Shoichi Fujita

Age-associated alterations in hepatic glutathione-S-transferase activities

Age-associated alterations of hepatic cytosolic glutathione-S-transferase (GST) activities towards sulfobromophthalein sodium tetrahydrate (BSP), styrene oxide (STOX), trans-4-phenyl-3-butene-2-one (PBO), 1,2-dichloro-4-nitrobenzene (DCNB), and 1-chloro-2,4-dinitrobenzene (CDNB) were investigated in Fischer-344 rats of both sexes with ages ranging from 1.5 to 28 months. The GST activities towards PBO and DCNB in male rats increased with age till 6-12 months when maximum values were attained, and then gradually decreased till 28 months when the values became the lowest. The GST activities towards STOX and BSP did not show any significant increase after 1.5 months and stayed at this level till 12 months, followed by a gradual decrease till 28 months when the values were the lowest. In contrast, the GST activity towards CDNB in male rats did not show much of an age-associated alteration. Age-associated alterations in GST activities in females were much smaller than those observed in males. Sex differences in GST activities (significantly higher male values than female values) were observed with all the substrates examined at least at some time of the animal life. The kinetic studies of GST activities indicated that alterations in the relative abundance as well as the total quantity of GST isozymes caused the substrate selective alterations of GST activities with age.

Cytochrome P450 isozymes catalyzing 4-hydroxylation of parkinsonism-related compound 1,2,3,4-tetrahydroisoquinoline in rat liver microsomes.

Microsomal 4‐hydroxylase of 1,2,3,4‐tetrahydroisoquinoline (TIQ), a possible candidate for causing Parkinson disease, was characterized by using rat hepatic microsomes and purified P450 isozymes. Kinetic analysis revealed that Km and Vmax values (mean ± se) for hepatic microsomal TIQ 4‐hydroxylase of male Wistar rats were 319.6 ± 26.8 μm and 12.13 ± 1.43 pmol·min–1·mg–1 protein, respectively. When TIQ 4‐hydroxylase activity was compared in Wistar (an animal model of extensive debrisoquine metabolizers) and Dark Agouti (an animal model of poor debrisoquine metabolizers) rats, significant strain (Wistar > Dark Agouti) and sex (male > female) differences were observed. The microsomal activity toward TIQ 4‐hydroxylation was increased by pretreatment of male Wistar rats with P448 inducers (β‐naphthoflavone and sudan I), but not with phenobarbital. Pretreatment with propranolol, an inhibitor of P450 isozymes belonging to the P450 IID gene subfamily, decreased TIQ 4‐hydroxylase activity. P450 BTL, a P450 isozyme belonging to the IID subfamily, showed TIQ 4‐hydroxylase activity of 64.1 pmol · min–1 · nmol P450–1, which was 3.2‐fold that of microsomes (20.9 pmol · min–1 · nmol P450–1). Antibody (IgG) against this isozyme suppressed microsomal TIQ 4‐hydroxylase activity concentration‐dependently. A male‐specific P450 ml (P450IIC11) catalyzed this reaction to a much lesser extent (10.0 pmol·min–1·nmol P450–1), and its antibody did not affect the microsomal activity. These results suggest that TIQ 4‐hydroxylation in hepatic microsomes are catalyzed predominantly by a P450 isozyme (or isozymes) belonging to the IID gene subfamily in non‐treated rats and its immunochemically related P450 isozyme (or isozymes), and that a P450 isozyme (or isozymes) belonging to the IA subfamily also participates in TIQ 4‐hydroxylation in rats pretreated with P448‐inducers.—Suzuki, T.; Fujita, S.; Narimatsu, S.; Masubuchi, Y.; Tachibana, M.; Ohta, S.; Hirobe M. Cytochrome P450 isozymes catalyzing 4‐hydroxylation of parkinsonism‐related compound 1,2,3,4‐tetrahydroisoquinoline in rat liver microsomes. FASEB J. 6: 771‐776; 1992.

Evaluation of the involvement of a male specific cytochrome P-450 isozyme in senescence-associated decline of hepatic drug metabolism in male rats

The major male specific species of cytochrome P-450 (P-450ml) was purified and an antibody against it used to evaluate the involvement of this isozyme in alterations of drug metabolism in senescence. P-450ml exhibited strikingly high imipramine (IM) N-demethylase activity while it showed no IM 2-hydroxylation, which is an alternate pathway of IM metabolism in rat liver microsomes. The antibody to P-450ml inhibited 80% of imipramine N-demethylation in young male rats. In old male rats, which have been shown to have lower IM N-demethylase activity, a 60% inhibition was observed. The inhibitable portion of this activity in old male rats is about one third of that in young rats, but the remaining portion not inhibited by this antibody is almost identical in young and old rats. IM 2-hydroxylation on the other hand was not inhibited by this antibody at all. It also inhibited about 30% of diazepam(DZ) N-demethylation in young rats but showed no inhibition in old rats, resulting in the loss of the age difference in the remaining portion. DZ 3-hydroxylation was not inhibited by this antibody, in spite of the fact that it showed a markedly higher activity in young male than in young female rats with a subsequent reduction in old age in male rats. This study provides the first direct evidence that differences in the amount of the major male specific P-450 isozyme (P-450ml) are responsible for the age- and sex-associated differences in some of the drug metabolizing activities. It also became apparent that P-450ml may not be the only isozyme responsible for these differences.

Impairment of debrisoquine 4-hydroxylase and related monooxygenase activities in the rat following treatment with propranolol.

The effect of repetitive oral administration of propranolol on hepatic microsomal drug metabolizing enzyme activities in the rat was investigated. Propranolol ring (4-, 5- and 7-)hydroxylase activities were markedly decreased, but, interestingly, N-desisopropylase activity was increased after propranolol administration. A marked decrease in enzyme activity after propranolol pretreatment was also observed with debrisoquine 4-hydroxylation. In addition, a similar decrease was observed with imipramine 2-hydroxylation which co-segregates with debrisoquine/sparteine type polymorphic drug oxidation, but not with imipramine N-demethylation. These results suggest the selective impairment of debrisoquine 4-hydroxylase by propranolol pretreatment.

Induction of hepatic microsomal drug metabolism by azo compounds: a structure-activity relationship.

The structure-activity relationship of 40 azo compounds in their ability to induce cytochrome P-448 and associated monooxygenase activities, as well as UDP-glucuronyltransferase (UDPGT) activity, was investigated. Regardless of their structure, hydrophilic azo dyes and lipophilic azobenzene derivatives were not able to induce these enzyme activities. Only those lipophilic azo dyes with 1-azo-2-naphthol or 1-azo-2-naphthylamine moieties were able to induce cytochrome P-448 and related monooxygenase activities, as well as UDPGT activity. The extent of induction is comparable to or greater than that caused by 3-methylcholanthrene (3-MC). It is suggested that those azo dyes capable of inducing P-450 type cytochromes can form coplanar structures with three fused, 6-membered rings through intramolecular hydrogen bonding. These structures are analogous to polycyclic aromatic hydrocarbons that can also induce.

Age associated alteration of lidocaine metabolism is position selective

Patterns of associated alterations in N-deethylation, 3-hydroxylation, and aromatic methylhydroxylation of a single substrate, lidocaine in liver microsomes from 0.7 - 28 months old Fischer 344 rats were examined. These three patterns were all different from one another. In addition to this position selectiveness, a clear sex difference was observed in the pattern of alteration in N-deethylation. These results are consistent with the hypothesis that age associated alterations in drug metabolism are caused by age associated alterations in relative abundance of multiple species of cytochrome P-450.

Induction of propranolol metabolism by the azo dye sudan III in rats

Effects of the azo dye sudan III, an inducer of cytochrome P450 isozymes belonging to the CYP1A subfamily, on propranolol (PL) in vitro and in vivo metabolism were investigated in rats. The kinetic parameters of the activity for each metabolic pathway were determined in liver microsomes from control and sudan III-treated rats. Sudan III pretreatment increased extensively PL 4-hydroxylase, 5-hydroxylase and N-desisopropylase activities at high but not at low PL concentrations. On the other hand, kinetic parameters of 7-hydroxylase activity were not affected by sudan III pretreatment. Sudan III pretreatment decreased blood concentrations of PL after intraportal infusion of PL at high doses (12.5 and 20 mg/kg), but not at a low dose (5 mg/kg). These observations were consistent with data obtained from the in intro studies showing that sudan III pretreatment induced low-affinity but not high-affinity cytochrome P450 isozymes involved in PL metabolism in rat liver microsomes.

Participation of the CYP2D subfamily in lidocaine 3-hydroxylation and formation of a reactive metabolite covalently bound to liver microsomal protein in rats

Lidocaine metabolism was investigated in rat liver microsomes and in a reconstituted system containing P450BTL, a cytochrome (P450) isozyme belonging to the CYP2D subfamily (Suzuki et al., Drug Metab Dispos 20: 367-373, 1992). P450BTL biotransformed lidocaine into 3-hydroxylidocaine (3-OH-LID) but not monoethylglycinexylidide and 2-methylhydroxylidocaine, in the reconstituted system including NADPH-P450 reductase and dilauroylphosphatidylcholine. An antibody against P450BTL inhibited microsomal lidocaine 3-hydroxylase activity by 97%. Thus, P450BTL and/or its immunorelated P450 isozyme(s) belonging to the CYP2D subfamily appear to be involved in lidocaine 3-hydroxylation. Furthermore, the antibody also suppressed the amounts of a lidocaine metabolite(s) bound to microsomal protein. These results suggest that the CYP2D subfamily biotransformed lidocaine into 3-OH-LID via an epoxy intermediate, which binds to microsomal macromolecules.

Substrate stereoselectivity and enantiomer/enantiomer interaction in propranolol metabolism in rat liver microsomes

The substrate stereoselectivity and enantiomer/enantiomer interaction of (S)- and (R)- propranolol for the formation of their metabolites were investigated in rat liver microsomal fractions. The enantiomers of primary metabolites of propranolol, 4-, 5-, 7-hydroxy- and N-desisopropyl-propranolol were separated and assayed by an HPLC method employing a chiral ovomucoid column. Regioselective substrate stereoselectivity (R < S for 4- and 5-hydroxylations; R > S for 7-hydroxylation; R = S for N-desisopropylation) was observed in the formation of propranolol metabolites when the individual enantiomers or a racemic mixture of propranolol were used as substrates. Concentration-dependent metabolic inhibition of propranolol enantiomers by their optical isomers was also observed. In addition, the inhibition of propranolol 4-, 5- and 7-hydroxylations between the enantiomers showed a typical competitive nature. These findings suggested that the propranolol enantiomers competed for the same enzyme, probably a cytochrome P450 isozyme in the CYP2D subfamily.

Enzymatic basis for the non-linearity of hepatic elimination of propranolol in the isolated perfused rat liver.

Propranolol (PL) metabolism was studied in the isolated perfused rat liver under single-pass and steady-state conditions. An attempt was made to predict the data observed in the isolated rat liver perfusion at PL infusion rates of 89-1317 nmol/min using the microsomal kinetic parameters obtained in our previous paper (Ishida et al., Biochem Pharmacol 43: 2489-2492, 1992) and the unbound PL fractions in rat liver microsomes and the perfusion medium. The values of kinetic parameters obtained in rat liver microsomes were corrected for the whole liver. Two groups of cytochrome P450 isozymes having high (Km < 0.5 microM)- and low (Km > 20 microM)-affinities participate in the metabolism of PL and sudan III pretreatment induces the low-affinity enzymes rather than the high-affinity enzymes in control rats. Of high-affinity isozyme(s) PL 4-hydroxylase and 7-hydroxylase made a major contribution to the overall activity, while for low-affinity isozymes PL 4-hydroxylase and N-desisopropylase did. A nonlinear relationship between the PL concentrations entering and leaving the liver was predicted from these corrected kinetic parameters using the venous equilibrium model. The outflow concentrations and the metabolic rates of PL for the predicted curves were over-estimated at higher inflow PL concentrations and under-estimated at higher substrate concentrations, respectively. On the other hand, the prediction for them was successfully carried out for the livers whose intrinsic clearance was altered due to the induction of low-affinity enzymes in PL metabolism by sudan III pretreatment. The outflow rates of 4-hydroxypropranolol showed a downward curvature at lower substrate concentrations, followed a linear rise in the livers from control rats, while the outflow rates of 5- and 7-hydroxypropranolol exhibited their respective limiting values. The outflow rates of 4-hydroxypropranolol and N-desisopropylpropranolol were enhanced markedly with increasing the outflow unbound concentration of PL by sudan III pretreatment. These results indicate that non-linear PL first-pass metabolism is due to the saturation of the reactions for the high-affinity enzymes among enzymes engaging in PL ring hydroxylations.