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ABSTRACT: Human fetal CYP3A7 and human NADPH-cytochrome P450 reductase were coexpressed in insect cells, TN-5, infected with a recombinant baculovirus carrying both cDNAs. The expression of reductase in TN-5 cells was shown to be sufficient for the CYP3A7 dependent 16 alpha-hydroxylation of dehydroepiandrosterone. However, the extra addition of cytochrome b5 and phospholipid was necessary to obtain a maximal activity of CYP3A7 catalyzing the reaction. CYP3A7 expressed in TN-5 cells was capable of metabolizing testosterone, cortisol and dehydroepiandrosterone 3-sulfate as well as dehydroepiandrosterone. The apparent Vmax for 6 beta-hydroxylations of testosterone was similar to that obtained for 6 beta-hydroxylation of cortisol (2.9 versus 2.5 nmol/nmolP450/min). In contrast, the apparent Vmax for 16 alpha-hydroxylation of dehydroepiandrosterone and its 3-sulfate were 20 and 2 times greater than those observed for steroid 6 beta-hydroxylations, respectively (67.5 and 5.8 versus 2.5-2.9 nmol/nmol P450/min). On the other hand, the apparent K(m) for 6 beta-hydroxylations of testosterone and cortisol were greater than those for 16 alpha-hydroxylations (120 and 860 versus 46-58 microM). Thus, CYP3A7 was active for steroid 6 beta-hydroxylations and 16 alpha-hydroxylations, but there were greater differences in Vmax/K(m) ratios between these reactions.
Research communications in molecular pathology and pharmacology 05/1998; 100(1):15-28.
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ABSTRACT: The GST Fpi and GST Ppi, pi class glutathione S-transferase (GST), were purified from human fetal livers and placentas, respectively. Both GST enzymes were indistinguishable each other in their subunit molecular weights, immunochemical properties and substrate specificities. Three clones (pFGP-1, pFGP-2 and pFGP-3) coding for the pi class GST purified from fetal livers were isolated from a human fetal liver cDNA library. The full-length clone encodes a polypeptide comprising 210 amino acid including the initiator methionine. All of these cDNA clones were nearly identical to a human placental cDNA clone, pGpi 2. The pFGP-1 cDNA had only a single base transition accompanied by an amino acid transition in the coding region, at position 313. The pFGP-2 and pFGP-3 cDNAs were also nearly identical to pGpi 2 cDNA, having only a single silent C-->T transition in the coding region, at position 555.
Research communications in molecular pathology and pharmacology 07/1997; 97(1):67-78.
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ABSTRACT: P450 HFLb purified from human fetal livers has been shown to be constitutively expressed in fetal livers. In the present study, the occurrence of proteins immunochemically related to P450 HFLb in extrahepatic tissues of human fetuses and their contribution to mutagenic activation of promutagens were investigated. The mutagenic activation of aflatoxin B1 (AFB1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and benzo[a]pyrene were observed in human fetal extrahepatic tissues, including adrenal glands, kidneys and lungs, at varying rates. Immunoblot analysis of homogenates of extrahepatic tissues with antibodies to P450 HFLb revealed the occurrence of proteins immunochemically related to P450 HFLb in adrenal glands, kidneys and lungs. Immuno-inhibition studies suggested that in fetal adrenal gland and kidney, the proteins cross-reactive with antibodies to P450 HFLb were capable of activating IQ and MeIQ to mutagens.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 11/1994; 310(1):73-7. · 2.85 Impact Factor
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ABSTRACT: Immunoblot analysis showed that alpha-class glutathione S-transferase (GST), which is one of the major forms in adult human liver, was expressed in human fetal liver. Mu-class GST was also expressed in fetal liver. The majority of mu-class GST expressed in adult liver consisted of a subunit with a molecular weight of 27 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two subunits of 27 and 26 kDa were detected in fetal liver as proteins immunochemically related to mu-class GST. On reverse-phase HPLC, these two subunits cross-reactive with antibodies to rat GST 3-3 in fetal liver were indistinguishable from each other in their retention time; though, they could be separated by chromatofocusing analysis. The molecular weights of GSTs immunochemically related to rat GST 3-3, eluted at pH 7.1, 6.4, and 5.7, were 27, 27 and 26, and 26 kDa, respectively. In addition, the N-terminal amino acid sequence of these subunits suggested that GSTs related to rat GST 3-3 expressed in fetal liver may be homodimeric and heterodimeric proteins. As expected, pi-class GST was found to be a major form of GST in fetal liver but not in adult liver. In contrast, the GST immunochemically related to rat GST Yrs-Yrs, which is classified as theta-class GST, was detected in adult liver but not in fetal liver. These results indicate that several isoenzymes of GST are expressed in human fetal liver, but they are not the same as those in adult liver.
Journal of Biochemistry 09/1994; 116(2):315-20. · 2.37 Impact Factor
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ABSTRACT: 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.
Asia-Oceania journal of obstetrics and gynaecology / AOFOG 10/1993; 19(3):329-41.
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ABSTRACT: 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.
The Journal of Toxicological Sciences 06/1993; 18(2):129-32. · 1.52 Impact Factor
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ABSTRACT: 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).
Biochimica et Biophysica Acta 11/1992; 1117(3):301-5. · 4.66 Impact Factor
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ABSTRACT: An acidic form of glutathione S-transferase (GST) was purified from human fetal livers by means of affinity chromatography and chromatofocusing. The major peak of the acidic form of GST was focused between pH 4.8 and 4.9. Judging by SDS-PAGE, the purified acidic GST was apparently homogeneous; the subunit molecular weight was estimated to be 23,000. The acidic GST catalyzed the conjugations of glutathione (GSH) with 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA). The immunochemical properties of the purified acidic GST were indistinguishable from those of human placental GST-pi. The N-terminal amino acid sequence of the acidic GST was identical with that of GST-pi from human placenta. The level of expression of the acidic form of GST was clearly different between human adult and fetal livers as examined on the levels of mRNA and protein.
Journal of Biochemistry 12/1991; 110(5):743-7. · 2.37 Impact Factor
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ABSTRACT: Four forms of cytochrome P-450 were separated and purified to electrophoretic homogeneity from human fetal livers. These forms of cytochrome P-450, termed P-450HFLa, P-450HFLb, P-450HFLc and P-450HFLd, were distinguishable from each other in their molecular weights, spectral properties, immunochemical properties and mutagen-producing activities from promutagens. The molecular weights of P-450HFLa, b, c and d were estimated to be 51,500, 49,000, 51,500 and 50,000, respectively. Antibodies to P-450HFLa recognized P-450HFLc but not P-450HFLb or d, and antibodies to rat P-448-H (P-450IA2) cross-reacted with P-450HFLb but not with other forms of cytochrome P-450. The N-terminal amino acid sequence of P-450HFLc was highly homologous, but not identical, to that of P-450HFLa. Each form of cytochrome P-450 catalyzed mutagenic activation of aflatoxin B1 (AFB1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole (Glu-P-1) at different rates. P-450 HFLa showed activities to produce mutagen(s) from AFB1, IQ and to a lesser extent from Glu-P-1. P-450 HFLb activated IQ at a faster rate than did the other forms. P-450 HFLc produced a mutagen from AFB1 and Glu-P-1 but not from IQ. P-450 HFLd did not activate these promutagens at significant rates.
Japanese journal of cancer research: Gann 05/1991; 82(4):426-32.
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ABSTRACT: The mutagenic activation of promutagens by human adult and fetal livers was investigated using the umu test system. Among the promutagens studied, aflatoxin B1 (AFB1) and 2-amino-3-methyl-imidazo[4,5-f] quinoline (IQ) were efficiently activated to mutagens by both adult and fetal livers. 7,8-Benzoflavone inhibited the activation of IQ by fetal livers, but the inhibition observed in fetal livers was much less than that observed in adult livers. Antibodies to P450HM1 (P450111A4) and P450HFLa markedly inhibited the activation of AFB1 by adult and fetal livers, respectively. The formation of genotoxic product(s) from IQ in human adult livers was almost completely inhibited by anti-P448H (P4501A2) antibodies but not by anti-P450HM1 antibodies, whereas that in fetal livers was inhibited by both anti-P450HFLa and anti-P450IA2 antibodies. P450HFLa catalyzed the mutagenic activation of both AFB1 and IQ in a reconstituted system. On the contrary, P450HM1 catalyzed the mutagenic activation of AFB1 but not IQ. A preparation of cytochrome P450 partially purified from human fetal livers and cross-reactive with anti-P450IA2 antibodies was found to be active for mutagenic activation of IQ in a reconstituted system. These results indicate that P450HFLa and P450HM1 are mainly involved in the genotoxic product formation from AFB1 in fetal and adult livers, respectively, and that the metabolic activation of IQ in fetal livers is catalyzed by two forms of cytochrome P450, P450HFLa, and cytochrome P450 immunochemically related to P450IA2 but that in adult livers it is mainly catalyzed by cytochrome P450 related to P450IA2.
Cancer Research 06/1990; 50(9):2641-5. · 7.86 Impact Factor
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ABSTRACT: The genotoxic and mutagenic activation of promutagens by human fetal livers was measured by the induction of umu gene expression in Salmonella typhimurium TA1535/pSk1002. Liver homogenates of human fetuses were the most active for the mutagenic activation of aflatoxin B1 (AFB1), followed by 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), and to a lesser extent by 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole (Glu-P-1). The amounts of P-450 HFLa immunochemically determined in human fetal livers correlated highly with the induction of umu gene expression by AFB1 (r = 0.98, n = 5). P-450 HFLa catalyzed the mutagenic activation of AFB1 in a reconstituted system: cytochrome b5 markedly stimulated the activation. Anti-P-450 HFLa antibodies inhibited the mutagenic activation of AFB1 in a dose-dependent manner. These results strongly support the idea that P-450 HFLa is responsible for the mutagenic activation of AFB1 in human fetal livers.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 10/1989; 227(1):53-8. · 2.85 Impact Factor
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ABSTRACT: From a human fetal liver cDNA library, a new cDNA clone (lambda HFL10) was isolated using an antiserum to P450 HFLa, which has been isolated from livers of human fetuses. Cytochrome P450 cDNAs, namely lambda hPA6, lamda hP2-1, and lambda hPD4 which were highly homologous to cDNA clones, pHY13, Hp1-1, and phP450j, respectively, were also isolated from the cDNA library of human adult livers. Using these cDNA clones as probes together with Lambda HFL10, Northern blot analysis was conducted to determine whether all of these cytochromes were expressed in human fetal livers. The results clearly showed that only P450 HFL10 mRNA was detected in human fetal livers. This result supports the allegation that there is a much more limited number of forms of cytochrome P450 in human fetal livers than in adult livers.
Archives of Biochemistry and Biophysics 08/1989; 272(1):219-25. · 2.93 Impact Factor
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ABSTRACT: Immunohistochemical localization of P-450 HFLa, a form of cytochrome P-450 in human fetal livers was investigated in human hepatocellular carcinoma. The cytoplasm of carcinoma cells positively reacted with anti-P-450 HFLa antibodies. It was found that the carcinoma cells showing a pseudoglandular pattern or poorly differentiated appearance exhibited a weaker reactivity with anti-P-450 HFLa antibodies than did relatively differentiated carcinoma cells. In the case of hepatoblastoma, the polygonal or round-shaped tumor cells which differentiated into epithelial structure exhibited a positive reaction with anti-P-450 HFLa antibodies, whereas the spindle-shaped tumor cells which showed a sarcomatous appearance did not react with the antibodies.
Journal of pharmacobio-dynamics 07/1989; 12(6):341-4.
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ABSTRACT: Proteins immunochemically reactive with anti-P-450 HFLa IgG were detectable in liver microsomes from all of the animals examined, although considerable variations of the amounts were observed among the animal species. The smallest amount was found in liver microsomes from female rats and the largest amount in monkey liver microsomes. Sex difference in the amounts was observed in rat liver microsomes but not in dog liver microsomes. No strain difference was observed among ddY, ICR and BALB/C mice. The activities of testosterone 6 beta-hydroxylases in liver microsomes from rats, dogs and monkeys were highly sensitive to inhibition by anti-P-450 HFLa IgG, but those in microsomes from guinea pigs and rabbits were less sensitive to inhibition by the antibodies.
Research communications in chemical pathology and pharmacology 11/1988; 62(1):31-40.
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ABSTRACT: A protein immunochemically related to P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, was detected in rat liver microsomes. The content of the immunoreactive protein in rat liver microsomes was increased by treatments with phenobarbital, pregnenolone 16 alpha-carbonitrile (PCN), erythromycin, erythromycin estolate, and oleandomycin but not with 3-methylcholanthrene, imidazole, ethanol, isosafrole, josamycin, midecamycin, or miocamycin. The activity of erythromycin N-demethylase correlated with the content of the immunoreactive protein in rat liver microsomes (r = 0.72). In addition, anti-P-450 HFLa IgG inhibited erythromycin N-demethylase in liver microsomes from erythromycin- or oleandomycin-pretreated rats. Furthermore, the content of the immunoreactive protein highly correlated with that of P-450 PB-1, which is distinct from Waxman's terminology, and is one of the forms of PCN-inducible cytochrome P-450s (r = 0.95). From these results and the results reported so far, it seems possible that P-450 HFLa is one of the forms of cytochrome P-450 inducible by glucocorticoids.
Archives of Biochemistry and Biophysics 08/1988; 264(1):61-6. · 2.93 Impact Factor
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ABSTRACT: Using conventional radioimmunoassay kits, we measured concentrations of two cancer-related antigens, tissue polypeptide antigen (TPA) and cancer antigen 125 (CA125) throughout gestation and at delivery. The maternal serum was collected from 147 pregnant women between 5 and 43 weeks gestation and 27 women were studied at delivery at which time samples of maternal blood, umbilical artery and vein blood as well as amniotic fluid were collected. The various concentrations of TPA and CA125 were compared with placental weight and infant birth weight. The results are summarized as follows: (1) Mean TPA levels in maternal serum increased with advancing gestation and rose above 110 U/l (upper non-pregnant limit) from 35 weeks onwards. Mean CA125 levels rose above 35 U/ml (normal non-pregnant upper limit) before 9 weeks gestation and thereafter fell. Both levels were markedly raised immediately after delivery. (2) In umbilical artery and vein serum, mean TPA levels were slightly raised. However, there were no significant differences between TPA levels in maternal serum and matched serum from the umbilical artery and vein. Mean umbilical CA125 levels were below 35 U/ml, while mean CA125 levels were significantly higher in the corresponding maternal serum. (3) The concentrations of TPA and CA125 were extremely high in amniotic fluid. The mean values reached 3604 U/l and 2187 U/ml, respectively. (4) None of the concentrations of TPA and CA125 in those pregnancy-related body fluids correlated significantly with birth weight, placental weight or fetal sex. These findings suggest that the production of these two cancer-related antigens is not by the fetus but the placenta.
Archives of Gynecology and Obstetrics 02/1988; 243(4):191-7. · 1.28 Impact Factor
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ABSTRACT: Tissue polypeptide antigen (TPA) and cancer antigen 125 (CA125) were studied immunohistochemically by the avidin-biotin immunoperoxidase technique in human and cynomolgus monkey placentae, membranes, umbilical cords and decidua. In early human placentae, TPA was localized mainly in the cell membranes of villous syncytio- and cyto-trophoblast. The cytoplasm of those trophoblastic cells were weakly stained with TPA. The membrane of basal chorionic trophoblast cells was strongly stained with TPA and the cytoplasm stained weakly. In early cynomolgus placentae, similar immunostaining results were obtained. However, the positive stainings for TPA was more marked in the cytoplasm of villous syncytiotrophoblast and basal chorionic trophoblast, and less marked in the cell membrane of villous cytotrophoblast. In early human and cynomolgus placentae, CA125 was not demonstrated immunohistochemically in the villi and basal chorion. In human and cynomolgus term placentae, the villous syncytiotrophoblast and basal and reflected chorionic trophoblast showed similar immunostaining as the early placentae. In addition, TPA was found in the amniotic epithelium in both sorts of placentae. TPA was not detected immunohistochemically in the umbilical cord and decidual cells. While weakly positive stains for CA125 were observed in decidual cells, CA125 was localized mainly in the membrane and cytoplasm of amniotic epithelium in both human and cynomolgus term placentae. TPA and CA125 are thus oncoplacental antigens and the monkey could serve as a model for their investigation.
Archives of Gynecology and Obstetrics 02/1988; 243(3):145-55. · 1.28 Impact Factor
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ABSTRACT: In a reconstituted system containing NADPH, dilauroyl-L-3-phosphatidylcholine, and NADPH-cytochrome P-450 reductase purified from rat liver microsomes, cytochrome P-450 (P-450 HFLa) purified from human fetal livers catalyzed the 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate (DHEA-sulfate). Addition of cytochrome b5 purified from rat liver microsomes to the reconstituted system resulted in a remarkable increase in the hydroxylase activity. The level of P-450 HFLa in liver homogenates from human fetuses highly correlated with the activity of DHEA-sulfate 16 alpha-hydroxylase. Antibodies to P-450 HFLa inhibited the 16 alpha-hydroxylation of DHEA-sulfate in a dose-dependent manner. The NH2-terminal amino acid sequence of P-450 HFLa was similar to that of P-450NF (Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S., and Guengerich, F. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8064-8068). We conclude that P-450 HFLa is a form of cytochrome P-450 involved in the 16 alpha-hydroxylation of DHEA-sulfate.
Journal of Biological Chemistry 11/1987; 262(28):13534-7. · 4.77 Impact Factor
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ABSTRACT: The purpose of this study was to clarify the pharmacological and physiological significance of P-450 HFLa. Thus, correlations between cytochrome P-450 (P-450 HFLa) level and different monooxygenase activities were investigated in liver homogenates from human fetuses. Poor correlation was seen between P-450 HFLa level and the activity of benzphetamine N-demethylation or aniline hydroxylation. In contrast, the content of P-450 HFLa was highly correlated with the activity of benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation or testosterone 6 beta-hydroxylation. In microsomes from human adult livers, a moderate relationship was also observed between testosterone 6 beta-hydroxylation and P-450 HFLa level. Furthermore, antibodies to P-450 HFLa inhibited testosterone 6 beta-hydroxylase activity in fetal and adult livers to similar extents. We conclude that P-450 HFLa is a form of cytochrome P-450 which catalyzes testosterone 6 beta-hydroxylation and limited drug oxidations in human fetal and adult livers.
Biochemical Pharmacology 03/1987; 36(4):453-6. · 4.70 Impact Factor
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ABSTRACT: P-450 HFLa is a form of cytochrome P-450 purified from human fetal livers. The amounts of P-450 HFLa in several fetal tissues were determined immunochemically. Detectable amounts presented in livers, kidneys, adrenals, lungs and some other tissues of human fetuses. The amounts were the highest in livers. Activities of 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase in livers but not in adrenals were inhibited by the anti-P-450 HFLa antibodies, probably suggesting that distinct forms of cytochrome P-450 are responsible for the oxidations in livers and adrenals.
Biochemical and Biophysical Research Communications 10/1985; 131(3):1154-9. · 2.48 Impact Factor
