Changes in the metabolic profiles of R- and S-warfarin and R- and S-phenprocoumon as a probe to categorize the effect of inducing agents on microsomal hydroxylases

The biotransformation of R- and S-warfarin was examined using liver microsomes prepared from both noninduced rats and rats pretreated with Arochlor 1254, β-napthoflavone (BNF), pregnenolone-16α-carbonitrile (PCN), phenobarbital (PB), and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). For comparison, the metabolism of a closely related coumarin anticoagulant, R- and S-phenprocoumon, was determined using the same microsomal preparations. In noninduced microsomes the overall metabolism of warfarin was about five times that of phenprocoumon, with 7-hydroxywarfarin as the principle metabolite followed by the 6-, 4′- and 8-hydroxy derivatives (benzylic hydroxylation was not examined). For phenprocoumon a different regioselectivity was observed with 4′ hydroxylation being the greatest followed by 6-, and 7 and 8 hydroxylation. In the case of warfarin, hydroxylation with noninduced microsomes was either nonstereoselective (4′ hydroxylation) or selective for the R-enantiomer. The metabolic pattern observed for phenprocoumon showed hydroxylation to be either nonstereoselective (7 and 8 hydroxylation) or, in contrast to warfarin, selective for the S-enantiomer. Induction of cytochrome P-450 by Arochlor, BNF, and TCDD produced a similar metabolic pattern for both substrates in which 6 and 8 hydroxylation were greatly increased over control levels. In keeping with the pattern obtained from noninduced microsomes, a reversed stereoselectivity was again observed after induction with these three agents, i.e. warfarin metabolism was selective for the R-enantiomer and phenprocoumon metabolism was selective for the S-enantiomer. Based on cytochrome P-450 levels PCN decreased the metabolism of both substrates while PB had no effect. However, the induction with PB was readily apparent when calculations were performed on a per mg protein basis.     

Warfarin—stereochemical aspects of its metabolism by rat liver microsomes

The R- and S-enantiomers of warfarin were differentially metabolized by hepatic microsomes prepared from male Wistar and Sprague-Dawley rats. These studies were not only carried out with two strains of rats but were conducted independently in two laboratories employing different techniques. Although minor differences were observed, the same stereoselectivity was found for the microsomal transformations produced by both strains. The formation of 7- and 8-hydroxywarfarin was stereoselective for the R-enantiomer and in addition this enantiomer was metabolized more rapidly than the S-enantiomer. The converse stereoselectivity was found for the process of 4′-hydroxylation in Sprague-Dawley rats but it could not be conclusively shown for Wistar rats. A Michaelis-Menten analysis of the metabolic products of R- and S-warfarin formed by liver microsomes from male Sprague-Dawley rats is reported. The K_m for the processes of 6-, 7-, and 4′-hydroxylation for both isomers and the K_m for 8-hydroxylation of the R-isomer were all of the order of 0.03 to 0.11 mM and were not statistically different. The K_m for 8-hydroxylation of the S-isomer, 0.20 mM, was significantly greater. The K_m for benzylic hydroxylation of both isomers appeared to be still greater but was less precisely determined. The V_max for each of the enantiomeric pairs of products was statistically different. The kinetic data are interpreted as being inconsistent with the supposition that an arene oxide (6–7 and/or 7–8) may serve as the intermediate in the formation of 7-hydroxywarfarin from either isomer. Further, if product formation is assumed to be rate limiting, the data provide evidence for at least three distinct enzymatic processes which may or may not be distinct hemoproteins. Reduction of the side-chain ketonic function of warfarin to the corresponding diastereomeric warfarin alcohols by the 105,000 g supernatant fraction displayed both a high degree of stereoselectivity (R-isomer) and stereospecificity (S-reduction). This reduction was best catalyzed by NADPH rather than by NADH. Determination and quantification of the metabolic products obtained after incubation of R- and S-warfarin with the 10,000 g supernatant were consistent with the summation of those independently produced by the microsomal pellet and the 105,000 g supernatant.     

Stereoselective metabolism of conformational analogues of warfarin by beta-naphthoflavone-inducible cytochrome P-450

Previous studies have shown that the structurally related oral anticoagulants warfarin and phenprocoumon are regioselectively hydroxylated in the 6- and 8-positions by hepatic microsomes obtained from 3-methylcholanthrene (3-MC) or beta-naphthoflavone (BNF) pretreated rats. Stereoselectivity for hydroxylation is also observed and favors (R)-warfarin but (S)-phenprocoumon. The possibility that the stereoselectivity of warfarin hydroxylation is a function of the solution conformation of the drug was tested with conformationally restricted analogues. In these experiments the analogues were incubated with microsomes obtained from BNF-pretreated rats and any stereoselectivity associated with 6- and 8-hydroxylation was determined. The R enantiomer of cyclocoumarol, the cyclic ketal analogue of warfarin, was found to be selectively hydroxylated, in contrast to the S enantiomer of warfarin 4-methyl ether, the ring-opened analogue. The latter compound is known to have a preferred solution conformation similar to that of phenprocoumon. The results suggest that at the active site of BNF-induced cytochrome P-450 (R)-warfarin is metabolized in its cyclic hemiketal tautomer, a form which spatially mimics the preferred solution conformation of (S)-phenprocoumon.     

Roles of two allelic variants (Arg144Cys and Ile359Leu) of cytochrome P4502C9 in the oxidation of tolbutamide and warfarin by human liver microsomes

1. Tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities were determined in liver microsomes of 39 Japanese and 45 Caucasians genotyped for the cytochrome P450 (P450 or CYP) 2C9 gene into three groups, namely the wild-type (Arg144·Ile359), and two heterozygous Cys allele (Cys144·Ile359) and Leu allele (Arg144·Leu359) variants. 2. Good correlations were found between tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities in liver microsomes of Japanese and Caucasians. Humans with the Cys allele CYP2C9 variant, which was detected in 22% of Caucasians, were found to have similar catalytic rates to those of the wild-type in the oxidations of tolbutamide and racemic warfarin, whereas humans with the Leu allele, which was detected in 8% Japanese and 7% Caucasian samples, had lower catalytic rates than those of other two groups. 3. The rates of 6- and 7-hydroxylation of racemic warfarin were correlated well with those of S-warfarin, but not R-warfarin, in human liver microsomes. 4. Both human liver microsomes and recombinant CYP2C9 catalysed 7-hydroxylation of S-warfarin more extensively than those of R-warfarin. K_m's for the 7-hydroxylation of S-warfarin were not very different in liver microsomes of humans with these three genotypes. Anti-CYP2C9 antibodies and sulphaphenazole inhibited the 6- and 7- hydroxylation of S-warfarin, but not R-warfarin,by > 90% and the methyl hydroxylation of tolbutamide by about 50%. 5. These results suggest that humans with Leu allele of CYP2C9 have lower V_max's for S-warfarin 7-hydroxylation and tolbutamide methyl hydroxylation than those with wildtype and Cys allele CYP2C9, although the K_m's are not very different in liver microsomes m of these three groups of humans. R-warfarin hydroxylation may be catalysed by P450 enzymes other than CYP2C9 in man.     

Cholesterol 7α-hydroxylase—Evidence for a distinct hepatic microsomal enzyme system

The interrelationship of microsomal 7α-hydroxylation and drug oxidation reactions was studied in liver microsomes obtained from the male Wistar rat. When several compounds were administered to rats, a variety of changes ensued concerning the rate of cholesterol 7α-hydroxylation, ethylmorphine N-demethylase activity, and alterations in electron transport components. Cholestyramine and d-thyroxine administration resulted in significant increases in cholesterol 7α-hydroxylase activity. Both phenobarbital (PB) and spironolactone pretreatment did not produce an elevation of cholesterol 7α-hydroxylation, but significant increases in ethylmorphine N-demethylation over controls were realized. There was a lack of congruence with the rate of cholesterol 7α-hydroxylation and the content or activity of electron transport components whereas there was a demonstrable dependency of ethylmorphine N-demethylation on the monitored electron transport components for PB- and spironolactone-treated rats. Along with an elevation of cytochrome P448 content, 3-methylcholanthrene (3-MC) pretreatment reduced cholesterol 7α-hydroxylase activity and the rate of ethylmorphine N-demethylation. Concomitant treatment with 3-MC and cholestyramine caused no alteration of cholesterol 7α-hydroxylase activity. The inhibitory action of 3-MC on cholesterol 7α-hydroxylase activity was not due to competition for reducing equivalents in liver microsomes. The inhibitory phenomenon caused by NADH on microsomal cholesterol 7α-hydroxylase activity was reproducible and was dose-dependent at both subsaturating and saturating levels of NADPH. In conclusion, the results of this study indicate that the cholesterol 7α-hydroxylase enzyme system is distinctly different from that which catalyzes drug oxidations.     

Effect of microsomal enzyme inducers on the soluble enzymes of hepatic phase II biotransformation

Numerous xenobiotics induce microsomal enzymes such as cytochrome P-450-dependent monooxygenases, epoxide hydrolase, and UDP-glucuronyltransferase by causing an increase in enzyme synthesis. Since induction of soluble enzymes involved in phase II biotransformation has not been thoroughly studied, effects of the following microsomal enzyme inducers on three important soluble enzymes were examined: phenobarbital (PB), 3-methylcholanthrene (3-MC), butylated hydroxyanisole (BHA), isosafrole (ISF), pregnenolone-16α-carbonitrile (PCN), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and trans-stilbene oxide (TSO). Representative microsomal enzymes of phase I pathways were examined simultaneously to indicate effective induction. The inducers selected produced the expected increases in hepatic cytochrome P-450 (75–170%), ethylmorphine (200–260%), and benzphetamine (100–260%) N-demethylation, benzo[a]pyrene hydroxylation (300%), ethoxyresorufin O-deethylation (2700%), and styrene oxide hydration (100–270%). The soluble conjugative enzymes studied were glutathione S-transferase, N-acetyltransferase, and sulfotransferase. Glutathione conjugation of 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, and sulfobromophthalein was increased by TSO (100–160%), BHA (60–80%), ISF (60–80%), and PB (60–80%). β-Naphthylamine N-acetyltransferase activity was increased by PCN and 3-MC (60 and 40%, respectively). Only PCN was able to enhance sulfotransferase. Sulfation of 2-naphthol, taurolithocholate, and dehydroepiandrosterone was increased by 28, 111, and 140%, respectively. In conclusion, while microsomal enzymes could be readily induced, activity of soluble phase II enzymes was increased to a much lesser extent. Of the inducers studied, PCN was the most effective at increasing activity of soluble enzymes.     

Stereochemical aspects of parachloroamphetamine metabolism: Rabbit liver microsomal metabolism of RS-, R(-)-, and S(+)-para-chloroamphetamine

With the aid of a chiral derivatizing reagent and a sensitive and specific nitrogen-phosphorus gas Chromatographic assay, metabolism of para-chloroamphetamine (PCA) enantiomers has been studied following incubation of racemic (RS)-, R(?) and S(+)-PCA with rabbit liver microsomal preparations. Significant metabolism of racemic PCA and the individual enantiomers was observed following incubation in rabbit liver microsomal preparations. Metabolism required viable microsomes, NADPH and molecular oxygen, and the rate of metabolism increased following pretreatment with phenobarbital. Incubation of racemic PCA resulted in more rapid metabolism of the S(+) enantiomer than of the R(?) enantiomer. When the enantiomers were incubated individually, each enantiomer was metabolized more rapidly than when incubated as part of a racemic mixture, and the R(?) enantiomer was metabolized more rapidly than the S(+) enantiomer.     

In vivo and in vitro effects of 3-methylcholanthrene on the microsome-mediated in vitro mutagenicity of 2-acetylaminofluorene

Pretreatment of rat, hamster or mouse by 3-methylcholanthrene (3-MC) largely induces the liver microsomal N-hydroxylase activity. The same pretreatment given simultaneously with 2-acetylaminofluorene (2-AAF) inhibits the hepatocarcinogenicity in the rat but not in the hamster.The present report compares the in vivo and in vitro effects of 3-MC on liver microsomal N-hydroxylation and liver microsome-mediated mutagenicity of 2-AAF in hamster, rat and mouse. The induction of hamster or mouse liver microsomal N-hydroxylase activity correlates well with the increase in the microsome-mediated mutagenicity of 2-AAF. With rat, however, even though the N-hydroxylase activity is largely enhanced, microsome-mediated mutagenicity is significantly reduced after pretreatment with 3-MC. Such a reduction parallels a decrease in enzyme affinity.Added in vitro to the incubation medium, 3-MC (μM concentration) inhibits both the N-hydroxylase activity and the microsome-mediated mutagenicity of 2-AAF. Those data are discussed in relationship with the biological interactions between 3-MC and 2-AAF.     

Differential effects of aging on hepatic microsomal monooxygenase induction by phenobarbital and β-naphthoflavone

The influence of aging on hepatic microsomal monooxygenase induction by phenobarbital (PB) or β-naphthoflavone (BNF) was investigated in male Fischer 344 rats maintained in a constant environment. PB-induced increases in microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity were similar in rats aged 3–5 months (young-adult) and 24–25 months (old), but increases in benzephetamine N-demethylase activity were markedly diminished in the old rats. Separation of hepatic microsomal proteins by sodium dodecylsulfate gel electrophoresis demonstrated that aging decreased the induction by PB of a polypeptide with a molecular weight of 52,500. BNF-induced increases in microsomal cytochrome P-450 and nitroanisole O-demethylase activity were greater in old than in young-adult rats, and BNF induction of 55,000 and 57,000 molecular weight microsomal polypeptides was increased slightly in livers from old rats. The results indicate that age-related effects on monooxygenase induction vary with different inducers of the hepatic microsomal enzyme system.     

Mixed function oxygenation of the lower fatty acyl residues--I. Hydroxylation of the acetic, propionic, butyric, and valeric residues by rabbit liver microsomes.

Rates of hydroxylation of C-atoms in various positions [ω-, (ω-1)-, (ω-2)-] of an increasing chain length have been measured with the 4-chloroanilides of acetic, propionic, butyric, and valeric acid as substrates. The hydroxylation products were separated by t.l.c, quantified by u.v. spectroscopy and further characterized by n.m.r. and o.r.d. spectroscopy. The hydroxylation products in which an asymmetric centre had been introduced by oxygenation, were shown to be optically active, the sign of the optical rotation indicating an excess of the S-over the R-isomer. From the alteration of the hydroxylation pattern caused by previous treatment of rabbits with either phenobarbital or 3-methylcholanthrene it can be deduced that the ω, (ω-1), and (ω-2)-hydroxylation of the lower fatty acyl residues is not only catalysed by PB- and 3-MCh-inducible forms of cytochrome P-450 but also by the forms not inducible by either PB or 3-MCh.     

Microsomal metabolism of arylamides by the rat and guinea pig—I.: Oxidation of N-3-fluorenylacetamide at carbon-atom-9—A major metabolic reaction

The N-hydroxylation of N-3-fluorenylacetamide (3-FAA), an isomer of the carcinogen, N-2-fluorenylacetamide (2-FAA), by hepatic microsomes of untreated and 3-methylcholanthrene (3-MC)-treated guinea pigs was found to be of a similar low order as that previously observed in the rat. Hepatic microsomes of the guinea pig and of the rat converted 3-FAA to N-(9-hydroxy)-3-FAA and to N-(9-oxo)-3-FAA. These new metabolites were separated and identified by high-pressure liquid chromatography (h.p.l.c.). N-(9-hydroxy)-3-FAA was the major product of the hydroxylation of 3-FAA by hepatic microsomes of the rat or guinea pig exceeding the formation of N-(7-hydroxy)-3-FAA, the principal phenolic metabolite of 3-FAA. The amounts of N-(9-oxo)-3-FAA formed were about one-third of the amounts of N-(9-hydroxy)-3-FAA produced. In contrast to the formation of phenolic metabolites, the hydroxylation of 3-FAA to N-(9-hydroxy)-3-FAA was not increased by pretreatment of guinea pigs or rats with 3-MC. Similarly, pretreatment of rats with PB did not enhance the yield of N-(9-hydroxy)-3-FAA. Co inhibited the formation of N-(9-hydroxy)-3-FAA by 80 per cent. These data lead us to conclude that the formation of N-(9-hydroxy)-3-FAA is catalyzed by a microsomal hemoprotein not identical with cytochrome P₁-450 or P-450. In contrast to 3-FAA, 2-FAA appeared to be a poor substrate for hydroxylation to the N-(9-hydroxy)-2-FAA. The susceptibility of 3-FAA to hydroxylation at carbon-atom 9 of the fluorene moiety may be rationalized by resonance structures in which carbon-atom 9 is positively charged and acts as a electrophilic center. Similar resonance structures cannot be written for 2-FAA.     

Additional routes in the metabolism of phenacetin

1. ω-Hydroxylation of the ethyl moiety of phenacetin by rabbit-liver microsomal preparations was slow, but was increased 10-fold by pretreatment of the animals with phenobarbitone (PB), and was decreased 2·8-fold by treatment with 3-methylcholanthrene (3-MC) or β-naphthoflavone (β-NF).

2. N-[4-(2-hydroxyethoxy)phenyl]acetamide (β-HAP), the ω-hydroxylation product, which was detected in trace amounts only in the urine of rabbits injected with phenacetin, was converted into [4-(acetylamino)phenoxy]acetic acid (4-APA) by the microsomal and cytosolic fraction of liver homogenate and NADP⁺ or NAD⁺. Rabbits excreted 56% of a dose of β-HAP as 4-APA in the 48?h urine.

3. Phenacetin, injected i.p. into rabbits previously treated with PB, was excreted in the urine as 4-APA (12·2% of dose).

4. β-HAP formed endogenously or added as substrate in vitro was recovered as the O-acetyl derivative, when ethyl acetate was used for extraction of metabolites from microsomal incubation mixtures.

5. (ω-1)-Hydroxylation of the ethyl moiety of phenacetin, which gave 4-acetamido-phenol, occurred rapidly with rabbit-liver microsomal preparations, and was not increased significantly after pretreatment of animals with either PB or 3-MC.

6. ω-Hydroxylation of the acetic moiety of phenacetin by rabbit-liver preparations to give N-(4-ethoxyphenyl)glycolamide (4-GAP) was slow, but was increased three-fold after pretreatment of animals with 3-MC or β-NF, whereas PB had no effect. 4-GAP was detected in trace amounts only in the urine of rabbits injected i.p. with phenacetin.

7. N-Hydroxylation of phenacetin by rabbit-liver microsomal preparations was slow, but increased three-fold after treatment of animals with 3-MC, and was unchanged by PB.

8. N-Hydroxylation of phenacetin by hepatic microsomes from 3-MC-treated rabbits was 26 times slower than that of 2-acetylaminofluorene; no N-hydroxy derivatives of N-(4-chlorophenyl)acetamide and propanil were detected in vitro.     

Microbial Models of Mammalian Metabolism: Stereoselective Metabolism of Warfarin in the Fungus Cunninghamella elegans

Biotransformation stereoselectivity of warfarin was studied in the fungus Cunninghamella elegans (ATCC 36112) as a model of mammalian metabolism. This organism was previously shown to produce all known phenolic mammalian metabolites of warfarin, including 6-, 7-, 8-, and 4-hydroxywarfarin, and the previously unreported 3-hydroxywarfarin, as well as the diastereomeric warfarin alcohols, warfarin diketone, and aliphatic hydroxywarfarins. Using S-warfarin and R-warfarin as substrates, and an HPLC assay with fluorescence detection to analyze metabolite profiles, the biotransformation of warfarin was found to be highly substrate and product stereoselective. Both aromatic hydroxylation and ketone reduction were found to be stereoselective for R-warfarin. Ketone reduction with the warfarin enantiomers exhibited a high level of product stereoselectivity in that R-warfarin was predominantly reduced to its S-alcohol, while S-warfarin was reduced primarily to the corresponding R-alcohol.     

Some characteristics of hamster liver and lung microsomal aryl hydrocarbon (biphenyl and benzo(a)pyrene) hydroxylation reactions

Aryl hydrocarbon hydroxylation (AHH) reactions were compared using liver and lung microsomes of corn oil- and 3-methylcholanthrene (3-MC)-treated hamsters, employing benzo(a)pyrene (BAP) and biphenyl as substrates. The predominant AHH activity of liver and lung microsomes from corn oil- or 3-MC-treated hamsters was biphenyl 4-hydroxylase. Biphenyl 2-hydroxylase and BAP-hydroxylase activities were approximately 50 per cent as active as biphenyl 4-hydroxylase in liver and approximately 1–3 per cent as active as biphenyl 4-hydroxylase in lung microsomes. Biphenyl 4-hydroxylase activity was 70–80 per cent as active in lung as in liver microsomes. Treatment with 3-MC in vivo induced the biphenyl 4-hydroxylation reaction in liver but not in lung microsomes, the biphenyl 2-hydroxylation reaction both in lung and liver microsomes, and the BAP hydroxylation reaction in lung but not in liver microsomes. Biphenyl 2- and 4-hydroxylase activities of liver microsomes displayed similar sensitivities to inhibition by a number of chemical inhibitors in vitro. Inhibition of biphenyl hydroxylation reactions by metyrapone or carbon monoxide did not distinguish between lung or liver microsomal mono-oxygenases of corn oil- or 3-MC-treated hamsters. While small differences were expressed by inhibition with ethylmorphine, large differences became apparent through inhibition studies with BAP or α-naphthoflavone. It is concluded that the major aromatic hydroxylase activity of lung microsomes from corn oil- or 3-MC-treated hamsters resembles the constitutive (uninduced) AHH of the liver microsomes and that the minor aromatic hydroxylase activity of lung microsomes from corn oil- or 3-MC-treated hamsters resembles the induced AHH of the liver microsomes.     

Metabolism of dichlorobiphenyls by hepatic microsomal cytochrome P-450

In vitro rat hepatic microsomal metabolism of ten individual dichlorobiphenyls (DCBs) has been investigated as part of a major study of the role of metabolism in the toxicity of polychlorinated biphenyl (PCB) pollutant mixtures. The DCBs were metabolized to monohydroxy and dihydrodiol metabolites and unstable metabolites of intermediate polarity. DCBs with both chloro substituents on the same ring, one or both of which were ortho substituents, were susceptible to the same regioselectivities for hydroxylation by control, phénobarbital (PB)- or β-naphthoflavone (BNF)-induced cytochromes P-450 (principally in the 4-position), with the greatest rates of hydroxylation arising with PB-induced cytochrome P-450. In contrast, DCBs with no ortho chlorosubstituents had regioselectivities for hydroxylation by control and PB-induced cytochrome P-450 which differed from that of BNF-induced cytochromes P-450; the greatest rates of hydroxylation were with BNF-induced systems. DCBs with one chloro substituent on each ring were metabolized, with the site of hydroxylation being under the electronic influence of the chloro substituent. With 4,4'-DCB, 60 per cent of the hydroxylated DCB metabolite underwent an NIH shift [G. Guroff, J. W. Daly, D. M. Jerina, J. Renson, B. Witkop and S. Udenfriend, Science157, 1524 (1967)]. The BNF-induced system produced the highest rates of dihydrodiol fomation that were eliminated by an epoxide hydratase inhibitor. The results indirectly prove that arene oxides are intermediates in DCB metabolism and are possibly the source of DCB mutagenicity. The PCBs 2,4,2'4'- and 3,4,3',4'-tetrachlorobiphenyl induced the same effects as PB and BNF respectively. Thus, PCBs differentially affect the metabolism of their individual components and are, possibly, responsible for enhancing their own toxicity by inducing enhanced rates of formation of arene oxide intermediates.     

Effects of induction of R- and S-warfarin and metabolite concentrations in rat plasma

The plasma half-lives of R- and S-warfarin were statistically indistinguishable (p < 0.05) for uninduced male Wistar rats, although plasma concentrations of S-warfarin were invariably higher at equivalent doses. Induction of hepatic mixed-function oxidase activity by phenobarbital significantly shortened the plasma half-lives and lowered the plasma zero-time concentrations of both enantiomers relative to uninduced rats. In contrast, induction with 3-methylcholanthrene produced a significantly lower than normal zero-time concentration of R-warfarin but not of S-warfarin. R- and S-warfarin plasma half-lives did not differ significantly from one another in phenobarbital or 3-methylcholanthrene-induced rats, but the half-lives differed significantly between the two induced groups. Concentrations of S-warfarin metabolites exceeded those of R-warfarin metabolites in the plasma of uninduced or induced rats. The major plasma metabolites were 4′-hydroxywarfarin from S-warfarin and, to a lesser extent, from R-warfarin. Plasma R- and S-warfarin metabolite patterns were markedly different from in vitro patterns catalyzed by hepatic microsomal enzymes of uninduced and phenobarbital- or 3-methylcholanthrene-induced rats. In uninduced rats a dose of 1 mg of S-warfarin/kg produced an anticoagulant response equivalent to 10 mg of R-warfarin/kg. The intrinsic activity of S warfarin was calculated to be 4.9 ± 0.4 times that of R-warfarin. Induction of rats with phenobarbital or 3-methylcholanthrene diminshed the magnitude of the normal anticoagulant response to R- and S-warfarin.     

Covalent binding in vitro of polychlorinated biphenyls to microsomal macromolecules. Involvement of metabolic activation by a cytochrome P-450-linked mono-oxygenase system.

Incubation of ¹⁴C-labeled polychlorinated biphenyls (PCBs) with rat, mouse or rabbit liver microsomes in the presence of an NADPH-generating system and molecular oxygen caused covalent binding of radioactive metabolites of PCBs to microsomal macromolecules. The binding was more pronounced with liver microsomes from animals pretreated with inducers of the microsomal mono-oxygenase system. The order of induction effect of the inducers used was KC-500 (a PCB preparation containing 55% chlorine) ≥ phenobarbital (PB) ? 3-methylcholanthrene (3-MC) in rats, PB > KC-500 > 3-MC in mice, and PB > KC-500 in rabbits. [¹⁴C]KC-300 (a PCB preparation containing 42% chlorine) was more effective than [¹⁴C]KC-500 as substrate for all the microsomal preparations. The binding reaction was dependent on both NADPH and oxygen, sensitive to carbon monoxide, glutathione, cysteine, hexobarbital, and aniline, and enhanced by EDTA, which inhibits lipid peroxidation. The addition of NADH, which was by itself a very poor electron donor, caused a synergistic increase of the NADPH-dependent binding of PCBs. It is concluded that the conversion of PCBs to active metabolites by the cytochrome P-450-linked mono-oxygenase system is prerequisite to the binding reaction. A survey of the effects of various inducers suggested that a cytochrome P-450 having a high aminopyrine N-demethylation activity is mainly responsible for the metabolic activation of PCBs in liver microsomes. Kidney and lung microsomes from untreated rats were virtually devoid of the PCB-binding capacity, but in kidney microsomes this capacity could be induced by pretreatment with 3-MC or KC-500, though not with PB.     

Drug Metabolism in Rats with Cancer Induced by N-Nitrosodiethylamine and Phenobarbital

Abstract: The metabolism of R- and S-warfarin in vivo and in vitro, bufuralol in vitro, and antipyrine and debrisoquine in vivo were studied in rats with cancer induced by N-nitrosodiethylamine and phenobarbital treatment. Microsomal cytochrome P-450 content was greatly reduced in both healthy and cancerous parts of the livers of tumour-bearing animals. The specific activities of R-warfarin and bufuralol 1′-hydroxylases were significantly elevated in rats with cancer. The activities of S-warfarin hydroxylases expressed per mg microsomal protein were reduced in animals with cancer, whereas those of R-warfarin and bufuralol 1′-hydroxylases were not. The urinary excretion of R-7-hydroxywarfarin was increased and those of S-6- and S-4′-hydroxywarfarin decreased in rats with cancer. The correlations between microsomal formation and urinary excretion of all warfarin metabolites were poor, except for R-7-hydroxywarfarin. Antipyrine oxidation was increased in the cancerous state but the urinary metabolic profiles were similar in rats with cancer and in controls. The metabolism of debrisoquine was decreased in tumour-bearing animals. Antipyrine metabolism did not show any correlation with either warfarin or debrisoquine metabolism, whereas several relationships were observed between warfarin and debrisoquine metabolism and between warfarin and bufuralol metabolism.     

Induction studies on the functional heterogeneity of rat liver UDP-glucuronosyltransferases

Differential induction with phenobarbital (PB) and 3-methylcholanthrene (3-MC) suggests at least two functionally distinct UDP glucuronosyltransferases (UDP-GT) which have different acceptor selectivities. One form is induced by 3-MC and preferentially conjugates group 1 acceptors, such as p-nitrophenol and 1-naphthol. Another UDP-GT is induced by PB and glucuronidates group 2 aglycones, morphine and chloramphenicol. To further study this functional heterogeneity, male Sprague-Dawley rats were pretreated with the following microsomal enzyme inducers: 7,8-benzoflavone (BF); benzo(a)pyrene (BP); 3-MC; 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); butylated hydroxyanisole (BHA); isosafrole; PB; pregnenolone-16α-carbonitrile (PCN); trans-stilbene oxide (TSO). The effect of induction on UDP-GT activity was determined with nine acceptors. Conjugation of group 1 aglycones, naphthol and p-nitrophenol, was increased by 3-MC (185 and 80%, respectively) whereas PB was ineffective. Conjugation of group 2 acceptors, morphine and chloramphenicol, was stimulated by PB (120 and 250%, respectively) while 3-MC had little effect. BP and TCDD enhanced glucuronidation of group 1 aglycones. ISF and TSO induced conjugation of both acceptor groups but were more effective for group 2. BF and BHA had negligible effects on UDP-GT activity. Since glucuronidation of valproic acid was increased only by PB and TSO treatment, this aglycone is probably a group 2 acceptor. Conjugation of digitoxigenin-monodigitoxoside (DIG) was stimulated by PB (200%) and PCN (1200%). PCN did not induce glucuronidation of group 1 acceptors but did have a slight effect on group 2 aglycones (130 and 40% for chloramphenicol and morphine, respectively). The 12-fold increase in DIG conjugation by PCN pretreated rats suggests that PCN may induce another group (form) of UDP-GT which preferentially glucuronidates DIG. Differential induction of UDP-GT activities within each group of acceptors indicates possible additional heterogeneity of the transferase.     

手性衍生化-反相高效液相色谱法测定大鼠肝微粒体中盐酸普罗帕酮对映体及其在代谢研究中的应用

目的　建立鼠肝微粒体中盐酸普罗帕酮消旋体(R/S-PPF)的手性拆分法,以研究大鼠肝微粒体中R/S-PPF体外代谢的立体选择性。方法　用GITC柱前手性衍生化、反相高效液相色谱法拆分R/S-PPF;外标法定量;体外微粒体孵育试验。结果　基线拆分了盐酸普罗帕酮两对映体,容量因子分别为7.9和9.5,分离系数α为1.2,分离度R为1.9,线性范围0.5～320 μg.mL^-1,检测限100 ng.mL^-1,定量限5 μg.mL^-1(RSD<15%)。平均绝对回收率S-PPF为77.1%,R-PPF为76.0%。平均日内、日间精密度均<10%。在DEX和BNF诱导鼠肝微粒体中,PPF的代谢呈显著的立体选择性,在空白对照微粒体中的代谢未呈立体选择性。结论　此法简便、经济,可用于鼠肝微粒体中R/S-PPF代谢的立体选择性研究。