Microarray analysis of hepatic gene expression during long-term cholestasis induced by common bile duct ligation in rats.

Cholestasis is one of the major liver diseases and results in progressive liver fibrosis and cirrhosis. In this study, the transcriptional response of the liver to common bile duct ligation in rats was examined by cDNA microarray analysis, and 134 genes for which expression was altered during long-term cholestasis were identified. Clustering analysis of these genes for multiple time-point data yielded 7 different patterns in which a large portion of the genes was classified into 3 clusters. Two clusters consisted of up-regulated genes, including genes that may be related to disruption of lipid metabolism and liver fibrosis observed in the early stage of cholestasis, and the other cluster consisted of down-regulated genes, including a gene that has been thought to be involved in the mechanism of cell protection against accumulation of bile acids. Since the expression patterns of these genes appear to reflect molecular features of cholestasis. Characterization of the genes identified in this study may shed further light on the physiological and pathological characteristics of long-term cholestasis.

[Actual conditions of occupational health administration of small-scale enterprises in Japan: (II). Occupational health controls for hazardous and musculo-skeletally stressful working factors].

In order to clarify the actual condition of occupational health management for hazardous and musculo-skeletally stressful work factors in small-scale enterprises (SSEs) in Japan, a questionnaire survey was conducted in an area near Osaka city. The hazardous work factors examined were dust, organic solvents, lead, specified chemical substances, anoxia, noise, hand-arm vibration, ionizing radiation, high and low temperatures, and high air pressure. The musculo-skeletally stressful work factors examined were VDT work, prolonged standing, unnatural postures, handling of heavy weights, and stress on neck, shoulders and arms. The number of SSEs that replied to the questionnaire was 765 (recovery rate: 69.3%). Enterprises with noise, dust, hand-arm vibration and organic solvents numbered 14.0%, 10.7%, 6.9% and 6.4%, respectively, and those with other hazardous factors numbered less than 3%. Special medical examinations and working environment measurements for hazardous factors were conducted in 0.0% to 26.7% and 0.0% to 13.3%, respectively, of the enterprises. Working environment controls were conducted in 0.0% to 40.2%. Enterprises with prolonged standing and VDT work, were 42.0% and 35.8%, whereas those with other stressful factors were approximately 30%. Special medical examinations for musculo-skeletally stressful factors were conducted in 3.0% to 5.1% of the enterprises, and work controls were conducted in 20.4% to 25.3%. Non execution of the special medical examinations and working environment measurements were mainly due to "lack of knowledge of the law (19.7% and 30.2%)" and "lack of time to perform (16.0% and 23.3%)". Non execution of the controls for the hazardous work factors was due to "lack of knowledge as to how to control (9.0%)", "high costs (7.4%)", "lack of time to perform (6.4%)" and "absence of a suitable adviser (5.9%)". Non execution of the controls for stressful work factors was due to "lack of knowledge as to how to control (15.6%)" and "lack of time to perform (10.2%)". Consequently, as a result of the survey, it was suggested that it is necessary to enlighten the employers of SEEs as to the importance of occupational health controls. It is also necessary to propose low-cost, feasible control methods.

Catalytic activity of three variants (Ile, Leu, and Thr) at amino acid residue 359 in human CYP2C9 gene and simultaneous detection using single-strand conformation polymorphism analysis.

This study evaluated the catalytic activity of three variants (Ile, Leu, and Thr) at codon 359 of CYP2C9 enzymes expressed in a yeast cDNA expression system, and then established single-strand conformation polymorphism (PCR-SSCP) analysis for simultaneous detection as a screening method. Diclofenac was used for the in vitro experiment, and its hydroxy metabolite (4'-hydroxydiclofenac) was measured by HPLC. To discuss the in vivo effect of the Thr359 variant on the pharmacokinetics of phenytoin, a case report is presented. The efficiency of the SSCP method was evaluated by analyzing DNA samples from a homozygote for Ile359 and a heterozygote for Leu359 or Thr359. To evaluate the interaction between the P450 level and reductase activity, two batches of the Thr359 variant with a different P450:reductase activity ratio (1:4.0 and 1:1.4) were used. The in vitro study revealed that recombinant Ile359, Leu359, and Thr359 (2 batches) possessed a mean Km of 2.0, 16.5 and (3.8 and 2.9) micromol and Vmax of 12.4, 17.9 and (4.4 and 5.1) nmol/min/nmol P450, respectively. Although the magnitude of the change in catalytic efficiency for the Thr359 variant was close to that of the Leu359 variant, the effect of the two variants on diclofenac 4'-hydroxylation appears to be different because Leu359 variant was associated with a high Km, and Thr359 with a low Vmax. No significant differences in the kinetic data were observed between the two Thr359 enzymes, suggesting that low reductase activity in the Thr359 enzyme was not a major determinant in the present in vitro experiment. Estimated pharmacokinetic parameters of phenytoin obtained by the Bayesian method in an epileptic patient who was a heterozygote carrier for Thr359 variant were: Km = 6.45 microg/mL, Vmax = 5.77 mg/kg/d, and Vmax/Km = 0.89 L/kg/day. The Vmax/Km value in this patient was similar to the population mean value (0.90 L/kg/day) in Japanese heterozygotes for the Leu359 variant. Results for PCR-SSCP were in complete agreement with those obtained using established methods. Thus, the PCR-SSCP approach is useful for identifying these three variants of the CYP2C9 gene.

CYP2C9 Ile359 and Leu359 variants: enzyme kinetic study with seven substrates.

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.

Potentiation of anticoagulant effect of warfarin caused by enantioselective metabolic inhibition by the uricosuric agent benzbromarone.

OBJECTIVE: To clarify the mechanism(s) for the interaction between warfarin and benzbromarone, a uricosuric agent, and to predict changes in the in vivo pharmacokinetics of (S)-warfarin from in vitro data. METHODS: Warfarin enantiomers and benzbromarone in serum, 7-hydroxywarfarin in urine, and serum unbound fractions of warfarin enantiomers were measured in patients with heart disease given warfarin with (n = 13) or without (n = 18) oral benzbromarone (50 mg/d). In vitro inhibition constants (K(i)) of benzbromarone for (S)-warfarin 7-hydroxylation were determined with use of human CYP2C9 and liver microsomes. The magnitude of changes in the formation clearance for 7-hydroxylation (CLf), the unbound oral clearance (CL(oral,u)), and the oral clearance (CL(oral)) for (S)-warfarin were predicted by equations incorporating the in vitro Ki, the theoretical maximum unbound hepatic benzbromarone concentration, and the fractions of warfarin eliminated through metabolism and of CYP2C9-mediated metabolic reaction susceptible to inhibition by benzbromarone. RESULTS: The patients given warfarin with benzbromarone required a 36% less (P < .01) warfarin dose than those given warfarin alone (2.5 versus 3.9 mg/d) to attain similar international normalized ratios (2.1 and 2.2, respectively), and the former had 65%, 53%, and 54% lower (P < .05 or P < .01) CLf, CL(oral),u, and CL(oral) for (S)-warfarin than the latter, respectively. In contrast, no significant differences were observed for (R)-warfarin kinetics between the groups. Benzbromarone was found to be a potent competitive inhibitor (Ki < 0.01 micromol/L) for (S)-warfarin 7-hydroxylation mediated by CYP2C9. The average changes in the in vivo CLf, CL(oral),u, and CL(oral)values for (S)-warfarin induced by benzbromarone were largely predictable by the proposed equations. CONCLUSION: Benzbromarone would intensify anticoagulant response of warfarin through an enantioselective inhibition of CYP2C9-mediated metabolism of pharmacologically more potent (S)-warfarin. The magnitude of changes in the in vivo warfarin kinetics may be predicted by in vitro data.

Pharmacokinetic interaction between warfarin and a uricosuric agent, bucolome: application of In vitro approaches to predicting In vivo reduction of (S)-warfarin clearance.

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.

[Actual conditions of occupational health activities in small-scale enterprises in Japan: system for occupational health, health management and demands by small-scale enterprises].

In order to clarify the real conditions of occupational health services (OHS) in small-scale enterprises (SSEs) in Japan, we analyzed questionnaires recovered from 765 SSEs in the area of a city neighboring Osaka City (recovery rate, 69.3%). The SSEs included 358 SSEs with 1 to 4 workers (46.8% of total SSEs), 203 with 5 to 9 (26.5%), 163 with 10 to 29 (21.3%) and 41 with 30 to 49 (5.4%). The main types of businesses were manufacturing (374, 48.9% of total SSEs), wholesale/retail trade/restaurants (153, 20.0%), community, social and personal services (132, 17.3%) and construction (72, 9.4%). Health examinations were performed in 47.7% of SSEs. The reason for the lack of examinations were "shortage of time" (33.3% of SSEs lacking health examination) and "employees do not want to be examined" (28.1%). Some health promotion measures were conducted in 29.2% of SSEs. Health examination (59.0% of SSEs), health promotion (36.5%), measure of mental health (25.9%) and information service for employers and employees (25.5%) were demanded by SSEs as OHS. Financial subsidies and economical incentives were demanded by 46.4% and 28.8% of SSEs, respectively. Regional occupational health center in this area was poorly known among SSEs (8.2%), but health examination (48.4%), information service (37.5%), assessment of work method and advice to improve (19.8%) and environment measurement (12.4%) are demanded of the center by SSEs.

[Load on the low back of teachers in kindergartens].

In order to evaluate the load on the low back of teachers in kindergartens, basic activity and working posture were analyzed for four teachers by means of video recording. The trunk inclination angle (TIA) was also measured continuously during full workshifts for 12 kindergarten teachers by means of an inclination monitor. The kindergarten teachers spent 67% of the workshift on activities in contact with children, "indoor group childcare", "indoor free playing", "outdoor childcare", "preparation and clearing away" and "help and care", and did not take a recess during the workshift. They spent 36% of the workshift in three working postures with the load on the low back, "standing bent forward", "squatting" and "kneeling". Cumulative time at a TIA of 20 degrees or more represented 43% of the workshift. The frequency of trunk-lifting from severe bending forward (TIA > 45 degrees) was 95 times/hr on average. A comparison of the kindergarten teachers and nursery teachers in 4-5 year age classes showed that the time distributions of basic activities were generally similar to each other. Although the time distributions of working postures were also similar, time spent "standing bent forward", "squatting" and "kneeling" was longer in the kindergarten teachers than in the nursery teachers. Cumulative time at a TIA of 45 degrees or more was significantly longer in the kindergarten teachers. Although the frequency of trunklifting was not significantly different, the kindergarten teachers tended to lift their trunk more frequently. The present study found that the load on the low back was considerably great in the kindergarten teachers.

Comparisons between in-vitro and in-vivo metabolism of (S)-warfarin: catalytic activities of cDNA-expressed CYP2C9, its Leu359 variant and their mixture versus unbound clearance in patients with the corresponding CYP2C9 genotypes.

To study whether an in-vitro model for three different genotypes of human CYP2C9*3 polymorphism would be useful for predicting the in-vivo kinetics of (S)-warfarin in patients with the corresponding genotypes, the intrinsic clearance (Cl(int) or Vmax/Km) for (S)-warfarin 7-hydroxylation obtained from recombinant human CYP2C9*1 [wild-type (wt)] and CYP2C9*3 (Leu359/Leu) expressed in yeast and the mixture of equal amounts of these were compared with the in-vivo unbound oral CI (CI(po,u)) of (S)-warfarin obtained from 47 Japanese cardiac patients with the corresponding CYP2C9 genotypes. The in-vitro study revealed that the recombinant CYP2C9*1 (wt/wt), 2C9*3 (Leu359/Leu) and their mixture (Ile359/Leu) possessed a mean Km of 2.6, 10.4 and 6.6 microM and Vmax of 280, 67 and 246 pmol/min/nmol P450, respectively. Thus, the mean in-vitro Cl(int) obtained from recombinant CYP2C9*3 (Leu359/Leu) and the mixture (Ile359/Leu) of 2C9*3 and 2C9*1 were 94% and 65% lower than that obtained from CYP2C9*1 (wt/wt) (6.7 versus 38 versus 108 ml/min/micromol P450, respectively). The in-vivo study showed that the median Cl(po,u) for (S)-warfarin obtained from patients with homozygous (Leu359/Leu, n = 1) and heterozygous (Ile359/Leu, n = 4) CYP2C9*3 mutations were reduced by 90% (62 ml/min) and 66% (212 ml/min, P < 0.05) compared with that obtained from those with homozygous 2C9*1 (625 ml/min, n = 42). Consequently, there was a significant correlation (r = 0.99, P < 0.05) between the in-vitro Cl(int) for (S)-warfarin 7-hydroxylation and the in-vivo Cl(po,u) for (S)-warfarin in relation to the CYP2C9*3 polymorphism. In conclusion, the in-vitro model for human CYP2C9*3 polymorphism using recombinant cytochrome P450 proteins would serve as a useful means for predicting changes in in-vivo kinetics for (S)-warfarin and possibly other CYP2C9 substrates in relation to CYP2C9*3 polymorphism.

Inhibition of S-warfarin metabolism by nonsteroidal antiinflammatory drugs in human liver microsomes in vitro.

We studied the inhibition of S-warfarin metabolism by nonsteroidal antiinflammatory drugs (NSAIDs) in human liver microsomes in vitro. After screening for potential inhibitors among ten NSAIDs using human recombinant cytochrome P450, inhibition kinetic parameters were estimated using human liver microsomes. Phenylbutazone and bucolome were suggested to increase the unbound steady-state level of S-warfarin about four- and five-fold, respectively, as estimated from these metabolic parameters.

[Workers' stress after Hanshin-Awaji earthquake in 1995--symptoms related to stress after 18 months].

Stress-related symptoms were investigated in workers living in the area hit by the Hanshin-Awaji earthquake. Questionnaires were distributed among taxi and truck drivers, longshoremen, workers in factories, hospitals and local government offices 18 months after the event. Nearly 40% of them, 3,015 answered. The multiple regressive analysis showed that the degree of housing damage was the factor most relevant to the symptoms. Although the symptoms decreased as time passed, the group with seriously damaged housing still complained of severe stress-related symptoms even 18 months after the event. Symptoms related to post traumatic stress disorder (PTSD) were observed in 48.0% in the male group with seriously damaged housing immediately after the quake. The figure was 34.2% of after 3 months and 12.6% after 18 months. 21.8%, 12.9% and 3.7%, respectively, were the figures in the male group with nil or slightly damaged housing. The number of workers who complained of the recurrence and worsening of chronic diseases peaked 3 months after the event and had not changed much even after 18 months. Workers also complained of anxiety over the loss of work, job pressure, overwork, difficulty of commuting to and from the area, danger at work, decline in income and changes of job location.

Load on the low back of teachers in nursery schools.

In order to evaluate the load on the low back of teachers in nursery schools, basic activity, working posture, child-lifting, and desk-lifting were analyzed for eight nursery teachers using video recording. The trunk inclination angle (TIA) was also measured continuously during full workshifts for 20 nursery teachers using an inclination monitor. The nursery teachers in the 0-1 (year) age class more often adopted low working postures, "sitting on the floor" and "kneeling," while teachers in the 4-5 age class more frequently adopted high working postures, "standing" and "sitting on a chair." The mean of TIA among all subjects was 20 degrees. The time spent at a TIA of more than 20 degrees represented 43% of the workshift. The mean and time distribution of TIA did not differ between the age classes. The frequency of trunk-lifting from severe bending forward (TIA > 45 degrees) was 86 times/hour on average. The frequency of trunk-lifting was highest in the 0-1 age class. The number of times of child-lifting was 46 in the 0-1 age class, while it was 1 in the 4-5 age class.

Modulation of ethanol-mediated CYP2E1 induction by clofibrate and L-carnitine in rat liver.

To understand the effects of lipid-lowering agents on the ethanol-induction of hepatic CYP2E1, clofibrate and L-carnitine were administered to adult male rats. The administration of ethanol in the diet (containing 21% calories as ethanol, given for 3 weeks) increased levels of hepatic CYP2E1 protein (1.9-fold that of untreated controls) and mRNA (2.1-fold). In contrast, the administration of clofibrate (0.1%v/v) in an ethanol-containing diet did not significantly increase either CYP2E1 protein (1.1-fold) or mRNA (0.8-fold), in spite of the significant increases in blood ketone bodies. Administration of L-carnitine alone had no clear effect on CYP2E1 and blood ketone body levels. Co-administration of L-carnitine, however, increased liver microsomal CYP2E1 protein (2.5-fold) in rats given an ethanol-containing diet. No difference was observed in the mRNA levels in rats receiving ethanol with and without L-carnitine. These results indicate that clofibrate and L-carnitine modulate ethanol-mediated induction of hepatic CYP2E1 independent of blood levels of ketone bodies. It is also suggested that these lipid-lowering agents affected hepatic CYP2E1 through particular mechanisms, suppression of the specific mRNA and post-translation stabilization.

Concentrations of blood and urine ethanol, acetaldehyde, acetate and acetone during experimental hangover in volunteers.

Blood and urine samples were analyzed for ethanol, acetaldehyde, acetate and acetone during experimental hangover in 6 healthy male volunteers (A, B, C, D, E, F). They drank freely for some 4 hr. In flushers (A, F) at 9 hr after ingestion (ethanol: 92 g, 1.2 g/kg and 1.3 g/kg), acetaldehyde levels were low in the blood, but high in the urine (37 microM, 45 microM). Heavy drinkers, non-flushers of B (ethanol: 176 g, 2.5 g/kg), C (157 g, 2.4 g/kg) and E (182 g, 2.9 g/kg) had a slightly high [lactate]/[pyruvate] ratio and 3-hydroxybutyrate in the blood at 11 hr after alcohol ingestion. Blood ethanol levels were dose dependent and blood acetaldehyde in B and C had a slightly high 6.3 microM and 8.0 microM 9 hr later, respectively. B, C and E had a high urine acetone concentration (100 microM over) in hangover. In C, in particular, urine acetate and acetone levels were unusually high. The ratio in blood (urine) among alcohol metabolites at 9 hr after drinking was approximately ethanol 1000 (1000): acetaldehyde 0.2-1.0 (0.1-5.9): acetate 36-163 (22-1554): acetone 1-11 (3-47).

Determinations of ethanol, acetaldehyde and acetate in blood and urine during alcohol oxidation in man.

Blood and urine samples were analyzed for ethanol, acetaldehyde and acetate during alcohol oxidation in Japanese men by head space gas chromatography, following the consumption of 16 ml/kg of beer during a 20 min period. The maximum level of blood/urine ethanol was found to be 15-17 mM (20-22 mM), while that of acetaldehyde in a flusher and in non-flushers was 20 microM (52 microM) and 2-5 microM (10-13 microM), respectively. Acetate levels in these groups ranged from 0.2 mM (0.1 mM) to 0.8 mM (1.0 mM). Blood ethanol levels were dose dependent, whereas acetaldehyde and acetate levels reflected individual metabolic rates. The relative concentrations of ethanol and acetaldehyde in blood and that of acetate in alcohol metabolism could be summarized as follows: 7500 (15 mM): 1-3 (2-5 microM); 250-400 (0.5-0.8 mM) for non-flushers; and 7500 (15 mM): 5-10 (10-20 microM): 250-400 (0.5-0.8 mM) for a flusher.