Liver microsomal cytochrome P-450-dependent O-dealkylation reaction in various animals

Liver microsomal O-dealkylation activity was determined using O-methyl, O-ethyl and O-propyl derivatives of p-nitrophenol, 7-hydroxycoumarin (umbelliferon) and 7-hydroxyphenoxazone (resorufin) as substrates. Microsomal O-dealkylation activities of p-nitrophenol and 7-hydroxycoumarin O-alkyl derivatives were of similar levels, but the activities of 7-hydroxyphenoxazone O-alkyl derivatives were very low compared with those of other substrates. Pretreatment of rats with beta-naphthoflavone resulted in the preferential increase of O-deethylation and O-depropylation activities regardless of the ring structure of the substrates, and the ratio of O-deethylation and O-depropylation activities to that of O-demethylation increased markedly. On the other hand, the O-dealkylase activity of all substrates increased generally upon pretreatment of the rats with phenobarbital, but the ratio of O-deethylase or O-depropylase activity to that of O-demethylase in the pretreated rats was not very different from that of the untreated animals. Hexobarbital inhibited competitively the O-dealkylation activity in control and phenobarbital-pretreated rat microsomes. On the other hand, the O-dealkylase activity in microsomes obtained from beta-naphthoflavone-pretreated rats was inhibited remarkably by alpha-naphthoflavone, but not in microsomes prepared from untreated and phenobarbital-pretreated rats. Based on these results, this report discusses the relationship between the alteration of O-dealkylation activity and the composition change of cytochrome P-450 in microsomal membrane. Species differences in the substrate specificity of the O-dealkylation reaction and in the responsiveness of the animals to typical inducers were also observed using liver microsomes obtained from several animals under various conditions.     

Cytochrome P-450-dependent monooxygenase activities in prenatal and postnatal human livers: comparison of human liver 7-alkoxycoumarin O-dealkylases with rat liver enzymes

Human liver samples from 17 embryos, 5 fetuses, 5 infants and 4 adults were used to investigate human liver cytochrome P-450-dependent 7-alkoxycoumarin O-dealkylase activities, and their drug-metabolizing activities were compared to those of rat livers. The O-dealkylase activities in human embryos and fetuses were very low, although detectable, similar to those in fetal rats. Both male and female rats showed a postnatal increase of hepatic O-dealkylase activities with a maximum at about 30-40 days after birth and then a decline in the activities which was marked in female rats. Adult female rats showed a marked decrease in the hepatic enzyme activity observed in the O-depropylation reaction rather than the O-demethylation and O-deethylation reactions. During the developmental period of human infants, the O-demethylase activity, but not O-depropylase activity, increased gradually. Enzymes in adult human livers metabolize the O-methyl derivative of 7-hydroxycoumarin in preference to the O-ethyl and O-propyl derivatives. The metabolic activities of human adult enzymes for 7-alkoxycoumarin resembled those in adult female rats and were quite different from those in male rats. The study demonstrated that caution must be exercised in extrapolating pharmacological results from animal to man in the field of drug metabolism.     

Comparison of the effects of various inducers on 7-alkoxycoumarin O-dealkylase activities in liver microsomes

The effects of six inducers and malotilate on 7-alkoxycoumarin O-dealkylase activities in rat liver microsomes were examined. Phenobarbital (PB) (100 mg/kg) was administered intraperitoneally to rats for 6 days; 3-methylcholanthrene (3-MC) (40 mg/kg), beta-naphthoflavone (beta-NF) (40 mg/kg), isosafrole (150 mg/kg) and polychlorinated biphenyls (PCB) (100 mg/kg) were administered intraperitoneally for 3 days; isoniazid (INH) (50 mg/kg) was administered intraperitoneally for 10 days; and malotilate (500 mg/kg) was administered orally for 3 days. The O-dealkylase activities toward 7-methoxycoumarin (7-MC), 7-ethoxycoumarin (7-EC) and 7-propoxycoumarin (7-PC) were examined 24 hr after the final administration of the drugs. The ratios of 7-EC O-deethylase and 7-PC O-depropylase to 7-MC O-demethylase activity in the control and six inducer-treated groups were compared. The ratios in the groups treated with the six compounds, each of which induces a different form(s) of cytochrome P-450 (P-450), were clearly different from each other. Therefore, the measurement of 7-alkoxycoumarin O-dealkylase activities should be extremely useful for the routine determination of the molecular species of P-450. On the other hand, the ratio in the malotilate-treated group was different from that in any other inducer-treated group, so that there might be a possibility that malotilate induced a form(s) of P-450 that is different from any of the already known species.     

7-Alkoxyquinolines: new fluorescent substrates for cytochrome P450 monooxygenases

A series of 7-alkoxyquinolines was synthesized and tested as substrates with hepatic microsomes prepared from male Wistar rats. Microsomal O-dealkylation rates and kinetic constants were determined for the 7-alkoxyquinolines with microsomes from control, 3-methylcholanthrene (MC)-pretreated, and phenobarbitone (PB)-pretreated rats. Structure-activity relationship studies indicated that the 7-benzyloxyquinoline was the most rapidly metabolized substrate for control microsomes and those from PB-pretreated rats, whereas the 7-ethoxy- and 7-propoxyquinolines were O-dealkylated more rapidly by microsomes of MC-pretreated animals. Differences in activities occurred in Vmax and apparent Km values; however, there does not appear to be a correlation between these two values for the different quinoline substrates. Apparent Km and Vmax values for the 7-alkoxyquinolines were: control microsomes, Km = 71-773 microM, Vmax = 0.37-8.4 nmol 7-quinolinol/min/mg protein; MC microsomes, Km = 0.5-14 microM, Vmax = 0.29-2.7 nmol 7-quinolinol/min/mg protein; PB microsomes, Km = 2.8-46 microM, Vmax = 0.9-12 nmol 7-quinolinol/min/mg protein. All of the quinoline substrates gave Type I binding spectra with control and MC microsomes. With PB microsomes, Type I. Reverse Type I, and a mixture of the two types of binding spectra were observed. Comparisons of the structure-activity relationships, levels of induction, and kinetic constants were made with 7-alkoxycoumarin and 7-alkoxyphenoxazone analogs. In addition, three new coumarin substrates (7-pentoxy-, 7-hexoxy-, and 7-benzyloxycoumarin) are described.     

Application of capillary electrophoresis with pH-mediated sample stacking to analysis of coumarin metabolites in microsomal incubations

A sensitive method for the analysis of metabolites of coumarin by capillary electrophoresis (CE), incorporating pH-mediated sample stacking, was developed. The analytes were detected in phosphate buffer (pH 7.5; 25 mM), the matrix of the microsomal incubations. Detection was by direct UV absorbance. The three metabolites studied were 7-hydroxycoumarin (7-OHC), 4-hydroxycoumarin (4-OHC) and 2-hydroxyphenylacetic acid (HPAA), and the limits of detection of the analytes were 0.1, 0.5 and 0.3 microM, respectively. The developed method was then applied to microsomal incubations of coumarin. Male Cynomologus monkey microsomes were used in the study and 7-OHC was detected in the incubation mixture.     

Interspecies differences in coumarin metabolism in liver microsomes examined by capillary electrophoresis

1. A simple, rapid method was developed for studying xenobiotic metabolism by cytochrome P450 in liver microsome preparations. Capillary electrophoresis was used to separate the metabolite from the metabolic mixture.

2. Coumarin is metabolized to 7-hydroxycoumarin by a cytochrome P450 isoenzyme. Human, bovine, gerbil, mouse (Schofield, CD1), rat, rabbit, porcine, and cynomologus monkey microsomal preparations were investigated for coumarin metabolism by determining the content of 7-hydroxycoumarin present after metabolism.

3. Separation of 7-hydroxycoumarin from the reaction mixture was carried out in 50 mM phosphate buffer, pH 6.8, on a fused silica capillary at 25°C and 15 kV. The metabolic matrix consisted of an NADPH regeneration system, 205.5 μM coumarin, and the microsomal preparation. Standard curves were prepared in the microsomal preparation and the limit of quantification was 6.17 μM, with a linear range from 0 to 308.5 μM.

4. The reaction was initiated by the addition of the microsomes. An aliquot of the reaction mixture was removed at specific timed intervals over 2?h and injected directly onto a capillary electrophoresis column and the concentration of 7-hydroxycoumarin determined. The metabolism of coumarin to 7-hydroxycoumarin is greatest in human and monkey microsomes.     

Effect of a new sleep inducer, 1H-1,2,4-triazolyl benzophenone derivative 450191-S on rat liver drug-metabolizing enzyme system

The effect of a new sleep inducer, 450191-S, on the hepatic drug-metabolizing enzyme system was examined using rats and compared with those of nitrazepam and phenobarbital. Cytochrome P-450-dependent 7-alkoxycoumarin O-dealkylation activity determined using liver homogenate and isolated microsomes increased after successive oral administrations of 450191-S, and induction of the enzyme system was observed by administration of 150 approximately 200 mg/kg of the drug for at least 3 approximately 5 days. Normal activity was recovered with withdrawal of the drug for 3 approximately 5 days after induction of the hepatic enzyme system. Administration of higher amounts of 450191-S (200 approximately 600 mg/kg/day) for 3 days caused remarkable increases in the O-dealkylase and UDPGA-glucuronyltransferase activities and cytochrome P-450 and b5 contents. Similar changes in the hepatic enzyme system were observed with administration of nitrazepam (200 approximately 600 mg/kg for 3 days, p.o.) or phenobarbital (10 approximately 40 mg/kg for 3 days, i.p.). We concluded that the inducing activity of 450191-S is almost the same as that of nitrazepam, but weaker than that of phenobarbital. When the hepatic enzyme system was induced by the administration of either 450191-S or phenobarbital, the pentobarbital-induced sleeping time was shortened with increasing doses of the drugs. On the other hand, sleeping time was prolonged by the administration of another type of inducer, beta-naphthoflavone. The results suggest that the inductive pattern of 450191-S is similar to that of phenobarbital.     

Safety evaluation and drug development based on biological fate of drugs -efforts made to overcome drug interaction in drug development-

1. Assay methods to detect drug interaction in toxicological samples were established by determining cytochrome P450 content and its activity in liver samples. The O-dealkylation reaction of 7-alkoxycoumarin was indicated to reflect changes in the molecular forms of P450s, and the enzyme induction or inhibition in the toxicological samples was easily detected by using the established methods. 2. During toxicological studies of 450191-S or the sleep inducer rilmazafone, a phenobarbital type-induction of hepatic drug metabolizing enzymes was observed in animals, and the doses required for the induction differed markedly between rats and dogs. Enzyme induction was caused by some specific metabolites of 450191-S, and the plasma concentrations of these metabolites were comparable when the enzyme induction was developed in both animals. 3. A nonsteroidal antiinflammatory compound 480156-S showed a slight or no effect on microsomal drug metabolizing activity in rats. On the other hand, repeated administration of this compound to humans resulted in a marked decrease in the oxidative metabolism of 480156-S, followed by a marked increase in the plasma concentrations of the compound. When volunteers were given 480156-S followed by several drugs, such as tolubutamide, the plasma clearance was delayed remarkably, indicating a severe drug interaction. 4. Cytochrome P450 belonging to the CYP2C family was indicated to participate in the oxidative metabolism of 480156-S in both rat and human liver microsomes. The preincubation of microsomes with 480156-S caused a concentration-dependent inhibition of CYP2C-dependent tolubutamide hydroxylation reaction in both rats and humans. There was a marked species difference in the susceptibility to the inhibitory effect of 480156-S, and the concentration required to inhibit rat CYP2C was almost 10 times higher than that required in humans. 5. The cephem antibiotics having N-methyltetrazolethiol (NMTT) at the 3'-position substituent were demonstrated to inhibit mitochondrial low K(m) aldehyde dehydrogenase (ALDH), and produced disulfiram-like (Antabuse) reaction during alcohol metabolism. Pharmacokinetic studies indicated that NMTT released from the antibiotics in bile duct or intestine cause the inhibitory action followed by the development of disulfiram-like reaction. 6. Attempts had been made to develop new cephem antibiotics lacking the disulfiram-like reaction by changing the chemical structure of 3'-position substituents, and a hydroxyethyltetrazolethiol was found not to inhibit the enzyme. Based on this result, together with the antibacterial activity, we have developed a new oxacephem antibiotic flomoxef (6315-S). Flomoxef showed no disulfiram-like reaction both in rats and human.     

Analysis of the glucuronidation of 7-hydroxycoumarin by HPLC

The {ce:italic}in-vitro{/ce:italic} metabolism of 7-hydroxycoumarin to 7-hydroxycoumarin-glucuronide was investigated in bovine liver homogenate. A metabolic reaction mixture was prepared that included a crude preparation of uridine diphosphate (UDP) glucuronyl transferase, 7-hydroxycoumarin and UDP-glucuronic acid. A HPLC method was developed to separate coumarin, 7-hydroxycoumarin, 7-hydroxycoumarin-glucuronide and an internal standard, 4-hydroxycoumarin. Samples were separated by reverse-phase HPLC, on a C18 column, with a 1 ml min ⁻¹ gradient elution with UV detection at 320 nm. The limit of quantification of the method, for 7-hydroxycoumarin-glucuronide, was 1.47 μM, and the linear range was from 0–295.7 μM. Concentrations of 7-hydroxycoumarin-glucuronide produced were calculated from a plot of 7-hydroxycoumarin-glucuronide concentration versus the mean absorbance ratio ({ce:inline-formula}n = 4{/ce:inline-formula}) (7-hydroxycoumarin-glucuronide absorbance/4-hydroxycoumarin absorbance). It was possible to monitor the decrease in the 7-hydroxycoumarin content as it was metabolised as well as the increase in 7-hydroxycoumarin-glucuronide as it was produced enzymatically. The identity of the compound produced was confirmed by photodiode array spectral analysis. A plot of time versus 7-hydroxycoumarin-glucuronide produced indicates that the metabolism is linear for the first 90 min and reached a plateau at 150 min. The rate of reaction in the first 90 min was {ce:inline-formula}2.96 ± 0.06{/ce:inline-formula} (RSD 1.7%, {ce:inline-formula}n = 3{/ce:inline-formula}) nmol of 7-hydroxycoumarin-glucuronide produced per minute per milligram of protein. After 150 min {ce:inline-formula}0.34 ± 0.005mM{/ce:inline-formula} (RSD 1.4%) 7-hydroxycoumarin-glucuronide was produced, from 0.77 mM 7-hydroxycoumarin introduced into the reaction mixture and {ce:inline-formula}58.0% ± 5.3%{/ce:inline-formula} (or {ce:inline-formula}0.44 ± 0.02mM{/ce:inline-formula}) of the 7-hydroxycoumarin remained. These results show that it is possible to monitor the production of the phase II metabolite of coumarin with minimal sample clean-up and without the need for deconjugation of the glucuronide moiety. The method was very reliable and applicable for the direct determination of 7-hydroxycoumarin-glucuronide in an {ce:italic}in-vitro{/ce:italic} metabolic assay.     

Daily fluctuation of 7-alkoxycoumarin O-dealkylase activities in the liver of male F344 rats under ad libitum-feeding or fasting condition.

It has been reported that drug metabolizing enzyme activities in the liver fluctuate daily, though the daily fluctuation of 7-alkoxycoumarin O-dealkylase (ACD) activities catalyzed by P450 has not been examined. The ACD activities are known to be useful to clarify the extensive induction of P450-dependent oxygenases. In the present study, the hepatic ACD activities, as well as P450 content, were investigated periodically in male F344 rats under ad libitum-feeding or fasting condition. The ACD activities in ad libitum-fed rats were found to follow obvious daily fluctuations with high values during dark periods and with low values during light periods. This agreed with the other previous results of drug metabolizing enzyme activities measured with other substrates. Because the daily fluctuation was also observed in fasted rats, the fluctuation in ACD activities was clearly not affected by feeding, an external factor. P450 content, however, showed different fluctuation patterns from the ACD activities. This suggests that the fluctuation of ACD activities might not be related to the quantitative variation of P450 proteins.     

西格列他钠的体外代谢

目的研究拟治疗2型糖尿病的创新化合物西格列他钠(chiglitazar)的体外代谢速率、代谢酶和代谢转化,为临床应用提供参考。方法采用高效液相-紫外检测(HPLC-UV)的方法测定肝微粒体孵育液中西格列他钠的浓度,用特异性抑制剂的方法分析化合物的代谢酶,用大鼠肝微粒体体外研究西格列他钠可能的代谢产物和代谢途径。结果建立了可靠的测定大鼠肝微粒体中西格列他钠的HPLC分析方法;体外半衰期方法求得西格列他钠的t1/2为27.2min,固有清除率(Clint)为50.9mL.min-1.g-1蛋白;代谢酶研究表明,西格列他钠主要被P450酶中的CYP3A亚型代谢;西格列他钠在大鼠肝微粒体中的代谢主要为羟基化和O-脱烷基化,采用LC/MSn分析共发现了8个代谢物。结论西格列他钠是代谢活跃的化合物,有必要研究代谢产物的活性及临床注意药物相互作用。     

Metabolism of paraldehyde to acetaldehyde in liver microsomes: Evidence for the involvement of cytochrome p-450

A concentration-dependent acetaldehyde (AcH) generation was observed when paraldehyde was incubated with the mouse liver microsomal fraction. The process, which exhibited a requirement for oxygen and NADPH and was inhibited by carbon monoxide, was found to have a K_m of 17.9mM with respect to paraldehyde and a V_max of 40.1 nmoles/mg protein/min with respect to AcH formation. NADH was much less effective as an electron donor than NADPH, though a more than additive increase in AcH generation was observed when both of these nucleotides were added to the incubation. The rate of microsomal AcH generation from paraldehyde was increased 2.5-fold by pretreatment of the mice with phenobarbital but only 0.6-fold by pretreatment with 3-methylcholanthrene. Pretreatment with 2-diethylaminoethyl-2, 2-diphenylvalerate hydrochloride (SKF-525A) resulted in 54% inhibition of the reaction rate. Addition of metopirone to the incubation inhibited AcH generation in a concentration-related fashion, the inhibition being greatest, proportionately, in microsomes from phenobarbital-pretreated animals. The above results conclusively indicate the involvement of cytochrome P-540 mixed function oxidase in the formation of AcH from paraldehyde by mouse liver microsomes. It is also postulated that this process may be accomplished in the reaction analogous to O-dealkylation.     

Liver microsomal uptake of (14C)vinyl chloride and transformation to protein alkylating metabolites in vitro.

[1,2-¹⁴C]Vinyl chloride gas was incubated with rat liver microsomes in an all-glass vacuum system. Microsomal uptake and irreversible protein binding of vinyl chloride radioactivity was determined. Both uptake of vinyl chloride by microsomes and alkylation of proteins by vinyl chloride metabolites were dependent on incubation time, enzymatically active microsomes, NADPH, oxygen, and the partial pressure of vinyl chloride in the atmosphere, and could be inhibited by carbon monoxide. During incubation in presence of NADPH, 10 times more vinyl chloride was taken up by microsomes than in absence of NADPH. Uptake of vinyl chloride by albumin solutions and liposomal suspensions was in a similar range compared to the microsomal uptake without NADPH. Addition of glutathione and cytoplasmic fractions to microsomal incubations with NADPH led to an increase in microsomal uptake of vinyl chloride and to a decrease in protein alkylation by vinyl chloride metabolites. If trichloropropene oxide was present in the microsomal incubation, the protein alkylation reaction by vinyl chloride metabolites was increased twofold, while the microsomal uptake of vinyl chloride was not influenced. Our results are consistent with the view that the microsomal uptake of vinyl chloride radioactivity is due to transformation of vinyl chloride gas to nonvolatile metabolites by microsomal enzymes and that chloroethylene oxide might be the primary microsomal metabolite of vinyl chloride capable of reacting with proteins.     

Biotransformation of 7-ethoxycoumarin in isolated perfused rainbow trout liver

A reliable technique for rainbow trout liver perfusion has been developed for studies on xenobiotic biotransformation. Normal function of the perfused liver was indicated throughout the perfusion experiment by 1) a proper oxygen consumption, 2) a low leakage of intracellular enzymes, 3) a stable pH value in the effluent, and 4) a stable release of metabolites into the effluent perfusate and bile for at least 2 hr after maximal rate of metabolism was attained. The main metabolite of 7-ethoxycoumarin in effluent perfusate was identified as 7-hydroxycoumarin glucuronide. Only trace amounts were identified as sulfates. When fish were pretreated with Clophen A50 or beta-naphthoflavone, the amount of metabolites released into the effluent perfusate increased 3.4- and 6.4-fold, respectively, when compared to control livers. Furthermore, in livers from Clophen A50- or beta-naphthoflavone-treated fish, only 80 and 67%, respectively, of excreted products were conjugated. Influence of temperature on 7-ethoxycoumarin metabolism was studied in perfused liver and isolated liver microsomes. Results indicate that the Q10 for the metabolism of 7-ethoxycoumarin in perfused liver deviates from that found in isolated microsomes. The amount of metabolites excreted into the bile consisted of about 25% of the amount found in effluent perfusate. The only metabolite detected in bile from perfused liver from control as well as treated fish was 7-hydroxycoumarin glucuronide.     

Characterization by liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry of two coupled oxidative-conjugative metabolic pathways for 7-ethoxycoumarin in human liver microsomes treated with alamethicin.

The microsomal metabolism of 7-ethoxycoumarin (7-EC) was investigated using liquid chromatography (LC)-NMR and liquid chromatography-mass spectrometry (LC-MS) to characterize the coupling of oxidative-conjugative metabolism events. Within microsomes, cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs) are spatially disparate, each having surface and luminal localization, respectively. To optimize cofactor and substrate transit to UGT without compromising P450 activity, the pore-forming peptide alamethicin was used for microsomal perforation. Aqueous extracts of microsomal incubations containing NADPH and UDP-glucuronic acid were injected for LC-NMR and LC-MS analysis. The analytical complementarity of LC-NMR and LC-MS permitted the identification of four metabolites (M1 to M4). The metabolites M1 and M2 are novel microsomal metabolites for 7-EC, consistent with 3-hydroxylation and subsequent glucuronidation, respectively. Metabolites M3 and M4 were 7-hydroxycoumarin (7-HC) and 7-HC glucuronide, respectively. Viewed collectively, these results illustrate the utility of alamethicin in the examination of coupled oxidative-conjugative metabolism and the synergy of LC-NMR and LC-MS in metabolite identification.     

A possible mechanism of the impairment of hepatic microsomal monooxygenase activities after multiple administration of propranolol in rats.

The mechanism of selective inhibition of propranolol hydroxylations after multiple administration of the drug was investigated by metabolic inhibition studies in rat liver microsomes. The time course of irreversible binding of a reactive metabolic intermediate(s) of propranolol to liver microsomal protein, which was proposed as the cause of the impairment of enzymatic activities, had a delayed phase followed by a rapid linear rise, while the unmetabolized propranolol remaining in the reaction mixture showed a rapid linear decrease immediately after the onset of incubation. Thus, it was conceivable that the reactive intermediate(s) was not always formed directly from the parent drug, propranolol. Among four primary metabolites of propranolol, 4-hydroxypropranolol was the most potent inhibitor of propranolol hydroxylase activities, and this inhibition was much enhanced by preincubation of 4-hydroxypropranolol with NADPH. The type of inhibition kinetics of propranolol 5- and 7-hydroxylase activities by 4-hydroxypropranolol was changed from a competitive type to a non-competitive type by the preincubation. These results suggest that a reactive metabolite(s) of propranolol which impaired propranolol hydroxylase activities is a further metabolite(s) of 4-hydroxypropranolol.     

Behavioral and immunohistological assessment of painful neuropathy induced by a single oxaliplatin injection in the rat

The in vitro metabolism of permethrin and its hydrolysis products in rats was investigated. Cis- and trans-permethrin were mainly hydrolyzed by liver microsomes, and also by small-intestinal microsomes of rats. trans-Permethrin was much more effectively hydrolyzed than the cis-isomer. When NADPH was added to the incubation mixture of the liver microsomes, three metabolites, 3-phenoxybenzyl alcohol (PBAlc), 3-phenoxybenzaldehyde (PBAld) and 3-phenoxybenzoic acid (PBAcid), were formed. However, only PBAlc was formed by rat liver microsomes in the absence of cofactors. The microsomal activities of rat liver and small intestine were inhibited by bis-p-nitrophenyl phosphate, an inhibitor of carboxylesterase (CES). ES-3 and ES-10, isoforms of the CES 1 family, exhibited significant hydrolytic activities toward trans-permethrin. When PBAlc was incubated with rat liver microsomes in the presence of NADPH, PBAld and PBAcid were formed. The NADPH-linked oxidizing activity was inhibited by SKF 525-A. Rat recombinant cytochrome P450, CYP 2C6 and 3A1, exhibited significant oxidase activities with NADPH. When PBAld was incubated with the microsomes in the presence of NADPH, PBAcid was formed. CYP 1A2, 2B1, 2C6, 2D1 and 3A1 exhibited significant oxidase activities in this reaction. Thus, permethrin was hydrolyzed by CES, and PBAlc formed was oxidized to PBAld and PBAcid by the cytochrome P450 system in rats.     

Inter-species comparison of microsomal reductive transformation of biologically active benfluron N-oxide

Benfluron N-oxide is an anti-neoplastic active metabolite of benfluron (B) /1/. It is generated by flavine-monooxygenase-catalysed reactions /2/ and immediately undergoes subsequent metabolic transformations, the most important of which are reductive reactions /3/. The products of reductive pathways catalysed by two different microsomal enzymatic systems are the tertiary amine benfluron (i.e. the original parent compound) and/or 7-dihydrobenfluron N-oxide. Our studies on the reductive transformation of B N-oxide in rat, mouse, guinea-pig, rabbit, mini-pig and human microsomes have revealed significant species differences both in the yields of respective reduced metabolites and in the conditions essential for the activity of the reductases involved. While B, the original tertiary amine, is the main product of aerobic incubation of B N-oxide with NADPH in rat, mouse and mini-pig, significantly higher activities of the enzymes catalysing the formation of 7-dihydro-B N-oxide have been detected in rabbit and human microsomes. In rat, mouse and mini-pig, NADPH rather than NADH is the preferred coenzyme for B formation, and NADPH is also the preferred coenzyme for the formation of 7-dihydro-B N-oxide in most of the species used. The yield of tertiary amine B is higher in anaerobic rather than aerobic conditions in most experimental species studied. Aerobic or anaerobic incubating conditions have an insignificant effect on the formation of 7-dihydro-B N-oxide. Based on the inhibitory effect of CO on the reductive transformation of B N-oxide, cytochromes P450 can be assumed to participate in the formation of B both in rat and mini-pig, and, in mini-pig only, also in the formation of 7-dihydro-B N-oxide. Inter-species comparison of the properties of the reductases participating in the transformation of B N-oxide shows that the rabbit is a suitable model to study reductive transformation of B N-oxide in man.     

Sex difference in the O-dealkylation activity of 7-hydroxycoumarin O-alkyl derivatives in liver microsomes of rats

Sex differences in the O-dealkylation activities of O-alkyl derivatives of 7-hydroxycoumarin were compared using liver microsomes from male and female rats. The sex difference (male>female) in the O-depropylation activity was found to be greater than the sex differences in O-demethylation and O-deethylation activities. The magnitude of sex difference seen in the O-depropylation activity was diminished after pretreatment of rats with spironolactone, phenobarbital, isosafrole or 3-methylcholanthrene. The latter two inducers were much more effective than the former in enhancing the activity. The sex differences in the O-dealkylation activities were also seen when the activities were measured in the presence of cumene hydroperoxide instead of NADPH. The sex difference of the O-depropylation activity remained essentially unchanged by the fortification of male and female microsomes with purified NADPH-cytochrome c (P-450) reductase. The difference was also seen when reconstituted with cyto- chrome P-450 partially purified from both male and female rats by means of ω-amino-n-octyl Sepharose 4B and hydroxylapatite columns. The addition of 7,8-benzoflavone to the incubation mixture resulted in the increased magnitude of sex difference in the O-depropylation activity.From these results, we confirm that one or more cytochrome P-450 species other than a cytochrome P-450 species sensitive to 7,8-benzoflavone are present in male microsomes in higher amounts than in female microsomes.     

Tissue distribution of coumarin, 7-hydroxycoumarin and their 7-hydroxy metabolites following parenteral administration of 14C-labeled compound in the DBA/lac mouse

The time related tissue distributions of coumarin (C), 7-hydroxycoumarin (7HC) and their metabolites were studied in DBA/lac mice following retro-orbital injection of 14C-labeled compound. The activity was determined as dpm/ml or g wet weight over a period of 120 min. C was found in blood, kidney, liver, muscle, brain, heart, lung and fat but not in the testes. Upon C dosing, 7HC was found in kidney, liver and lung; 7-hydroxycoumarin glucuronide (7HCG) was found in blood, kidney and liver; 7-hydroxycoumarin sulfate (7HCS) was found in the blood, kidney and lung. Unidentified glucuronide and sulfate metabolites were detected in blood, kidney, liver and lung. Two unidentified unconjugated metabolites were detected in the kidney and liver. Unidentified water soluble metabolites were detected in kidney, liver, muscle, heart and testes. Upon 7HC dosing only 7HC, 7HCG and 7HCS were detected in any organ. 7HC and 7HCG were found in blood, kidney, liver, muscle and lung. 7HCS was detected in blood, kidney, liver and lung. Upon both C and 7HC dosing the kidney and liver contained the highest levels of radioactivity.