Morio Fukuhara

Effects of co-administration of butylated hydroxytoluene, butylated hydroxyanisole and flavonoids on the activation of mutagens and drug-metabolizing enzymes in mice.

Effects of co-administration of food additives and naturally occurring food components were studied on the activation of mutagens. Male mice (ddY) were given diets containing butylated hydroxytoluene (BHT) or butylated hydroxyanisole (BHA) and flavone or flavanone (2,3-dihydroflavone) for two weeks and the ability of hepatic microsomes to activate aflatoxin B1, benzo[a]pyrene and N-nitrosodimethylamine was determined by the mutagenicity test. Co-administration of an antioxidant (0.1% BHT or 0.2% BHA in diet) and a flavonoid (0.1% flavone or 0.1% flavanone) resulted in additive effects on the activation of aflatoxin B1 and benzo[a]pyrene, while the activation of N-nitrosodimethylamine was not elevated significantly by the co-administration. To understand the mechanism for the additive effects, induction of specific isozymes of cytochrome P450 involved in the activation of the mutagens was studied. Co-administration of BHT (0.1%) and flavone (0.1%) increased markedly the levels of proteins and the activities of the enzymes related to the isozymes of CYP2A and CYP2B, while co-administration of BHA (0.2%) and flavanone (0.1%) elevated those related to CYP1A. Further, the activation of aflatoxin B1 and benzo[a]pyrene in hepatic microsomes was inhibited by the antibodies against these isozymes, which suggested that the enhanced activation of the mutagens by the co-administration might be mediated by the induction of these isozymes.

The effect of locally injected anti-inflammatory drugs on the synthesis of collagen and non-collagen protein of carrageenin granuloma in rats

Abstract The effect of steroid and non-steroid anti-inflammatory drugs was studied on protein synthesis of the granulation tissue induced by carrageenin on the dorsum of rats. The drugs were injected daily into the pouch of preformed granuloma for 4 days from day 5 up to day 8. Immediately after the last injection of the drugs the rats were injected s.c. with 3H-proline, and 24 hr later the granuloma was harvested on day 9. The total content, specific activity and total radioactivity of collagen and non-collagen protein of the granuloma were determined. Betamethasone disodium phosphate, a steroid drug, markedly inhibited the incorporation of 3H-proline into collagen hydroxyproline as well as into non-collagen protein. Among the non-steroids tested, indomethacin and phenylbutazone failed to show any significant changes in the synthesis of these proteins. Sodium salicylate significantly reduced the synthesis of collagen in the granulomatous tissue without affecting non-collagen protein synthesis.

Complete cDNA sequence of a major 3-methylcholanthrene-inducible cytochrome P-450 isozyme (P-450AFB) of Syrian hamsters with high activity toward aflatoxin B1

Cytochrome P-450AFB is major isozyme inducible by 3-methylcholanthrene in Syrian golden hamsters and shows high potency toward aflatoxin B1 activation. We have isolated and sequenced cDNA clones to P-450AFB by immunoscreening a hamster liver cDNA library in lambda gt11. The longest clone contains an open reading frame of 1482 nucleotides and encodes a protein of 494 amino acids with a molecular weight of 57,420. The sequence of P-450AFB shares a 73% and 65% homology with that of mouse P-450 15 alpha (IIA3) and rat P-450a (IIA1), respectively, indicating that P-450AFB is a unique gene of the P-450IIA subfamily. The apparent concentration of a mRNA species hybridizable to the clone as well as the concentration of a protein immunoreactive to P-450AFB was increased significantly by the treatment with 3-methyl-cholanthrene, which indicates that the increase in P-450AFB protein is due mainly to an elevation of the mRNA.

Hepatotoxic action of a poisonous mushroom, Amanita abrupta in mice and its toxic component

An aqueous extract of a poisonous mushroom, Amanita abrupta was injected intraperitoneally into male ICR mice and the acute effects on the liver were studied. Contents of serum glucose and liver glycogen decreased to 60% and 10% of the control levels, respectively, 6 h after injection. Activities of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase increased to 3- and 8-fold, respectively, 12 h after injection, and the elevated activities were maintained up to 24 h. Activities of the hepatic drug metabolizing enzymes were also reduced 15 h after injection. Histological examination demonstrated massive liver cell necrosis and disappearance of glycogen granules in the liver of the treated animals. Two amino acids, L-2-amino-4-pentynoic acid and L-2-amino-4,5-hexadienoic acid were identified in the mushroom extract. The former caused similar biochemical effects to those of the mushroom extract.

Characterization of benzo[a]pyrene metabolism and related cytochrome P‐450 isozymes in Syrian hamster livers

Cytochrome P-450 monooxygenases of golden Syrian hamsters were characterized with respect to benzo[a]pyrene metabolism. Male hamsters were treated with phenobarbital, 3-methylcholanthrene, dexamethasone, benzoflavone, or ethanol, and the activity of aryl hydrocarbon hydroxylase and benzo[a]pyrene activation was determined by mutagenicity testing in hepatic microsomes. Aryl hydrocarbon hydroxylase activity was induced markedly by treatment with phenobarbital but not with 3-methylcholanthrene, nor with other chemicals. The degree of benzo[a]pyrene activation on mutagenicity testing was significantly elevated by treatment with 3-methylcholanthrene and phenobarbital but was reduced with dexamethasone. Immunoinhibition of these activities and Western blotting of hepatic microsomes using antibodies against cytochrome P-450 isozymes suggested that the isozymes responsible for benzo[a]pyrene metabolism in Syrian hamsters belong to the CYP1A, CYP2A, and CYP3A families, a result that differs from observations in rats.

Cytochrome P450 isoforms catalyzing benzo[a]pyrene metabolism in the Chinese hamster liver.

Cytochrome P450 isoforms involved in the benzo[a]pyrene metabolism in the Chinese hamster liver were characterized. The activity of benzo[a]pyrene hydroxylase in male hamster livers increased markedly by treatment with 3-methylcholanthrene (25 mg/kg per day, i.p., 3 days) and moderately with phenobarbital (60 mg/kg per day) and dexamethasone (100 mg/kg per day). In contrast, the ability for the mutagenic activation of benzo[a]pyrene determined by the mutagenicity test was increased most markedly by treatment with phenobarbital and significantly with 3-methylcholanthrene, but not with dexamethasone. These observations are similar to those in the rat rather than in the Syrian hamster. Western blot analysis and assay of the enzymes associated with cytochrome P450 isoforms showed that the 3-methylcholanthrene treatment elevated markedly the level of CYP1A2, but not that of CYP1A1, while the phenobarbital treatment elevated markedly the level of CYP2A and CYP3A, but not that of CYP2B. Further, immunoinhibition study demonstrated that, in Chinese hamster livers, CYP2A and CYP1A2 were mainly involved in the mutagenic activation of benzo[a]pyrene and CYP3A in the benzo[a]pyrene hydroxylase activity, respectively.

Characterization of cytochrome P450-monooxygenases of Chinese hamsters with respect to aflatoxin B1 activation

Monooxygenases of Chinese hamster livers were characterized with respect to aflatoxin B1 activation and cytochrome P450 isozymes. Polychlorinated biphenyl (PCB)-treatment induced higher activity for aflatoxin B1 activation in Chinese hamsters than in rats and this activity was more elevated by the treatment with phenobarbital than with 3-methylcholanthrene, which is contrary to the observation in Syrian hamsters and rather similar to that in rats. Western blot analysis demonstrated that the treatment of Chinese hamsters with phenobarbital markedly elevated the level of cytochrome P450 isozyme belonging to the CYP2A family and only slightly that immuno-related to CYP2B1. In parallel with this, the activities of testosterone 15 alpha-hydroxylase and 7-ethoxycoumarin O-deethylase were significantly induced by phenobarbital treatment. These suggest that phenobarbital mainly induces an isozyme related to CYP2A2 which is responsible for aflatoxin B1 activation in Chinese hamsters.

In vitro inhibitory action of cadmium on microsomal monooxygenases of rabbit lung

In vitro addition of cadmium (Cd) salts to pulmonary microsomes isolated from male rabbits decreased the cytochrome P-450 levels and the activity of benzo[a]pyrene hydroxylase and aminopyrine N-demethylase but not that of NADPH-cytochrome c reductase. The Cd-induced reduction of pulmonary monooxygenase activity was enhanced when microsomes were preincubated in the presence of Cd and inhibition increased as the time of preincubation progressed, attaining its maximum rate at 20 min of preincubation. When hepatic microsomes were used, this delayed effect of Cd on monooxygenase was less apparent. The presence of NADH and/or NADPH in the preincubation did not markedly enhance the Cd-induced inhibition rate of monooxygenase activity. The addition of Cd-acetate to pulmonary microsomes produced a concentration-dependent inhibition of the monooxygenase activity and the estimated IC50 values of Cd-acetate (i.e. the concentration required to inhibit control enzyme activity by 50%) were 3.8 x 10(-5)M, 6.5 x 10(-6)M and 5.3 x 10(-5)M for cytochrome P-450, benzo[a]pyrene hydroxylase and aminopyrine N-demethylase, respectively. The inhibitory action of Cd-acetate on the monooxygenase activity was also observed with microsomes isolated from the lungs of male guinea-pigs and rats.

cDNA cloning and expression of a novel CYP3A from the Syrian hamster, CYP3A31

A clone, encoding a cytochrome P450 protein consisting of 501 amino acids, was isolated from a cDNA library constructed from mRNA of Syrian hamster liver. The deduced amino acid sequence of this clone showed a high homology (65 to 81%) with other mammalian CYP3As and hence, this novel isozyme was named CYP3A31. By Northern blotting, using an oligonucleotide specific to CYP3A31, the mRNA for this isozyme was shown to be expressed constitutively in liver and induced by treatment with phenobarbital but repressed by 3-methylcholanthrene or dexamethasone treatments. The increase in mRNA expression by phenobarbital and decrease by dexamethasone corresponded to changes in CYP3A protein as analysed by Western blotting. These indicate that CYP3A31 might constitute one of the major CYP3A isozymes in the hamster.

Molecular cloning, heterologous expression, and characterization of a novel member of CYP2A in the Syrian hamster

The cDNA clone coding for a novel cytochrome P-450 2A subfamily member (CYP2A16) was isolated from a Syrian hamster liver cDNA library. The deduced amino acid sequence of CYP2A16 showed more than 90% identity with those of rat CYP2A3 and mouse CYP2A4/5. The catalytic activity of CYP2A16 was determined by transient expression of its cDNA in transfected COS7 cells and CYP2A16 was found to have the testosterone 2 beta-, 15 alpha-, and 15 beta-hydroxylases, coumarin 7-hydroxylase, and ethoxycoumarin O-deethylase activities. These enzymatic characteristics of CYP2A16 are different from those of other Syrian hamster CYP2A subfamily members, CYP2A8 and CYP2A9. Northern blot analysis showed that CYP2A16 was expressed in kidney and lung while most of the other CYP2A subfamily members have been reported to be expressed in liver and olfactory. These observations indicated that the Syrian hamster CYP2A16 had unique properties compared with those of other CYP2A subfamily members.