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Objective: Drug-induced extrapyramidal symptoms (DIEPS) often substantially compromise quality of life (QOL) of patients receiving drugs with central antidopaminergic activities. A lack of comprehensive screening method based upon patients' subjective symptoms for detecting DIEPS appears to have prevented pharmacists from delivering satisfactory pharmaceutical care for these patients. Thus, we have attempted to develop a comprehensive questionnaire for screening patients having higher risks of developing DIEPS., Methods: One hundred fourteen outpatients taking gastroprokinetic drugs (itopride, cisapride, trimebutine, domperidone and metoclopramide) at least 2 weeks participated in the study. One patient with familial Parkinson disease served as a positive reference. They undertook a questionnaire consisting of 9 comprehensive questions written in non-technical words that were aimed to detect typical symptoms of Parkinsonism including akathisia and dyskinesia. Each symptom was scored in a semiquantitative scale [i.e., from 1 (not at all) to 5 (very much)] by the patients., Results: Of the 108 subjects who successfully completed the questionnaires, 43 gave scores 2 or greater indicating the presence of DIEPS. However, no statistically significant correlations were observed between the scores of any possible pairs of the questionnaire items. Five subjects had a mean questionnaire score of equal to or greater than 1.6, and the patient with familiar Parkinsonism had the highest mean score of 1.9., Conclusion: The questionnaire presented herein detected 4 patients with suspected DIEPS. Further studies should be warranted to assess whether it would be useful for pharmacists as a screening tool for DIEPS in patients having higher risks of DIEPS.

Objectives: In recent years, the therapeutic implications of dyslipidemias have been clarified in large-scale epidemiologic surveys, and the validity of pharmacotherapy has been established. We investigated the practical realities of pharmacotherapy for dyslipidemias at a community hospital in Japan., Methods: Medical chart surveys were performed retrospectively on 451 dyslipidemic outpatients who visited a community hospital in Japan in July 1997. We collected clinical data from medical charts regarding selected drugs for dyslipidemias, serum lipid levels before drug treatment and after one year of treatment, and risk factors for coronary heart disease (CHD)., Results: Regardless of dyslipidemia phenotype, approximately 80% of patients were administered statins. The possibility was raised that physicians recorded risk factors in medical charts incompletely, particularly with regard to family CHD history, smoking, and obesity. Based on Japanese and us guidelines for dyslipidemias, low-density lipoprotein (LDL) cholesterol levels fully satisfied the requirements for initiating pharmacotherapy in the present study. However, the higher the risk of CHD, the lower the percentage of subjects who met the treatment goals defined by both guidelines. Only 23% of patients at high risk for CHD controlled LDL cholesterol sufficiently based on Japanese guidelines., Conclusion: To optimize pharmacotherapy for dyslipidemias, medical staff should assess risk factors for CHD more completely and attempt to achieve full control of serum lipids, particularly in patients at high risk for CHD.

The objective of this study was to develop a new simplified method using thin-layer chromatography (TLC) for determining isoniazid (INH) compliance in patients receiving antituberculosis chemotherapy. TLC was performed on silica gel plates using a standard solution of INH and acetylisoniazid (AcINH) and ethyl acetate-methanol (70:30 v/v) as the developing solvent. The spots of compound were detected by iodine. In the human study, fractional urine samples were collected over 24 hours from 4 healthy human subjects genotyped for NAT2* and to whom 400 mg of INH were administered orally. These samples were used for TLC analysis. The results of TLC were compared with those of high-performance liquid chromatography (HPLC). This method indicated good separation between INH and AcINH in standard solutions. The detection limits for INH and AcINH (applied volume; 20 microl of standard solution) were 2.2 nmole and 5 nmole, respectively, as detected by iodine. In the human study, the INH spot in urine was not detected on the TLC plate, except in one sample over the 0- to 4-hour period from 1 volunteer. However, the AcINH spot was detected in all urine samples from all volunteers. The total experimental time from application of the urine sample to analysis on TLC was 30 minutes. The results suggest that this method for detecting AcINH on TLC is an excellent, convenient, and simple method for determining INH compliance in patients receiving standard antituberculosis chemotherapy regimen or INH preventative therapy, regardless of the patient's NAT2* genotype.

Persistent dry cough is well known as the most common side-effect of angiotensin-converting enzyme (ACE) inhibitors. We examined the relationship between a cough and ACE gene polymorphism, plasma bradykinin (BK), substance P (SP) and ACE inhibitor concentrations in patients with hypertension or chronic nephritis. First, ACE genotyping was carried out in 96 patients, 42 with coughs and 54 without coughs, which had been treated with various kinds of ACE inhibitors. However, no significant difference in the ACE genotypes was observed between the two groups. Second, the plasma concentrations of BK, SP and ACE inhibitor were measured in 12 patients, which were treated with trandolapril at a daily dose of 1 mg for 4-33 weeks. In 3 patients, the cough was induced during the trandolapril therapy, while it was induced not in 9 patients. The plasma levels of BK and SP did not significantly change after trandolapril administration in the patients with and without coughs. Between the two groups, there were no significant differences in the plasma levels of BK and SP either before or after the trandolapril therapy. Also the plasma concentrations of trandolapril and trandolaprilat, the active metabolite of trandolapril, did not significantly differ between the two groups. These results suggest that there is no significant relationship between the ACE inhibitor-induced cough and ACE gene polymorphism, plasma BK, SP and ACE inhibitor concentrations in patients with hypertension or chronic nephritis.

Serum from an individual who was chronically infected with hepatitis C virus (HCV) was inoculated into chimpanzees and a human T cell line. The serum contained a quasispecies population of HCV consisting of at least six genetic sequences. However, only two sequences (the same two sequences) were recovered both in chimpanzees and in cultured human T cell line, indicating that infection of the culture system reflects infection in vivo.

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.

The reductive metabolism of 1,2-benzisoxazole-3-methanesulfonamide (zonisamide) to 2-sulfamoylacetylphenol (SMAP) was observed in liver microsomes from female rats, as well as male rats, but the SMAP-producing activity in female rats was 4-fold lower than that found in male rats. In addition, the reductive metabolism of zonisamide in liver microsomes was induced by the treatment of male rats with phenobarbital. However, the SMAP-producing activity did not correlate positively with the amounts of P-450 2B1 and P-450 2C11 immunochemically determined. In contrast, the reductive metabolism of zonisamide was also found to be induced by the pretreatment of male rats with pregnenolone 16 alpha-carbonitrile, triacetyloleandomycin, and dexamethasone. Furthermore, the SMAP-producing activity correlated highly with the amount of P-450 cross-reactive with antihuman P-450 3A4 antibody, suggesting that P-450 3A1/2 may function in the reductive metabolism of zonisamide. In addition, the P-450 PCNa (3A2) exhibited the SMAP-producing activity in a reconstituted system. The antihuman P-450 3A4 antibody inhibited markedly the formation of SMAP from zonisamide in male rat liver microsomes. These results indicate that cytochrome(s) P-450 belonging to P-450 3A subfamily may be predominantly responsible for the reductive metabolism of zonisamide in rat 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.

An epidemic outbreak of hepatitis E occurred in an army recruit camp of Yangon, Myanmar, in October 1989. One hundred and eleven patients among 600 residents were hospitalized. As high as 83.7% of these patients were positive for the acute phase antibody against hepatitis E virus by an enzyme-linked immunosorbent assay developed in our laboratory. Also, 30.6% of 49 symptom-free residents examined were positive for the antibody. We prepared a stool extract from six patients and inoculated it into 10 rhesus monkeys for a series of three sub-passages. All of them developed acute biochemical hepatitis along with an elevation of antibody levels. A rechallenge with viruses of the present outbreak failed to provoke hepatitis in two monkeys that had previously recovered from acute hepatitis caused by an isolate of sporadic hepatitis E of the same area. Similarly, the rechallenge of the sporadic strain did not induce hepatitis in two monkeys that had been previously infected with the epidemic virus. These data suggested that the subjects would obtain neutralizing antibodies against the hepatitis E virus once infected, and many adult inhabitants of the endemic area had no protective antibodies and were still susceptible to hepatitis E infection.

Clarithromycin and its metabolites have been examined for their abilities to induce specific form(s) of cytochrome P-450 and metabolite complex formation in rats. Pretreatment of rats with clarithromycin,N-demethyl clarithromycin, clarithromycin N-oxide, and decladinosyl clarithromycin resulted in 48-75% decreases in the amount of 2C11 and 100-600% increases in the amount of 3A1. Clarithromycin and N-demethyl clarithromycin, but not decladinosyl clarithromycin, produced a metabolite complex with cytochrome P-450 in vivo. Activities of testosterone 2 beta- and 6 beta-hydroxylases were increased by administration of clarithromycin and N-demethyl clarithromycin when these activities were measured in the presence of ferricyanide, but not significant induction was observed when measured in the absence of ferricyanide. Clarithromycin N-oxide treatment resulted in the increase in hydroxylation of testosterone not only at the 2 beta- and 6 beta-positions (430 and 190%, respectively), but also at 7 alpha-position (60%), regardless of the presence or absence of ferricyanide. In vitro experiments with hepatic microsomes of dexamethasone-pretreated rats indicated that the metabolites that were modified at the tertiary amino group more efficiently produced the metabolite complex with cytochrome P-450 compared with the parent compound. In contrast, decladinosyl and hydroxylated metabolites had similar or lower capacities for metabolite complex formation than did the parent compound. Clarithromycin and its N-modified metabolites were able to induce 3A1 and form a metabolite complex with cytochrome P-450 in vivo in varying extents. Decladinosyl clarithromycin has a weak inducibility of 3A1 and did not form a metabolite complex with cytochrome P-450 in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatitis E virus (HEV) is a causative agent of enterically transmitted non-A, non-B hepatitis. Hepatitis E occurs not only in sporadic forms but also in epidemic outbreaks in the developing world. We have revealed the nucleotide and predicted amino acid sequences of full cDNA of HEV isolated from sporadic hepatitis E of Myanmar. The genome is 7194 nucleotides long, followed by a poly(A) tail, and has three open reading frames. The nonstructural gene is located in the 5' terminus, while the structural gene is situated in the 3' terminus. Our HEV strain has 98.5% nucleic acid identity with the HEV strain cloned by workers at Genelabs Incorporated from Myanmar. The difference is point nucleotide substitutions. There is a high degree of nucleotide relatedness among HEVs isolated from the same geographical location.

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).

Zonisamide (1,2-benzisoxazole-3-methanesulfonamide) was metabolized to its reductive product, 2-sulfamoylacetylphenol, in rat liver microsomes under anaerobic conditions. The rate of NADPH-dependent reaction was much more rapid than that of NADH-dependent reaction. Furthermore, synergistic effect of NADH on NADPH-dependent reaction was not observed. The optimal formation of 2-sulfamoylacetylphenol from zonisamide in the presence of NADPH was observed around pH 7.0. Cimetidine showed an inhibitory effect on the formation of 2-sulfamoylacetylphenol in a dose-dependent manner. The reductive metabolism of zonisamide was almost completely inhibited by carbon monoxide, and was increased by pretreatment of rats with phenobarbital and pregnenolone 16 alpha-carbonitrile but not by pretreatment with ethanol, 3-methylcholanthrene and imidazole. These results suggest that phenobarbital- and pregnenolone 16 alpha-carbonitrile-inducible form(s) of cytochrome P-450 is responsible for the reductive metabolism of zonisamide to 2-sulfamoylacetylphenol in rat liver microsomes.

Viral hepatitis E is endemic, frequently provoking epidemic outbreaks in many developing countries. We have attempted to clone the viral genome and to develop an antibody assay system. A lambda gt11 cDNA library was constructed from the bile juice containing putative causative viruses and was immunoscreened by the antisera obtained from patients and monkeys infected with hepatitis E. Three virus-specific clones were isolated and were revealed to overlap one another in sequence, with 1,459 nucleotides in total length. These clones direct the synthesis of polypeptides probably having common immunological epitope(s). Immunoplaque assay revealed the occurrence of antibodies against this epitope in the sera from experimental monkeys with the convalescent phase and from patients of Myanmar, Nepal and India. The data indicate that the cDNA fragments are useful for immunodiagnosis of hepatitis E.

Pharmacokinetics of theophylline were determined in patients with liver cirrhosis and idiopathic portal hypertension with reference to estimated hepatic blood flow assessed by indocyanine green (ICG). Decreased plasma clearance of theophylline was noted in patients with liver cirrhosis and the clearance was significantly lower in Child C group than in Child A, B groups (17.5 +/- 3.4 ml/Kg/hr vs 27.6 +/- 8.7, p less than 0.05). Theophylline has been classified as a drug with a low hepatic extraction ratio and it has been believed that changes in hepatic blood flow have little effect on its clearance. The results of present study indicate that theophylline clearance is basically not related to ICG clearance but to theophylline extraction ratio, supporting the common belief. However, it is noteworthy that the clearance was related to decreased hepatic blood flow rather than extraction ratio in a cirrhotic patient with huge extrahepatic shunt, suggesting that hepatic clearance of this drug could be affected by hepatic blood flow under some circumstances.

We have developed animal models of viral hepatitis E using cynomolgus and rhesus monkeys. They developed acute biochemical and histological hepatitis after the inoculation of virus particles with identical kinetics and magnitude for the sixth subpassage. Virus particles multiplied in hepatocytes and were excreted into feces via bile. Additionally, a transient viremia was recognized. Molecular cloning of virus gene cDNA was successfully accomplished from two separate libraries (HT3 and NE). These clones were expressed into polypeptides having immunological epitopes, which were used for antibody assay of sera of monkeys and patients with positive results.

Eisai-hyperbilirubinuria rats (EHBR) is a mutant originated from Sprague Dawley rats. The activities of UDP-glucuronyltransferase and drug metabolizing enzymes in EHBR were compared with those in Sprague Dawley rats as the control. The activity of aniline hydroxylase was significantly increased in liver microsomes of EHBR whereas the activity of ethylmorphine N-demethylase was found to be significantly decreased in EHBR as compared to control rats. In addition, the activity of testosterone 7 alpha-hydroxylase was increased in EHBR whereas the activity of testosterone 6 beta-hydroxylase was significantly decreased in EHBR as compared to control rats. Western blot analysis of liver microsomes of EHBR with antibodies to P-450IA2, P-450IIB1, P-450IIC11 and P-450IIIA2 showed that the amounts of P-450IIB1 and P-450IIIA2 in liver microsomes were significantly lower in EHBR than in control rats. These results indicated the form-specific alteration in the amounts of cytochrome P-450 in liver microsomes of EHBR.

The fragment gene of enterically-transmitted non-A, non-B hepatitis virus (ET-NANBHV) was cloned as a cDNA and inserted into an expression vector pUEX2. The recombinant protein was expressed in Escherichia coli HB101 as a fusion protein with beta-galactosidase (beta-Gal). The fusion protein reacted with the sera of infected cynomolgus monkeys and of patients from Myanmar. This reaction was highly related with ET-NANBHV infection, and obviously demonstrates in that the recombinant protein can be used for the detection of ET-NANBHV infection.

The present investigation confirms the possibility that the etiological agent of enterically-transmitted non-A, non-B (ET-NANB) hepatitis (type E hepatitis), multiplied in hepatocytes, is excreted into the feces via bile. The fecal extract was inoculated into 7 cynomolgus monkeys. Bile juice was collected directly from the gallbladder by needle puncture after abdominal operation 3 to 6 times during the experimental course. All 7 monkeys developed elevated serum aminotransferases, which began gradually approximately 2 weeks postinoculation and reached a peak at 3 to 5 weeks. In parallel with this elevation, both in time and magnitude, necroinflammation was observed in the livers. The virus-like particles (VLPs) were found in the bile juice of all 7 monkeys and the serial occurrence of VLPs was typified as follows: the VLPs were negative on day 7, appeared on day 10 after inoculation, and were present until the 3rd week when the subjects were sacrificed. While the particles were individually dispersed on day 10, they started to exhibit spontaneous aggregation on and after week 2. Also, empty particles were very rare at first, but increased in ratio compared to full ones over time. Thus, the putative causative virus of ET-NANB hepatitis was demonstrated to be excreted through bile. The spontaneous aggregation of VLPs might be due to the specific antibody secreted into the bile juice and was closely correlated with hepatitis activity. The increase in empty particles might indicate an increase in disorganized assembly of the nucleic acid and protein during virus proliferation.

In order to establish an animal model and to identify a causative virus of enterically transmitted non-A, non-B hepatitis, Macaca fascicularis was inoculated with a fecal extract obtained from Myanmar patients with acute sporadic non-A, non-B hepatitis. The primates developed acute hepatitis exhibited by a transient elevation of aminotransferases in the sera and occurrence of hepatic necroinflammation between 2 and 4 weeks postinoculation. Subsequent second passage of the fecal extract made from first-passage primates into another Macaca fascicularis and Macaca mulatta induced acute hepatitis. Likewise, third passage was also successfully performed. Immune electron microscopy of the stool extract incubated with the primate serum at the acute phase of hepatitis showed an aggregation of virus-like particles. These particles consisted of full and empty round particles without an envelope, measuring approximately 27 nm in diameter. A dispersion of similar particles was found ultrastructurally in the hyaloplasm of hepatocytes surrounding the focal necrosis. This putative causative virus appears to be a new hepatitis virus.

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.

Cytochrome b5 did not exert any effect on NADPH-dependent aniline hydroxylation in the absence of acetone or 2,2'-bipyridine, whereas cytochrome b5 exhibited a stimulatory effect on the reaction in the presence of acetone or 2,2'-bipyridine. In addition, cytochrome b5 did not have any significant effect on the cumene hydroperoxide-dependent reaction in the presence of acetone or 2,2'-bipyridine.

The mean hepatic extraction ratio (ER) of propranolol was estimated directly by simultaneous measurements of arterial and hepatic venous blood concentrations of the drug following systemic venous and portal venous administration in the rat. The ER was greater than 0.9 in the dose range of 2.5 to 12.5 mg/kg following rapid infusion of propranolol into the femoral vein and was not dependent on infusion rate. On the other hand, the ER following intraportal constant-rate infusion decreased progressively with increasing dose, although the ER at an intraportal dose of 2.5 mg/kg was as high as that found after administration into the femoral vein. In addition, it was found that the ER at an intraportal dose of 12.5 mg/kg of propranolol was significantly influenced by infusion rate. The unusual AUC-dose relationship of propranolol previously reported in the rat could be explained on the basis of the present nonlinear hepatic extraction depending on the route and rate of administration which was clarified in vivo. The nonlinear hepatic extraction was further confirmed by determining the remarkably decreased ER of (14)C-propranolol given intravenously after pretreatment or during portal venous administration of unlabelled propranolol.

The activities of drug oxidation in a reconstituted system which contains two forms of cytochrome P-450 and a limiting amount of NADPH-cytochrome P-450 reductase were determined. Cytochrome P-450 (termed MC P-4481 and MC P-4482) purified from liver microsomes of 3-methyl-cholanthrene-treated rats was active in both 2- and 4-hydroxylation of biphenyl but cytochrome P-450 (termed PB P-450) purified from liver microsomes of phenobarbital-treated rats was active in 4-hydroxylation of biphenyl only. PB P-450, MC P-4481 and MC P-4482 were most active toward benzphetamine N-demethylation, aniline hydroxylation and 7-ethoxycoumarin O-deethylation, respectively. PB P-450 inhibited the activity of biphenyl 2-hydroxylation supported by MC P-4481 or MC P-4482. On the contrary, no inhibition of PB P-450 supported benzphetamine N-demethylation was observed when MC P-4481 or MC P-4482 was added to the system containing PB P-450 and limited amount of the reductase. The apparent Km of PB P-450 for the reductase obtained from double reciprocal plot of the reductase concentration and the activity of biphenyl hydroxylase or benzphetamine N-demethylation was lower than that of MC P-4481 or MC P-4482. These and other results suggest that there is a certain hierarchy among the cytochrome P-450 species for receiving electrons from reductase.

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.

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.

Two cynomolgus monkeys were inoculated with a stool extract originally derived from patients suffering from enterically transmitted non-A, non-B hepatitis. Subsequently, the primates developed self-limiting acute hepatitis and their liver tissues were obtained sequentially by needle biopsy or at sacrifice. Histologically, the liver tissues exhibited necroinflammation which appeared in parallel, both in time and magnitude, with elevation in serum aminotransferases. Necroinflammation was characterized by focal dropout of hepatocytes with accumulation of lymphocytes and macrophages. These lymphocytes were positive for a cytotoxic/suppressor immunophenotype. The hepatocytes surrounding these focal necroses showed depletion of glycogen granules and decrease in glucose-6-phosphatase and succinic dehydrogenase activities. Ultrastructurally, damaged hepatocytes around the focal necroses revealed marked dilatation of both rough and smooth endoplasmic reticula, swelling and disruption of the mitochondria and leakage of nuclear materials into the cytoplasm. Frequently, direct contact between the damaged hepatocytes and lymphocytes was noted. Virus-like particles measuring about 27 nm in diameter were observed singly or in small groups within the cytoplasm of damaged hepatocytes. Primary hepatocyte culture of a cynomolgus monkey, inoculated with a transmissible stool extract did not show any cytopathic change, although similar virus-like particles were recognized ultrastructurally in the cultured hepatocytes. Morphological analysis of in vitro and in vivo transmission studies in cynomolgus monkeys strongly supported the hypothesis of immune-mediated hepatocytolysis rather than a direct cytopathic effect of this hepatitis virus.

The effect of cyanide on 7-ethoxycoumarin O-deethylation by two cytochrome P-450 isozymes obtained from 3-methylcholanthrene treated rat liver microsomes was investigated. 7-Ethoxycoumarin O-deethylation was stimulated by the addition of cyanide to a reconstituted monooxygenase system consisting of NADPH, dilauroyl 3-L-phosphatidylcholine, NADPH-cytochrome P-450 reductase and MC P-448(2) (low spin form of cytochrome). In contrast, a weak inhibitory effect of cyanide on 7-ethoxycoumarin O-deethylation was observed when MC P-448(1) (high spin form of cytochrome) was used in the reconstituted system. Cyanide did not influence the apparent Km for 7-ethoxycoumarin when either form of cytochrome P-450 was used in the reconstituted system and did not stimulate the cumene hydroperoxide dependent O-deethylation by MC P-448(2). The stimulatory effect of cyanide on O-deethylation by MC P-448(2) was decreased with increasing the concentration of the reductase added to the reconstituted system. On the other hand, the effect of cyanide on O-deethylation by MC P-448(1) was virtually independent on the amount of the reductase added.

Effects of cyanide, acetone, 2,2'-bipyridine and paraoxon on aniline hydroxylation by human liver microsomes were studied. The activity of aniline hydroxylation was inhibited by cyanide when low concentrations of substrate were used, whereas the activity was increased by the addition of cyanide when higher concentrations of substrate were used. Although there were large variences among different liver samples in the extent of the activation of aniline hydroxylation, acetone, 2,2'-bipyridine and paraoxon also exerted stimulatory effects on aniline hydroxylation in human liver microsomes. The degree of the stimulation was not related with the activities observed in the absence of these compounds.

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.

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.