Hepatic Microsomal Preparations Research Articles

A Comparative Study of Phase I and II Hepatic Microsomal Biotransformation of Phenol in Three Species of Salmonidae: Hydroquinone, Catechol, and Phenylglucuronide Formation.

The in vitro biotransformation of phenol at 11 °C was studied using pre-spawn adult rainbow (Oncorhynchus mykiss) (RBT), brook (Salvelinus fontinalis) (BKT), and lake trout (Salvelinus namaycush) (LKT) hepatic microsomal preparations. The incubations were optimized for time, cofactor concentration, pH, and microsomal protein concentration. Formation of Phase I ring-hydroxylation and Phase II glucuronidation metabolites was quantified using HPLC with dual-channel electrochemical and UV detection. The biotransformation of phenol over a range of substrate concentrations (1 to 180 mM) was quantified, and the Michaelis-Menten kinetics constants, Km and Vmax, for the formation of hydroquinone (HQ), catechol (CAT), and phenylglucuronide (PG) were calculated. Species differences were noted in the Km values for Phase I enzyme production of HQ and CAT, with the following rank order of apparent enzyme affinity for substrate: RBT > BKT = LKT. However, no apparent differences in the Km for Phase II metabolism of phenol to PG were detected. Conversely, while there were no apparent differences in Vmax between species for HQ or CAT formation, the apparent maximum capacity for PG formation was significantly less in LKT than that observed for RBT and BKT. These experiments provide a means to quantify metabolic activation and deactivation of xenobiotics in fish, to compare activation and deactivation reactions across species, and to act as a guide for future predictions of new chemical biotransformation pathways and rates in fish. These experiments provided the necessary rate and capacity (Km and Vmax) inputs that are required to parameterize a fish physiologically based toxicokinetic (PB-TK) model for a reactive chemical that is readily biotransformed, such as phenol. In the future, an extensive database of these rate and capacity parameters on important fish species for selected chemical structures will be needed to allow the effective use of predictive models for reactive, biotransformation chemicals in aquatic toxicology and environmental risk assessment.

Research on Some Aspects of the Cytochrome P450 Proteins

The Cytochrome P450 was first described in 1958 as a brown pigment when carbon monoxide was added to hepatic microsomal preparations, which had been reduced with dithionite or NADH. This hemeprotein had probably arisen very early in chemical evolution (the origin of life). One of its early functions was probably the detoxification of molecular oxygen, which was poisonous to primordial life, which had not evolved other protective mechanisms. Since its initial discovery, it has been identified in a very large variety of animal tissues, plants and microorganisms. It is now known that there are various cytochromes P450, which perform a large variety of enzymatic functions. The cytochromes P450 have among other functions the metabolism or detoxification of ‘xenobiotics’ or ‘foreign chemicals’, which include drugs and environmental pollutants. The functioning of the steroid hydroxylases in adrenocortical mitochondria is influenced (regulated) by the dielectric constant in the immediate vicinity of the enzyme. Sodium and potassium ions influence the functioning of these hydroxylases differently in view of the different effect their ionic radii have on the dielectric constant. Spices have been found to bind to cytochrome P450. This may modify the activity of the enzyme. This may account for certain herbal medicinal preparations being effective in members of some communities but not in others, which have different dietary habits or preparations. Spices are also added as a component of some herbal medicinal preparations. More investigations need to be performed to ascertain the effect the consumption of spices may have on the efficacy of medicines in general.

Identification of Metabolite Biomarkers of the Designer Hallucinogen 25I-NBOMe in Mouse Hepatic Microsomal Preparations and Human Urine Samples Associated with Clinical Intoxication.

'NBOMe' (dimethoxyphenyl-N-[(2-methoxyphenyl)methyl]ethanamine) derivatives are a new class of designer hallucinogenic drugs widely available on the Internet. Currently, 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) is the most popular abused derivative in the USA. There are little published data on the absorption, metabolism and elimination of 25I-NBOMe, or any of the other NBOMe derivatives. Therefore, there are no definitive metabolite biomarkers. We present the identification of fifteen 25I-NBOMe metabolites in phase I and II mouse hepatic microsomal preparations, and analysis of two human urine samples from 25I-NBOMe-intoxicated patients to test the utility of these metabolites as biomarkers of 25I-NBOMe use. The synthesis of two major urinary metabolites, 2-iodo-4-methoxy-5-[2-[(2-methoxyphenyl) methylamino]ethyl]phenol (2-O-desmethyl-5-I-NBOMe, M5) and 5-iodo-4-methoxy-2-[2-[(2-methoxyphenyl)methylamino]ethyl]phenol (5-O-desmethyl-2-I-NBOMe), is also presented. Seven phase II glucuronidated metabolites of the O-desmethyl or the hydroxylated phase I metabolites were identified. One human urine sample contained 25I-NBOMe as well as all 15 metabolites identified in mouse hepatic microsomal preparations. Another human urine sample contained no parent 25I-NBOMe, but was found to contain three O-desmethyl metabolites. We recommend β-glucuronidase enzymatic hydrolysis of urine prior to 25I-NBOMe screening and the use of M5 as the primary biomarker in drug testing.

The influence of methyl substitution on the N-demethylation and N-oxidation of normethadone in animal species.

Abstract N-Monodemethylation and N-oxidation were shown to be the major routes of metabolism of normethadone, (—)-methadone and (—)-isomethadone in vitro by hepatic microsomal preparations from rat, rabbit, guinea-pig, mouse and hamster. The rate of N-oxidation was decreased and the rate of N-demethylation was increased by the introduction of the methyl substituent into normethadone; the configuration of the methyl substituent influenced these processes. Km and Vmax values were determined for liver microsomal N-demethylation of normethadone, (—)-methadone and (—)-isomethadone by rat and guinea-pig and for the N-oxidation of normethadone by guinea-pig. The use of selective inhibitors showed that N-demethylation was not preceeded by N-oxidation in these compounds.

The recognition of a diarylimine as a metabonate produced during incubation of N-benzyl-4-chloroaniline with hepatic microsomal preparations.

Evidence is presented for the formation of N-benzylidene-4-chloroaniline as a metabonate during the metabolism of N-benzyl-4-chloroaniline. Control studies suggest that the diarylimine is formed as a chemical artifact from the debenzylation products (benzaldehyde and 4-chloroaniline). This novel observation indicates a possible pathway to amide formation from N-benzylanilines via diarylimines as intermediates.

Quantitative protein determination for CYP induction via LC‐MS/MS

The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel-free, high-throughput LC-MS approach to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific peptides released by enzymatic digestion of the hepatocyte incubations. The method uses synthetic stable isotope-labeled peptides as internal standards and allows both relative and absolute quantification to be performed from hepatic microsomal preparations. CYP protein determined by this LC-MS method correlated well with the mRNA and activity for induced levels of CYP1A2, CYP2B6 and CYP3A4. Interestingly, a small fold change was observed for the induction of 3A5 with phenobarbital. The results were reproducible with an average CV less then 10% for repeat analysis of the sample. This LC-MS method offers a robust assay for CYP protein quantitation for use in CYP induction assays.

2,2′,3,3′,6,6′-Hexachlorobiphenyl (PCB 136) Atropisomers Interact Enantioselectively with Hepatic Microsomal Cytochrome P450 Enzymes

2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is a chiral and highly neurotoxic PCB congener of environmental relevance. (+)-PCB 136 was previously shown to be enriched in tissues from mice treated with racemic PCB 136. We investigated the spectral interactions of (+)-, (-)-, and (+/-)-PCB 136 with mouse and rat hepatic microsomal cytochrome P450 (P450) enzymes to test the hypothesis that enantioselective binding to specific P450 enzymes causes the enrichment of (+)-PCB 136 in vivo. Hepatic microsomes prepared from C57BL/6 mice or Long Evans rats treated with beta-naphthoflavone or 3-methylcholanthrene, phenobarbital, and dexamethasone (prototypical inducers of CYP1A, CYP2B, and CYP3A, respectively) were used to determine first, whether the (+)-PCB 136 atropisomer binds to hepatic microsomal P450 enzymes to a greater extent than does the (-)-PCB 136 atropisomer and second, whether P450 enzymes of one subfamily bind the two PCB 136 atropisomers more efficiently than do P450 enzymes of other subfamilies. Increasing concentrations of (+)-, (-)-, or (+/-)-PCB 136 were added to hepatic microsomes, and the difference spectrum and maximal absorbance change, a measure of PCB binding to P450 enzymes, were measured. A significantly larger absorbance change was observed with (+)-PCB 136 than with (-)-PCB 136 with all four hepatic microsomal preparations in mice and rats, indicating that (+)-PCB 136 interacted with microsomal P450 enzymes to a greater degree than did (-)-PCB 136. In addition, binding of the PCB 136 atropisomers was greatest in microsomes from PB-treated mice and rats and was inhibited by CYP2B antibodies, indicating the involvement of CYP2B enzymes. Together, these results suggest preferential binding of (+)-PCB 136 to P450 enzymes (such as CYP2B and CYP3A) in hepatic microsomes, an observation that may explain the enantioselective enrichment of the (+)-PCB 136 atropisomer in tissues of mice.

Midazolam Metabolism in Cytochrome P450 3A Knockout Mice Can Be Attributed to Up-Regulated CYP2C Enzymes

The cytochrome P450 3A (CYP3A) enzymes represent one of the most important drug-metabolizing systems in humans. Recently, our group has generated cytochrome P450 3A knockout mice to study this drug-handling system in vivo. In the present study, we have characterized the Cyp3a knockout mice by studying the metabolism of midazolam, one of the most widely used probes to assess CYP3A activity. We expected that the midazolam metabolism would be severely reduced in the absence of CYP3A enzymes. We used hepatic and intestinal microsomal preparations from Cyp3a knockout and wild-type mice to assess the midazolam metabolism in vitro. In addition, in vivo metabolite formation was determined after intravenous administration of midazolam. We were surprised to find that our results demonstrated that there is still marked midazolam metabolism in hepatic (but not intestinal) microsomes from Cyp3a knockout mice. Accordingly, we found comparable amounts of midazolam as well as its major metabolites in plasma after intravenous administration in Cyp3a knockout mice compared with wild-type mice. These data suggested that other hepatic cytochrome P450 enzymes could take over the midazolam metabolism in Cyp3a knockout mice. We provide evidence that CYP2C enzymes, which were found to be up-regulated in Cyp3a knockout mice, are primarily responsible for this metabolism and that several but not all murine CYP2C enzymes are capable of metabolizing midazolam to its 1'-OH and/or 4-OH derivatives. These data illustrate interesting compensatory changes that may occur in Cyp3a knockout mice. Such flexible compensatory interplay between functionally related detoxifying systems is probably essential to their biological role in xenobiotic protection.

Antioxidant activity of polyphenolic extract of monofloral honeybee-collected pollen from mesquite ( Prosopis juliflora, Leguminosae)

The antioxidant capacity related to the phenolic composition of monospecific honeybee-collected pollen extract from the mesquite tree ( Prosopis juliflora) from Durango, Mexico, was evaluated in an in vitro-biological system (as inhibitor of lipid peroxidation on mouse hepatic microsomal preparations) and in an in vivo system (on homogenized liver of bromobenzene-intoxicated mice) by quantification of thiobarbituric acid-reactive substances (TBARS). The comparison of results obtained from these two different systems was also made. The results obtained suggest that pollen of P. juliflora is an important source of flavonoids, which can be considered as natural antioxidants. Mesquite pollen extracts showed antioxidant activity related to the flavonol concentration in both the in vitro-biological system and the in vivo system with a lower activity in the latter of these systems. Under in vivo conditions and in those in which a state of oxidation in not induced, a high concentration of flavonols in the extract of mesquite pollen can have a pro-oxidant effect.

Identification of a genotoxic mechanism for the carcinogenicity of the environmental pollutant and suspected human carcinogeno-anisidine

2-methoxyaniline (o-anisidine) is an industrial and environmental pollutant and a bladder carcinogen for rodents. The mechanism of its carcinogenicity was investigated with 2 independent methods, 32P-postlabeling and 14C-labeled o-anisidine, to show that o-anisidine binds covalently to DNA in vitro after its activation by human hepatic microsomes. We also investigated the capacity of o-anisidine to form DNA adducts in vivo. Rats were treated i.p. with o-anisidine (0.15 mg/kg daily for 5 days) and DNA from several organs was analyzed by 32P-postlabeling. Two o-anisidine-DNA adducts, identical to those found in DNA incubated with o-anisidine and human microsomes in vitro, were detected in urinary bladder (4.1 adducts per 10(7) nucleotides), the target organ, and, to a lesser extent, in liver, kidney and spleen. These DNA adducts were identified as deoxyguanosine adducts derived from a metabolite of o-anisidine, N-(2-methoxyphenyl)hydroxylamine. This metabolite was identified in incubations with human microsomes. With 9 human hepatic microsomal preparations, we identified the specific CYP catalyzing the formation of the o-anisidine metabolites by correlation studies and by examining the effects of CYP inhibitors. On the basis of these analyses, oxidation of o-anisidine was attributed mainly to CYP2E1. Using recombinant human CYP (in Supersomes) and purified CYPs, the participation of CYP2E1 in o-anisidine oxidation was confirmed. In Supersomes, CYP1A2 was even more efficient in oxidizing o-anisidine than CYP2E1, followed by CYP2B6, 1A1, 2A6, 2D6 and 3A4. The results, the first report on the potential of the human microsomal CYP enzymes to activate o-anisidine, strongly suggest a carcinogenic potential of this rodent carcinogen for humans.

Down-regulation of cytochrome P450 proteins and its activities by Shiga-like toxin II from Escherichia coli O157:H7

Escherichia coli O157:H7 infection frequently induces clinical complications such as hemolytic uremic syndromes and intestinal dysfunctions. These changes could alter the disposition of drugs, consequently changing their efficacy. However, the possible changes of drug-metabolizing activities by E. coli O157:H7 infection have not been addressed. Thus, we have investigated the effect of Shiga-like toxin type II (SLT-II), derived from E. coli O157:H7, on the hepatic cytochrome P450 (CYP) content and its activity in rats. SLT-II (2 μg per animal, i.v.) time-dependently decreased total CYP content and the contents of CYP2C11 and CYP3A2 in hepatic microsomal preparations up to 24 hr following injection. Consistently, SLT-II time-dependently decreased CYP activity in vivo, as represented by systemic clearance of antipyrine. An inhibitor of inducible nitric oxide synthase, S-methylisothiourea, restored the decreased systemic clearance of antipyrine by SLT-II, suggesting the involvement of the overproduction of nitric oxide by SLT-II. Moreover, dexamethasone restored the decreased systemic clearance of antipyrine by SLT-II. In the hepatic microsomal preparation, dexamethasone restored the SLT-II-induced decrease of CYP3A2 whereas S-methylisothiourea did not affect both CYP subtypes. Taken together, these results suggest that SLT-II might alter hepatic drug-metabolizing function during E. coli O157 infection and that more than one cytokines induced by SLT-II, including nitric oxide, might make a critical contribution to the decrease of CYP content and its activity.

Spectral interactions of tetrachlorobiphenyls with hepatic microsomal cytochrome P450 enzymes

This study investigated the spectral interactions of hepatic microsomal cytochrome P450 (CYP) enzymes with four symmetrical polychlorinated biphenyls (PCBs): 2,2′,4,4′-tetrachlorobiphenyl (PCB 47); 2,2′,5,5′-tetrachlorobiphenyl (PCB 52); 2,2′,6,6′-tetrachlorobiphenyl (PCB 54); and 3,3′,4,4′-tetrachlorobiphenyl (PCB 77). The PCBs were selected to explore structure–activity relationships and the effect of the chlorination pattern on PCB–CYP spectral interactions. To examine CYP enzyme specificity, difference spectra were measured with hepatic microsomes prepared from control, phenobarbital (PB)-, 3-methylcholanthrene (MC)-, and dexamethasone (DEX)-treated rats in the absence and presence of CYP-specific antibodies. The four PCB congeners elicited a type I spectral change with all hepatic microsomal preparations. The binding efficiency of the PCBs was highest with microsomes from PB-treated rats. The largest absorbance change and highest binding efficiency were observed with PCB 54, the most non-coplanar congener tested. Antibody inhibition and CYP immunoquantitation data showed that the PCBs bind to CYP1A, CYP2B, CYP2C and CYP3A enzymes to varying degrees. For example, PCB 47, 52, and 54 bind preferentially to CYP2B and to a lesser extent to CYP3A enzymes in microsomes from PB-treated male rats; PCB 52 binds primarily to CYP3A enzymes in microsomes from DEX-treated female rats; and PCB 54 binds to CYP3A and to CYP2C enzymes in microsomes from control male rats. The study demonstrated that the extent of PCB–CYP binding interaction was dependent on the chlorination pattern of the PCB and on the relative abundance of individual CYP enzymes in hepatic microsomes.

Rapid and simple quantitative assay method for diastereomeric flurbiprofen glucuronides in the incubation mixture

Acyl glucuronides of nonsteroidal anti-inflammatory drugs having a chiral center are known to be chemically very active and form covalently bound adducts with proteins, such as human serum albumin, which may be the cause of hypersensitive reactions. Hepatic acyl glucuronosyltransferase catalyzes the transformation of α-aryl propionates into these diastereoisomeric acyl glucuronides, and, hence, its activity needs to be characterized. From this point of view, we developed a rapid, accurate and reproducible analytical method for the separation and determination of diastereoisomeric glucuronides of flurbiprofen, one of the nonsteroidal anti-inflammatory drugs, in the incubation mixture of the hepatic microsomal preparation by high-performance liquid chromatography with a simple column-switching technique for deproteinization. The glucuronides were separated on a TSKgel ODS-80Ts column with 20 m M ammonium acetate buffer (pH 5.6)–ethanol–acetonitrile as the mobile phase and monitored with a UV detector at 246 nm. The detection limit of the proposed method was 600 fmol/injection at a signal-to-noise ratio of 10. The validation results indicated that this method would be very useful for the determination of diastereomeric acyl glucuronides formed from flurbiprofen in an incubation mixture.

Oxidative monensin metabolism and cytochrome P450 3A content and functions in liver microsomes from horses, pigs, broiler chicks, cattle and rats.

The oxidative metabolism of monensin, an ionophore antibiotic extensively used in veterinary practice as a coccidiostat and a growth promoter, was studied in hepatic microsomal preparations from horses, pigs, broiler chicks, cattle and rats. As assayed by the measurement of the amount of the released formaldehyde, the rate of monensin O-demethylation was nearly of the same order of magnitude in all species, but total monensin metabolism, which was estimated by measuring the rate of substrate disappearance by a high-performance liquid chromatography (HPLC) method, was highest in cattle, intermediate in rats, chicks and pigs, and lowest in horses. When expressed as turnover number (nmol of metabolized monensin/min nmol cytochrome P450-1), the catalytic efficiency (chick >> cattle >> pig approximately rat > horse) was found to correlate inversely with the well known interspecies differences in the susceptibility to the toxic effects of the ionophore, which is characterized by an oral LD50 of 2-3 mg/kg bodyweight (bw) in horses, 50-80 mg/kg bw in cattle and 200 mg/kg bw in chicks. Chick and cattle microsomes also displayed both the highest catalytic efficiency toward two P450 3A dependent substrates (erythromycin and triacetyloleandomycin) and the highest immunodetectable levels of proteins cross-reacting with anti rat P450 3A1/2. Further studies are required to define the role played by this isoenzyme in the oxidative biotransformation of the drug in food producing species.

Sex-dependent toxicity of a novel acyl-CoA:cholesterol acyltransferase inhibitor, YIC-C8-434, in relation to sex-specific forms of cytochrome P450 in rats.

YIC-C8-434 is a novel inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT). To clarify the toxicity of YIC-C8-434, the compound was given orally to Sprague-Dawley rats for 28 days at 0, 4, 20, 100, or 500 mg/kg/day. The toxicity of the drug differed significantly between male and female rats. In female rats treated at 500 mg/kg, many symptoms including moribund condition, suppression of weight gain and food consumption, abnormal blood chemistry, and decreases in organ weights (thymus, ovaries, and uterus) were observed. In male rats by contrast, no significant toxicity was observed at any dose. After a single administration of YIC-C8-434 at 500 mg/kg, female rats had a higher blood concentration of the compound than male rats. Little elimination of YIC-C8-434 was observed in female rats on analysis of drug-elimination kinetics. Furthermore, the metabolism of YIC-C8-434 was analyzed using rat hepatic microsomal preparations from both sexes. Consistent with the observations in vivo, hepatic microsomes from male rats better metabolized YIC-C8-434 than those from females. In addition, the metabolism of YIC-C8-434 by hepatic microsomes from male rats was blocked by SKF525A, a P450 inhibitor. Inhibition experiments using anti-rat CYP1A1, CYP1A2, CYP2B1, CYP2C11, CYP2E1, CYP3A2, and CYP4A1 antisera indicated that CYP3A2 played the predominant role in the metabolism of YIC-C8-434 in rats. Since there is less CYP3A2 in the liver of female than male rats, the involvement of CYP3A2 in YIC-C8-434 metabolism has implications for the sex-related metabolic activity and toxicity of YIC-C8-434.

METABOLISM OF THE STYRENE METABOLITE 4-VINYLPHENOL BY RAT AND MOUSE LIVER AND LUNG

Styrene is a widely used chemical in the reinforced plastics industry and in polystyrene production. Its primary metabolic pathway to styrene oxide and then to styrene glycol, which is further metabolized to mandelic acid and phenylglyoxylic acid, has been well studied. However, a few studies have reported finding a minor metabolite, 4-vinyl-phenol (4-VP), in rat and human urine. The present studies sought to determine if the formation and metabolism of 4-VP in rat and mouse liver and lung preparations could be measured. When styrene was incubated with hepatic and pulmonary microsomal preparations, 4-VP formation could not be measured in these preparations. However, considerable 4-VP metabolizing activity, as determined by the loss of 4-VP, was observed in both mouse and rat liver and lung microsomal preparations. 4-Vinylphenol metabolizing activity in mouse liver microsomes was three times greater than that in rat liver microsomes, and activity in mouse lung microsomes was eight times greater than that in rat lung microsomes. This activity was completely absent in the absence of NADPH. Studies with cytochrome P-450 inhibitors indicated the involvement of CYP2E1 and CYP2F2. Induction of CYP2E1 by pyridine resulted in an increase in 4-VP metabolism by mouse hepatic microsomes but not by pulmonary microsomes. The metabolite(s) formed as a result of this oxidative pathway remain to be identified. In additional studies, glutathione conjugation appeared to be involved in 4-VP metabolism with the highest activity being in mouse lung, with or without the addition of NADPH.

Benzylic hydroxylation of 1-methylpyrene and 1-ethylpyrene by human and rat cytochromes P450 individually expressed in V79 Chinese hamster cells.

Alkyl-substituted polycyclic aromatic hydrocarbons may be metabolized to highly reactive benzylic sulfuric acid esters via benzylic hydroxylation and subsequent sulfonation. We have studied the benzylic hydroxylation of 1-methylpyrene (MP), a hepatocarcinogen in rodents, and 1-ethylpyrene (EP), whose benzylic hydroxylation would produce a secondary alcohol (alpha-HEP), in contrast to the primary alcohol (alpha-HMP) formed from MP. The hydrocarbons were incubated with hepatic microsomal preparations from humans and rats, as well as with V79-derived cell lines engineered for the expression of individual cytochrome P450 (CYP) forms from human (1A1, 1A2, 1B1, 2A6, 2E1, 3A4) and rat (1A1, 1A2, 2B1). All microsomal systems and CYP-expressing cell lines used, but not CYP-deficient V79 cells, showed biotransformation of both hydrocarbons. Formation of the benzylic alcohol was detected in each case. alpha-HMP and its oxidation product, 1-pyrenylcarboxylic acid (COOH-P), accounted for a major part of the total amount of the metabolites formed from MP in the presence of human liver microsomes (38-64%) and cells expressing human 3A4, 2E1 or 1B1 (80-85%). Likewise, cells expressing human 1A1 showed a higher contribution of alpha-HMP and COOH-P to the total metabolites (45%) than cells expressing the orthologous enzyme of the rat (3%). EP was metabolized at a higher rate and with modified regioselectivity compared with MP, although omega-hydroxylation of the side chain was not detected with the cell lines and only accounted for a small percent of the biotransformation by the microsomal preparations. The highest contributions of alpha-HEP to the total metabolites from EP were detected with the cells expressing human 1A1, 1B1 and 3A4 (38-51%). alpha-HEP accounted for 16% of the metabolites formed in the presence of human hepatic microsomes. Thus, benzylic hydroxylation is a major initial step in the metabolism of MP and EP. This pathway appears to be even more important in humans than in rats. Previously, we had shown that the second step of the activation, the sulfonation of alpha-HMP and alpha-HEP, is also efficiently catalysed by various forms of human sulfotransferases.

In vitro metabolism of (S)-(-)-[2'-14C]nicotine, using various tissue preparations of marmoset.

The nicotine metabolite profile produced by marmoset liver, lung and kidney preparations was investigated after 30 minutes incubation of (S)-(-)-[2'-14C]nicotine. Cation-exchange high performance liquid radiochromatography was employed to separate and quantify nicotine and its metabolites. Cotinine-N-oxide (CNO, 0.7%), 3'-hydroxy-cotinine (3'-OH-C, 0.2%), norcotinine (NORC, 0.9%) and nornicotine (NORN, 0.4%) were formed in the incubates of marmoset lung homogenates; when marmoset kidney homogenates were used, CNO, 0.4%; 3'-OH-C, 0.2%; NORC, 0.7%; NORN, 0.7%; and cotinine (COT, 0.4%) were detected in the incubates. These nicotine metabolites constituted only approximately 2.2% and 2.4% of the original nicotine substrate used by lung and kidney homogenates respectively. When marmoset hepatic homogenates and microsomes were used, both COT and NORN were detected as the major nicotine metabolites. In addition, traces of CNO and 3'-OH-C were also detected in both incubates. The amounts of COT (6.4%) and NORN (1.8%) in the hepatic homogenates were approximately twice that of those formed by hepatic microsomes (3.8% and 0.9%, respectively). Nicotine-1'-N-oxide (NNO, 1.1%) was only detected in the latter preparation. Under the experimental conditions, these nicotine metabolites constituted only 8.2% and 5.8% of the substrate nicotine used in the respective incubates. The present results showed that both primary C-oxidation pathways, i.e. cotinine formation and N-demethylation of nicotine, occurred in the lung, kidney and liver of marmoset in vitro. However, N-oxidation of nicotine was only observed when a marmoset hepatic microsomal preparation was used.

The in vitro hepatic microsomal metabolism of N-benzyladamantanamine in rats.

The metabolism of N-benzyladamantanamine (NBAD) was studied in vitro using rat hepatic microsomal preparations. The substrate and proposed metabolites were synthesized and characterized using spectroscopic techniques and separated using a reverse phase HPLC system. NBAD was incubated with rat microsomal preparations, extracted into DCM in the presence of NaCl and evaporated under a stream of nitrogen. The results from HPLC studies showed that NBAD produced the corresponding nitrone and hydroxylamine. This experiment also revealed that dealkylation occurred. No metabolites were observed which corresponded to authentic amide or oxaziridine. The reactions required a microsomal enzyme source and NADPH as a cofactor. The results indicate that the nitrone observed as a metabolite of NBAD is not an intermediate leading to the formation of an oxaziridine and hence an amide, under careful experimental conditions excluding light.

The in vitro hepatic microsomal metabolism of 3,5-dimethyl-4-(phenylazo)-(1H)-pyrazole in rats.

The in vitro hepatic microsomal metabolism of 3,5-dimethyl-4-(phenylazo)-(1H)-pyrazole (DMPAP) was studied using washed rat hepatic microsomal preparations fortified with NADPH. The substrate, DMPAP, and its potential metabolites, i.e. the corresponding reduction product, 3,5-dimethyl-4-amino-(1H)-pyrazole (DMAP), and the oxidation product, 3,5-dimethyl-4-(phenylazoxy)-(1H)-pyrazole (DMAPO), were synthesized and their structures elucidated by use of their spectral characteristics. DMPAP and its potential metabolites were then separated using a reverse phase HPLC system consisting of a C18 column and a mobile phase of acetonitrile:water (50:50) at a flow rate of 1 ml/min with UV detection at 254 nm. DMPAP was incubated with rat microsomal preparations, extracted into DCM in the presence of NaCl, and finally evaporated under a stream of nitrogen. The results from HPLC studies showed that DMPAP was metabolised to the corresponding reduction and oxidation products in the presence of NADPH.