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Study Design: Prospective observational study., Objectives: Classification of spinal-cord injury and prediction of independence in activities of daily living (ADL) based on performance evaluations such as upper-limb function have not been reported. Therefore, this study aimed to establish a severity classification and calculate cutoff values for independence in ADL using the Capabilities of Upper Extremity Test (CUE-T) for individuals with cervical spinal-cord injury (CSCI)., Setting: A spinal-cord injury rehabilitation center in Japan., Methods: This study included individuals with subacute CSCI. Collected data included the CUE-T and Spinal Cord Independence Measure III (SCIM III) scores. The severity classification was used for the hierarchical cluster analysis using the CUE-T. The cutoff values of CUE-T scores for independence in ADL were calculated using an adjustment model with logistic regression analysis. The dependent variable was binary (independent/non-independent) for each SCIM III Self-care item, and the independent variable was CUE-T., Results: A total of 71 participants were included in the analysis. The severity of upper-limb dysfunction was classified into four categories using CUE-T. Significant differences in upper-limb function and ADL were observed between clusters. The cutoff values for CUE-T score for independence in ADL ranged from 37 to 91 points. All cutoff values showed good results in the internal validation, sensitivity analysis., Conclusions: This study determined the severity of upper limb function in CSCI and the cutoff values of CUE-T scores for independence in ADL. These results may help set criteria and goals for interventions in the clinical and research fields., Sponsorship: None., (© 2024. The Author(s), under exclusive licence to International Spinal Cord Society.)

Context: The number of patients with cervical spinal cord injury (CSCI) is increasing, and the Capabilities of Upper Extremity Test (CUE-T) is recommended for introduction in clinical trials. We calculated the minimal clinically important difference (MCID) of the CUE-T using an adjustment model with an interval of 1 month., Design: This was a prospective study., Setting: This study was conducted with participants from the Chiba Rehabilitation Center in Japan., Participants: The participants were patients with subacute CSCI., Interventions: The CUE-T and spinal cord independence measure (SCIM) III were performed twice within an interval of 1 month., Outcome Measures: The MCID was calculated using an adjustment model based on logistic regression analysis. The participants were classified into an improvement group and a non-improvement group based on the amount of change in the two evaluations using the 10-point SCIM III MCID as an anchor., Results: There were 52 participants (56.8 ± 13.5 years old, 45 men/7 women) with complete or incomplete CSCI: 18 in the improvement group and 34 in the non-improvement group. A significant regression equation was obtained when calculating the MCID, and the total, hand, and side scores were 7.7, 2.0, and 3.7 points, respectively., Conclusion: The calculated MCID of the CUE-T in this study was 7.7 points. The results of this study provide useful criteria for implementation in clinical trials. Future studies should use patient-reported outcomes, a more recommended anchor, and calculate the MCID using methods such as the patient's condition.

Objective: A formula is needed that is practical for current livestock breeding methods and that predicts the approximate rate of inbreeding (∆F) in populations where selection is performed according to four-path programs (sires to breed sons, sires to breed daughters, dams to breed sons, and dams to breed daughters). The formula widely used to predict inbreeding neglects selection, we need to develop a new formula that can be applied with or without selection., Methods: The core of the prediction is to incorporate the long-tern genetic influence of the selected parents in four-selection paths executed as sires to breed sons, sires to breed daughters, dams to breed sons, and dams to breed daughters. The rate of inbreeding was computed as the magnitude that is proportional to the sum of squared long-term genetic contributions of the parents of four-selection paths to the selected offspring., Results: We developed a formula to predict the rate of inbreeding in populations undergoing four-path selection on genomically enhanced breeding values and with discrete generations. The new formula can be applied with or without selection. Neglecting the effects of selection led to underestimation of the rate of inbreeding by 40% to 45%., Conclusion: The formula we developed here would be highly useful as a practical method for predicting the approximate rate of inbreeding (ΔF) in populations where selection is performed according to four-path programs.

Intensive use of a few elite sires has increased the risk of the manifestation of deleterious recessive traits in cattle. Substantial genotyping data gathered using single-nucleotide polymorphism (SNP) arrays have identified the haplotypes with homozygous deficiency, which may compromise survival. We developed Japanese Black cattle haplotypes (JBHs) using SNP array data (4843 individuals) and identified deleterious recessive haplotypes using exome sequencing of 517 sires. We identified seven JBHs with homozygous deficiency. JBH_10 and JBH_17 were associated with the resuming of estrus after artificial insemination, indicating that these haplotypes carried deleterious mutations affecting embryonic survival. The exome data of 517 Japanese Black sires revealed that AC_000165.1:g.85341291C>G of IARS in JBH_8_2, AC_000174.1:g.74743512G>T of CDC45 in JBH_17, and a copy variation region (CNVR_27) of CLDN16 in JBH_1_1 and JBH_1_2 were the candidate mutations. A novel variant AC_000174.1:g.74743512G>T of CDC45 in JBH_17 was located in a splicing donor site at a distance of 5 bp, affecting pre-mRNA splicing. Mating between heterozygotes of JBH_17 indicated that homozygotes carrying the risk allele died around the blastocyst stage. Analysis of frequency of the CDC45 risk allele revealed that its carriers were widespread throughout the tested Japanese Black cattle population. Our approach can effectively manage the inheritance of recessive risk alleles in a breeding population.

The objectives of this study were to better understand the genetic architecture and the possibility of genomic evaluation for feed efficiency traits by (i) performing genome-wide association studies (GWAS), and (ii) assessing the accuracy of genomic evaluation for feed efficiency traits, using single-step genomic best linear unbiased prediction (ssGBLUP)-based methods. The analyses were performed in residual feed intake (RFI), residual body weight gain (RG), and residual intake and body weight gain (RIG) during three different fattening periods. The phenotypes from 4,578 Japanese Black steers, which were progenies of 362 progeny-tested bulls and the genotypes from the bulls were used in this study. The results of GWAS showed that a total of 16, 8, and 12 gene ontology terms were related to RFI, RG, and RIG, respectively, and the candidate genes identified in RFI and RG were involved in olfactory transduction and the phosphatidylinositol signaling system, respectively. The realized reliabilities of genomic estimated breeding values were low to moderate in the feed efficiency traits. In conclusion, ssGBLUP-based method can lead to understand some biological functions related to feed efficiency traits, even with small population with genotypes, however, an alternative strategy will be needed to enhance the reliability of genomic evaluation., (© 2019 Japanese Society of Animal Science.)

Background: Growth curves have been widely used in genetic analyses to gain insights into the growth characteristics of both animals and plants. However, several questions remain unanswered, including how the initial phenotypes affect growth and what is the duration of any such impact. For beef cattle production in Japan, calves are procured from farms that specialize in reproduction and then moved to other farms where they are fattened to achieve their market/purchase value. However, the causal effect of growth, while calves are on the reproductive farms, on their growth during fattening remains unclear. To investigate this, we developed a model that combines a structural equation with a growth curve model. The causal effect was modeled with B-splines, which allows inference of the effect as a curve. We fitted the proposed structural growth curve model to repeated measures of body weight from a Japanese beef cattle population (n = 3831) to estimate the curve of the causal effect of the calves' initial weight on their trajectory of growth when they are on fattening farms., Results: Maternal and reproduction farm effects explained 26% of the phenotypic variance of initial weight at fattening farms. The structural growth curve model was fitted to remove the effects of these factors in growth curve analysis at fattening farms. The estimated curve of causal effects remained at approximately 0.8 for 200 d after the calves entered the fattening farms, which means that 64% of the phenotypic variance was explained by the initial weight. Then, the effect decreased linearly and disappeared approximately 620 d after entering the fattening farms, which corresponded to an average age of 871.5 d., Conclusions: The proposed model is expected to provide more accurate estimates of genetic values for growth patterns because the confounding causal factors such as maternal and reproduction farm effects are removed. Moreover, examination of the inferred curve of the causal effect enabled us to estimate the effect of a calf's initial weight at arbitrary times during growth, which could provide suitable information for decision-making when shifting the time of slaughter, building models for genetic evaluation, and selecting calves for market.

Objective: Models for genomic selection assume that the reference population is an unselected population. However, in practice, genotyped individuals, such as progeny-tested bulls, are highly selected, and the reference population is created after preselection. In dairy cattle, the intensity of selection is higher in males than in females, suggesting that cows can be added to the reference population with less bias and loss of accuracy. The objective is to develop formulas applied to any genomic prediction studies or practice with preselected animals as reference population., Methods: We developed formulas for calculating the reliability and bias of genomically enhanced breeding values (GEBV) in the reference population where individuals are preselected on estimated breeding values. Based on the formulas presented, deterministic simulation was conducted by varying heritability, preselection percentage, and the reference population size., Results: The number of bulls equal to a cow regarding the reliability of GEBV was expressed through a simple formula for the reference population consisting of preselected animals. The bull population was vastly superior to the cow population regarding the reliability of GEBV for low-heritability traits. However, the superiority of reliability from the bull reference population over the cow population decreased as heritability increased. Bias was greater for bulls than cows. Bias and reduction in reliability of GEBV due to preselection was alleviated by expanding reference population., Conclusion: Cows are easier in expanding reference population size compared with bulls and alleviate bias and reduction in reliability of GEBV of bulls which are highly preselected than cows by expanding the cow reference population.

Objective: The purpose of this study was to compare intended and actual yearly genetic gains for milk production and conformation traits and to investigate the simple selection criterion practiced among milk production and conformation traits during the last two decades in Japan. Learning how to utilize the information on intended and actual genetic gains during the last two decades into the genomic era is vital., Methods: Genetic superiority for each trait for four paths of selection (sires to breed bulls [SB], sires to breed cows [SC], dams to breed bulls [DB], and dams to breed cows [DC]) was estimated. Actual practiced simple selection criteria were investigated among milk production and conformation traits and relative emphasis on milk production and conformation traits was compared., Results: Selection differentials in milk production traits were greater than those of conformation traits in all four paths of selection. Realized yearly genetic gain was less than that intended for milk production traits. Actual annual genetic gain for conformation traits was equivalent to or greater than intended. Retrospective selection weights of milk production and conformation traits were 0.73:0.27 and 0.56:0.44 for intended and realized genetic gains, respectively., Conclusion: Selection was aimed more toward increasing genetic gain in milk production than toward conformation traits over the past two decades in Japan. In contrast, actual annual genetic gain for conformation traits was equivalent to or greater than intended. Balanced selection between milk production and conformation traits tended to be favored during actual selection. Each of four paths of selection (SB, SC, DB, and DC) has played an individual and important role. With shortening generation interval in the genomic era, a young sire arises before the completion of sire's daughters' milk production records. How to integrate these four paths of selection in the genomic era is vital.

We evaluated the genetic relationships (1) among feed efficiency traits with different fattening periods, (2) between feed efficiency traits and growth traits, and (3) between feed efficiency traits and carcass traits, to determine the influence of genetic factors on feed efficiency traits. In total, 4,578 Japanese Black cattle from a progeny testing program were used. Residual feed intake (RFI), residual BW gain (RG), and residual intake and BW gain (RIG) were defined as feed efficiency traits, and were measured for the first half (approximately 9 to 15 months of age), latter half (approximately 15 to 21 months of age), and total period of fattening (approximately 9 to 21 months of age). A single-trait animal model for estimating heritability and a two-trait animal model for estimating genetic and phenotypic correlations were used. The heritability estimates for RFI, RG, and RIG were different in each fattening period, ranging from 0.36 to 0.46, 0.19 to 0.28, and 0.28 to 0.34, respectively, and the heritability estimates for the total fattening period were greater than those for the first and latter halves separately. RIG showed the greatest preferred genetic correlation, with a greater feed conversion ratio than the other feed efficiency traits (ranging from -0.84 to -0.96). RG in the first and latter halves of the fattening period had different genetic correlations with the growth starting point (0.82 and -0.06, respectively) and maturity rate (0.49 and -0.51, respectively) of the Gompertz growth curve parameters, and is strongly dependent on the different fattening periods. Feed efficiency traits in different fattening periods had low genetic correlations with the carcass traits (from -0.05 to 0.19 for RFI; from 0.02 to 0.31 for RG; and from -0.11 to 0.20 for RIG). This study indicated the possibility for genetic improvement through the selection of high-RIG animals to decrease feed intake and increase BW gain without any unfavorable correlated responses affecting mature (asymptotic) weight and carcass grade.

Objective: In practical breeding, selection is often performed by ignoring the accuracy of evaluations and applying economic weights directly to the selection index coefficients of genetically standardized traits. The denominator of the standardized component trait of estimated genetic evaluations in practical selection varies with its reliability. Whereas theoretical methods for calculating the selection index coefficients of genetically standardized traits account for this variation, practical selection ignores reliability and assumes that it is equal to unity for each trait. The purpose of this study was to clarify the effects of ignoring the accuracy of the standardized component trait in selection criteria on selection responses and economic weights in retrospect., Methods: Theoretical methods were presented accounting for reliability of estimated genetic evaluations for the selection index composed of genetically standardized traits., Results: Selection responses and economic weights in retrospect resulting from practical selection were greater than those resulting from theoretical selection accounting for reliability when the accuracy of the estimated breeding value (EBV) or genomically enhanced breeding value (GEBV) was lower than those of the other traits in the index, but the opposite occurred when the accuracy of the EBV or GEBV was greater than those of the other traits. This trend was more conspicuous for traits with low economic weights than for those with high weights., Conclusion: Failure of the practical index to account for reliability yielded economic weights in retrospect that differed from those obtained with the theoretical index. Our results indicated that practical indices that ignore reliability delay genetic improvement. Therefore, selection practices need to account for reliability, especially when the reliabilities of the traits included in the index vary widely.

The two most frequently observed single nucleotide polymorphisms (SNPs) of the human multidrug resistance 1 (MDR1) gene are 2677G/T/A (893Ala/Ser/Thr) and 3435C/T (no amino acid substitution). In this study, six forms of MDR1 cDNAs with the SNPs were expressed in LLC-PK1 cells and their transport activities were determined. Nearly identical amounts of the recombinant MDR1 proteins were expressed in the established cell lines using the Flp recombinase, which integrates a gene of interest at a specific genomic location. Four structurally diverse compounds: verapamil, digoxin, vinblastine and cyclosporin A, were examined for transcellular transport activities and intracellular accumulation. No significant differences were observed between cells expressing five polymorphic types of the MDR1 cDNAs (2677G/3435T, 2677A/3435C, 2677A/3435T, 2677T/3435C, 2677T/3435T) and cells expressing the wild-type (2677G/3435C). These results suggested that the two frequently observed MDR1 SNPs had no effect on the transport activities of MDR1 proteins expressed in LLC-PK1 cells in vitro, and other genetic or environmental factors might control the expression of MDR1 and the in vivo activity of MDR1.

Purpose: Hepatobiliary excretions of drugs from the blood to the bile include two essential transmembrane processes: uptake into hepatocytes and secretion from hepatocytes. The purpose of this study was to clarify the transport mechanisms underlying these processes for a new non-peptide endothelin antagonist, (+)-(5S,6R,7R)-2-butyl-7-[2((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxy-phenyl)cyclopentenol[1,2-b]pyridine-6-carboxylic acid (J-104132)., Methods: Biliary excretion of J-104132 was assessed in rats after intravenous injection. To evaluate the hepatic uptake process, J-104132 was incubated with freshly isolated rat hepatocytes and the uptake of J-104132 was calculated. To evaluate the biliary secretion process, the uptake of J-104132 into rat canalicular membrane vesicles that were isolated from normal Sprague-Dawley rats or Eisai hyperbilirubinemic rats was measured., Results: After intravenous injection, J-104132 was recovered from the bile quantitatively (99.7 +/- 1.3%) as its intact form. J-104132 was taken up by isolated rat hepatocytes in a time- and temperature-dependent manner. The uptake was saturable with Km and Vmax of 5.7 microM and 564 pmol/min/10(6) cells, respectively. The uptake was Na+ independent and was reduced in the presence of ATP depleters (rotenone and carbonyl cyanide-p-(trifluoromethoxy)-phenylhydrazone), organic anions (dibromosulfophthalein, indocyanine green, BQ-123, and pravastatin), and bile acids (taurecholate and cholate). In Sprague-Dawley rats, J-104132 was taken up by canalicular membrane vesicle ATP-dependently with Km and Vmax values of 6.1 microM and 552 pmol/min/mg protein, respectively. However, ATP-dependent uptake disappeared in Eisai hyperbilirubinemic rats., Conclusions: These data suggest that energy-dependent and carrier-mediated transport systems play important roles in hepatobiliary excretion of J-104132 (both uptake and secretion processes), which is the main excretion route in rats. As for the secretion process of J-104132, an involvement of mrp2 was demonstrated.

The glucuronidation of 6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13-(beta-D-glucopyranosyl)5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione (compound 1), a potent inhibitor of DNA topoisomerase I, and its related indolocarbazole compounds was studied using human liver microsomes. Compound 1 and its structurally related compounds with the NHCHO moiety at the N-6 position were glucuronidated even if the positions of the phenolic hydroxy moiety were different in these molecules. Compounds that have the NHCH(CH(2)OH)(2) moiety at the N-6 position, however, were not glucuronidated. The three-dimensional structure of these substrates was determined by the semiempirical molecular-orbitals calculation method. Computer-modeling studies, however, revealed that the O-glucuronidation of indolocarbazole analogs depended on the molecular size of the substrates. Compounds larger than 14.5 A in diameter perpendicular to the phenolic hydroxy moiety were not glucuronidated. The chemical reactivity of the hydroxy moiety, evaluated by the atom electron density and the electrostatic potential charges, was very similar in these substrates. These results suggest that a molecular length less than 14.5 A may be required for a substrate to interact with the active site of UDP-glucuronosyltransferase (UGT). To further characterize the glucuronidation of indolocarbazole analogs, compound 1 was used as a representative compound to assess expressed human UGTs. The glucuronidation of compound 1 was catalyzed by recombinant UGT1A9 and UGT1A10 among UGT isoforms tested.

To assess the effects of Ile359 to Leu359 change on CYP2C9-mediated metabolism, we performed site-directed mutagenesis and cDNA expression in yeast for CYP2C9 and examined in detail the kinetics of seven metabolic reactions by wild-type CYP2C9 (Ile359) and its Leu359 variant. For the metabolism of all the substrates studied, the Leu359 variant exhibited smaller Vmax/Km values than did the wild-type. The differences in the Vmax/Km values between the wild-type and the Leu359 variant varied from 3.4-fold to 26.9-fold. The Leu359 variant had higher Km values than did the wild-type for all the reactions studied. Among the seven reactions studied, the greatest difference in the Vmax values between the wild-type and the Leu359 variant was for piroxicam 5'-hydroxylation (408 versus 19 pmol/min/nmol P450), whereas there were no differences in the Vmax values between the wild-type and the Leu359 variant for diclofenac 4'-hydroxylation and tolbutamide methylhydroxylation. These results indicate that the Ile359 to Leu359 change significantly decreases the catalytic activity of all the CYP2C9-mediated metabolisms studied, whereas the extent of the reduction in activity and changes of the kinetic parameters varies between substrates. Moreover, the amino acid substitution decreased the enantiomeric excess in the formation of 5-(4-hydroxyphenyl)-5-phenylhydantoin from phenytoin.

A uricosuric agent, bucolome, has been shown to intensify the anticoagulant effect of warfarin. The aims of the present study were to clarify its mechanism(s) and to apply in vitro approaches for predicting this potentially life-threatening in vivo interaction. An in vivo study revealed that Japanese patients given warfarin with bucolome (300 mg/day, n = 21) showed a 1.5-fold greater international normalized ratio than those given warfarin alone (n = 34) despite that the former received a 58% smaller warfarin dose than the latter. Enantioselective assays revealed that bucolome increased plasma unbound fractions of (S)- and (R)-warfarin by 2-fold (p <.01), reduced unbound oral clearances of (S)- and (R)-warfarin by 84 (p <.01) and 26% (p <.05), respectively, and inhibited the unbound formation clearance for (S)-warfarin 7-hydroxylation by 89% (p <.01). In contrast, bucolome elicited no appreciable changes in the plasma unbound (S)-warfarin concentration versus the international normalized ratio relationship. In vitro studies with recombinant human cytochrome P-450 2C9 and liver microsomes showed that bucolome was a potent mixed-type inhibitor for (S)-warfarin 7-hydroxylation, with K(i) of 8.2 and 20.2 microM, respectively. An in vitro model incorporating maximum unbound bucolome concentration in the liver estimated as a sum of hepatic artery and portal vein concentrations and in vitro K(i) made an acceptable prediction for bucolome-induced reductions in in vivo total (bound + unbound) oral clearance, unbound oral clearance, and unbound formation clearance for (S)-warfarin. In conclusion, the augmented anticoagulant effect of warfarin by bucolome due to the metabolic inhibition for pharmacologically more potent (S)-warfarin may be predictable from in vitro data.

To investigate the interaction between endothelin (ET) and the nitric oxide system, we examined the effects of ET-1 and ET-3 on the induction of inducible nitric oxide synthase (iNOS) and guanosine triphosphate cyclohydrolase I (GTP:CHI), the rate-limiting enzyme of de novo synthesis of the cofactor tetrahydrobiopterin (BH4), in rat mesangial cells. ET-1 inhibited the nitrite accumulation induced by a combination of interleukin-1 beta, tumor necrosis factor-alpha, and lipopolysaccharide in a concentration-dependent manner. The inhibitory effect of ET-3 was less potent than that of ET-1. A selective ETA antagonist, BQ-485, and an ETA and ETB antagonist, TAK-044, abolished the inhibitory effects of ET-1, whereas the selective ETB antagonist BQ-788 had no effect on the inhibition produced by ET-1. These observations indicate that ET-1 inhibits cytokine-stimulated nitrite accumulation through the ETA receptor. Western blot analysis showed that the suppression of nitrite accumulation was accompanied by a decrease in iNOS protein. Northern blot analysis showed that ET-1 inhibited the expression of both iNOS and GTP:CHI mRNA. In conclusion, ET-1 inhibits cytokine-stimulated nitric oxide production through the ETA receptor by suppressing the expression of iNOS and GTP:CHI mRNA in rat mesangial cells.

Metabolism of diazepam was studied in vitro to identify the forms of cytochrome P450 (CYP) responsible for N-demethylation (nordazepam formation) and 3-hydroxylation (temazepam formation), using liver microsomes obtained from extensive (EM) and poor metabolizers (PM) for S-mephenytoin 4'-hydroxylation. Involvement of at least two P450 forms in diazepam N-demethylation was suggested by a biphasic pattern in Lineweaver-Burk and Eadie-Hofstee plots from the EM, whereas a monophasic pattern was observed from the PM liver microsomes. The kinetic parameters for the N-demethylation in the EM group were: Km 1, 19.4 +/- 0.4 microM; Vmax 1, 0.27 +/- 0.04 nmol min-1 per mg protein; Km 2, 346 +/- 34 microM; Vmax2, 1.82 +/- 0.63 nmol min-1 per mg protein (n = 3, mean +/- SD). The PM group showed the mean values of Km and Vmax (Km, 319 +/- 30 microM; Vmax, 1.49 +/- 0.62 nmol min-1 per mg protein) (n = 3) similar to those of Km2 and Vmax2 in the EM group. An antibody raised against CYP2C9 (anti-human CYP2C) strongly inhibited diazepam N-demethylation in EM liver microsomes at a low substrate concentration (20 microM). However, the anti-human CYP2C showed no clear inhibition of N-demethylation in EM liver microsomes at a high substrate concentration (200 microM). Diazepam N-demethylation in PM liver microsomes was not clearly inhibited by the anti-human CYP2C at either the low or high substrate concentrations. These data suggest that different P450 forms mediated diazepam N-demethylation in EM and PM liver microsomes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of diazepam (DZP) was studied in vitro to clarify the involvement of different forms of hepatic cytochrome P450 (CYP) in rats, and humans of Japanese and Caucasian origin. Microsomal 3-hydroxylation was the major pathway of DZP metabolism in rats and was inhibited by anti-CYP3A antibodies. Purified CYP3As and CYP2C11 catalysed 3-hydroxylation and N-demethylation, respectively, in the reconstituted systems. The rates of both reactions in human liver microsomes depended on the substrate concentration: the rate of 3-hydroxylation was 3-4 times higher than N-demethylation at 0.2 mM; the two activities were essentially the same at a lower substrate concentration (0.02 mM). Inhibitions of the N-demethylation by anti-CYP2C antibody and S-mephenytoin also depended on the substrate concentration and was detectable only at a low substrate concentration. Kinetic studies revealed the presence of two distinct catalytic activities for the N-demethylation; low Km and low Vmax, and high Km and high Vmax. The former activity seems to be mediated by a CYP2C P450 form. On the other hand, DZP 3-hydroxylation was rather selectively catalysed by a CYP3A P450 at the low and high substrate concentrations. These results were consistent with the observation in vivo that DZP N-demethylation and S-mephenytoin 4'-hydroxylation are closely correlated in humans. These results also suggest that the apparent discrepancy on the role of CYP forms in DZP metabolism in vitro and in vivo may reside in the difference in substrate concentration.

Microsomal hydroxylation of tolbutamide in Japanese livers was studied in vitro to ascertain the enzyme catalysing this reaction. Rates of tolbutamide hydroxylation differed individually 33-fold and 42-fold at 0.1 mM and 2.4 mM tolbutamide concentrations, respectively, and were segregated into two groups, rapid and slow metabolizers. An antibody raised against P450 human-2 (a form of CYP2C9) strongly inhibited the hydroxylation in livers of rapid metabolizers but only weakly inhibited in the slow metabolizer. Kinetic experiments further demonstrated a clear distinction in tolbutamide hydroxylation between two groups; the mean of apparent Km values for tolbutamide was 0.25 mM (n = 3) in the rapid group and 2.58 mM (n = 2) in the slow, respectively. These data suggest that different enzymes are involved in the hydroxylation in both metabolizer groups. Furthermore, CYP2C9 produced by cDNA expression in yeasts, catalysed tolbutamide hydroxylation at rates similar to the rapid metabolizer group at both the 0.1 mM and 2.4 mM concentrations. The apparent Km value of the expressed protein for tolbutamide, 0.26 mM, was similar to that determined for the rapid group of microsomal samples. Clear correlations were observed between the rate of microsomal tolbutamide hydroxylation at 0.1 mM and CYP2C9 protein content or the rate of S-mephenytoin 4'-hydroxylation in human liver. These results indicate that considerable portions of microsomal tolbutamide hydroxylation are catalysed by CYP2C9 or the closely related form in the rapid metabolizers.

Effect of pretreatment of rats with FK506((-)-(1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-17-allyl-1,14- dihydroxy-12-[(E)-2-[(1R,3R,4R)-4-hydroxy-3methoxycyclohexyl]-1- methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4- azatricyclo-[22.3.1.0(4.9)]octacos-18-ene-2,3,10,16-tetrone hydrate, CAS 104987-11-3) on microsomal cytochrome P-450 system and oxidations of the administered drug and other model substrates were studied and compared with those of a pharmacologically related drug, ciclosporin (cyclosporin A). Oral treatment of male Sprague-Dawley rats with FK506 (0.4, 2 or 10 mg/kg/d) for 7 days did not decrease microsomal content of total cytochrome P-450 in livers, but rather increased the content in groups with the dose of 0.4 or 10 mg/kg to the levels of 126-130% of the control. Microsomal NADPH-cytochrome c reductase activities were decreased up to 67% of the control with the increasing dose of FK506 and to 62% in a group treated orally with cyclosporin A (25 mg/kg/d for 7 days), although another microsomal electron-transport component, cytochrome b5, was rather increased in all the treated groups. Treatment with FK506 or cyclosporin A did not reduce but slightly increased microsomal activities of aniline hydroxylation, p-nitroanisole O-demethylation and O-ethoxyresorufin O-deethylation. Microsomal depropylation of 7-propoxycoumarin, a typical P-450IIIA-substrate, was also not reduced in all dose groups of FK506, while it was decreased by the treatment with 25 mg/kg cyclosporin A.(ABSTRACT TRUNCATED AT 250 WORDS)

The cDNA of a P-450 human-2 and the two other closely related cDNAs, MP-8 (two deduced amino acids substituted) and lambda hPA6 (two deduced amino acids deleted) were expressed in Saccharomyces cerevisiae cells, and their catalytic and chemical properties were compared to identify which cDNA encodes a major S-mephenytoin 4'-hydroxylase in human livers. In immunoblots, P-450 human-2 cDNA-derived protein in yeasts was stained at the position identical with P-450 human-2 purified from liver and a major protein in microsomes of 19 Japanese livers. MP-8- and lambda hPA6-derived proteins were immunostained at positions near, but distinct from P-450 human-2, and were not detected in those 19 livers. All three proteins expressed in yeasts catalyzed hydroxylation of mephenytoin, hexobarbital, benzo[a]pyrene and tolbutamide, although the rates of the hydroxylation of most of the drugs by P-450 human-2 were higher than those of the two others. In addition, these expressed proteins showed clear differences in the hydroxylation of chiral substrates: P-450 human-2 catalyzed the hydroxylation of S-mephenytoin five times faster than that of the R-enantiomer. Similar high enantioselectivities were also observed on the hydroxylation of R- and S-hexobarbital. However, MP-8- and lambda hPA6-derived proteins catalyzed hydroxylation of these two drugs with less or almost no stereoselectivity. These results indicate that only a few amino acid alterations cause dramatic changes in both the chemical and catalytic properties of P-450 human-2.

Stereoselective 4'-hydroxylations of R-(-)-mephenytoin and S-(+)-mephenytoin and 3'-hydroxylation of R-(-)-hexobarbital and S-(+)-hexobarbital were determined in liver microsomes of 14 Japanese subjects who were extensive metabolizers of mephenytoin and in five Japanese subjects who were poor metabolizers of mephenytoin. Content of P-450 human-2 assessed by Western blots was correlated to microsomal S-(+)-mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' alpha-hydroxylation, and S-(+)-hexobarbital 3' beta-hydroxylation, and was less correlated to R-(-)mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' beta-hydroxylation, and S-(+)-hexobarbital 3' alpha-hydroxylation. Antibodies raised against P-450 human-2 inhibited microsomal S-(+)-mephenytoin 4'-hydroxylation efficiently but was less efficient on R-(-)-mephenytoin 4'-hydroxylation in extensive metabolizers and on 4'-hydroxylation of mephenytoin enantiomers in poor metabolizers. The antibodies also inhibited R-(-)-hexobarbital 3' alpha-hydroxylation and S-(+)-hexobarbital 3' beta-hydroxylation but did not effectively inhibit the hydroxylation of the two other optical isomers of hexobarbital in extensive metabolizers and of four stereoisomers in poor metabolizers. These findings indicate the close relationship between polymorphic mephenytoin 4'-hydroxylation and two stereospecific hexobarbital hydroxylations, and they suggest that P-450 human-2 is a typical S-(+)-mephenytoin 4'-hydroxylase and a major hexobarbital 3'-hydroxylase in the livers of extensive metabolizers. The findings were further supported by the experiments that used P-450 human-2 complementary dexoyribonucleic acid-derived protein in yeast microsomes.

Stereoselective hydroxylation of mephenytoin is expressed polymorphically in the Japanese population: the occurrence of the poor metabolizer was approximately 20% in Japanese, which was 5 to 8 times higher than that in Caucasians. Microsomal 4'-hydroxylation of S-mephenytoin, but not of R-mephenytoin, was correlated to the content of cytochrome P-450 human-2. P-450 human-2 cDNA was cloned and expressed in yeast cells. The expressed P-450 showed stereoselectivities similar to those in human livers for hydroxylations of mephenytoin and hexobarbital enantiomers. The P-450 catalyzed benzo[a]pyrene and tolbutamide hydroxylations and cyclophosphamide oxidation, but showed low activity for the mutagenic activation of IQ (2-amino-3-methylimidazo [4, 5-f]quinoline). Two cDNAs (MP-8 and lambda hPA6), which are two amino acids mutated and deleted, respectively, from P-450 human-2 cDNA were expressed in yeasts. These two expressed P-450s revealed complete loss of the stereoselectivity in the hydroxylation of mephenytoin and hexobarbital. These results indicate that many xenobiotics and carcinogens are detoxicated and activated by the human liver in quite different manners among individuals by reason of genetic differences.

Two forms of cytochrome P-450 (P-450 human-1 and P-450 human-2) have been purified from human liver microsomes to electrophoretic homogeneity. P-450 human-1 and P-450 human-2 differ in their apparent molecular weights (52,000 and 56,000, respectively) and Soret peak maxima in the CO-binding reduced difference spectrum (447.6 and 450.3 nm, respectively). In the reconstituted system using rat liver NADPH-cytochrome c (P-450) reductase, P-450 human-2 more effectively oxidized benzo(a)pyrene (80-fold), ethylmorphine (2-fold), and 7-ethoxycoumarin (2-fold) than did P-450 human-1. However, P-450 human-1 showed higher testosterone 6 beta-hydroxylase activity, and the activity was markedly increased by the inclusion of cytochrome b5 or spermine in the reconstituted system. Antibodies raised against P-450 human-1 inhibited more than 80% of microsomal testosterone 6 beta-hydroxylase activity in human liver. Immunoblotting analysis using anti-P-450 human-1 IgG revealed a single immuno-staining band near Mr 52,000 in all human liver samples examined. The amount of immunochemically determined P-450 human-1 varied in parallel with the testosterone 6 beta-hydroxylase activity in human liver. These results indicate that P-450 human-1 is a major form of cytochrome P-450 responsible for microsomal testosterone 6 beta-hydroxylation. Thus, this paper is the first report on human cytochrome P-450 responsible for testosterone 6 beta-hydroxylation, which is the major hydroxylation pathway in human liver microsomes.

P-450 human-2 is a human cytochrome P-450 that is immunochemically related to a constitutive male-specific cytochrome P-450 (P-450-male) and the phenobarbital-inducible P-450b/e in rat liver. By screening a human liver cDNA library in bacteriophage lambda gt11, we isolated a clone with an insert length of 1,847 bases (pHY13). The clone was sequenced and shown to code for a protein of 487 amino acids. The N-terminal 11-amino-acid sequence was in agreement with the protein sequence of P-450 human-2. The nucleotide sequence of pHY13 showed less than 50% similarity with those of human cytochrome P-450s, pHP-450(1), HLp, P-450NF, P1-450 4, and P3(450), but the nucleotide sequence of pHY13 is 80% similar to the reported sequence of rat cytochrome P-450, P-450(M-1). In addition, the coding sequence of pHY13 showed close similarity to that of MP-8, which was recently reported as the sequence corresponding to human cytochrome P-450MP, although no apparent similarity was observed in their 3' non-coding sequences except for the first 75 bases and the expected length of the complete sequences. These results, together with the immunochemical data, indicate that P-450 human-2 is closely related, but not identical, to P-450MP, and may belong to the category of developmentally regulated constitutive cytochrome P-450s.