK2, which had been purified from liver microsomes of untreated and polychlorinated biphenyl (PCB)-treated crab-eating monkeys, respectively. MKj1 was 1508 bp long, encoding a polypeptide of 449 residues, which is presumed to lack N-terminal 45 residues as compared with the sequence for human P450 2E1. Northern blot analysis indicated that monkey P450 2C, 2E and 3A mRNAs were expressed constitutively in monkey livers. P450 2E and 3A mRNAs were induced by both 3MC and PCB, while P450 2C mRNA was induced only by PCB. The deduced amino acid sequences of four monkey cytochrome P-450 cDNAs, including P450 1A1 (MKah1) which we isolated previously, were more than 92% identical with those of corresponding human cytochrome P-450 cDNAs.

Amino Acid Sequence↗

PubMed1992

Immunochemical characterization and toxicological significance of P-450HFLb purified from human fetal livers.

Immunochemical properties of P-450HFLb purified from human fetal livers were investigated. P-450HFLb cross-reacted with antibodies to rat P-4501A1 but not with antibodies to CYP2A6, CYP2C9, CYP3A7 (P-450HFLa) and rat CYP2B1. In addition, P-450HFLb also cross-reacted with both monospecific antibodies to rat CYP1A1 and CYP1A2. However, P-450HFLb was shown to be an immunochemically distinct form of cytochrome P-450 from P-450PA (human CYP1A2). Immunoblot analysis of human fetal livers with the antibodies to P-450HFLb showed that P-450HFLb was expressed in all fetal livers studied although there appeared to be individual differences in the amounts of P-450HFLb expressed in fetal livers. The formation of mutagens from IQ (but not from AFB1) in fetal liver homogenates was inhibited by the antibodies to P-450HFLb in a dose dependent manner. These results suggest that P-450HFLb may be a form of human cytochrome P-450 classified into CYP1 gene family, and that the cytochrome P-450 is, in part, responsible for the mutagenic activation of IQ in human fetal livers as well as CYP3A7 (P-450HFLa).

Aflatoxin B1↗

PubMed1992

Evaluation of adriamycin-induced lipid peroxidation.

Lipid peroxidation is known to be a mechanism for Adriamycin-induced toxicity. In the present study, two methods which detect fluorescent substances and high molecular weight protein aggregates in peroxidized membranes were applied to Adriamycin-induced lipid peroxidation in liver microsomes. A rat liver microsomal suspension containing an NADPH-generating system was incubated with Adriamycin. Thiobarbituric acid reactive substances (TBA-RS), formed during this incubation, were transferred from the microsomes to the medium. Fluorescent substances determined by the fluorescence emitted from both the microsomes themselves and the chloroform/methanol extracts of the microsomes, were found to be formed during this incubation. High molecular weight protein aggregates determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were also formed. Fluorescent substances and high molecular weight protein aggregates were found in microsomal membranes themselves and increased time dependently. These substances retained in membranes can be of great use to delineate the site of Adriamycin-induced lipid peroxidation in vitro and in vivo and to determine how this lipid peroxidation affects the membrane.

Animals↗

PubMed1992

Molecular cloning of monkey P450 1A1 cDNA and expression in yeast.

Monkey P450 1A1 cDNA (MKah1) was isolated from the lambda gt11 cDNA library of a liver from a 3-methylcholanthrene (3MC)-treated crab-eating monkey using a dog P450 1A1 cDNA fragment as a probe. MKah1 was 2453 bp long and contained an entire coding region for a polypeptide of 512 residues. The nucleotide and deduced amino acid sequences of MKah1 displayed 95% and 94% identity with those of the human P450 1A1 gene, respectively. Even in the 3' noncoding region, MKah1 showed 94% homology with human P450 1A1, whereas it showed less than 69% homology with other mammalian P450 1A1. Monkey P450 1A1 mRNA was not detectable in untreated livers, but was induced by polychlorinated biphenyl and 3MC. The expression plasmid (designated as pMKC-1) was constructed by introduction of the coding region of MKah1 into a yeast expression vector (pAM82) containing the promoter of acid phosphatase (APase). Northern blot analysis revealed that monkey P450 1A1 mRNA was expressed in yeast under the control of the APase promoter. Microsomes from yeast transformed by pMKC-1 catalyzed 7-ethoxycoumarin O-deethylation, benzo(a)pyrene hydroxylation and the mutagenic activation of 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), 3-amino-1-methyl-5H-pyrido(4,3-b)-indole acetate (Trp-P-2) and 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole acetate (Glu-P-1).

Amino Acid Sequence↗

PubMed1992

A new form of cytochrome P-450 responsible for mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline in human livers.

Antibodies to P-450IA2 strongly inhibited the mutagenic activation of 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate but not aflatoxin B1 in human liver microsomes. The anti-rat P-450IA2 antibodies were capable of recognizing two proteins which show different mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of human liver microsomes. A new form of cytochrome P-450 (designated P-450-HM4) cross-reactive with anti-rat P-450IA2 antibodies showing that the smaller molecular weight was purified from human liver microsomes by means of the fast-performance liquid chromatography system. The molecular weight of P-450-HM4 was estimated to be 49,000, which was apparently different from that of P-450PA (human P-450IA2). The antibodies to P-450-HM4 did not cross-react with P-450PA (human P-450IA2) but inhibited to various extents the mutagenic activation of IQ in microsomes from human livers. In addition, P-450-HM4 showed significant mutagen-producing activity from IQ in a reconstituted system. Together with these and other results reported previously, it is concluded that at least two forms of cytochrome P-450 [P-450-HM4 and P-450PA (human P-450IA2)] are involved in the mutagenic activation of IQ in human liver.

Antibodies↗

PubMed1992

Stable expression of cytochrome P450IIIA7 cDNA in human breast cancer cell line MCF-7 and its application to cytotoxicity testing.

A mammalian cell expression plasmid containing cytochrome P450IIIA7 complementary DNA was constructed. Breast cancer cells (MCF-7) were transfected with the plasmid and neomycin-resistant selection marker plasmid. We established three cell lines, termed M13, M21, and M27, which expressed the cytochrome P450IIIA7 as examined by RNA blot and immunoblot analyses. These cell lines showed 8- to 10-fold higher sensitivity against aflatoxin B1 compared to parental MCF-7 cells, suggesting that cytochromes P450IIIA7 expressed in the cells were responsible for the production of the cytotoxic metabolite of aflatoxin B1.

Adult↗