Koshiro Itahashi

Purification and properties of cytochrome P-450 from homogenates of human fetal livers

A form of cytochrome P-450, namely P-450HFLa of human fetal livers, was purified to a specific content of 12.6 nmol/mg protein. The cytochrome P-450 preparation was electrophoretically homogeneous and had an apparent monomeric molecular weight of 51,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cytochrome showed catalytic activities as oxidations of N-methylaniline, ethylmorphine, N,N-dimethylaniline, N,N-dimethylnitrosamine, benzphetamine, aminopyrine, aniline, p-nitroanisole, and 7-ethoxycoumarin to various extents. In fetal liver homogenate, the amount of cytochrome P-450 that reacted with the antiserum to P-450HFLa accounted for more than 36% of the total cytochrome P-450 in three different fetal livers. On the other hand, the amount of P-450HFLa was less than 5% of the total cytochrome P-450 in adult liver microsomes.

Significance of cytochrome P-450 (P-450 HFLa) of human fetal livers in the steroid and drug oxidations.

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.

Isolation of a new human fetal liver cytochrome P450 cDNA clone: evidence for expression of a limited number of forms of cytochrome P450 in human fetal livers.

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.

Four Forms of Cytochrome P‐450 in Human Fetal Liver: Purification and Their Capacity to Activate Promutagens

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 promntagens. 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 Bl (AFB1), 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ) and 2‐amino‐6‐methyldipyrido‐[l,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.

Immunochemical examinations of cytochrome P-450 in various tissues of human fetuses using antibodies to human fetal cytochrome P-450, P-450 HFLa

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.

Immunochemical similarity of P-450 HFLa, a form of cytochrome P-450 in human fetal livers, to a form of rat liver cytochrome P-450 inducible by macrolide antibiotics

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 Waxmans 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.

Immunoradiometrical measurement of tissue polypeptide antigen (TPA) and cancer antigen 125 (CA125) in pregnancy and at delivery

SummaryUsing 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.

Immunohistochemical study of tissue polypeptide antigen (TPA) and cancer antigen 125 (CA125) in the human and cynomolgus monkey placenta, umbilical cord and decidua

SummaryTissue 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.

Possible occurrence of P450 related to P450 HFLb in extrahepatic tissues of human fetuses and its contribution to metabolic activation of promutagens

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.