Showing papers in "Xenobiotica in 1992"

Antioxidant and pro-oxidant properties of active rosemary constituents: carnosol and carnosic acid

Abstract: 1. Carnosol and carnosic acid have been suggested to account for over 90% of the antioxidant properties of rosemary extract.2. Purified carnosol and carnosic acid are powerful inhibitors of lipid peroxidation in microsomal and liposomal systems, more effective than propyl gallate.3. Carnosol and carnosic acid are good scavengers of peroxyl radicals (CCl3O2) generated by pulse radiolysis, with calculated rate constants of 1–3 × 106M-1 S-1 and 2.7 × 107M-1 S-1 respectively.4. Carnosic acid reacted with HOCl in such a way as to protect the protein α1-antiproteinase against inactivation.5. Both carnosol and carnosic acid stimulated DNA damage in the bleomycin assay but they scavenged hydroxyl radicals in the deoxyribose assay. The calculated rate constants for reaction with ·OH in the deoxyribose system for carnosol and carnosic acid were 8.7 × 1010M-1 and 5.9 × 1010M-1 S-1 respectively.6. Carnosic acid appears to scavenge H2O2, but it could also act as a substrate for the peroxidase system.7. Carnosic acid a...

Cytochromes P-450 in rats : structures, functions, properties and relevant human forms

Abstract: (1992). Cytochromes P-450 in rats: structures, functions, properties and relevant human forms. Xenobiotica: Vol. 22, No. 1, pp. 83-103.

The metabolism of cypermethrin in man: differences in urinary metabolite profiles following oral and dermal administration

Abstract: 1 The pyrethroid insecticide cypermethrin was administered orally to six male volunteers as a single dose of 33 mg (cis: trans 1 : 1) and dermally to six volunteers at a dose of 31 mg/800 cm2 (cis : trans 56 : 44) as a soya oil-based formulation Urine samples were collected for up to 5 days and analysed for the metabolites cis and trans 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (DCVA), 3-phenoxybenzoic acid (3PBA) and 3-(4′-hydroxyphenoxy) benzoic acid (4OH3PBA) following an acid hydrolysis procedure2 Following oral dosing approx equal amounts of (cis + trans DCVA) and (3PBA + 4OH3PBA) were excreted with peak excretion rates occurring between 8 and 24 h after dosing The ratio of trans:cis DCVA was on average 2:1 Based on DCVA measurements the amount of cypermethrin absorbed was estimated to be between 27% and 57% (mean 36%) of the administered dose3 Peak urinary excretion rates of metabolites occurred between 12 and 36h after dermal dosing The amount of metabolites derived

The pharmacokinetics of propofol in laboratory animals

Abstract: 1. The pharmacokinetics of propofol in an emulsion formulation ('Diprivan') have been studied after single bolus doses to rats, dogs, rabbits and pigs, and after single and multiple infusions to dogs. Venous blood propofol concentrations were determined by h.p.l.c. with u.v. or fluorescence detection. Curve fitting was performed using ELSFIT. 2. The distribution of propofol in blood and its plasma protein binding have been studied in rat, dog, rabbit and man. Protein binding was high (96-98%), and in most species propofol showed appreciable association with the formed elements of blood. 3. Where an adequate sampling period was employed the pharmacokinetics of propofol were best described by a three-compartment open 'mammillary' model. Propofol was distributed into a large initial volume (1-21/kg) and extensively redistributed (Vss = 2-10 x body weight) in all species. Clearance of propofol by all species was rapid, ranging from about 30-80 ml/kg per min in rats, dogs and pigs to about 340 ml/kg per min in rabbits.

Measurement of oxybutynin and its N-desethyl metabolite in plasma, and its application to pharmacokinetic studies in young, elderly and frail elderly volunteers.

Abstract: 1. A quantitative h.p.l.c. plasma assay for oxybutynin (OB) and its active metabolite, N-desethyl oxybutynin (DEOB) is described. The method is linear with coefficients of variation ranging between 4 and 11.8% for OB and 4.6-9.1% for DEOB over the typical concentration range measured. Minimum detectable levels were 0.5 and 5 ng/ml for OB and DEOB respectively from a 2 ml sample. 2. Pharmacokinetic parameters were obtained after a single oral dose of OB and after administration two or three times daily to frail elderly and elderly volunteer groups respectively. Single dose results were also compared with data from young healthy volunteers. 3. There was a wide range in peak blood levels and high levels of parent drug were matched by high DEOB metabolite levels. Plasma levels on repeated administration were as would be predicted from the single dose kinetics. 4. Area under the plasma time course curve for DEOB metabolite was less than or equal to 5 than that of the parent drug. 5. A trend of increasing peak plasma levels and bioavailability was observed with increasing age and frailty, with the differences more apparent between the active elderly and frail elderly groups than between the active elderly and young volunteers. 6. Results indicate that for frail elderly patients a lower initial starting dose of 2.5 mg OB given two or three times a day may provide adequate therapeutic blood levels of the drug.

Interspecies variations in caffeine metabolism related to cytochrome P4501A enzymes.

Abstract: 1 Interspecies (including man, monkey, rabbit, rat and mouse) variations in caffeine metabolism by liver microsomes were studied While N-3 demethylation was the major pathway in man (81% of total dimethylxanthines), N-7 demethylation was predominant in monkey (89%), and the three demethylation pathways were about equal in mouse, rabbit and rat2 Three monooxygenase activities (methoxyresorufin O-demethylase, phenacetin O-deethylase and acetanilide 4-hydroxylase) correlated significantly with the rate of metabolism of caffeine3 P4501A1 and 1A2 enzymes were immunodetected in different species P4501A2 was the only isoform detected in liver of man, rat and mouse, while no polypeptide immunorelated to P4501A was detected in monkey and only a minor band of P4501A1 was detected in rat and rabbit4 All in vitro data indicate that paraxanthine formation is mediated mainly by P4501A2 in mammals while theophylline formation is mediated mainly by cytochromes P-450 other than those of the 1A family

Biological fate of sulphur mustard, 1,1'-thiobis(2-chloroethane): isolation and identification of urinary metabolites following intraperitoneal administration to rat.

Abstract: 1. The metabolism of sulphur mustard, 1,1'-thiobis(2-chloroethane), in vivo was investigated following i.p. administration to rat.2. Approx. 60% of dose was excreted in the 24 h urine. Many metabolites were present; nine have been isolated by h.p.l.c. and characterized by mass spectrometry. Structural assignments were confirmed by comparison with authentic synthetic standards.3. Some metabolites result from initial hydrolysis of the sulphur mustard, but the majority are formed by conjugation with glutathione. These are further metabolized to N-acetylcysteine conjugates, or to methylthio/methylysulphinyl derivatives by a pathway probably involving β-lyase, accompanied by oxidation of the mustard sulphur atom to sulphoxide or sulphone.4. Thiodiglycol sulphoxide, 1,1'-sulphonylbis[2-S (N-acetylcysteinyl)ethane] and 1,1'-sulphonylbis[2-methylsulphinyl)ethane] or 1-methylsulphinyl-2-[2-(methylthio ethylsulphonyl]ethane were the most prevalent metabolites resulting from the three major pathways. Metabolic pathw...

Methylation pharmacogenetics: Thiopurine methyltransferase as a model system

Abstract: 1. Methyl conjugation is an important pathway in the biotransformation of many drugs and xenobiotic compounds. 'Pharmacogenetic' variation exists in the activities of many methyltransferase enzymes, and experiments with the drug-metabolizing enzyme thiopurine methyltransferase (TPMT) offer a model for one approach that has proven useful in the study of methyltransferase pharmacogenetics. 2. TPMT catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. This enzyme activity is present in the human red blood cell (RBC), and RBC TPMT activity is controlled by a common genetic polymorphism that regulates also the enzyme activity in all other human tissues that have been studied. 3. Subjects with inherited low levels of TPMT activity are at increased risk for thiopurine drug-induced myelotoxicity, while patients with high TPMT activities may be 'undertreated' with these drugs. 4. TPMT activity in tissue from selected strains of inbred mice also is regulated by a genetic polymorphism. These mice provide an animal model for use in the study of pharmacological or toxicological consequences of inherited differences in TPMT activity. 4. Other methyltransferase enzymes including thiol methyltransferase, catechol O-methyltransferase, and histamine N-methyltransferase also are present in the human RBC, are regulated by inheritance, and are responsible for individual variation in drug metabolism. Enhanced understanding of the pharmacogenetics of methylation may make it possible to understand and predict individual variation in the biotransformation, toxicity and therapeutic effect of compounds that undergo methyl conjugation.

Pharmacokinetics and metabolism of dofetilide in mouse, rat, dog and man.

Abstract: 1. Pharmacokinetics of dofetilide were studied in man, dog, rat and mouse after single i.v. and oral doses of dofetilide or 14C-dofetilide.2. Dofetilide was absorbed completely in all species. Low metabolic clearance in man resulted in complete bioavailability following oral administration. Higher metabolic clearance in rodents, and to a lesser extent dogs, resulted in decreased bioavailability because of first-pass metabolism.3. Following i.v. administration, the volume of distribution showed only moderate variation in all species (2˙8–6˙3***1/kg). High plasma clearance in rodents resulted in short half-life values (mouse 0˙32, male rat 0˙5 and female rat 1˙2 h), whilst lower clearance in dog and man gave longer terminal elimination half-lives (4˙6 and 7˙6 h respectively).4. After single i.v. doses of 14C-dofetilide, unchanged drug was the major component excreted in urine of all species with several metabolites also present.5. Metabolites identified in urine from all species were formed by N-oxidation o...

Metabolism of albendazole and albendazole sulphoxide by ruminal and intestinal fluids of sheep and cattle

Abstract: 1. The metabolism of albendazole (ABZ), albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO2) by ruminal, abomasal and ileal fluids of sheep and cattle was investigated under anaerobic conditions in vitro.2. None of the compounds was metabolically changed by incubation with abomasal fluids of sheep and cattle.3. ABZ and ABZSO were extensively metabolized by sheep and cattle ruminal and ileal fluids. ABZSO2 was unaffected by incubation with these gastrointestinal fluids.4. The rate of ABZ oxidation into ABZSO was greater for cattle ruminal and ileal fluids than for sheep fluids.5. ABZSO was reduced back to ABZ by ruminal and ileal fluids of both species. This reducing activity was significantly higher for both ruminal and ileal fluids of sheep compared with those of cattle.

Propofol metabolism in man during the anhepatic and reperfusion phases of liver transplantation.

Abstract: 1. An i.v. dose of 14C-propofol (0.53 mg/kg) was administered to three male and three female patients during the anhepatic phase of liver transplantation, which lasted 30-56 min after dosing. Arterial and venous blood samples, bile (T-tube drainage) and urine were collected at various times afterwards and submitted to h.p.l.c. and radioassay or specific fluorescence detection for the unchanged drug. 2. Extrahepatic metabolism was apparent during the anhepatic phase, since at 30 min post-dose, unchanged propofol comprised only 42-89% of the blood radioactivity. 3. Examination of the plasma radioactivity during the anhepatic phase in two subjects showed evidence of propofol glucuronide and 4-quinol sulphate, confirming extrahepatic metabolism of the drug. Quinol glucuronides were only detected in the liver reperfusion phase. 4. There was no evidence that the lungs contribute to the extrahepatic metabolism of propofol, since drug concentrations in the arterial blood were not less than in central venous samples. 5. During the first 24 h period, urine collected from five patients contained 7-74% dose, whilst the bile contained 0.1-0.9%. In three patients with normal renal function recovery in urine was 66-74% dose. Examination of urinary radioactivity in one subject showed the main component to be propofol glucuronide during the anhepatic phase.

The metabolism of benzo(a)pyrene by English sole (Parophrys vetulus): comparison between isolated hepatocytes in vitro and liver in vivo.

Abstract: 1. Metabolites and DNA adducts of 3H-benzo(a)pyrene (BaP) formed by isolated hepatocytes from English sole (Parophrys vetulus) in vitro were compared to those in bile and liver of sole exposed i.m. to 3H-BaP.2. English sole liver was perfused with a collagenase solution and hepatocytes were isolated with greater than 95% viability. Determination of kinetic parameters for metabolism of 3H-BaP showed a Km of 29 ± 10 μM and an apparent Vmax of 1300 pmol BaP metabolized/106 cells per h.3. Analysis of medium from hepatocyte cultures and bile by ion-pair h.p.l.c. showed significant amounts of radioactivity in regions where glucuronide and glutathione conjugates of BaP metabolites elutc. No sulphate conjugates of BaP metabolites were detected. The major unconjugated metabolite formed by hepatocytes was the BaP-9,10-dihydrodiol.4. Hydrolysis of glucuronide conjugates by β-glucuronidase and reversed-phase h.p.l.c. analysis of chloroform-soluble metabolites showed the presence of BaP-7,8-dihydrodiol, 1-hydroxyBaP a...

Pharmacokinetics of ticlopidine during chronic oral administration to healthy volunteers and its effects on antipyrine pharmacokinetics

Abstract: 1 The pharmacokinetics of ticlopidine, a novel antithrombotic agent, have been investigated in 10 healthy volunteers dosed orally with the drug (250 mg 12 hourly for 21 days), to determine the basic pharmacokinetic parameters in humans, to investigate its accumulation during repeated administration, and to assess its effects on hepatic drug-metabolizing enzymes2 After the first dose, peak plasma concentrations (median 031, range 008–080 mg/l) were generally found at 2 h The levels decreased rapidly to a median concentration of 0087 mg/l by 4 h then declined to 0022 (range<0005–0128) mg/l at 12 h after administration, with apparent half-lives of approx 4 h The median AUC value for this first dosage interval (AUCτ) was 097 (range 041–349) mg h l−13 Pre-dose plasma concentrations indicated that steady state was reached after 5–10 days, and then remained essentially unchanged through to the end of the study From 30 h after the final dose, drug levels declined exponentially with a median hal

Metabolism of coumarin by rat, gerbil and human liver microsomes.

Abstract: 1. o-Hydroxyphenylacetaldehyde was the major metabolite of coumarin (1 mM) in rat, gerbil and human liver microsomes.2. Treatment of rats with phenobarbitone (PB) or β-naphthoflavone increased the o-hydroxyphenylacetaldehyde formed. 3-Hydroxycoumarin was the other main metabolite produced by rat liver microsomes.3. Liver microsomal metabolism of coumarin in gerbil was extensive with 3-, 5-, 6-, 7-and 8-hydroxycoumarins, and 3,7- and 6,7-dihydroxycoumarins produced, in addition to o-hydroxyphenylacetaldehyde. The profile of the hydroxy metabolites was altered by in vivo treatment of gerbils with cytochrome P-450 inducers, but there was no increase of coumarin metabolism.4. Coumarin was metabolized by human liver microsomes to o-hydroxyphenylacetaldehyde, 7-hydroxycoumarin, 3-hydroxycoumarin, and trace amounts of 5-, 6-and 8-hydroxycoumarins.5. At low substrate concentrations (0-10 μM) hepatic microsomal metabolism of coumarin in gerbil resembled that in man, with 7-hydroxycoumarin being a major metabolite....

Microsomal formation of a pyrrolic alcohol glutathione conjugate of the pyrrolizidine alkaloid senecionine.

Abstract: 1. Pyrrolizidine alkaloids (PAs) are metabolized primarily to putative dehydroalkaloid (PA pyrrole) metabolites and to PA N-oxide by rat liver microsomal monooxygenases. 2. The dehydroalkaloids are highly reactive and either bind covalentely to tissue nucleophiles or are hydrolysed to the more stable pyrrole, (R,S)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), and the corresponding necic acid. 3. Addition of glutathione (GSH 1 mM) to incubation mixtures containing rat liver microsomes and the PA senecionine (SN), resulted in the formation of a conjugate of DHP with GSH. 5. The mass spectrum of this DHP-GSH conjugate was identical to that of the chemically-synthesized dehydroretronecine (the R enantiomer of the racemic DHP) and GSH. 6. Only negligible amounts of DHP-GSH conjugate were formed when DHP itself was incubated with GSH at physiological pH. 7. These findings provide strong evidence for the microsomal conversion of SN to a highly reactive metabolite, presumably dehydrosenecionine, which then reacts with GSH to form the DHP-GSH conjugate. 8. It is likely that a similar mechanism is responsible in vivo for the formation of GSH conjugates of DHP from SN and other PAs.

Metabolism of 2,4,5,2',4',5'-hexachlorobiphenyl with liver microsomes of phenobarbital-treated dog; the possible formation of PCB 2,3-arene oxide intermediate.

Abstract: 1. Metabolism of 2,4,5,2',4',5'-hexachlorobiphenyl (HCB) was investigated in vitro using liver microsomes of one male beagle dog after phenobarbital treatment.2. Three major metabolites were isolated and identified as 3-hydroxy-2,4,5,2',4',5'-HCB, 2-hydroxy-4,5,2',4',5'-pentachlorobiphenyl (PenCB), and 2-hydroxy-3,4,5,2',4',5'-HCB, by comparison of g.l.c.-mass spectrometry and 1H-n.m.r. data with those of authentic samples.3. 2-Hydroxy-3,4,5,2',4',5'-HCB was found as a metabolite of 2,4,5,2',4',5'-HCB for the first time using dog liver microsomes. Present results indicate that this metabolite and the dechlorinated PenCB are derived from a metabolic intermediate, namely, 2,3-epoxy-2,4,5,2',4',5'-HCB. 2,3-Epoxide formation is a new metabolic pathway of PCB.

Reductive metabolism of the anticonvulsant agent zonisamide, a 1,2-benzisoxazole derivative

Abstract: 1. The metabolism of zonisamide in vitro was characterized through aerobic and anaerobic incubations with rat liver subcellular fractions and cultured gastrointestinal microflora.2. Zonisamide reacted with rat hepatic microsomal cytochrome P-450 and exhibited a Type I binding spectrum.3. Metabolism of zonisamide in vitro by hepatic subcellular fractions and cultured gastrointestinal flora produced a single metabolite, 2-(sulphamoylacetyl)-phenol (2-SMAP), by reductive cleavage of the 1,2-benzisoxazole ring.4. The reductive metabolism of zonisamide was primarily mediated by microsomal cytochrome P-450. The soluble fraction enhanced reduction when combined with the microsomal fraction but itself possessed only weak reductive activity.5. Reduction of zonisamide by the most enzymically active liver fractions required NADPH, was stimulated by FMN and SKF-525A, and was inhibited by CO or air, as well as by n-octylamine.6. Unlike their involvement in the reduction of numerous nitro, azo, and N-oxide compounds, c...

Polymorphisms of N-acetyltransferase genes.

Abstract: 1. A genetic polymorphism of human liver arylamine N-acetyltransferase (NAT) enzyme activity leads to wide variation in the disposition of many drugs and potential carcinogens, resulting in differential susceptibility to chemical-induced toxicity. 2. During studies to determine the biochemical and molecular mechanisms underlying this pharmacogenetic defect, we cloned two human genes, NAT1 and NAT2, which encode the functional acetylating enzymes NAT1 and NAT2. 3. NAT1 and NAT2 are both expressed in human liver cytosol, the latter as two closely related isoforms NAT2A and NAT2B. 4. NAT2 gene locus is the site of the human acetylation polymorphism, because its products NAT2A and NAT2B selectively acetylate 'polymorphic' arylamine substrates (e.g. sulphamethazine), and since the liver content of these isozymes is markedly reduced in genetically slow acetylator subjects. 5. NAT1 shows marked kinetic selectivity for 'monomorphic' substrates (e.g. p-aminobenzoic acid) whose in vivo acetylation rates do not correlate with the acetylation polymorphism. 6. Despite the drastic reduction in NAT2A/B proteins in livers from phenotypically slow acetylators, levels of the NAT2 gene transcript are not altered. 7. Three common mutant alleles at the NAT2 gene locus have so far been identified, which may be detected by restriction fragment length polymorphism (RFLP) analysis on Southern blots or by allele-specific polymerase chain reaction amplification.

Glutathione-dependent toxicity.

Abstract: 1. Recent studies show that glutathione conjugate formation is an important bioactivation mechanism for several groups of compounds with implications for organ-selective toxicity and carcinogenicity.2. Vicinal dihaloalkanes, such as 1,2-dihaloethanes, yield S-(2-haloalkyl)glutathione conjugates that give rise to highly electrophilic episulphonium ions, which are involved in the cytotoxicity and mutagenicity of 1,2-dihaloethanes.3. Nephrotoxic haloalkenes are metabolized to S-(haloalkenyl)- or S-(haloalkyl)-glutathione conjugates which, after metabolism to the corresponding cysteine conjugates, are bioactivated by renal cysteine conjugate β-lyase to yield cytotoxic or mutagenic metabolites.4. Finally, hepatic glutathione conjugate formation with hydroquinones and amino-phenols yields conjugates that are directed to γ-glutamyltransferase-rich tissues, such as the kidney, where they undergo alkylation or redox cycling reactions, or both, that cause organ-selective damage.

Comparative metabolism of flunarizine in rats, dogs and man: an in vitro study with subcellular liver fractions and isolated hepatocytes

Abstract: 1. The biotransformation of 3H-flunarizine ((E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propenyl)piperazine dihydrochloride, FLUN) was studied in subcellular liver fractions (microsomes and 12,000 g fraction) and in suspensions or primary cell cultures of isolated hepatocytes of rats, dogs and man. The major in vitro metabolites were characterized by h.p.l.c. co-chromatography and/or by mass spectrometric analysis. 2. The kinetics of FLUN metabolism was studied in microsomes of dog and man. The metabolism followed linear Michaelis-Menten kinetics over the concentration range 0.1-20 microM FLUN. 3. A striking sex difference was observed for the in vitro metabolism of FLUN in rat. In male rats, oxidative N-dealkylation at one of the piperazine nitrogens, resulting in bis(4-fluorophenyl) methanol, was a major metabolic pathway, whereas aromatic hydroxylation at the phenyl of the cinnamyl moiety, resulting in hydroxy-FLUN, was a major metabolic pathway in female rats. In incubates with hepatocytes, these two metabolites were converted to the corresponding glucuronides. 4. In human subcellular fractions, aromatic hydroxylation to hydroxy-FLUN was the major metabolic pathway. In primary cell cultures of human hepatocytes, oxidative N-dealkylation at the 1- and 4-piperazine nitrogen and glucuronidation of bis(4-fluorophenyl)methanol were observed. The in vitro metabolism of FLUN in humans, resembled more than in female rats and in dogs than that in male rats. 5. The present in vitro results are compared with data of previous in vivo studies in rats and dogs. The use of subcellular fractions and/or isolated hepatocytes for the study of species differences in the biotransformation of xenobiotics is discussed.

Effects of various medium formulations and attachment substrata on the performance of cultured ruminant hepatocytes in biotransformation studies.

Abstract: 1. A procedure for the isolation and primary culture of hepatocytes from goat and cattle is described. Hepatocyte culture performance was monitored for 51 h by measuring viability, cytochrome P-450 maintenance, dealkylation of scoparone and ethylmorphine, and glucuronidation of phenol red.2. Culture medium composition is discussed in relation to differences between splanchnic blood composition of ruminant and monogastric animal species. Main differences are in glucose and volatile fatty acid concentrations. Modified Williams’ E culture medium did not yield higher culture performance than non-modified Williams’ E.3. Coating of culture dishes with either collagen or fibronectin did not improve culture performance.4. Williams’ E, although developed for rodent cells, proves to be a suitable basal medium for ruminant hepatocytes. In this medium, culture quality is high for at least several days.5. In cultured goat hepatocytes, biotransformation rate for scoparone amounted to 20 nmol/mg protein per h, for ethyl...

Effects of arsenite on hepatic mixed-function oxidase activity in rats.

Abstract: 1. Injection of arsenite (As3+) to control rats results in losses of total hepatic cytochrome P-450 and significant decreases of ethoxycoumarin O-deethylase (ECOD) and ethoxyresorufin O-deethylase (EROD) activities. However, As3+ appears to decrease the activity of these enzymes differentially, with EROD showing greater sensitivity than ECOD.2. Injection of As3+ to rats treated with phenobarbital and isosafrole significantly decreases the total content of hepatic cytochrome P-450 and various mixed function oxidase (MFO) activities, with the exception of ECOD which appears to be insensitive to As3+.3. 3-Methylcholanthrene administration apparently protects against the effects of As3+ on the cytochrome P-450 system, since total content of the cytochrome P-450 and various MFO activities were all insensitive to this treatment.

The role of the flavin-containing monooxygenase (EC 1.14.13.8) in the metabolism and mode of action of agricultural chemicals.

Abstract: 1. The flavin-containing monooxygenase (FMO) (EC 1.14.13.8) is a versatile enzyme that catalyses the monooxygenation of a large number of xenobiotic soft nucleophiles ranging from inorganic ions to organic compounds with nitrogen, sulphur, phosphorus or selenium heteroatoms. 2. The substrate specificity relative to agricultural chemicals is discussed and compared with that of the cytochrome P-450-dependent monooxygenase system. The relative activity of these two enzymes towards common substrates varies from substrate to substrate and from tissue to tissue as is shown in the case of the insecticide, phorate and the hepatotoxicant, thiobenzamide. 3. The products of FMO action may be chemically different (e.g. nicotine) to those from P-450, or the two enzymes may produce different isomers of the same product (e.g. phorate). 4. Recent studies have demonstrated that, in the rabbit, the FMOs from liver and lung are different gene products which differ not only in primary sequence but also in physical, catalytic and immunochemical properties. These studies are being extended to include other tissues such as skin and brain. 5. Immunocytochemical localization of FMO in lung and skin correlates well with measurements of the oxidation of methimazole, a specific FMO substrate.

Hep G2 cell line as a human model for sulphate conjugation of drugs.

Abstract: 1. The objective of this study was to examine the usefulness of the hepatoma cell line Hep G2 as a model for human sulphoconjugation of drugs, in particular stereoselective conjugation.2. Using the substrates p-nitrophenol and dopamine, we found sulphation activities consistent with the presence of both the phenol (P) and the monoamine (M) form of the human phenolsulphotransferases in these cells.3. The Kmapp was 3.0γM for the sulphation of p-nitrophenol. This activity was inhibited selectively by 2,6-dichloro-4-nitrophenol, IC50 6γM. The Kmapp was 39 γM for the sulphation of dopamine. This activity was selectively inhibited by elevated temperature.4. The chiral adrenergic drugs (±)-terbutaline and (±)-4-hydroxypropranolol were both sulphated stereoselectively with Kmapp and Fmaxapp values for each enantiomer virtually identical to previous observations with human liver cytosol.5. In a direct comparison, the estimated activity of the P form of phenolsulphotrans-ferase in the Hep G2 cell line was 30% of th...

Induction of rat hepatic mixed-function oxidases by acetone and other physiological ketones: Their role in diabetes-induced changes in cytochrome P450 proteins

Abstract: 1. To evaluate the role of ketone bodies in diabetes-induced changes in hepatic cytochrome P450 composition, rats were treated with acetone, 3-hydroxybutyrate or 1,3-butanediol. 2. Treatment with acetone enhanced the rat hepatic 0-dealkylations of ethoxyresorufin and methoxyresorufin, and the hydroxylation of p-nitrophenol, but had no effect on lauric acid hydroxylation and ethylmorphine N-demethylation. Neither 3-hydroxybutyrate nor 1,3-butanediol modulated the metabolism of the above substrates. 3. Immunoblot analysis of hepatic microsomal proteins revealed that treatment with acetone increased the apoprotein levels of P4501A2, P4502B1/2 and P4502E1. 4. It is concluded that acetone is responsible, at least partly, for the diabetes-induced increase in hepatic microsomal P4501A2, P4502B1/2 and P4502E1 proteins but does not mediate the increases in the P4503A1 and P4504A1 proteins. On the basis of work from our own and other laboratories a mechanism for the diabetes-induced changes in hepatic cytochrome P450 proteins is proposed.

Biotransformation of mianserin in laboratory animals and man

Abstract: 1. The biotransformation and excretion of the antidepressant mianserin were studied after oral administration of the labelled drug to rats, mice, rabbits, guinea pigs and humans. Mianserin was well absorbed and almost completely metabolized in all five species. 2. Major metabolic pathways of mianserin were p-oxidation of the N-substituted aromatic ring followed by conjugation, and oxidation and demethylation of the N-methyl moiety, followed by conjugation. Direct conjugation of the N-methyl moiety was observed as a metabolic pathway specific for man. 3. Conjugated metabolites were isolated by h.p.l.c. and identified by 1H-n.m.r. and FAB spectrometry. Novel metabolites such as an N-O-glucuronide in the guinea pig and an N-sulphonate in rat and guinea pig, were identified using these techniques. A quaternary N-glucuronide was found only in man.

Single- and multiple-dose kinetics of d-fenfluramine in rats given anorectic and toxic doses.

Abstract: 1. High parenteral doses of a twice-daily schedule of d,l-fenfluramine (d,l,-F) may cause long-lasting decrease of functional indices of brain serotoninergic neurones in rats. The single- and multiple-dose (b.i.d. x 4 days) kinetics of low (1.25 mg/kg) and high (12.5 mg/kg) subcutaneous (s.c.) doses of d-F, which accounts of the anorectic effects of the racemate, and its deethylated metabolite d-norfenfluramine (d-NF), were therefore examined and compared with those of pharmacologically effective oral doses (0.3–1.25 mg/kg) in rats.2. There were dose-dependent alterations of kinetic parameters after s.c. and oral dosing, indicating that hepatic clearance of d-F in the rat can be saturated either by increasing the size of the single dose or during repeated dosing. Nonlinearity was also observed for d-NF. Consequently at high doses exposure of rat to the drug, as measured by the sum of area under the plasma concentration-time curve (AUC) of d-F and d-NF considerably exceeded that expected from simple dosage...

Investigations of mechanisms of reactive metabolite formation from (R)-(+)-pulegone

Abstract: 1. (R)-(+)-Pulegone is a monoterpene that is oxidized by cytochromes P-450 to reactive metabolites that initiate events in the pathogenesis of hepatotoxicity in mice, rats and humans. 2. Selective labelling of (R)-(+)-pulegone with deuterium revealed that menthofuran was a proximate hepatotoxic metabolite formed by oxidation of the allylic methyl groups of pulegone. Incubations of pulegone with mouse liver microsomes in an atmosphere of 18O2 resulted in the formation of menthofuran that contained only oxygen-18 in the furan moiety. These results are consistent with oxidation of pulegone to an allylic alcohol that reacts intramolecularly with the ketone moiety to form a hemiketal that subsequently dehydrates to generate menthofuran. 3. Studies on the metabolism of menthofuran revealed that it is oxidized by cytochromes P-450 to an electrophilic gamma-ketoenal that reacts with nucleophilic groups on proteins to form covalent adducts. In addition, diastereomeric mintlactones are formed. Investigations with H2(18)O and 18O2 are indicative of a furan epoxide intermediate, or a precursor, in the formation of the gamma-ketoenal and mintlactones.

Role of metabolism and pharmacokinetic studies in the discovery of new drugs--present and future perspectives.

Abstract: 1. The impact which pharmacokinetics and drug metabolism studies have made to drug discovery programmes is reviewed with examples from the anti-infective, cardiovascular, anti-inflammatory and CNS therapeutic areas.2. Contributions that advances in analytical technology have made to the early application of pharmacokinetics and drug metabolism are discussed.3. Some future perspectives are given on the advances being made in basic science and technology and how this will provide the basis for further growth in the contribution to the drug discovery process.

The interaction of phosphine with haemoglobin and erythrocytes.

Abstract: 1. Phosphine progressively converts oxyhaemoglobin to methaemoglobin and hemichrome species, with the product formed being time- and concentration-dependent. 2. The reaction of phosphine with oxyhaemoglobin leads to the formation of phosphite and phosphate. 3. Incubation of rat erythrocytes with various concentrations of phosphine results in the progressive uptake of phosphine by the erythrocytes in a temperature-dependent first-order process. 4. Uptake of phosphine by erythrocytes causes crenation, but conversion of oxyhaemoglobin to methaemoglobin and hemichrome could not be demonstrated.