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.

Role for enhanced faecal excretion of bile acid in hydroxysteroid sulfotransferase-mediated protection against lithocholic acid-induced liver toxicity.

The efficient clearance of toxic bile acids such as lithocholic acid (LCA) requires drug-metabolizing enzymes. We therefore assessed the influence of pregnenolone 16alpha-carbonitrile (PCN) treatment on LCA-induced hepatotoxicity and disposition of LCA metabolites using female farnesoid X receptor (FXR)-null and wild-type mice. Marked decreases in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, and hepatic tauroLCA (TLCA) concentrations were found in LCA-fed wild-type mice co-treated with PCN. Whereas induction of Cyp3a and hydroxysteroid sulfotransferase (Sult2a) proteins was observed in FXR-null and wild-type mice, clear increases in biliary 3alpha-sulfated TLCA but not total 6alpha-hydroxy LCA (taurohyodeoxycholic acid and hyodeoxycholic acid) were only observed in PCN-treated wild-type mice. Biliary 3alpha-sulfated TLCA output rate was increased 7.2-fold, but accounts for only 4.2% of total bile acid output rate in LCA and PCN-co-treated wild-type mice. Total 3alpha-sulfated LCA (LCA and TLCA) was, however, the most abundant bile acid component in faeces suggesting that efficient faecal excretion of biliary 3alpha-sulfated TLCA through escape from enterohepatic circulation. FXR-null mice, which have constitutively high levels of the Sult2a protein, were fed a diet supplemented with 1% LCA and 0.4% dehydroepiandrosterone (DHEA), a typical Sult2a substrate/inhibitor. The faecal total 3alpha-sulfated bile acid excretion was reduced to 62% of FXR-null mice fed only the LCA diet. Hepatic TLCA concentration and serum AST activity were significantly higher in FXR-null mice fed DHEA and LCA diet than in FXR-null mice fed the LCA diet or DHEA diet. These results suggest that hepatic formation of 3alpha-sulfated TLCA is a crucial factor for protection against LCA-induced hepatotoxicity.

Prediction of oral clearance from in vitro metabolic data using recombinant CYPs: comparison among well-stirred, parallel-tube, distributed and dispersion models.

Intrinsic clearances (CLint-HLM-total) for the metabolism of NE-100, metoprolol, clarithromycin (CAM), lornoxicam and tenoxicam were predicted from in vitro data with recombinant cytochorme P450s (CYPs) using relative activity factor (RAF) and then compared with CLint-HLM observed in human liver microsomes (HLM). The predicted CLint-HLM-total correlated well with the observed CLint-HLM in HLM. When oral clearances (CLoral) of low-clearance drugs such as metoprolol, CAM, lornoxicam and tenoxicam were predicted from the in vitro data using four physiological models (well-stirred, parallel tube, distributed and dispersion models), the predicted CLoral corresponded well with the observed CLoralin vivo and were similar among the four models. For a high-clearance drug, the predicted CLoral of NE-100 in extensive CYP2D6 metabolizers (EMs) was substantially different between individual models, although the predicted CLoral in a poor metabolizer of CYP2D6 (PMs) was similar. The CLoral ratio of NE-100 between the EMs and the PMs predicted from the dispersion model, which leads to a reliable prediction for the high-clearance drug, was 48.4, but the ratio decreased depending on the increase of the NE-100 plasma concentration. The results suggest that the CLoral decrease in the EMs is caused by saturation of NE-100 metabolism mediated by CYP2D6 and is based on increases in plasma NE-100 concentrations dependent on the dose of NE-100. The study suggests that the RAF and the in vitro-in vivo scaling approaches are useful for predicting CLoral from in vitro data with recombinant CYPs without using HLM and hepatocytes.

Isolation and characterization of a new major intestinal CYP3A form, CYP3A62, in the rat.

Based on information of the nucleotide sequence obtained from rat genome clones, a new CYP3A (CYP3A62) cDNA was isolated from the cDNA library of a rat liver. The CYP3A62 cDNA was 1746 base pairs (bp) in length, which included 1491 bp of an open reading frame and 93 bp and 209 bp of the respective 5'- and 3'-noncoding regions. Amino acid sequence deduced from CYP3A62 cDNA shared the highest similarity with rat CYP3A9 (79.9%) among human and rat CYP3A forms previously reported. CYP3A62 mRNA and protein were consistently detected in small intestines as well as livers. CYP3A62 was a major form in small intestines of both sexes but was a female-predominant form in livers of adult rats. CYP3A62 in both tissues of male and female rats were clearly enhanced by the treatment with dexamethasone. These expression profiles resembled those of CYP3A9. Despite clear detection of CYP3A62, no detectable levels of CYP3A1 and CYP3A2 proteins, as well as those of mRNAs, were found in the intestinal tract. Therefore, CYP3A62 may play major roles together with CYP3A9 and CYP3A18 in endogenous or exogenous detoxification at the absorption site.

Prediction of differences in in vivo oral clearance of N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl] ethylamine monohydrochloride (NE-100) between extensive and poor metabolizers from in vitro metabolic data in human liver microsomes lacking CYP2D6 activity and recombinant CYPs.

1. It has previously been reported that N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) was predominantly metabolized by cytochrome P450 (CYP) 2D6 in human liver microsomes (HLM). In the present study, the contribution of CYP forms involved in the formation of the major metabolites of NE-100 in human liver lacking CYP2D6 activity (PM-HLM) has been predicted by use of in vitro kinetic data on recombinant CYPs microsomes (rCYPs). 2. In PM-HLM, NE-100 is predicted to be metabolized to N-despropyl-NE-100 (NE-098), p-hydroxy-NE-100 (NE-152) and m-hydroxyl-NE-100 (NE-163), but not to O-demethy-NE-100 (NE-125), which is a major metabolite in pooled human liver microsomes (EM-HLM). The relative activity factor approach assumed that NE-098 formation is predominantly catalysed by CYP3A4 and CYP2C9 and the NE-152+163mix (a mixture of two hydroxylated metabolites, NE-152 and NE-163) formation is only catalysed by CYP3A4. 3. The predicted contribution rates of CYP3A4 and CYP2C9 for NE-098 formation were 58.1 and 34.6%, respectively, in PM-HLM. These predicted results were strongly supported by kinetic and inhibition studies using PM-HLM. The intrinsic clearance of NE-100 predicted from rCYPs (the predicted CLint-HLM-total) corresponded to those observed from EM- and PM-HLM (the observed CLint-HLM). 4. The in vivo oral clearance (CLoral) of NE-100 in extensive metabolizers and poor metabolizers of CYP2D6 was predicted to be 50times higher in extensive metabolizers than poor metabolizers using in vitro-in vivo scaling method based on the dispersion model. These data suggest that polymorphism of CYP2D6 might greatly affect NE-100 metabolism in vivo.

In vitro inhibition of human small intestinal and liver microsomal astemizole O-demethylation: different contribution of CYP2J2 in the small intestine and liver.

1. The effects of chemical agents on the metabolism of the antihistamine drug astemizole were investigated to evaluate drug-drug interactions. 2. Chemical inhibitors of astemizole O-demethylation were screened using the small intestinal and liver microsomes from rabbit as an animal model for the first-pass metabolism of humans. In the rabbit small intestine, astemizole O-demethylation was clearly inhibited by ebastine, arachidonic acid, alpha-naphthoflavone, ketoconazole, tranylcypromine, troglitazone and terfenadine. 3. In humans, these inhibitors also reduced microsomal astemizole O-demethylation in both the small intestine and liver. However, the inhibition rate of almost all these chemicals were clearly greater in the small intestine than in the liver. Thus, a different contribution of cytochrome p450 in each tissue is suggested. 4. All the chemicals inhibited astemizole O-demethylation in recombinant CYP2J2 microsomes. The results suggest that CYP2J2 is involved in astemizole O-demethylation in both the human small intestine and liver; however, the contribution in the liver is lower than in the small intestine. The effects of the CYP2J2 inhibitors during first-pass metabolism may be more important in the small intestine than in the liver. Since all the inhibition profiles of astemizole O-demethylation were different in the liver and small intestine, involvement of another p450 in astemizole O-demethylation in human liver may be speculated. 5. In the rabbit microsomal systems, the same metabolites found in humans were qualitatively detected and the inhibition profiles of the chemical agents in the microsomes resembled that of humans.

Identification of ST2A1 as a rat brain neurosteroid sulfotransferase mRNA.

A hydroxysteroid sulfotransferase (ST2A1) was identified as a form mediating neurosteroid sulfation in rat brain. The sole expression among known rat ST2A forms was indicated by brain RT-PCR. All nucleotide sequences of seven ST2A cDNA clones isolated from brain matched completely with that of hepatic ST2A1. The recombinant ST2A1 protein mediated neurosteroid sulfation. These data strongly suggest a functional role of ST2A1 as a neurosteroid sulfotransferase in rat brain.

Mechanism of 7,12-dimethylbenz[a]anthracene-induced immunotoxicity: role of metabolic activation at the target organ.

The polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene (DMBA), is an immunosuppressor as well as a potent organ-specific carcinogen. To understand the organ-specific mechanism of DMBA-induced lymphoid toxicity, aryl hydrocarbon-nonresponsive mice and microsomal epoxide hydrolase (mEH)-null mice were analyzed. DMBA caused a dose-dependent decrease in spleen weights, but not the thymus weights in aryl hydrocarbon-nonresponsive mice. On the other hand, both spleen and thymus weights were decreased to less than a half in wild-type mice exposed to 30 mg/kg of DMBA. In contrast, no decrease was detected in spleen weights of mEH-null mice exposed to up to 100 mg/kg of DMBA, while thymus weights were markedly lower. Responses to the B-cell mitogen lipopolysaccharide and to T-cell mitogen phytohemagglutinin were nearly completely abolished in splenocytes isolated from wild-type mice treated with 100 mg/kg of DMBA. These responses were decreased, but maintained in splenocytes isolated from mEH-null mice treated with DMBA. Two DMBA metabolites dependent on mEH including DMBA-3,4-diol were detected in an HPLC chromatogram of spleen microsomes isolated from wild-type mice, but not those from mEH-null mice. These results suggest the involvement of mEH in splenic activation of DMBA for immunotoxicity and the difference for the DMBA-induced lymphoid toxicity between spleen and thymus.

[Current availability of genetic information of drug metabolizing enzymes, particularly on cytochrome P450(CYP)].

Genetic polymorphisms of drug metabolizing enzymes often associated with clinical alteration among individuals. Current understanding of the molecular basis of the polymorphism of drug metabolizing enzymes is reviewed, particularly on cytochrome P450(CYP). Pharmacogenetic/pharmacogenomic studies are started for the estimation of pharmacokinetics of drugs, but now expanding for a tool of individual optimization of drug therapy for both drug efficacy and safety, and also for the development of new drugs. Genotyping is a powerful tool for these purposes, but not always simple because of the existence of more than 20 alleles for a single enzyme in some cases. Accumulated evidence, however, shows that occurrences of ethnic difference are rather common for allele distribution of drug metabolizing enzymes. These data indicate the necessity of the genetic analyses of Japanese population prior to the application of the genetic information for drug therapy.

Inhibitory potential of herbal medicines on human cytochrome P450-mediated oxidation: properties of umbelliferous or citrus crude drugs and their relative prescriptions.

To investigate the possible drug interaction with herbal medicine, hot water decoctions or 40% ethanol infusions of several Umbelliferous or Citrus crude drugs and their prescriptions were examined in vitro for their abilities to inhibit human cytochrome P450 3A (CYP3A). Addition of each decoction or infusion from Baizhi (Angelica dahurica and varieties), Qianghuo (Notopterygium incisum or N. forbesii), Duhuo (Angelica biserrata), Fangfeng (Saposhnikovia divaricata), Danggui (Angelicasinensis), Zhishi or Zhiqiao (Citrus aurantium) resulted in various degrees of human CYP3A inhibition as determined by microsomal testosterone 6beta-hydroxylation. The inhibitory potency was consistent with the abundance of the hydrophobic components for each sample. Experiments on the infusion of a Japanese Baizhi (BZ1) showed the major role of furanocoumarins on human CYP3A inhibition. Some of the crude drugs and a related prescription showed increased inhibition after the preincubation, suggesting the involvement of a mechanism-based inhibition. Some formulated prescriptions, however, showed intense inhibition with their hydrophobic fractions rather than with their hydrophobic fractions, suggesting that components other than furanocoumarins in herbal prescriptions may also cause CYP3A inhibition. These results indicate the necessity of intensive investigations on the possible drug interaction with traditional medicines.

Involvement of multiple human cytochromes P450 in the liver microsomal metabolism of astemizole and a comparison with terfenadine.

AIMS: The aims of the present study were to investigate the metabolism of astemizole in human liver microsomes, to assess possible pharmacokinetic drug-interactions with astemizole and to compare its metabolism with terfenadine, a typical H1 receptor antagonist known to be metabolized predominantly by CYP3A4. METHODS: Astemizole or terfenadine were incubated with human liver microsomes or recombinant cytochromes P450 in the absence or presence of chemical inhibitors and antibodies. RESULTS: Troleandomycin, a CYP3A4 inhibitor, markedly reduced the oxidation of terfenadine (26% of controls) in human liver microsomes, but showed only a marginal inhibition on the oxidation of astemizole (81% of controls). Three metabolites of astemizole were detected in a liver microsomal system, i.e. desmethylastemizole (DES-AST), 6-hydroxyastemizole (6OH-AST) and norastemizole (NOR-AST) at the ratio of 7.4 : 2.8 : 1. Experiments with recombinant P450s and antibodies indicate a negligible role for CYP3A4 on the main metabolic route of astemizole, i.e. formation of DES-AST, although CYP3A4 may mediate the relatively minor metabolic routes to 6OH-AST and NOR-AST. Recombinant CYP2D6 catalysed the formation of 6OH-AST and DES-AST. Studies with human liver microsomes, however, suggest a major role for a mono P450 in DES-AST formation. CONCLUSIONS: In contrast to terfenadine, a minor role for CYP3A4 and involvement of multiple P450 isozymes are suggested in the metabolism of astemizole. These differences in P450 isozymes involved in the metabolism of astemizole and terfenadine may associate with distinct pharmacokinetic influences observed with coadministration of drugs metabolized by CYP3A4.

Enzymatic characterization and interspecies difference of phenol sulfotransferases, ST1A forms.

Cytosolic sulfotransferases, which mediate activation and detoxification of both endogenous and exogenous compounds, consist of at least five different gene families (ST1 to ST5) in mammals. Several cDNAs corresponding to ST1A forms have been reported, but their functional properties are not well characterized. In addition, only a single form of ST1A sulfotransferase has been reported in each experimental animal species despite the expressions of plural forms in humans. Therefore, enzymatic properties of human ST1A3, ST1A5, rat ST1A1, mouse St1a4, and newly isolated rabbit ST1A8 have been characterized and compared by use of their recombinant proteins to clarify the functional difference between human and experimental animal ST1A forms. From the results using more than 25 phenolic chemicals, all the experimental animal ST1A forms showed substrate specificities similar to human ST1A3 rather than ST1A5. They showed high affinities toward p-nitrophenol and 6-hydroxymelatonin as found in human ST1A3. These forms also showed high activities toward umbelliferone and naringenin, but very low activities toward catecholamines, representative substrates of human ST1A5. Hepatic contents of experimental animal ST1A forms varied (66-250 pmol/mg of cytosolic protein) but showed the same order as observed with human ST1A3 (120 pmol/mg). Hepatic content of human ST1A5 was about 19-fold less than that of ST1A3. Therefore, ST1A forms identified in experimental animal species correspond to human ST1A3 functionally. For chemicals such as troglitazone and 2-amino-4'-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine, clear species differences were detected among the ST1A forms examined.

Hepatotoxicity of acetaminophen and N-acetyl-p-benzoquinone imine and enhancement by fructose.

1. Although oral administration of 400 mg/kg acetaminophen (APAP) or 1.8-3.4 g/kg sucrose had no effect on serum levels of alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH), their co-administration resulted in 20-fold increases in ALT/SDH activities. APAP alone (1250 mg/kg, p.o.) caused the elevation hepatotoxicity parameters, but the levels were lower than observed with co-administration of APAP (400 mg/kg) and sucrose (2.6 or 3.4 g/kg). 2. Sucrose-associated increase in serum ALT/SDH activities was selective with APAP and not detected with carbon tetrachloride (160 mg/kg, i.p.), D-galactosamine (400 mg/kg, i.p.) or alpha-naphthyl isothiocyanate (100 mg/kg, p.o.). 3. To verify the synergistic mechanism of sucrose, a major reactive intermediate of APAP, N-acetyl-p-benzoquinone imine (NAPQI), was given via the portal vein to rat pretreated with sucrose. Clear elevation of ALT/SDH activities was detected in the co-treated group. These results, together with an allopurinol-inhibition experiment, suggest the involvement of high-dose sucrose at a step(s) occurring after the metabolic activation of APAP. 4. Co-administration of glucose or fructose as well as sucrose elevated APAP-induced hepatotoxicity parameters in rat. Fructose but not glucose elevated APAP- or NAPQI-induced LDH leakage in a primary hepatocyte system. The results suggest the primary role of fructose is on the sucrose enhancement of APAP toxicity in rat.

Intestinal cytochrome P450 and response to rifampicin in rabbits.

Properties of cytochrome P450 (P450) in rabbit intestines have been investigated to assess the possibility of an experimental model for human intestinal oxidation of drugs. Significant amounts of P450 and cytochrome b5 and activities of NADPH-cytochrome P450 reductase were detected in microsomes from rabbit duodenal, jejunal, ileac and colon mucosa. All the small intestinal fractions mediated phenytoin, dextromethorphan and testosterone oxidations. Several P450 forms belonging to the CYP1A, CYP2C, CYP2D and CYP3A, but not CYP2B and CYP2E, subfamilies were detected in these tissues by Western blotting. A good correlation was observed between immunodetectable levels of CYP3A and activities of testosterone 6beta-hydroxylation. Small intestine, but not colon, CYP3A levels were increased by the pretreatment of rabbits with rifampicin (50 mg/kg for 4 days, p.o.). The extent of the increase was similar between duodena and livers. These properties of rabbit intestinal P450s were comparable to those of human intestine. These phenomena suggest the possibility that the rabbit is a beneficial in vivo model for the assessment of drug interaction occurring at the first pass of drugs ingested.

Inhibitory effect of natural furanocoumarins on human microsomal cytochrome P450 3A activity.

To investigate the possible drug interaction with herbal medicine, furanocoumarin derivatives isolated from several Umbelliferous crude drugs were examined for their inhibitory effects on a typical human drug metabolizing enzyme, cytochrome P450 3A (CYP3A). Most furanocoumarins tested at 0.1 mM reduced microsomal testosterone 6beta-hydroxylation as an index of CYP3A activity to less than 50% of the control. In particular, the dimer and trimer derivatives of furanocoumarins showed striking inhibition, whose potencies were similar to that of a typical CYP3A inhibitor, ketoconazole. Preincubation of dimer types of furanocoumarins increased suppression but not most of the monomer derivatives, suggesting that the inhibition on CYP3A activity was caused by at least plural mechanisms. These results raised the possibility that the furanocoumarin containing herbal medicines may alter pharmacokinetics of co-ingested drugs similar to the case with grapefruit juice.

Pharmacogenetics of sulfotransferase.

Cytosolic sulfotransferase catalyzes sulfoconjugation of relatively small lipophilic endobiotics and xenobiotics. At least 44 cytosolic sulfotransferases have been identified from mammals, and based on their amino acid sequences, these forms are shown to constitute five different families. In humans, 10 sulfotransferase genes have been identified and shown to localize on at least five different chromosomes. The enzymatic properties characterized in the recombinant forms indicate the association of their substrate specificity with metabolisms of such nonpeptide hormones as estrogen, corticoid, and thyroxine, although most forms are also active on the sulfation of various xenobiotics. Genetic polymorphisms are observed on such human sulfotransferases as ST1A2, ST1A3, and ST2A3.

Role of furanocoumarin derivatives on grapefruit juice-mediated inhibition of human CYP3A activity.

With juices of grapefruit and related fruits, possible relationships between contents of six different furanocoumarins and extents of inhibition of microsomal CYP3A activity have been studied in vitro. Microsomal CYP3A-mediated testosterone 6beta-hydroxylation was inhibited by the addition of a fruit juice (2.5%, v/v) from eight different grapefruit sources, two sweeties, three pomelos, and one sour orange, whereas no clear inhibition was observed with two sweet orange juices. The inhibitory component in grapefruit juice resides mainly in the precipitate rather than in the supernatant after centrifugation. Higher amounts of (R)-6',7'-dihydroxybergamottin (DHB) were distributed in the supernatant, whereas GF-I-1, GF-I-2, GF-I-4, and the newly isolated GF-I-5 and GF-I-6 were detected predominantly in the precipitate. Mixing of five representative furanocoumarins at their detectable levels in grapefruit juice reproduced roughly the inhibitory potencies of grapefruit juice, but omission of any of the components resulted in decreased potencies. These results suggested that all the major furanocoumarins contributed to the CYP3A inhibitory properties of grapefruit juice. Furthermore, all six furanocoumarins showed stronger CYP3A inhibitory potencies after preincubation in the presence of NADPH, suggesting that both competitive and mechanism-based inhibition occur in a grapefruit juices-drug interaction.

Amounts and variation in grapefruit juice of the main components causing grapefruit-drug interaction.

A method for the determination of three furocoumarins containing two new chemicals (GF-I-1 and GF-I-4) in commercially available grapefruit juice and grapefruit itself was developed using high-performance liquid chromatography (HPLC). These components isolated from grapefruit juice have 5-geranyloxyfurocoumarin dimer structures showing extremely high affinities for a form of cytochrome P450 (CYP3A4). Considerable differences were observed on the contents among commercial brands and also batches. The contents were determined to be 321.4+/-95.2 ng/ml GF-I-1, 5641.2+/-1538.1 ng/ml GF-I-2 and 296.3+/-84.9 ng/ml GF-I-4 in twenty-eight white grapefruit juices. These chemicals were not detected in beverages from orange, apple, grape and tangerine, except that trace amount of GF-I-2 and GF-I-4 were found in lemon juice. The average levels of these furocoumarins were lower in the juice from red grapefruit than a white one. The highest level of these components were found in the fruit meat.

Inhibition selectivity of grapefruit juice components on human cytochromes P450.

Five compounds including furanocoumarin monomers (bergamottin, 6', 7'-dihydroxybergamottin (DHB)), furanocoumarin dimers (4-¿¿6-hydroxy-71-¿(1-hydroxy-1-methyl)ethyl-4-methyl-6-(7-oxo-7H- furo¿3,2-g1benzopyran-4-yl)-4-hexenyl]oxy]-3,7-dimethyl- 2-octenyl]oxy]-7H-furo[3,2-g]¿1benzopyran-7-one (GF-I-1) and 4-¿¿6-hydroxy-7¿¿4-methyl-1-(1-methylethenyl)-6-(7-oxo-7H-furo¿3, 2-g1benzopyran-4-yl)-4-hexenyloxy-3, 7-dimethyl-2-octenyloxy-7H-furo¿3,2-g1benzopyran-7-one (GF-I-4)), and a sesquiterpene nootkatone have been isolated from grapefruit juice and screened for their inhibitory effects toward human cytochrome P450 (P450) forms using selective substrate probes. Addition of ethyl acetate extract of grapefruit juice into an incubation mixture resulted in decreased activities of CYP3A4, CYP1A2, CYP2C9, and CYP2D6. All four furanocoumarins clearly inhibited CYP3A4-catalyzed nifedipine oxidation in concentration- and time-dependent manners, suggesting that these compounds are mechanism-based inhibitors of CYP3A4. Of the furanocoumarins investigated, furanocoumarin dimers, GF-I-1 and GF-I-4, were the most potent inhibitors of CYP3A4. Inhibitor concentration required for half-maximal rate of inactivation (K(I)) values for bergamottin, DHB, GF-I-1, and GF-I-4 were calculated, respectively, as 40.00, 5. 56, 0.31, and 0.13 microM, whereas similar values were observed on their inactivation rate constant at infinite concentration of inhibitor (k(inact), 0.05-0.08 min(-1)). Apparent selectivity toward CYP3A4 does occur with the furanocoumarin dimers. In contrast, bergamottin showed rather stronger inhibitory effect on CYP1A2, CYP2C9, CYP2C19, and CYP2D6 than on CYP3A4. DHB inhibited CYP3A4 and CYP1A2 activities at nearly equivalent potencies. Among P450 forms investigated, CYP2E1 was the least sensitive to the inhibitory effect of furanocoumarin components. A sesquiterpene nootkatone has no significant effect on P450 activities investigated except for CYP2A6 and CYP2C19 (K(i) = 0.8 and 0.5 microM, respectively).