Formation of globules and aggregates of DNA chains in DNA/polyethylene glycol/monovalent salt aqueous solutions.

It has been known that giant DNA shows structural transitions in aqueous solutions under the existence of counterions and other polymers. However, the mechanism of these transitions has not been fully understood. In this study, we directly observed structures of probed (dye-labeled), dilute DNA chains in unprobed DNA/polyethylene glycol (PEG)/monovalent salt (NaCl) aqueous solutions with fluorescent microscopy to examine this mechanism. Specifically, we varied the PEG molecular weight and salt concentration to investigate the effect of competition between the depletion and electrostatic interactions on the coil-globule transition and the aggregate formation. It was found that the globules coexist with the aggregates when the unprobed DNA chains have a concentration higher than their overlap concentration. We discuss the stability of the observed structures on the basis of a free energy model incorporating the attractive depletion energy, the repulsive electrostatic energy, and the chain bending energy. This model suggested that both of the globules and aggregates are more stable than the random coil at high salt concentrations/under existence of PEG and the transition occurs when the depletion interaction overwhelms the electrostatic interaction. However, the coexistence of the globule and aggregate was not deduced from the thermodynamic model, suggesting a nonequilibrium aspect of the DNA solution and metastabilities of these structures. Thus, the population ratio of globules and aggregates was also analyzed on the basis of a kinetic model. The analysis suggested that the depletion interaction dominates this ratio, rationalizing the coexistence of globules and aggregates.

Possible mechanism of Sudan III-induced prevention of chemical carcinogenesis in rats.

The effect of treatment of rats with Sudan III on the ability of the liver to transform benzo(a)pyrene (BP) to a mutagen was investigated to elucidate a possible mechanism of the Sudan III-induced prevention of chemical carcinogenesis (Huggins et al., Proc. Natl. Acad. Sci., 75:4524-4527, 1978) using a modified Ames mutagenesis test system. Preincubation of BP with liver 9000 x g supernatant (S-9) from rats treated with Sudan III markedly increased the mutagenicity of BP in the conventional Ames method. The increase in number of revertant colonies was proportional to the aryl hydrocarbon hydroxylase activity of the S-9 fractions used (r greater than 0.9). This azo dye was found to induce both cytochrome P-448 which is an activating enzyme of procarcinogens and the activities of UDP glucuronyl transferase and glutathione-S-transferase (GST), which are detoxifying enzymes of active carcinogenic intermediates. The addition of anti-cytochrome P-448 antibody or the cofactors for UDP glucuronyl transferase and glutathione-S-transferase to the liver S-9 preincubation mixture with BP caused a marked decrease in BP mutagenicity when liver S-9 fractions from Sudan III-treated rats were used. The decrease obtained by the addition of the cofactors was proportional to the induced levels of the UDP glucuronyl transferase or glutathione-S-transferase. It was concluded that Sudan III-induced prevention of chemical carcinogenesis is due to an accelerated elimination of the carcinogen by the induction of both cytochrome P-448 and detoxifying enzymes with a limited increase in levels of active metabolic intermediates.

Processing of dielectric oxynitride perovskites for powders, ceramics, compacts and thin films.

Oxynitride perovskites, having oxide and nitride anions together in a compound, are a new class of dielectric material. The shaping process in either bulk ceramics or thin films is an essential factor for investigating and utilizing the dielectric properties of these materials. In this perspective, recent studies on the shaping of dielectric oxynitride perovskites are reviewed with a consideration of the powder preparation and thermal stability for sintering, several sintering methods, ultra-high pressure compaction, and thin-film formation.

Oxidative metabolism of bunitrolol by complementary DNA-expressed human cytochrome P450 isozymes in a human hepatoma cell line (Hep G2) using recombinant vaccinia virus.

We examined the oxidative metabolism of a beta-blocker bunitrolol (BTL) by 10 human cytochromes P450 (CYP) (1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, 3A3, 3A4 or 3A5), which were individually expressed in Hep G2 cells with a vaccinia virus complementary DNA expression system. Among the 10 isozymes, only CYP2D6 and 1A2 at a substrate concentration of 5 microns, and CYP2C8 and 2C9 in addition to the two isozymes at a BTL concentration of 1 mM, exhibited detectable BTL 4-hydroxylase activities. The activities at 1 mM BTL were on the order of CYP2D6 (100% as relative activity) > CYP1A2 (86%) >> CYP2C8 and 2C9 (7-8%). Enzyme kinetic parameters of CYP2D6 were calculated to be 4.41 microns as a Km value and 0.442 nmol min-1 per nmol CYP as a Vmax value. Kinetic parameters of CYP1A2 were calculated as 295 microns and 0.411 nmol min-1 per nmol CYP for Km and Vm values, respectively. These results suggest that both CYP2D6 and 1A2 primarily catalyse BTL 4-hydroxylation, but that the former is a predominant isozyme responsible for the reaction at a low substrate concentration range of BTL in human liver.

Diphenylamine as an important structure of nonsteroidal anti-inflammatory drugs to uncouple mitochondrial oxidative phosphorylation.

A marked difference has been observed in the inhibitory effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on oxidative phosphorylation of rat liver mitochondria. It should be noted that some of the potent inhibitors, N-phenylanthranilic acids and diclofenac, have a similar "skeleton" structure, diphenylamine. Diphenylamine itself was found to inhibit oxidative phosphorylation significantly, although its inhibition potency was weaker than that of NSAIDs with a diphenylamine structure. In addition to decreases in the respiration control index (ratio of state 3 to state 4 respiration), these compounds released oligomycin-inhibited state 3 respiration. These results demonstrated that diphenylamine, as well as N-phenylanthranilic acids and diclofenac, was an uncoupler of oxidative phosphorylation of rat liver mitochondria. Thus, diphenylamine was suggested to play an important role in the uncoupling effects of NSAIDs with a diphenylamine skeleton.

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.

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.

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.

Metabolic activation of lidocaine and covalent binding to rat liver microsomal protein.

Incubation of [14C]lidocaine with rat liver microsomes in the presence of an NADPH-generating system resulted in covalent bindings of a 14C-labelled material to microsomal protein. The covalent binding of radioactivity needed NADPH and atmospheric oxygen, and was diminished by purging of carbon monoxide and the addition of SKF-525A. Hence the covalent binding of a 14C-labelled material resulting from a reactive metabolite of lidocaine formed by cytochrome P450-dependent monooxygenation. The covalent binding measured at various concentrations of lidocaine (2.5-30 microM) followed Michaelis-Menten kinetics, and the Km value (4.52 microM) of the activation reaction was close to the Km value (1.78 microM) of lidocaine 3-hydroxylation. The metabolism-dependent covalent binding of lidocaine to microsomal protein as well as lidocaine 3-hydroxylase activity was much lower in the Dark Agouti strain rat, which is known as a poor-metabolizer animal model of debrisoquine 4-hydroxylation, than in the Wistar rat for the corresponding sexes. The covalent binding in male rats was greater than that in females of both strains, but the extent of the sex difference in the binding was smaller than that of the lidocaine N-deethylase activity in Wistar rats. Propranolol and quinidine, specific inhibitors of debrisoquine 4-hydroxylase, markedly inhibited lidocaine 3-hydroxylase activity of Wistar male rats, but not N-deethylase activity. These compounds also inhibited the metabolism-dependent covalent binding of lidocaine to microsomal protein. These strain difference and inhibition studies showed that the reaction converting lidocaine to a reactive metabolite capable of binding covalently to microsomal protein was related to lidocaine 3-hydroxylation, and may be catalysed by cytochrome P450 isozyme(s) belonging to the CYP2D subfamily. The covalent binding of radioactivity to rat liver microsomal protein was diminished by nucleophiles, reduced glutathione and cysteine, indicating that the reactive metabolic intermediate of lidocaine is an electrophilic metabolite such as an arene oxide.

Selective 3-hydroxylation deficiency of lidocaine and its metabolite in Dark Agouti rats.

Selective and marked 3-hydroxylase deficiency of lidocaine and its N-deethylated metabolite, MEGX, were observed in male and female DA rats. These findings suggest that cytochrome P450 isozymes metabolizing debrisoquine may be involved in the 3-hydroxylations of lidocaine and MEGX in rats and humans.

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

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.

Possible pharmacokinetic and pharmacodynamic factors affecting parkinsonism inducement by cinnarizine and flunarizine.

Potentialities of cinnarizine [1-(diphenylmethyl)-4-(3-phenyl-2-propenyl)piperazine, CZ] and its fluorine derivative flunarizine [1-[bis(4-fluorophenyl)-methyl]-4-(3-phenyl-2-propenyl)piperazine, FZ] to induce parkinsonism as an adverse effect were evaluated pharmacokinetically and pharmacodynamically in rats. In multiple-dose experiments, CZ or FZ was given to rats at a daily dose of 20 mumol/kg for 1, 5, 10, 15, and 30 days, and CZ, FZ, and the ring-hydroxylated metabolites of their cinnamyl moiety [1-(diphenylmethyl)-4-[3-(4'-hydroxyphenyl)-2-propenyl]piperazine, C-2 and 1-[bis(4-fluorophenyl)methyl]-4-[3-(4'- hydroxyphenyl)propenyl]piperazine, F-2] in the plasma and striatum were determined 24 hr after the final dose. Plasma and striatum concentrations of the above compounds except for FZ reached steady state after 10 doses, but their concentrations of FZ continued to increase throughout the experiments. The concentrations obtained after the 30 doses were in the order of FZ > F-2 > CZ > C-2 for the plasma and of F-2 > FZ > CZ > C-2 for the striatum. The ratios of striatum to plasma concentrations of C-2 and F-2 were 2.4 and 3 times higher than those of the parent drugs. Binding affinities of CZ, FZ, and their 10 metabolites for rat striatal dopamine D-2 receptors (D2-R) were assessed by competitive radioligand-binding studies using [3H]-N-[(2RS,3RS)-1-benzyl-2-methyl-3-pyrrolidinyl]-5-chloro-2-met hoxy- 4-methylamino-benzamide ([3H]-YM-09151-2). The IC50s calculated from their Ki values were in the order of F-2 < C-2 < FZ < CZ < C-4 << F-1, indicating that C-2 and F-2 exhibit higher affinities for D2-R than the parent drugs, whereas affinities of other metabolites were 1 to 2 orders of magnitude less than those of C-2 and F-2. These results suggest some important roles of C-2 and F-2 in the development of parkinsonism as active metabolites during chronic medication with CZ and FZ, respectively.

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.

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.

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.

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.

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.

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.

Docosahexaenoic acid exhibits a potent protection of small intestine from methotrexate-induced damage in mice.

Oral administration of methotrexate (MTX) to mice causes the damage of small intestine. The permeability of poorly absorbable compound fluorescein isothiocyanate (FITC)-labeled dextran (average molecular weight, 4,400) through the small intestine was studied in vitro using everted segments of the small intestine. The permeability of FITC-dextran increased remarkably in the MTX-treated mice and oral administration of docosahexaenoic acid ethyl ester (DHA) protected the small intestine from the increase in the small intestinal permeability induced by the MTX treatment. The MTX treatment decreased retinol concentration in plasma of mice and the coadministration of DHA maintained its concentration to the level of control mice. The present study showed that DHA protected the small intestine of mice from the MTX-induced damage.

Characterization of the oxidation reactions catalyzed by CYP2D enzyme in rat renal microsomes.

Monooxygenase activities in rat renal microsomes were determined with the substrates of hepatic CYP2D enzymes. Seven kinds of CYP2D-mediated monooxygenase activities and immunochemically determined CYP2D contents in kidneys corresponded to approximately 3% of those in livers. Debrisoquine 4-hydroxylase and bunitrolol 4-hydroxylase in renal microsomes were inhibited almost completely by the antibody against a CYP2D enzyme purified from rat liver. A marked strain difference (Wistar > Dark Agouti) in these activities was observed in kidney like in liver. The two hydroxylases were inhibited stereoselectively by quinine and quinidine both in renal and hepatic microsomes. Substrate stereoselectivity in (+)- and (-)-bunitrolol 4-hydroxylase activities in kidneys was also consistent with that in livers. These results suggested that the CYP2D enzyme(s) was expressed in the kidney at levels much less than in the liver but had similar functions to those in the liver.