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Investigator-initiated trials (IIT) are important aspects of medical research and have contributed substantially to modern oncology. IIT using post-approval drugs have been conducted by domestic institutions in Japan. Data from the present study were obtained by all IIT registered clinical trials for five cancers (lung, colorectal cancer, gastric cancer, liver cancer, and breast cancer) using drugs approved from 1999 to 2009 in Japan. Kaplan-Meier method, analysis of variance (anova), and Kruskal-Wallis test were used to estimate time to enrolment completion (TTEC) and time to enrolment per patient (TTEP). Of 1222 trials eligible for analysis, 465 trials (38%) completed enrolment to the studies, and 203 trials (17%) published results. In the distribution according to trial phase, 98 (8%) were phase I, 1058 (87%) were phase I/II + II, and 66 (5%) were phase II/III + III. Accrual achievement and publication rates were higher in late-phase than in early-phase trials. Median TTEC was 1387 days (95% confidence interval [CI], 1302-1472). Median TTEP was 38.5 days (95% CI, 34.5-42.5). The median TTEC and TTEP were significantly different in each trial phase (P < 0.01), funding source (P < 0.01), and publication status (median TTEC published trials versus unpublished trial; 720 days vs 1672 days, median TTEP; 16 days vs 55.8 days; P < 0.001). Many IIT using approved cancer drugs have been conducted; however, the quality of the clinical trials was low in terms of accrual achievement, publication rate, and time to publication of trial results., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Excitation spectra of ^{11}C are measured in the ^{12}C(p,d) reaction near the η^{'} emission threshold. A proton beam extracted from the synchrotron SIS-18 at GSI with an incident energy of 2.5 GeV impinges on a carbon target. The momenta of deuterons emitted at 0° are precisely measured with the fragment separator (FRS) operated as a spectrometer. In contrast to theoretical predictions on the possible existence of deeply bound η^{'}-mesic states in carbon nuclei, no distinct structures are observed associated with the formation of bound states. The spectra are analyzed to set stringent constraints on the formation cross section and on the hitherto barely known η^{'}-nucleus interaction.

Crizotinib is a standard treatment for advanced ALK-positive non-small-cell lung cancer (NSCLC). We undertook this study to investigate the pharmacokinetics of crizotinib and clinical and pharmacogenomic factors that may increase the risk of adverse events (AEs). We defined clinically significant AEs as grade 4 hematological toxicity, grade ≥3 non-hematological toxicity, and any grade of interstitial lung disease. Eight subjects with ALK-positive NSCLC scheduled to receive crizotinib 250 mg twice daily were studied. Six patients were female and two were male, and most of the patients had low body weight with a median body weight of 46.8 kg (range, 42.4-61.0 kg). All patients developed AEs, five developing six clinically significant AEs. Six patients required dose reduction. In pharmacokinetic analysis, blood samples were obtained on days 1 and 15. The mean area under the plasma concentration-time curve from 0-12 h (AUC0-12 ) on day 15 was significantly increased in patients with clinically significant AEs (n = 5) compared with those without (n = 3) (P = 0.04). Genetic polymorphisms of ABCB1 were analyzed. One patient with the ABCB1 1236TT-2677TT-3435TT genotype was an outlier, with an AUC0-12 and peak concentrations on day 15 of 2.84× and 2.61× the mean, respectively, compared with those with other genotypes. Our results suggest that some Japanese NSCLC patients treated with crizotinib developed clinically significant toxicities that were related to altered pharmacokinetics parameters due to genotype and body weight factors., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Influenza vaccination guidelines have recommended that pediatricians should consult with allergists in the case of for children with histories of anaphylaxis to hen's eggs. On the other hand, whether such children can be safely vaccinated is unclear., Objective: To evaluate the safety of influenza vaccination for children severely allergic to eggs., Methods: The study population consisted severe egg-allergic children that had never been immunized with influenza vaccine. The inclusion criteria of severe egg-allergic children included evidence of serum specific IgE antibodies to egg white or an ovomucoid level of class4 or more and complete elimination of dietary intake of eggs, or occurrence of Sampson Grade 3 to 5 anaphylactic reactions upon egg ingestion. Patients underwent skin prick tests, and received 0.1ml of influenza vaccine, followed in 30 minutes if no reaction with the remainder of an age-appropriate dose. We observed the subjects for 30 minutes afterwards, and they were observed by their patients during the subsequent 24 hours., Results: A total of 17 patients were enrolled. All patients received influenza vaccination without an allergic reaction., Conclusions: Influenza vaccination is safe even in children with histories of severe egg allergy. influenza vaccination without an allergic reaction.

Background: Cheek support and subject's arm position can influence on lung function values measured with forced oscillation technique. We examined resistance and reactance by different methods using a new forced oscillation technique machine called MostGraph (Chest Co. Ltd., Tokyo, Japan)., Methods: We evaluated nineteen adults. Four methods are measured. 1: the subjects support their cheeks with their hands. 2: the tester support the subject's cheeks with their hands. 3: the subjects stiffen their cheeks without any cheek support. 4: subject relax their cheeks without any cheek support. R5, R20, R5-R20, X5, Fres and ALX were compared between four methods., Results: In resistance values, R20 was the lowest in the fourth method, and it was significantly different from the first (p<0.01) and the second (p<0.01) method. R5-R20 was the lowest in the second method, and it was significantly different from the third (p<0.01) and forth (p<0.01) methods. In reactance values, X5 was the highest in the second method. All ractance values in the second method were significantly different from the first and the fourth method., Conclusion: Measurement values using Mostgraph was influenced by methods of supporting cheeks and subject's arm position. Measurement method should be unify and the first method is ideal and practical.

Background: Lung function measurements are useful objective indices to monitor asthma control in children. Recently, a new forced oscillation technique machine called MostGraph (Chest Co. Ltd., Tokyo, Japan) has been developed to diagnose and monitor asthma. We assessed the utility of MostGraph in asthmatic children., Methods: We evaluated 66 well controlled asthmatic children (11±3 yrs). For each subject, we measured respiratory function using MostGraph and spirometry and assessed the relationship between the two indices., Results: MostGraph measurements were significantly correlated with age and body height. R5 was remarkably associated with FEV1% by analyzing partial correlation that excluded the factor of height. In addition, R5-R20 and Fres were significantly related to FEV1% and MMF. Furthermore, R5-R20 and Fres were significantly associated with MMF through multiple regression analyses for MostGraph parameters, the height and spirometory parameters. We classified asthmatic children into two groups with 70% of V25 cut point. Group with V25 below 70% had higher scores for R5-R20, Fres and ALX, compared to group with V25 above 70%., Conclusion: MostGraph could be effectively used for the R5-R20 and Fres lung function measurements to assess asthma control in children. The results also suggested that R5-R20 and Fres might be parameters of small airway dysfunction.

The Θ(+) pentaquark baryon was searched for via the π(-)p→K(-)X reaction with a missing mass resolution of 1.4 MeV/c(2) (FWHM) at the Japan Proton Accelerator Research Complex (J-PARC). π(-) meson beams were incident on the liquid hydrogen target with a beam momentum of 1.92 GeV/c. No peak structure corresponding to the Θ(+) mass was observed. The upper limit of the production cross section averaged over the scattering angle of 2° to 15° in the laboratory frame is obtained to be 0.26 μb/sr in the mass region of 1.51-1.55 GeV/c(2). The upper limit of the Θ(+) decay width is obtained to be 0.72 and 3.1 MeV for J(Θ)(P)=1/2(+) and J(Θ)(P)=1/2(-), respectively, using the effective Lagrangian approach.

We report on the first spectroscopic study of the N=22 nucleus 32Ne at the newly completed RIKEN Radioactive Ion Beam Factory. A single gamma-ray line with an energy of 722(9) keV was observed in both inelastic scattering of a 226 MeV/u 32Ne beam on a carbon target and proton removal from 33Na at 245 MeV/u. This transition is assigned to the deexcitation of the first Jpi=2+ state in 32Ne to the 0+ ground state. Interpreted through comparison with state-of-the-art shell-model calculations, the low excitation energy demonstrates that the "island of inversion" extends to at least N=22 for the Ne isotopes.

An animal model of otitis media using chinchillas was developed to evaluate the efficacy of tebipenem pivoxil (TBM-PI) against experimental otitis media. Chinchillas inoculated via the transbullar approach with Streptococcus pneumoniae serogroup 6 were included in the efficacy study with TBM-PI, amoxicillin (AMX) or untreated as controls. TBM-PI resulted in survival rate of 83%, compared with 25% survival for AMX and 0% survival for controls (p<0.01). Quantitative cultures in the middle ear effusions at day 5 of the TBM-PI group yielded 3.5+/-2.4log(10)CFUs/ml. TBM-PI is a promising antibiotic for the treatment of acute otitis media.

Deeply bound 1s states of pi(-) in (115,119,123)Sn were preferentially observed using the Sn(d,3He) pion-transfer reaction under the recoil-free condition. The 1s binding energies and widths were precisely determined and were used to deduce the isovector parameter of the s-wave pion-nucleus potential to be b1=-(0.115+/-0.007)m(-1)(pi). The observed enhancement of |b(1)| over the free piN value (b(free)1/b1=0.78+/-0.05) indicates a reduction of the chiral order parameter, f*pi(rho)2/f2pi approximately 0.64, at the normal nuclear density, rho=rho(0).

Susceptibility testing of 288 clinical isolates of P. aeruginosa in 2000 was performed by the disk diffusion method with observation at regular time course. Detailed analysis of the shape of the zones of inhibition gave interesting results, i.e. double zone of inhibition was found in MEPM specifically, unlike BIPM and IPM. The incidence was 50%. Moreover same phenomenon was detected in CAZ. After analyzing this phenomenon from the results of short-time-killing curve and inducibility of AmpC beta-lactamase it seems that there is a close relationship between formation of double zone of inhibition and bactericidal activity in sub-MIC drug concentration.

[reaction: see text] Synthesis of aromatic ring substituted (E)-1,1'-biindenylidene derivatives was achieved by a domino-Heck type double cyclization of (Z,Z)-1,6-diaryl-1,5-hexadien-3-ynes.

We observed well-separated 1s and 2p pi(-) states in 205Pb in the 206Pb(d,3He) reaction at T(d) = 604.3 MeV. The binding energies and the widths determined are B(1s) = 6.762+/-0.061 MeV, Gamma(1s) = 0.764(+0.154)(-0.171) MeV, B(2p) = 5.110+/-0.045 MeV, and Gamma(2p) = 0.321(-0.062)(+0.060) MeV. They are used to deduce the real and imaginary strengths of the s-wave part of the pion-nucleus interaction, which translates into a positive mass shift of pi(-) in 205Pb.

Sjögren's syndrome (SS) is an autoimmune disease, and some patients have been found to have SS complicated with rheumatoid arthritis (RA), in which IgG is known to carry abnormal N-linked oligosaccharides. In order to investigate the relationship between SS and RA, the structures of N-linked oligosaccharides of IgG from 12 primary SS patients without RA, 9 RA patients, and 8 healthy individuals were analyzed using reversed-phase high-performance liquid chromatography, in combination with sequential exoglycosidase treatment and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. All of the IgG samples obtained from primary SS patients, RA patients, and healthy individuals contained the same series of biantennary complex-type oligosaccharides, but the ratio of each oligosaccharide differed among these 3 groups. The incidence of galactose-lacking N-linked oligosaccharides obtained from the IgG of RA patients was significantly higher than that from healthy individuals, but that from the serum IgG of primary SS patients varied among individuals. The patients with primary SS were classified into two groups based on the galactosylation levels of IgG oligosaccharides; one group exhibits galactosylation levels as low as those of RA patients and another exhibits levels similar to those of healthy individuals. Measurement of levels of rheumatoid factor (RF) revealed that primary SS patients with a high incidence of RF belonged to the low galactosylation group, as did RA patients. These results suggest that appearance of IgG carrying abnormal N-linked oligosaccharides in primary SS may be related to future complication with RA.

Background: Doxapram is contraindicated for newborn infants in Japan because of its serious side effects. However, because of encouraging results of recent studies regarding the efficacy and safety of therapy for apnea of prematurity (AOP) with lower doses of doxapram than those previously proposed, approximately 60% of Japanese neonatologists continue to use doxapram at small doses. Caution is warranted because the sample sizes of the former studies are inadequate to evaluate doxapram for both its beneficial and harmful effects. Therefore, we conducted the present study in order to investigate the efficacy and harmful events of low-dose doxapram therapy for idiopathic AOP in very low-birth weight (VLBW) infants in a larger population., Methods: One hundred and six VLBW infants with idiopathic AOP were treated with doxapram at a dose of 0.2-1.0 mg/kg per h in combination with methylxanthines and the frequency of apnea and secondary outcomes were compared with a group of control infants., Results: An approximate 80% reduction in the frequency of apnea was found with only minimal side effects following low-dose doxapram. Although there were no significant differences in secondary outcomes between the doxapram-treated and control groups, mortality in doxapram-treated infants was significantly lower than that in control infants., Conclusions: Patients with AOP unresponsive to treatment with methylxanthines may benefit from the addition of low-dose doxapram.

LJC 11,036 is the active metabolite of L-084, a novel oral carbapenem that exhibits potent broad-spectrum activity. Antibacterial activities of LJC 11,036 against clinical isolates from respiratory infections, such as Streptococcus pneumoniae (n = 52), Streptococcus pyogenes (n = 19), Haemophilus influenzae (n = 50), Klebsiella pneumoniae (n = 53), and Moraxella catarrhalis (n = 53), and from urinary-tract infections, such as Escherichia coli (n = 53) (MICs at which 90% of the isolates were inhibited [MIC(90)s], 0.1,

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

An extremely rare abnormality, a variant of omphalomesenteric duct, was observed in the umbilical cord 1 cm from the entry point into the abdominal wall, which itself was as an isolated mass of well-formed intestinal tissue free of structural defects. This intestinal tissue seems to be the remnant of an intestinal loop from caudal limb of midgut following physiological herniation during the third month of gestation.

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.

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.

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.