Soft-hydrothermal processing of red cedar bedding reduces its induction of cytochrome P450 in mouse liver.

Red cedar-derived bedding materials cause changes in cytochrome P450-dependent microsomal enzyme systems in laboratory animals. We examined the effect of essential oil of red cedar (EORC), as well as the effect of bedding from which it had been removed, on the hepatic expression cytochrome P450s in mice. EORC was obtained from liquid extracts of red cedar bedding by a soft-hydrothermal process and was administered orally to mice. Between days 1 and 2 after administration, hepatic P450s were significantly induced as follows: CYP3As, 7.1x; CYP1As, 1.6x; CYP2E1, 1.5x; CYP2Cs, 1.6x. A housing study of mice indicated that red cedar bedding increased the levels of these P450s in mouse liver, whereas mice housed in cedar bedding from which EORC had been removed (ST-cedar bedding) showed significantly lower levels of P450s, especially CYP3As, CYP1As and CYP2E1. Soft-hydrothermal processing partially removed many components of EORC. In particular, several volatile sesquiterpenes, naphthalene-derived aromatics and 4,4-dimethyl-13alpha-androst-5-ene were decreased in the ST-cedar bedding, suggesting that these may be responsible for P450 induction. This study demonstrated that the removal of these volatile compounds by soft-hydrothermal processing can decrease the hepatic P450-inducing effect of red cedar bedding.

Expression of a skin cholesterol sulfotransferase, St2b2, is a trigger of epidermal cell differentiation.

1. St2b2, a mouse cytosolic sulfotransferase, predominantly catalyses epidermal cholesterol sulfation. St2b2 was found in the basement layer by immunohistochemical analysis of normal mouse skin. The highest expression level was detected in epidermis from 3-day-old mice and then decreased before maturation. There was a good correlation between expression levels of skin St2b2 and a differentiation marker, involucrin. 2. To understand the role of St2b2 in epidermal cell differentiation, recombinant St2b2 was expressed in primary epidermal cells. The expression of St2b2 enhanced the involucrin expression with an increase of cholesterol sulfate. Furthermore, by down-regulation of the St2b2 gene expression, involucrin was decreased in dorsal skin of 1-3-day-old mice by 67% of the control. 3. These results strongly suggest a possibility that St2b2 expression plays a trigger of epidermal cell differentiation by controlling cholesterol sulfate level in the cells.

Species difference in N-hydroxylation of a tryptophan pyrolysis product in relation to mutagenic activation.

Metabolic activation of a tryptophan pyrolysate, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), in liver microsomes from rats, mice, hamsters, guinea pigs, and rabbits was studied to know whether N-hydroxylation is a common obligatory step for mutagenic activation of Trp-P-2. Among hepatic microsomes obtained from untreated animals, the highest activity of Trp-P-2 N-hydroxylase was observed in microsomes from hamsters, followed by those from guinea pigs, mice, and rabbits. In rats, the activity was low, and there was no appreciable difference between the sexes. The activity of Trp-P-2 N-hydroxylase in microsomes was increased by pretreating the animals with a polychlorinated biphenyl mixture. The induction was most profound in rats, and the activity was enhanced 257-fold, as compared to that of untreated animals. The activity was also enhanced in microsomes from polychlorinated biphenyl mixture-treated rabbits, mice, and hamsters, while the activity was increased only slightly in guinea pigs and was thereby lowest among microsomes from the polychlorinated biphenyl mixture-treated animals. In bacterial mutagenesis test systems using Salmonella typhimurium TA 98, the number of revertants induced by Trp-P-2 was increased in parallel with the microsomal ability of the N-hydroxylation. These results indicate that in all species examined N-hydroxylation is an essential metabolic step for mutagenic activation of Trp-P-2.

Microsomal activation of 2-amino-3-methylimidazo[4,5-f]quinoline, a pyrolysate of sardine and beef extracts, to a mutagenic intermediate.

The mechanism involved in the metabolic activation of 2-amino-3-methylimidazo[4,5-f]quinoline, which is a pyrolysate isolated from broiled foods, to a mutagenic intermediate was studied in vitro. In a system containing hepatic microsomes and reduced nicotinamide adenine dinucleotide phosphate, 2-amino-3-methylimidazo[4,5-f]quinoline was converted to a product which was directly mutagenic to Salmonella typhimurium. The structure of the mutagenic metabolite was determined as the 2-N-hydroxy derivative on the basis of the chemical properties and the mass spectral evidence of the azoxy adduct with o-nitrosotoluene. The activation reaction was mediated by microsomal enzymes and was inhibited by carbon monoxide, 7,8-benzoflavone, and other chemicals which were known to inhibit the cytochrome P-450-dependent reaction. With the use of four forms of purified cytochrome P-450, the N-hydroxylation of 2-amino-3-methylimidazo[4,5-f]quinoline and the induction of the reverse mutation of the bacteria were clearly demonstrated to be catalyzed mainly by a high-spin form of cytochrome P-450, P-448 II-a.

Metabolic activation of mutagenic tryptophan pyrolysis products by rat liver microsomes.

In an attempt to determine whether cytochrome P-450 metabolizes tryptophan pyrolysis products, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) to active forms, studies were done using microsomes and the 9000 x g supernatant (S-9) from polychlorinated biphenyl mixture-treated rat livers together with Salmonella typhimurium TA 98 as a tester strain. The number of revertants increased with increments in the amount of S-9 fraction added. The highest mutation was seen with an amount of S-9 that is equivalent to 2.5 mg of liver tissue; however, further increase in the S-9 fraction resulted in a sharp decline in the number of revertants. There was a similar biphasic response when a relatively large amount of S-9 fraction was incubated for an increasing length of time. Microsomes showed parallel biphasic responses, and, in addition, the 105,000 x g supernatant fraction was ineffective in increasing the number of revertants. These results suggested that most if not all of the ability of the S-9 fraction to convert Trp-1 and Trp-P-2 to active forms resided in the microsomes. The involvement of microsomal cytochrome P-450 in this process was further confirmed by the following evidence. The treatment of rats with polychlorinated biphenyl mixture and 3-methylcholanthrene resulted in a marked increase in the ability of microsomes to activate Trp-P-1 and Trp-P-2, and the activation reaction required reduced nicotinamide adenine dinucleotide phosphate as a cofactor and was inhibited by carbon monoxide, 7,8-benzoflavone, n-octylamine, and 2-diethylaminoethyl-2,2-diphenylvalerate. The mutagenic metabolite formed as a result of microsomal metabolism of Trp-P-2 was fairly stable and survived heating at 90 degrees for 7 min. With a high-performance liquid chromatography, an active metabolite of Trp-P-2 was purified. A preliminary analysis showed this molecule to be an N-hydroxylated derivative of Trp-P-2.

Acetyl coenzyme A dependent activation of N-hydroxy derivatives of carcinogenic arylamines: mechanism of activation, species difference, tissue distribution, and acetyl donor specificity.

Acetyl coenzyme A dependent activation of 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-OH-Glu-P-1) and 3-hydroxyamino-1-methyl-5H-pyrido [4,3-b]indole (N-OH-Trp-P-2) was investigated using cytosols from hepatic and extrahepatic tissues of various animal species in comparison with that of N-hydroxy-2-aminofluorene. N-OH-Glu-P-1 and N-OH-Trp-P-2 were metabolized to the reactive species capable of binding to transfer RNA through a putative O-acetylation process by liver cytosols. Kidney, small intestinal mucosa, lung, and bladder from hamsters and rats also mediated the reaction, although their activities were lower than that in the liver. Marked species differences in the enzymatic activities of livers were observed. Hamsters showed the highest ability in the activation for N-OH-Glu-P-1 and N-OH-Trp-P-2, followed by rats. Rabbits with a rapid acetylator phenotype, which showed a high activity in the N-acetylation of arylamines, activated N-OH-Glu-P-1 but scarcely N-OH-Trp-P-2. A rabbit with a slow acetylator phenotype, mice, guinea pigs, and a dog showed marginal or nondetectable activities with N-OH-Glu-P-1 and N-OH-Trp-P-2. A typical nonheterocyclic N-hydroxyarylamine, N-hydroxy-2-aminofluorene was also activated by the acetyl coenzyme A dependent system to an intermediate which bound to transfer RNA. However, the acetyl-CoA dependent binding of N-hydroxy-2-aminofluorene was markedly different from those observed with N-OH-Glu-P-1 and N-OH-Trp-P-2 concerning the order of activities among animal species used. In addition to short chain acyl coenzyme As, N-hydroxy-2-acetylaminofluorene also served as an acetyl donor for the activation of N-OH-Glu-P-1 and N-OH-Trp-P-2 in liver cytosol systems. The formation of N-acetyl-N-OH-Glu-P-1, however, was not detected in the cytosolic system of N-OH-Glu-P-1 with acetyl-CoA, suggesting the direct O-acetylation at the N-hydroxy group as a major pathway for the activation of N-hydroxyarylamines.

Evidence for the involvement of N-hydroxylation of 3-amino-1-methyl-5H-pyrido[4,3-b]indole by cytochrome P-450 in the covalent binding to DNA.

The involvement of N-hydroxylation of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) by cytochrome P-450 in the formation of covalent binding of Trp-P-2 to DNA, the induction of his+ revertant in the Ames test, and the formation of the active metabolite were confirmed. Among four cytochrome P-450 preparations, PCB-P-448 and MC-P-448 purified from liver microsomes of polychlorinated biphenyl (PCB)- and 3-methylcholanthrene-treated rats, respectively, showed higher activities for induction of mutation by Trp-P-2 than did the other two preparations, PCB-P-450 and PB-P-450 purified from PCB-and phenobarbital (PB)-treated rats, respectively. PCB-P-448 was more active than was PB-P-450 in metabolizing Trp-P-2 to N-hydroxylated Trp-P-2 (N-hydroxy-Trp-P-2). Cytochrome P-450 with higher capacity to form the N-hydroxylated metabolite induced a larger number of his+ revertants. Larger amounts of [1-14C]Trp-P-2 bound covalently to DNA were also seen when PCB-P-448 was incubated with calf thymus DNA and reduced nicotinamide adenine dinucleotide phosphate than with PCB-P-450 and PB-P-450. The direct binding of N-[ring-3H]hydroxy-Trp-P-2 isolated by high-performance liquid chromatography to calf thymus DNA was also demonstrated. These results indicate that N-hydroxylation of Trp-P-2 is an obligatory step for the covalent binding to DNA and mutagenesis of Trp-P-2. Based on these results, we propose that N-hydroxy-Trp-P-2 produced by cytochrome P-450 is important in the exertion of the mutagenicity of Trp-P-2 as it binds to DNA.

Catalysis of the covalent binding of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole to DNA by a L-proline- and adenosine triphosphate-dependent enzyme in rat hepatic cytosol.

An enzymatic mechanism involved in the activation of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-hydroxy-Trp-P-2), a mutagenic intermediate of a tryptophan pyrolysate, was studied in vitro. In hepatic cytosol supplemented with adenosine triphosphate and L-proline, N-hydroxy-Trp-P-2 was converted to a form which reacts readily with DNA. The enzyme responsible for the activation was partially purified and identified as prolyl transfer RNA synthetase as judged by their cofactor requirements, inhibition by pyrophosphate or adenosine monophosphate, and copurification of their activities. The prolyl transfer RNA-dependent covalent binding of N-hydroxy-Trp-P-2 to DNA of hepatic cytosol was highest in rats, followed by mice, hamsters, rabbits, and guinea pigs in that order. The capacity for the binding of N-hydroxy-Trp-P-2 was largely consistent with their prolyl transfer RNA synthetase activity. With regard to the ultimate form of N-hydroxy-Trp-P-2 for the covalent binding, a possible formation of N,O-prolyl-3-amino-1-methyl-5H-pyrido[4,3-b]indole was proposed.

Arachidonic acid-dependent peroxidative activation of carcinogenic arylamines by extrahepatic human tissue microsomes.

Prostaglandin H synthase (PHS), an arachidonic acid-dependent peroxidase, has been implicated in the peroxidative activation of carcinogenic aromatic amines in extrahepatic carcinogen target tissues of experimental animals. We have examined the arachidonic acid-dependent activation of [3H]benzidine to DNA-bound products by microsomal preparations from 75 normal human tissues obtained during necessary surgical procedures. For several samples of urinary bladder epithelium, prostatic epithelium, colonic mucosa, and peripheral lung tissue, an arachidonic acid-dependent, microsomal-catalyzed activation of benzidine was observed; and the activity could be inhibited appreciably by indomethacin, a known inhibitor of PHS. Little or no arachidonic acid-dependent activity was detected in human placenta, breast, or liver microsomes or the majority of colon microsomes. Substrate specificity was also examined with purified ram PHS and with human bladder and with active colon preparations. Purified PHS catalyzed the activation of benzidine much greater than 2-naphthylamine, 2-amino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole greater than 4-aminobiphenyl greater than 2-amino-3-methylimidazo[4,5-f]quinoline greater than 3-amino-1-methyl-5H-pyrido[4,3-b] indole. In comparison, human bladder and colon microsomes catalyzed the activation of benzidine greater than 4-aminobiphenyl, 2-amino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole, 2-naphthylamine greater than 2-amino-3-methylimidazo[4,5-f]quinoline, 3-amino-1-methyl-5H-pyrido[4,3-b]indole. To confirm the occurrence of PHS antigen in human extrahepatic tissues, an avidin/biotin-amplified competitive enzyme-linked immunoabsorbent assay was developed with purified ram PHS and a commercially available monoclonal antibody known to cross-react with human platelet PHS. The avidin/biotin-amplified enzyme-linked immunosorbent assay, which detected ng quantities of ram PHS, clearly established the presence of the PHS protein in human bladder, prostate, and lung microsomes. In contrast, PHS antigen was not detected in the liver or placental microsomes. The interindividual and tissue-dependent variability of PHS and its role in aromatic amine carcinogenesis are discussed.

Suppression of clofibrate-induction of peroxisomal and microsomal fatty acid-oxidizing enzymes by growth hormone and thyroid hormone in primary cultures of rat hepatocytes.

Using primary cultures of rat hepatocytes on a matri-gel, effects of peroxisome proliferator and omega-hydroxydodecanoic acid on cellular levels of acyl-CoA oxidase and CYP4A have been studied to determine the hormonal influence in serum-free media. Peroxisomal acyl-CoA oxidation, microsomal CYP4A content and laurate omega-hydroxylation were increased in rat hepatocytes by the addition of 100 microM clofibrate or Wy14,643 for two days. omega-Hydroxydodecanoic acid (100 microM) also increased peroxisomal acyl-CoA oxidation, but had no clear effect on microsomal CYP4A level and laurate omega-hydroxylation. CYP4A-mediated laurate omega-hydroxylation in hepatocytes was suppressed by the addition of pituitary growth hormone (0.05 mU/ml), but was not altered by the addition of triiodothyronine (30 nM). In contrast, clofibrate-mediated induction of acyl-CoA oxidase activity was decreased by the addition of either one of the hormones in hepatocytes. Suppression by those hormones was also observed with omega-hydroxydodecanoic acid-mediated induction of acyl-CoA oxidase activity. These results indicate the possibility that GH and T3 exert the suppressive effects on peroxisomal acyl-CoA oxidation through plural mechanisms with and without the alteration of CYP4A levels in livers.

2-Methoxy-4-nitroaniline and its isomers induce cytochrome P4501A (CYP1A) enzymes with different selectivities in the rat liver.

We reported previously that 2-methoxy-4-nitroaniline (2-MeO-4-NA) is a selective inducer of cytochrome P4501A2 (CYP1A2) in the rat liver, and its molecular size is the smallest among known CYP1A2-selective inducers. In the present study, a structure-activity relationship on the CYP1A2-selective induction has been investigated using isomeric nitroanisidines and their related chemicals. Western blot analyses revealed that the chemicals removed a substituent (amino, methoxyl or nitro group) from a 2-MeO-4-NA molecule had no capacity for inducing CYP1A enzymes in rat livers. On the other hand, isomeric nitroanisidines such as 2-MeO-4-NA, 2-MeO-5-NA and 4-MeO-2-NA induced both CYP1A2 and CYP1A1 enzymes with different selectivities. As judged from the induced levels of CYP1A proteins, 2-MeO-4-NA (CYP1A2/CYP1A1 ratio; 9.5) and 4-MeO-2-NA (0.3) were the most selective inducers of CYP1A2 and CYP1A1, respectively, among the isomeric nitroanisidines (0.44 mmol/kg) used. The induced level of CYP1A2 protein was in the order 2-MeO-4-NA > 2-MeO-5-NA > 4-MeO-2-NA, although no significant difference was observed on their CYP1A2 mRNA level. On the contrary, increases in the levels of CYP1A1 mRNA and protein were in the order 4-MeO-2-NA > 2-MeO-5-NA > 2-MeO-4-NA. The present findings indicate that all three substituents (amino, methoxyl and nitro groups) are necessary components of nitroanisidines for induction of CYP1A enzymes, and also show that regio-isomeric positions of these substituents determine the selectivity in the induction of CYP1A enzymes.

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.

Hepatic microsomal tolbutamide hydroxylation in Japanese: in vitro evidence for rapid and slow metabolizers.

Microsomal hydroxylation of tolbutamide in Japanese livers was studied in vitro to ascertain the enzyme catalysing this reaction. Rates of tolbutamide hydroxylation differed individually 33-fold and 42-fold at 0.1 mM and 2.4 mM tolbutamide concentrations, respectively, and were segregated into two groups, rapid and slow metabolizers. An antibody raised against P450 human-2 (a form of CYP2C9) strongly inhibited the hydroxylation in livers of rapid metabolizers but only weakly inhibited in the slow metabolizer. Kinetic experiments further demonstrated a clear distinction in tolbutamide hydroxylation between two groups; the mean of apparent Km values for tolbutamide was 0.25 mM (n = 3) in the rapid group and 2.58 mM (n = 2) in the slow, respectively. These data suggest that different enzymes are involved in the hydroxylation in both metabolizer groups. Furthermore, CYP2C9 produced by cDNA expression in yeasts, catalysed tolbutamide hydroxylation at rates similar to the rapid metabolizer group at both the 0.1 mM and 2.4 mM concentrations. The apparent Km value of the expressed protein for tolbutamide, 0.26 mM, was similar to that determined for the rapid group of microsomal samples. Clear correlations were observed between the rate of microsomal tolbutamide hydroxylation at 0.1 mM and CYP2C9 protein content or the rate of S-mephenytoin 4'-hydroxylation in human liver. These results indicate that considerable portions of microsomal tolbutamide hydroxylation are catalysed by CYP2C9 or the closely related form in the rapid metabolizers.

Lack of low Km diazepam N-demethylase in livers of poor metabolizers for S-mephenytoin 4'-hydroxylation.

Metabolism of diazepam was studied in vitro to identify the forms of cytochrome P450 (CYP) responsible for N-demethylation (nordazepam formation) and 3-hydroxylation (temazepam formation), using liver microsomes obtained from extensive (EM) and poor metabolizers (PM) for S-mephenytoin 4'-hydroxylation. Involvement of at least two P450 forms in diazepam N-demethylation was suggested by a biphasic pattern in Lineweaver-Burk and Eadie-Hofstee plots from the EM, whereas a monophasic pattern was observed from the PM liver microsomes. The kinetic parameters for the N-demethylation in the EM group were: Km 1, 19.4 +/- 0.4 microM; Vmax 1, 0.27 +/- 0.04 nmol min-1 per mg protein; Km 2, 346 +/- 34 microM; Vmax2, 1.82 +/- 0.63 nmol min-1 per mg protein (n = 3, mean +/- SD). The PM group showed the mean values of Km and Vmax (Km, 319 +/- 30 microM; Vmax, 1.49 +/- 0.62 nmol min-1 per mg protein) (n = 3) similar to those of Km2 and Vmax2 in the EM group. An antibody raised against CYP2C9 (anti-human CYP2C) strongly inhibited diazepam N-demethylation in EM liver microsomes at a low substrate concentration (20 microM). However, the anti-human CYP2C showed no clear inhibition of N-demethylation in EM liver microsomes at a high substrate concentration (200 microM). Diazepam N-demethylation in PM liver microsomes was not clearly inhibited by the anti-human CYP2C at either the low or high substrate concentrations. These data suggest that different P450 forms mediated diazepam N-demethylation in EM and PM liver microsomes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome P450 mediated metabolism of diazepam in human and rat: involvement of human CYP2C in N-demethylation in the substrate concentration-dependent manner.

Metabolism of diazepam (DZP) was studied in vitro to clarify the involvement of different forms of hepatic cytochrome P450 (CYP) in rats, and humans of Japanese and Caucasian origin. Microsomal 3-hydroxylation was the major pathway of DZP metabolism in rats and was inhibited by anti-CYP3A antibodies. Purified CYP3As and CYP2C11 catalysed 3-hydroxylation and N-demethylation, respectively, in the reconstituted systems. The rates of both reactions in human liver microsomes depended on the substrate concentration: the rate of 3-hydroxylation was 3-4 times higher than N-demethylation at 0.2 mM; the two activities were essentially the same at a lower substrate concentration (0.02 mM). Inhibitions of the N-demethylation by anti-CYP2C antibody and S-mephenytoin also depended on the substrate concentration and was detectable only at a low substrate concentration. Kinetic studies revealed the presence of two distinct catalytic activities for the N-demethylation; low Km and low Vmax, and high Km and high Vmax. The former activity seems to be mediated by a CYP2C P450 form. On the other hand, DZP 3-hydroxylation was rather selectively catalysed by a CYP3A P450 at the low and high substrate concentrations. These results were consistent with the observation in vivo that DZP N-demethylation and S-mephenytoin 4'-hydroxylation are closely correlated in humans. These results also suggest that the apparent discrepancy on the role of CYP forms in DZP metabolism in vitro and in vivo may reside in the difference in substrate concentration.

Primary structure and molecular basis of polymorphic appearance of an acetyltransferase (AT-II)* in hamsters.

Three hamster clones (clones 1, 2 and 3) were isolated from a genomic library constructed from a homozygote rapid acetylator using a cDNA (hamAT-101) of a monomorphic acetyltransferase (AT-I) as a probe. Clone 1 (13 kbp) was found to contain a gene corresponding to AT-I. The entire coding region was located in an exon and completely identical to that of AT-I cDNA. Clones 2 and 3 (14.5 and 15 kbp) each contained identical information to the AT-I-related protein (AT-B protein). The intronless coding region shared 83.7% of sequence similarly to the AT-I cDNA, and its length was identical to that of the AT-I cDNA. Clone 2 also included a nucleotide sequence identical to the 3'-portion of the AT-I gene, which is located 5'-upstream of the AT-B gene. Restricted fragment lengths of clone 3, which encompassed the entire coding region was expressed in COS-1 cells. The expressed protein migrated at a position identical to that of AT-II purified from a hamster liver on Western blots. AT-B-expressed protein catalysed acetyl CoA-dependent N-acetylation of 2-aminofluorene and p-aminobenzoic acid, but had marginal activities for O-acetylation of 2-N-hydroxyamino-6-methyl-6-methyldipyrido[1,2-a:3',2'-d]imidazole and N-hydroxyarylacetamide-dependent N-acetylation of 4-aminoazobenzene. These results are in good agreement with the data of AT-II purified from hamster livers, indicating that the AT-B gene encodes a polymorphic acetyltransferase (AT-II) in hamsters. Although the AT-II protein was undetectable in slow acetylators, specific mRNA, hybridizing with a selective oligonucleotide probe for the AT-II gene (AT-B), was detected in livers of both homozygous acetylators. Analysis of genomic DNA of a homozygous slow phenotype hamster indicates that AT-II DNA from the slow phenotype has a point mutation which causes premature termination at the 243th (Arg to stop codon) position of the deduced amino acid sequence. PCR-RFLP analysis further confirmed that the point mutation conferred a defective AT-II protein in slow phenotype hamsters.

Primary structures and properties of two related forms of aryl sulfotransferases in human liver.

cDNAs and genes of ST1A2 and ST1A3 section were isolated from human liver cDNA and genomic libraries and shown to encode arylsulfotransferases. Studies on their mRNAs and proteins expressed indicate the existence of two distinct phenol-sulfating enzymes which are involved in the metabolic activation of carcinogenic arylamines in human livers.

Isolation and characterization of a new rat P450 (CYP3A18) cDNA encoding P450(6)beta-2 catalyzing testosterone 6 beta- and 16 alpha-hydroxylations.

A cytochrome P450 cDNA, encoding a new form of CYP3A protein, was isolated from a liver cDNA library of a male rat using anti-P450(6)beta-1 and anti-P450(6)beta-2 antibodies and the CYP3A2 cDNA. The cDNA (CYP3A18 cDNA) consisted of 1987 nucleotides, in which were contained an open reading frame of 1491 bp (corresponding to 497 amino acids), 5'-(59 bp) and 3'-noncoding regions (437 bp). The deduced amino acid sequence of CYP3A18 cDNA was completely identical in the first 27 N-terminal residues of P450(6)beta-2 previously isolated by us (Nagata et al. J Biochem 1990: 107, 718-725) from livers of rats treated with dexamethasone, and also shared higher extents of similarity with hamster CYP3A10 (78.5%) than with rat CYP3As previously sequenced (66.3-69.3%). Northern blot analyses indicated a male-dominant expression of this new CYP3A mRNA and enhanced expression in dexamethasone-or pregnenolone-16 alpha-carbonitrile (PCN)-treated, but not phenobarbital-or 3-methylcholanthrene-treated rats. Expressed CYP3A18 protein in COS-1 cells migrated at a position identical to that of purified P450(6)beta-2 on sodium dodecyl sulfate-acrylamide gel electrophoresis and catalyzed 16 beta- and 6 alpha-hydroxylations of testosterone. In contrast to CYP3A1 and CYP3A2, cytochrome b5 was not essential for maximal catalytic activities of recombinant CYP3A18 protein. These results, together with ontogenic profiles of CYP3A18 mRNA and P450(6)beta-2 protein, indicate that the newly isolated CYP3A18 cDNA encodes P450(6)beta-2 in rat liver.

Specific CYP3A4 inhibitors in grapefruit juice: furocoumarin dimers as components of drug interaction.

Four components were isolated from grapefruit juice that inhibit human CYP3A-mediated drug oxidation. The structures of these compounds were identified as furocoumarin derivatives by absorption spectra, APCI-liquid chromatography/tandem mass spectrometry and nuclear magnetic resonance after their purification by reversed-phase high performance liquid chromatography. They include two new furocoumarins, 4-[[6-hydroxy-7-[[1-[(1-hydroxy-1-methyl)ethyl]-4-methyl-6- (7-oxo-7H-furo[3,2-g][1]benzopyran-4-yl)-4-hexenyl]oxy]-3,7-dimeth yl- 2-octenyl] oxy]-7H-furo[3,2-g][1]benzopyran-7-one (GF-I-1) and 4-[[6-hydroxy-7-[[4-methyl-I- (1-methylethenyl)-6-(7-oxo-7H-furo[3,2-g][1]benzopyran-4-yl)-4- hexenyl] oxy]-3,7-dimethyl-2-octenyl]oxy]-7H-furo[3,2-g][1]benzopyran-7-one (GF-I-4). These furocoumarins are strong candidates for causative agents of grapefruit juice-mediated drug interaction, because of an inhibition potential that is equal to or stronger than the prototypical CYP3A4 inhibitor, ketoconazole, on liver microsomal testosterone 6 beta-hydroxylation.

Polymorphism in hydroxylation of mephenytoin and hexobarbital stereoisomers in relation to hepatic P-450 human-2.

Stereoselective 4'-hydroxylations of R-(-)-mephenytoin and S-(+)-mephenytoin and 3'-hydroxylation of R-(-)-hexobarbital and S-(+)-hexobarbital were determined in liver microsomes of 14 Japanese subjects who were extensive metabolizers of mephenytoin and in five Japanese subjects who were poor metabolizers of mephenytoin. Content of P-450 human-2 assessed by Western blots was correlated to microsomal S-(+)-mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' alpha-hydroxylation, and S-(+)-hexobarbital 3' beta-hydroxylation, and was less correlated to R-(-)mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' beta-hydroxylation, and S-(+)-hexobarbital 3' alpha-hydroxylation. Antibodies raised against P-450 human-2 inhibited microsomal S-(+)-mephenytoin 4'-hydroxylation efficiently but was less efficient on R-(-)-mephenytoin 4'-hydroxylation in extensive metabolizers and on 4'-hydroxylation of mephenytoin enantiomers in poor metabolizers. The antibodies also inhibited R-(-)-hexobarbital 3' alpha-hydroxylation and S-(+)-hexobarbital 3' beta-hydroxylation but did not effectively inhibit the hydroxylation of the two other optical isomers of hexobarbital in extensive metabolizers and of four stereoisomers in poor metabolizers. These findings indicate the close relationship between polymorphic mephenytoin 4'-hydroxylation and two stereospecific hexobarbital hydroxylations, and they suggest that P-450 human-2 is a typical S-(+)-mephenytoin 4'-hydroxylase and a major hexobarbital 3'-hydroxylase in the livers of extensive metabolizers. The findings were further supported by the experiments that used P-450 human-2 complementary dexoyribonucleic acid-derived protein in yeast microsomes.