Shizuo Narimatsu

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 damaged small intestine of mouse following methotrexate administration.

Purpose: Methotrexate (MTX) treatment causes damage to the small intestine, resulting in malabsorption and diarrhea. The active and passive transport capacities of the small intestine are decreased by the treatment. The purpose of this study was to evaluate the damage to the small intestine of mice caused by MTX administration by examining the permeability of the paracellular pathway of the small intestinal epithelium. Methods: MTX was administered orally to male ddY mice once daily for 1–6 days. The permeability of the small intestine to the nonabsorbable markers phenol red (PR) and fluorescein isothiocyanate (FITC) dextrans was examined using everted segments of the intestine. Results: PR and FITC dextran permeation through the small intestine increased significantly in parallel with changes in body weight of the mice, wet weight of the small intestine and chemical composition of the small intestinal epithelium. Conclusions: In addition to changes in permeation through the transcellular pathway reported previously, this study revealed that MTX treatment disorders the paracellular barrier function of the small intestinal epithelium, resulting in increased permeation of nonabsorbable markers via the paracellular pathway of the small intestinal mucosa. The present approach to the examination of the barrier function of the intestinal epithelium could be of great use in evaluating the damage to the small intestine and malabsorption.

Role of the CYP2D subfamily in metabolism-dependent covalent binding of propranolol to liver microsomal protein in rats.

In vitro covalent binding of a chemically reactive metabolite of propranolol to microsomal macromolecules, which is presumed to cause inhibition of its own metabolism in rats, was diminished in liver microsomes from rats pretreated with propranolol. Covalent binding was suppressed by the addition of an antibody against P450BTL, which is a cytochrome P450 (P450) isozyme belonging to the CYP2D subfamily. SDS-PAGE of microsomal proteins after incubation with [3H]propranolol and NADPH indicated that the binding was non-selective but prominent at the molecular mass of approx. 50 kDa, corresponding to those of the P450 protein. The radioactivity peak was markedly but not completely diminished by the addition of reduced glutathione. In a reconstituted system containing P450BTL, NADPH-cytochrome P450 reductase (fp2) and dilauroylphosphatidylcholine, propranolol 4-, 5- and 7-hydroxylase activities decreased time dependently following preincubation with propranolol in the presence of NADPH, indicating time-dependent inactivation of P450BTL. The covalent binding of a reactive metabolite of [3H]propranolol to the proteins was also observed in this system. SDS-PAGE showed that among the three proteins in the reconstituted system, fp2 and P450BTL consisting of two polypeptides with molecular masses of 49 and 32 kDa, the binding was specific for a polypeptide corresponding to the P450 isozyme with a molecular mass of 49 kDa. In addition, the ratio of the amount of covalently bound radiolabelled materials to that of P450BTL which was estimated from each impaired propranolol hydroxylase activity under the same reconstitutional conditions was calculated to be approx. 1.0. These findings indicate that propranolol is a mechanism-based inactivator of a cytochrome P450 isozyme(s) belonging to the CYP2D subfamily.

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.

Rat liver microsomal lipid peroxidation produced during the oxidative metabolism of ethacrynic acid.

Thiobarbituric acid reactive substances (TBARS) were produced in rat liver microsomal suspension incubated with ethacrynic acid (loop diuretic drug) and NADPH. Two oxidative metabolites of ethacrynic acid with dicarboxylic acid and hydroxylated ethyl group, respectively, were formed in the reaction mixture. The oxidative metabolism of ethacrynic acid was inhibited by cytochrome P450 inhibitors. The formation of TBARS was remarkably depressed by inhibitors like diethyldithiocarbamate and disulfiram. These results indicate that lipid peroxidation occurred in rat liver microsomes through the oxidative metabolism of ethacrynic acid.

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.

Kinetic analysis of mutual metabolic inhibition of lidocaine and propranolol in rat liver musomes

The metabolic interaction between lidocaine (LD) and propranolol (PL) was analysed kinetically in rat liver microsomes. Employing a very short incubation time of 30 sec, we demonstrated that PL competitively inhibited liver microsomal 3-hydroxylation of LD, but did not affect either the formation of monoethylglycinexylidide or methylhydroxylidocaine from LD in PL concentrations up to 1 microM. On the other hand, LD competitively inhibited PL 4-, 5- and 7-hydroxylations, but the inhibition type of LD for PL N-desisopropylation could not be clarified. Comparison of the kinetic data for liver microsomes from Wistar and Dark Agouti rats indicated that among the primary metabolic pathways of LD, the Vmax value for 3-hydroxylation was markedly less in female Dark Agouti rats. The results suggest that LD 3-hydroxylation and PL ring hydroxylations are mediated by the same isozyme(s) belonging to the CYP2D subfamily.

Activation of propranolol and irreversible binding to rat liver microsomes: strain differences and effects of inhibitors

In summary, strain difference and inhibition studies showed that an enzyme(s) converting propranolol to a reactive metabolite capable of irreversible binding to microsomal macromolecules appeared to be a P450 isozyme(s) which catalyses debrisoquine 4-hydroxylation in rats. It seems likely that cytochrome P450 isozymes responsible for debrisoquine 4-hydroxylation activate propranolol and may be impaired after chronic use of propranolol also in human subjects. The findings obtained in the present study provide a clue for the elucidation of the mechanism of propranolol-induced impairment of the drug metabolizing enzyme system. Further studies using purified debrisoquine 4-hydroxylase are required to identify a P450 isozyme(s) responsible for the metabolic activation of propranolol. We are now performing experiments along this line.