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

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

Sex difference in responsiveness to Aztreonam of monooxygenase system in liver microsomes from rats.

Effect of successive administration of Aztreonam on microsomal monooxygenase system was investigated in male and female Sprague-Dawley rats. The activities of benzphetamine N-demethylase, aminopyrine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes from male rats were decreased dose-dependently by Aztreonam. On the contrary, the activities in liver microsomes from female rats were slightly increased rather than decreased by the administration of Aztreonam. In addition, Aztreonam was found to decrease the specific content of microsomal cytochrome P-450 in male rats but not in female rats. The decreases in the activities observed in male rats were accompanied by a parallel decrease in the specific content of cytochrome P-450. Furthermore, the results of quantitation of P-450 (M-1), one of the male specific forms of cytochrome P-450, indicated that the administration of Aztreonam resulted in a dose-dependent decrease in the content of P-450 (M-1) in liver microsomes from male rats.

The interaction between two forms of cytochrome P-450 during drug oxidation in the reconstituted system containing limited amount of NADPH-cytochrome P-450 reductase.

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.

Different effects of cyanide on the activities of 7-ethoxycoumarin O-deethylation catalyzed by two forms of cytochrome P-450 purified from 3-methylcholanthrene-treated rats.

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.

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.

[Theophylline pharmacokinetics in patients with liver diseases with reference to estimated hepatic blood flow].

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.

[A survey on the current status and trends in bladder irrigation].

Questionnaires were sent to pharmacies and clinics in 592 Japanese hospitals from July to November, 1983 to investigate the use of bladder irrigation. The questionnaires were completed and returned by 418 hospitals, the rate of response being 70.8%. Fifty eight kinds of irrigants were prepared in hospital pharmacies and were reported to have been used in 1,171 patients. Of these irrigants, chlorhexidine gluconate accounted for 18.8%, Polymyxin B for 17.1%, normal saline for 13.3%, potassium permanganate for 6.9%, and boric acid for 5.7%. Chlorhexidine gluconate was usually used at a concentration of 0.02% (in 79% of the total use of the agent), Polymyxin B at 0.01%, 0.02% and 0.05%, potassium permanganate at 0.01% and 0.02% (in 73% of the total use), and acrinol at 0.01%, 0.05% and 0.1%. To questions about side effects, such as irritation, 65 hospitals answered that irritation had occurred. Irrigants were most frequently supplied to non-disposable 500 ml containers used exclusively for this purpose, 38% of all the hospitals using this method. Non-disposable 1 liter containers for exclusive use with bladder irrigants were used in 23% of the hospitals. Purified water was most frequently used to prepare the bladder irrigants (in 35% of hospitals) followed in descending order, by sterile purified water in 33%, and distilled water for injection in 26%. Water for irrigants was prepared at the hospital in 83% of the hospitals. The time of storage of the prepared bladder irrigants was mostly one week to one month, and was within one month in 78% of the hospitals. Seventeen percent of the hospitals answered that the preparation of bladder irrigants was a cause of overwork in their pharmacies. Irrigants against Pseudomonas infections were prepared separately in 40% of the hospitals, and there was no such regulation in 37%. Forty four agents were used to prepare such irrigants, and Polymyxin B accounted for 50%. The use of these agents significantly (P less than 0.001) differed between hospitals. Forty eight percent of the hospitals made special preparations for patients with clouded urine, and there was no such regulation in 46%. Thirty nine agents were used for this purpose. Polymyxin B was used in 30%, and chlorhexidine gluconate in 19%. The use of these agents differed significantly (P less than 0.001) between hospitals.(ABSTRACT TRUNCATED AT 400 WORDS)

[Advanced medical pharmacy].

This paper reviews the progress of Medical Pharmacy for about the past 30 years. Nowadays, Medical Pharmacy is becoming an important and essential part of pharmacist's business and pharmaceutical education.

Enhancement of aniline hydroxylation in human liver microsomes.

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.

The proteins immunochemically related to P-450 HFLa, a major form of cytochrome P-450 in human fetal livers, are present in liver microsomes from various animal species.

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.

Purification and some properties of NADPH-cytochrome P-450 reductase from crab-eating monkey liver microsomes.

NADPH-cytochrome P-450 reductase was purified to 30.8 units/mg from monkey liver microsomes. The purified reductase showed one major protein band (78,000) and two minor ones (58,000 and 20,000) on analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Monkey, rat, and guinea pig reductases were not immunochemically identical to each other judged from Ouchterlony double diffusion analysis and immunotitration with regard to NADPH-cytochrome c reductase activity.

Polyamine lowered the hepatic lipid peroxide level in rats.

This is the first report for the hepatic lipid peroxide lowering effect of spermine in vivo. The influence of administration of polyamines on hepatic lipid peroxide level has been investigated by using normal or carbon tetrachloride (CCl4)-treated rats. Spermine was found to lower the hepatic lipid peroxide level most efficiently among polyamines used in CCl4-treated rats. In addition, the extent of liver enlargement caused by CCl4 treatment was reduced by spermine administration. Lipid peroxide lowering effect of spermine was also observed in normal rats. Hepatic spermine content was significantly increased in both normal and CCl4-treated rats after administration of spermine. Clear inverse relationship between the content of lipid peroxide and the concentration of spermine was observed. In reconstituted system containing NADPH-cytochrome P-450 reductase and extracted hepatic microsomal lipid, spermine inhibited the NADPH-dependent lipid peroxidation effectively at the concentration of 0.1 mM. From these results, we concluded that spermine exerted an inhibitory effect of lipid peroxidation in vivo as well as in vitro.

Purification and properties of cytochrome P-450 from untreated monkey liver microsomes.

Untreated monkey liver cytochrome P-450 (monkey P-450) has been purified to a specific content of 14.9 n mole/mg protein. The purified preparation was apparently homogeneous and the minimum molecular weight was estimated to be 50,000 by SDS-PAGE. Absolute spectrum of the oxidized form showed peaks at 565, 535 and 417 nm. The monkey P-450 was active in the mixed function oxidation of benzphetamine, aminopyrine, ethylmorphine, aniline and 7-ethoxycoumarin in the presence of rat liver NADPH-cytochrome P-450 reductase and DLPC. Anti monkey P-450 IgG could not inhibit rat P-450s (PB P-450, MC P-448(1) and MC P-448(2] catalyzed 7-ethoxycoumarin O-deethylation activities.

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.

Formation of reductive metabolite, 2-sulfamoylacetylphenol, from zonisamide in rat liver microsomes.

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.

6-Fluoro-2-methylspiro(chroman-4,4'-imidazolidine)-2',5'-dione and related compounds as inducers of monooxygenase in rat liver microsomes.

The relationship between the structure of spirohydantoin derivatives and their inducing effects on hepatic monooxygenase system was studied. At the dose of 1 mumol/kg (0.3 mg/kg), 6-fluoro-2-methylspiro(chroman-4,4'-imidazolidine)-2',5'-dione (M79175) was found to exhibit an inducing effect on benzphetamine N-demethylase. On the contrary, the inducing effect of Sorbinil, in which the 2-methyl group on the chroman ring of M79175 was replaced by hydrogen atom, on benzphetamine N-demethylase was 100 times less than that of M79175. The substitution of the methyl group of M79175 with the dimethyl group did not affect the inducing effect, whereas the substitution with the hexyl group resulted in the loss of the inducing effect on drug oxidation activities tested at a dose of 10 mg/kg. Furthermore, cyclohexane spiro-2,6-chloro-1'-(3-dimethylaminopropyl)spiro(chroman-4,4'-imidazolid ine)-2' , 5'-dione (M79193) had an inducing effect on total cytochrome P-450 content, but had no inducing effect on benzphetamine N-demethylase. In addition, M79175 was found to induce cytochrome P-450 which is immunochemically related to one of the major forms of phenobarbital-inducible cytochrome P-450 (P-450(PB-1], but not cytochrome P-450 which is immunochemically related to the major form of 3-methylcholanthrene-inducible cytochrome P-450 (P-450(MC-1]. On the other hand, M79193 was found to induce neither P-450(PB-1) nor P-450(MC-1).

P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, is the 16 alpha-hydroxylase of dehydroepiandrosterone 3-sulfate.

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.

Possible mechanism of inhibition by polyamines of lipid peroxidation in rat liver microsomes.

The rate of malondialdehyde formation in rat liver microsomes decreased as concentration of spermine added to the reaction mixture was increased. Inhibitory effect on lipid peroxidation was observed even when spermine was added at 15 min after the onset of reaction. Microsomal lipid peroxidation was markedly increased when extracted lipids were added to the reaction mixture. It was, moreover, found that in the presence of exogeneous microsomal lipids, spermine inhibition was weakened. The character of spermine inhibition with respect to the amounts of exogeneous and endogeneous lipids was shown to be competitive. From these results, it was suggested that spermine interaction with microsomal lipids may be responsible for the inhibition of lipid peroxidation.