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This paper reviews the design challenges that current and future processors must face, with stringent power limits, high-frequency targets, and the continuing system integration trends. This paper then describes the architecture, circuit design, and physical implementation of a first-generation Cell processor and the design techniques used to overcome the above challenges. A Cell processor consists of a 64-bit Power Architecture processor coupled with multiple synergistic processors, a flexible IO interface, and a memory interface controller that supports multiple operating systems including Linux. This multi-core SoC, implemented in 90-nm SOI technology, achieved a high clock rate by maximizing custom circuit design while maintaining reasonable complexity through design modularity and reuse.

FTY720, a novel immunosuppressant, prolonged the survival of WKAH skin allografts transplanted into MHC-incompatible F344 rats. In this allograft model, the median survival time of the control group was 7 days, whereas those of the groups given FTY720 orally at 0.1 mg/kg and cyclosporin A (CsA) at 10 mg/kg were 10.5 and 11 days, respectively. In contrast, FTY720 (0.1 mg/kg) combined with CsA (10 mg/kg) synergistically prolonged allograft survival with a median survival time exceeding 70 days. To elucidate the mechanisms of this remarkable synergistic effect, mRNA expressions of IL-2 and IFN-γ and that of CD3 (δ-chain), which reflects T cell infiltration, in allografts were temporally analyzed using a semiquantitative PCR method. In WKAH skin allografts, mRNA levels of IL-2, IFN-γ, and CD3 were increased as compared with isograft controls, peaking on days 4 to 5. CsA (10 mg/kg) significantly inhibited elevations of IL-2 and IFN-γ mRNA, while slightly inhibiting that of CD3 mRNA in allografts. On the contrary, FTY720 (0.1 mg/kg) markedly inhibited the elevation of CD3 mRNA, while slightly inhibiting those of IL-2 and IFN-γ mRNA. FTY720 (0.1 mg/kg) combined with CsA (10 mg/kg) almost completely suppressed the intragraft expressions of mRNA for IL-2, IFN-γ, and CD3. Immunohistochemical staining and flow cytometric analysis also confirmed that FTY720 decreased T cell infiltration into allografts. From these results, the synergistic effect of FTY720 combined with CsA on prolongation of allograft survival is presumably based on the respective inhibitions of T cell infiltration and cytokine production in grafts.

Processor performance and functional improvement has been driven by innovations in various areas, such as architecture, circuit, device, and software, but we are now facing or will face hard challenges, such as power consumption and process technology. What would the next step be in processor evolution? This panel will discuss with experts from HPC, server and embedded area what the requirements of the processors would be for future applications, what technology would give solutions to them, and what the challenges would be.

We propose a method for calculating the reproducing outputs of magnetic heads from their impedance characteristics. The efficiency of a magnetic head is first calculated from the impedance characteristics, and the reproducing output is then calculated from this efficiency. In a study using ferrite heads and Sendust multilayer heads, the calculated reproducing outputs agreed with the measured ones; the differences between the values were generally lower than 1 dB. Using our method, we were able to evaluate the reproducing output characteristics in various conditions from the impedance characteristics of the magnetic heads.

In this paper, we use a magnetic circuit method to analyze an electromagnetic shield for reducing the inductance of magnetic heads. First, we constructed a magnetic circuit model of a magnetic head having an electromagnetic shield. The frequency characteristics of the inductance and resistance calculated by using the model agreed with the measured ones. Next, the head inductance and resistance as functions of the shield thickness were analyzed according to the model, and their characteristics were clarified.

We here report on some modifications of the Primitive Chain Network (PCN) model, originally proposed in [Y. Masubuchi et al., J. Chem. Phys. 115, 4387 (2001)], which both refine the model and make it suitable for predicting nonlinear rheological response in fast flows. Simulation results are compared with some existing viscoelastic data on monodisperse polystyrene melts [Schweizer et al., J. Rheol. 48, 1345 (2004); Bach et al., Macromolecules 36, 5174 (2003)], by using as single adjustable parameter (to be assigned once and for all) a basic relaxation time which relates to the coarse graining of the PCN model. Essentially quantitative prediction of linear viscoelasticity is achieved. Without further parameters, the nonlinear behavior of the polystyrene melts in start‐up of shear and uniaxial elongational flow is also reproduced by the simulations. Specifically, the viscosity growth functions are quantitatively predicted, whereas transient elongational viscosities are reasonably reproduced only up to extensional rates of the order of the reciprocal Rouse time. At higher elongation rates, although the strain hardening effect is correctly captured by the simulations, steady state values are not. Notwithstanding this latter limitation, the simulation model appears adequate to portray the rheological behavior of entangled melts, both in the linear and the nonlinear range.

This paper reviews the design challenges that current and future processors must face with stringent power limits and high frequency targets, and the design methods required to address the continuing system integration trends. This paper then describes the implementation of a first-generation CELL processor and the design methods used to overcome the above challenges. A CELL processor consists of a 64 bit power architecture processor coupled with multiple synergistic processors, a flexible IO interface, and a memory interface controller that supports multiple operating systems including Linux. This multicore SoC, implemented in 90nm SOI technology, achieved a high clock rate by maximizing custom circuit design while maintaining reasonable complexity through design modularity and reuse.

A CELL processor is a multi-core chip consisting of a 64b power architecture processor, multiple streaming processors, a flexible IO interface, and a memory interface controller. This SoC is implemented in 90nm SOI technology. The chip is designed with a high degree of modularity and reuse to maximize the custom circuit content and achieve a high-frequency clock-rate.

7-ethoxycoumarin was not found to induce lipid peroxidation in rat liver microsomes, although it was reported to cause lipid peroxidation in rat cultured hepatocytes. The productions of thiobarbituric acid reactive substances (TBARS) and chemiluminescence (CL) in the presence of some drugs including tolbutamide, naproxen and salicylic acid and their oxidation activities were determined. There was a negative correlation between extent of oxidative stress and oxidation activities. These results suggested that the oxidative metabolism of these drugs did not directly contribute to the microsomal peroxidation reaction. In addition, naproxen-induced microsomal lipid peroxidation was found to occur in rat, but did not in guinea pig.

The implementation of a first-generation CELL processor that supports multiple operating systems including Linux consists of a 64 bit power processor element (PPE) and its L2 cache, multiple synergistic processor elements (SPE) (B. Flachs et al.) each with its own local memory (LS) (T. Asano et al.), a high bandwidth internal element interconnect bus (EIB), two configurable non-coherent I/O interfaces, a memory interface controller (MIC), and a pervasive unit that supports extensive test, monitoring, and debug functions. In conclusion, special circuit techniques, rules for modularity and reuse, customized clocking structures, and unique power and thermal management concepts were applied to optimize the design.

Retinol binding protein (RBP) in plasma of rats treated with carbon tetrachloride (CCl4) was monitored to clarify if RBP is available for the evaluation of the drug-induced hepatotoxicity. Blood was withdrawn by heart puncture at 0 hr and 12 hr after i.p. administration of CCl4 (0.2 ml/kg) to rats. Lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) in plasma significantly increased at 12 hr after CCl4 administration, compared with the control, while RBP in plasma significantly decreased. On the other hand, albumin in plasma was unaffected at 12 hr after CCl4 administration. Thus RBP seems to monitor the different aspects in the drug-induced hepatotoxicity from LDH and ALT, and from the viewpoint of protein synthesis in the liver, to be more sensitively affected by the drug-induced hepatotoxicity than albumin.

A numerical method for investigating the two-dimensional behaviour of a fuel pellet in inertial confinement fusion (ICF) is presented. In this method, a severe mesh distortion or mesh overlapping is avoided by introducing an arbitrary mesh velocity. Example computations are presented for the implosion phase of an ICF pellet irradiated by a light-ion beam (LIB). >

A 4 GOPS 3-way VLIW image-recognition processor for an automobile system is based on a configurable media-processor which enables design-time configuration to optimize for a specific application. It uses a 0.25 /spl mu/m CMOS process with a standard-cell design method.

It has been known that diclofenac is biotransformed into chemically reactive metabolites, which bind covalently to liver microsomal proteins, including cytochrome P450 enzyme(s). We have investigated the ability and selectivity of diclofenac to inactivate P450 enzymes. Preincubation of microsomes of untreated rats with diclofenac in the presence of NADPH resulted in time-dependent loss of testosterone 2alpha- and 16alpha-hydroxylation activities. No effect of the preincubation was observed on ethoxyresorufin O-deethylase, pentoxyresorufin O-depentylase, or testosterone 6beta-hydroxylation activity. The time-dependent decreases in testosterone 2alpha- and 16alpha-hydroxylation activities followed the pseudo-first order kinetics and were saturable with increasing diclofenac concentrations. Reduced glutathione was not capable of protecting against the decrease in the enzyme activities. These data establish that a mechanism-based inactivation of CYP2C11 occurs during the oxidative metabolism of diclofenac. The diclofenac concentrations required to achieve the half-maximal rate of inactivation (K(I)) were 3 to 4 microM, which were close to K(m) for the low-K(m) components for diclofenac 4'- and 5-hydroxylation activities (7.29 and 4.43 microM, respectively). Anti-CYP2C11 IgG inhibited diclofenac 4'- and 5-hydroxylation activities, indicating that CYP2C11 is a major isozyme responsible for these aromatic oxidations. The preincubation of microsomes with 4'- or 5-hydroxydiclofenac did not cause a decrease in testosterone 2alpha- or 16alpha-hydroxylation activity, suggesting that neither of the primary metabolites is a precursor of the metabolite that inactivates CYP2C11. Therefore, a highly reactive intermediate(s) inactivating CYP2C11, probably arene-oxide, appears to be generated during the process of diclofenac 4'- and/or 5-hydroxylation. Diclofenac metabolism in human liver microsomes did not cause inactivation of CYP2C9, a major isozyme involved in diclofenac 4'-hydroxylation. Because the human microsomes have high diclofenac 4'-hydroxylation but not 5-hydroxylation activity, importance of the latter pathway in the inactivation is suggested.

Years ago it was reported that, as lung volume increased, there was a corresponding increase in scalene EMG activity (Raper et al. J Appl Physiol 21: 497-502, 1966). Otherwise, the relationship between changing lung volume and the EMG of the respiratory muscles has not been defined. We therefore inserted fine wire electrodes into the scalene (SCLN), sternocleidomastoid (STERNO), and trapezius (TRAPEZ) muscles in 6 healthy subjects under direct vision using high-resolution ultrasound. Maximum EMG activity (EMGmax) was obtained for each muscle by a variety of respiratory and postural maneuvers. Then, in the standing posture, air flow, raw and moving average EMG signals were sampled and input to a computer during quasi-static inspiration from functional residual capacity (FRC) to total lung capacity (TLC). We found that the relationship between EMG and lung volume for SCLN, but not for STERNO or TRAPEZ, was expressed by exponential curves. The onset of SCLN, STERNO and TRAPEZ EMG occurred at 13.3 +/- 7.4 (mean +/- SE), 67.8 +/- 14.6, and 89.2 +/- 3.9% of inspiratory capacity. The EMG of SCLN, STERNO, TRAPEZ, reached 85.7 +/- 2.6, 60.7 +/- 8.6, and 11.8 +/- 5.2% of EMGmax, respectively, at TLC. We conclude that: 1) SCLN is the most, and TRAPEZ the least, active neck accessory inspiratory muscle, while STERNO is intermediate, and 2) there is a lung volume dependency of the neck accessory muscle EMG activity.

Diphenylamine is a common structure of nonsteroidal anti-inflammatory drugs (NSAIDs) to uncouple mitochondrial oxidative phosphorylation and to cause a decrease in hepatocellular ATP content and hepatocyte injury. The mechanism for acute cell injury induced by diphenylamine and its structurally related NSAIDs was investigated with rat liver mitochondria and freshly isolated hepatocytes, focusing on the relation to the uncoupling of oxidative phosphorylation. Incubation of mitochondria with diphenylamine as well as mefenamic acid and diclofenac caused pseudoenergetic mitochondrial swelling, indicating that these compounds induce mitochondrial membrane permeability transition. Diphenylamine also caused changes in safranine-binding spectra to mitochondria that was energized by succinate oxidation. This spectral shift indicates the loss of mitochondrial membrane potentials, which is known as one of the characteristics for uncouplers of oxidative phosphorylation, and also was caused by mefenamic acid and diclofenac. Incubation of hepatocytes with mefenamic acid, diclofenac, and diphenylamine diminished cellular ATP content, followed by leakage of lactose dehydrogenase from hepatocytes. Fructose, a low K(m) substrate for glycolysis, partially protected against the ATP depletion and hepatocyte injury induced by these compounds. Further addition of oligomycin, which blocks ATPase, pronounced the protection against cell injury. These results suggested that decreases in cellular ATP content, mainly caused by uncoupling of mitochondrial oxidative phosphorylation, were responsible for acute hepatocyte injury induced by diphenylamine and structurally related NSAIDs.

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.

FTY720, a novel immunosuppressant, sequesters circulating mature lymphocytes, especially T cells, within lymph nodes and Peyer's patches by accelerating lymphocyte homing, and thereby causes lymphocyte depletion in the blood. The FTY720-induced acceleration of lymphocyte homing appears to be mediated by lymphocyte homing receptors including CD62L, CD49d/beta7, and CD11a/CD18. In this study, expressions of CD62L, CD49d and CD11a on T cells in the peripheral blood, lymph nodes and Peyer's patches were analysed by flow cytometry in rats given FTY720 (1 mg/kg) orally. FTY720 markedly decreased the number of peripheral blood T cells, while not affecting CD62L, CD49d and CD11a expressions at 1-3 hr after administration. In contrast, both the frequency of CD62L-positive T cells and intensity of CD62L expression on T cells were increased in Peyer's patches but not lymph nodes at 3 hr after administration of FTY720. CD49d and CD11a expressions on T cells were unaffected by FTY720 in both Peyer's patches and lymph nodes at the same point in time. On the other hand, analysis of lymphocyte homing with calcein-labelled lymphocytes and anti-CD62L monoclonal antibody (mAb) confirmed that FTY720 predominantly increased CD62L-dependent lymphocyte homing to Peyer's patches. These findings indicate that FTY720 increases the frequency of CD62L-positive T cells by accelerating CD62L-predominant homing in Peyer's patches.

Hepatotoxicity is one of the common side effects of nonsteroidal anti-inflammatory drugs (NSAIDs). We investigated the cytotoxicity of 18 acidic NSAIDs (3 salicylic acids, 3 anthranilic acids, 6 arylacetic acids, 6 arylpropionic acids) to freshly isolated rat hepatocytes as assessed by the NSAID-induced leakage of lactate dehydrogenase (LDH) in order to determine structural requirements for the direct hepatotoxicity of the NSAIDs. Diflunisal (salicylic acids), flufenamic acid, mefenamic acid, tolfenamic acid (anthranilic acids), diclofenac, indomethacin, acemetacin (arylacetic acids) and flurbiprofen (arylpropionic acids) caused significant LDH leakage, indicating that substituent position of a carboxyl group does not relate to the hepatotoxicity of the NSAIDs. Because the cytotoxic NSAIDs were of two types as classified by their "skeleton," diphenyl and diphenylamine, we tested the cytotoxicity of the compounds. Diphenyl did not cause LDH leakage, but diflunisal, which has the diphenyl structure, was cytotoxic. On the other hand, diphenylamine induced LDH leakage to the same degree as diclofenac, which has the diphenylamine structure. Therefore, diphenylamine itself was suggested to be responsible for the cytotoxicity of diclofenac and anthranilic acids, whereas a substituted group(s) in addition to diphenyl structure seems to be important for diflunisal cytotoxicity. All of the cytotoxic NSAIDs and diphenylamine extensively decreased hepatocellular ATP content, whereas the noncytotoxic NSAID did not, indicating that the NSAID-induced decrease in ATP, probably by their uncoupling effects on mitochondrial oxidative phosphorylation, is involved in the hepatotoxicity of the NSAIDs.

Rat liver microsomes (1 mg protein/ml) were incubated at 37 degrees C with non-steroidal anti-inflammatory drug, salicylic acid (5 mM) in the presence of 0.2 mM NADPH. The amounts of thiobarbituric acid reactive-substances (TBARS) were increased remarkably by the incubation for 30 min. TBARS formation was dependent on salicylic acid concentration. Salicylic acid-induced lipid peroxidation was not observed in the absence of NADPH. It was also inhibited by the presence of cytochrome P450 inhibitor, SKF-525A. The singlet oxygen's scavengers such as histidine suppressed salicylic acid-induced lipid peroxidation. These results suggested that salicylic acid-induced lipid peroxidation was coupled with the metabolism through cytochrome P450 in rat liver microsomes. The singlet oxygen may be involved in the reaction.

Dihydralazine is known to induce immunoallergic hepatitis, and the anti-liver microsome (anti-LM) autoantibodies found in the serum of the patients have been reported to react with cytochrome P450 1A2 (CYP1A2). It is thus suggested that a reactive metabolite of dihydralazine covalently binds to the P450 protein and triggers an immunological response as a neoantigen. We investigated the selectivity of inactivation of P450 enzymes during the metabolism of dihydralazine to evaluate the target protein of its reactive metabolite. Liver microsomes from male Wistar rats were preincubated with dihydralazine in the presence of NADPH, followed by assays of several monooxygenase activities. Preincubation of microsomes of beta-naphthoflavone-treated rats with dihydralazine resulted in time-dependent loss of phenacetin O-deethylase activity (an indicator of CYP1A2 activity), showing inactivation of CYP1A2 during the dihydralazine metabolism. The preincubation with dihydralazine was less effective on ethoxyresorufin O-deethylase activity in microsomes of beta-naphthoflavone-treated rats (CYP1A1) and pentoxyresorufin O-depentylase activity in microsomes of phenobarbital-treated rats (CYP2B). On the other hand, preincubation of microsomes of untreated rats with dihydralazine caused time-dependent loss of testosterone 2alpha-, 16alpha- (CYP2C11), and 6beta- (CYP3A) hydroxylase activities. These results demonstrated that dihydralazine was metabolically activated by CYP1A2, and the chemically reactive metabolite bound to the enzyme itself and inactivated it, as was suggested by the appearance of anti-LM antibodies in dihydralazine-hepatitis, whereas CYP2C and -3A enzymes were also suggested to be the enzymes that activate dihydralazine and lead to the target of the reactive intermediates.

Cytochrome P450 enzymes belonging to the CYP2D subfamily have been shown to be one of determinants of the polymorphic drug oxidations in the human and the rat. Debrisoquine 4-hydroxylation is a typical reaction catalyzed by these enzymes. However, various strains of mice were observed to have much lower debrisoquine 4-hydroxylase activity than Wistar rats, whereas other monooxygenase activities in mice toward bunitrolol, propranolol, imipramine and amitriptyline, which are mediated by the CYP2D enzymes in the rat, were comparable to those of the rats. Immunoblot analysis of mouse liver microsomes with an antibody raised against a rat CYP2D enzyme indicated that the mouse liver contained a P450 enzyme(s) immunochemically related to the rat CYP2D enzyme. The antibody inhibited propranolol ring-hydroxylase and imipramine 2-hydroxylase activities, as well as testosterone 16alpha-hydroxylase activity, a typical reaction of mouse CYP2D9, but not debrisoquine 4-hydroxylase activity in mouse liver microsomes. We partially purified a P450 enzyme (designated P450 ML2d) from livers of male ddY mice by monitoring the cross-reactivity with the antibody. The partially purified enzyme was indicated to belong to the CYP2D subfamily from its N-terminal amino acid sequence, but the homology of the sequence to other CYP2D enzymes of the mouse (CYP2D9-11) was 62%, suggesting that P450 ML2d is a novel P450 enzyme. P450 ML2d had the oxidation activities for the rat CYP2D-substrates, such as propranolol 4-hydroxylation and imipramine 2-hydroxylation, in higher rates than those of the microsomes, but did not exhibit debrisoquine 4-hydroxylase activity. Our result is the first finding that a mouse CYP2D enzyme also metabolizes substrates for the rat CYP2D enzyme, in addition to steroids, but the enzyme had a limited specificity for the substrates of the CYP2D enzymes of the rat and the human.

Preincubation of microsomes from male Wistar rats with imipramine (IMI) in the presence of NADPH caused a time-dependent loss of bunitrolol 4-hydroxylase activity, indicating that the CYP2D enzyme is inactivated during IMI metabolism, which has also been observed after in vivo administration of IMI. A similar effect was obtained when desipramine, an N-demethylated metabolite of IMI, was used as an inhibitor, whereas 2-hydroxy-IMI had no effect on the activity. Thus, it seems likely that the inactivation of the CYP2D enzyme is related to 2-hydroxylation process of IMI. Incubation of microsomes with [3H]IMI in the presence of NADPH resulted in covalent binding of a 3H-labeled material to microsomal protein. Formation rates of the reactive metabolites covalently bound to protein followed Michaelis-Menten kinetics, and the K(m) value (1.1 microM) was close to that for microsomal IMI 2-hydroxylation. The metabolism-dependent covalent binding of [3H]IMI was lower in Dark Agouti rats, which is an animal model of CYP2D deficiency, than in Wistar rats. The binding was inhibited by propranolol and quinidine, a substrate and an inhibitor of CYP2D, respectively, and by an antibody against CYP2D. Similar strain difference (Dark AgoutiWistar) and inhibitory effects by the compounds and the antibody were observed in IMI 2-hydroxylase but not in N-demethylase activity. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of microsomal protein incubated with [3H]IMI and NADPH showed that the binding was prominent at the molecular mass of approximately 50 kDa, which would be consistent with the P450 protein being a target for the binding. Furthermore, proteins to which [3H]IMI metabolites covalently bound were immunoprecipitated with the anti-CYP2D antibody. These results suggest that IMI is biotransformed into a chemically reactive metabolite (probably arene-oxide) through its 2-hydroxylation step by the CYP2D enzyme in rat liver microsomes, and the metabolite binds covalently to the enzyme itself, resulting in the inactivation.

Repetitive oral administration of imipramine (100 mg/kg/day for 5 days) caused a decrease in rat liver microsomal debrisoquine 4-hydroxylase activity, a characteristic reaction catalyzed by cytochrome P450 (CYP) 2D1. Other CYP2D-dependent reactions (such as bunitrolol 4-hydroxylation, lidocaine 3-hydroxylation, and propranolol 4-, 5- and 7-hydroxylations) were also impaired by the treatment, but not those catalyzed by other CYP isozymes. Imipramine pretreatment did not change the immunochemically determined content of the CYP2D protein, suggesting that CYP2D is inactivated. Imipramine pretreatment also resulted in an increase in total CYP content and in formation of a ferrous CYP metabolic intermediate (MI)-complex absorbing at 454 nm. Although the total CYP content was increased by the treatment of these microsomes with ferricyanide to dissociate the MI-complex, the CYP2D-dependent activities were not restored, suggesting that the MI-complex was not the primary cause of CYP2D inhibition. This pretreatment regimen caused marked increases in immunochemically determined levels of CYP2A1, CYP2B1, CYP2B2, CYP2C6, and CYP3A2, and in the activities of 2 alpha-, 2 beta-, 6 beta-, 7 alpha-, 16 alpha-, and 16 beta-hydroxylation and 17-oxidation of testosterone. These results indicate that imipramine has two actions on the liver CYP system (i.e. as an inhibitor of the CYP2D enzyme and as a phenobarbital-type inducer).

Oxidative metabolic pathways of propranolol consist of naphthalene ring-hydroxylations (at the 4-, 5-, and 7-positions) and side-chain N-desisopropylation in mammals. We characterized cytochrome P450 isozymes responsible for propranolol metabolism, especially N-desisopropylation and 5-hydroxylation, in human liver microsomes. 4-Hydroxy, 5-hydroxy-, and N-desisopropylpropranolol were detected as primary metabolites, whereas 7-hydroxypropranolol was in trace amounts. Good correlations were obtained for activities of propranolol 4- and 5-hydroxylases with immunochemically determined CYP2D6 content, whereas correlations of these activities with CYP1A2, CYP2C, or CYP3A4 content were relatively low. The activities also correlated highly with debrisoquine 4-hydroxylase, compared with other metabolic activities such as phenacetin O-deethylase, hexobarbital 3'-hydroxylase, and testosterone 6 beta-hydroxylase, which are typical reactions for CYP1A2, CYP2C, and CYP3A4, respectively. Propranolol N-desisopropylase activity in the samples highly correlated with CYP1A2 content and phenacetin O-deethylase activity, but not with the other P450 isozyme contents or metabolic activities. Quinidine, a specific inhibitor of CYP2D6, inhibited propranolol 4- and 5-hydroxylase activities selectively and in a concentration-dependent manner. alpha-Naphthoflavone, a potent inhibitor of CYP1A2, inhibited all of the propranolol oxidation activities, and the IC50 value for N-desisopropylase activity was much smaller than the values for ring-hydroxylase activities. Antibody directed to CYP2D inhibited propranolol 4- and 5-hydroxylase activities by 70% at an antibody/microsomal protein ratio of 1.0. Anti-CYP2C9 antibody did not inhibit any activity determined. These results indicate that propranolol 5-hydroxylation, as well as 4-hydroxylation, is mainly catalyzed by CYP2D6 in human liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolism of propranolol was examined by using microsomes from Dark Agouti rats known as a poor-metabolizer animal model for debrisoquine 4-hydroxylation and Wistar rats. Propranolol 4- and 5-hydroxylations followed biphasic Michaelis-Menten kinetics, and 7-hydroxylation and N-desisopropylation were monophasic in both strains. The kinetic studies showed that the Vmax for propranolol 7-hydroxylase activity and the Vmax of high-affinity phases for propranolol 4- and 5-hydroxylase activities were markedly low in Dark Agouti rats compared with those in Wistar rats. The antibody against a cytochrome P-450 isozyme, P-450BTL (Suzuki, T., et al., Drug Metab. Dispos. 20, 367-373, 1992), belonging to the CYP2D subfamily, inhibited by 90% propranolol 4-, 5-, and 7-hydroxylase activities in liver microsomes from male Wistar rats at a low propranolol concentration (5 microM). However, less inhibitory effects of the antibody on propranolol 4- and 5-hydroxylase activities were observed at a high propranolol concentration (1 mM), whereas a similar inhibitory effect of the antibody on propranolol 7-hydroxylase activity was shown. The antibody inhibited propranolol N-desisopropylase activity, but less extent of the inhibition on this activity than those on ring-hydroxylase activities was observed at the low and high propranolol concentrations. These results indicate that a polymorphic cytochrome P-450 isozyme(s) belonging to the CYP2D subfamily is involved predominantly in propranolol 4-, 5-, and 7-hydroxylations at low substrate concentrations in the rat.

A cytochrome P-450 isozyme, P-450 bunitrolol (BTL), catalyzing bunitrolol 4-hydroxylation was partially purified from liver microsomes of adult male Sprague-Dawley rats by hydrophobic affinity chromatographic (omega-aminooctyl-Sepharose 4B) and high-performance liquid chromatographic (anion-exchange diethylaminoethyl-5PW) techniques. The specific content of the final preparation was 5.02 nmol/mg protein, which was 7.8-fold that of microsomes. It showed two protein bands of 49 and 32 kDa in sodium dodecylsulfate-polyacrylamide gel electrophoresis. N-Terminal 20 amino acid sequence of the protein of a higher molecular mass (49 kDa) isolated by an electroblotting technique is 94% homologous with that of CYP2D2. In a reconstituted system including NADPH-cytochrome P-450 reductase and an NADPH-generating system, the final preparation had the highest activity toward BTL and debrisoquine 4-hydroxylation among 12 isozymes of cytochrome P-450 examined. Kinetic parameters, KM and Vmax values, of P-450 BTL calculated for BTL 4-hydroxylation were 10.7 microM and 19.68 nmol/min/nmol P-450, respectively, whereas those values (mean +/- SE) of rat liver microsomes were 0.84 +/- 0.05 microM and 2.05 +/- 0.11 nmol/min/nmol P-450. When preincubated with rat liver microsomes, the antibody against the final P-450 BTL preparation suppressed bunitrolol and debrisoquine 4-hydroxylase activities dose-dependently and almost completely. These results suggest that cytochrome P-450 BTL and its immunochemically related P-450 isozyme(s) play a major role in debrisoquine 4-hydroxylation as well as in BTL 4-hydroxylation in rat liver microsomes.

Although considerable information is available concerning steroidogenesis in the human fetus, the function of the different steroids formed during pregnancy and the factors regulating this delicate hormones balance are poorly understood. During human pregnancy, the placenta synthesizes large quantities of progesterone, estradiol, estrone and estriol and secretes these hormones into both the maternal and fetal circulations; progesterone from maternal lipoprotein-cholesterol, estradiol and estrone from maternal and fetal dehydroepiandrosterone sulfate (DHAS), and estriol largely from fetal 16 alpha-OH-DHAS. It has been demonstrated that preimplantation blastocysts of several animal species have the capacity to accumulate steroids to pregnenolone to progesterone, and to interconvert estrone and estradiol. Estetrol (E4), 15 alpha-hydroxy derivative of estriol is an interesting compound, since its formation is relatively unique to fetal liver function. Of special interest is that placental sulfatase deficiencies result in an extension of the gestation, and Cesarean section has to be done. This raises the question of the role of estrogens in determining the onset of labor, much as in the case of anencephaly. In general, progesterone may decline prior to an abortion, but there has not been a direct application to clinical practice. Estrogen levels during pregnancy are influenced by factors other than fetal well-being and include fetal weight, placental enzyme function, fetal adrenal function, maternal intestinal flora, maternal renal excretion and maternal liver function. Although not yet extensively utilized, such a dynamic test as the infusion of DHAS may yield useful information within a short period in otherwise complicated cases related to fetal and placental function.