Long interspersed element-1 is differentially regulated by food-borne carcinogens via the aryl hydrocarbon receptor.

A single human cell contains more than 5.0 × 10(5) copies of long interspersed element-1 (L1), 80-100 of which are competent for retrotransposition (L1-RTP). Recent observations have revealed the presence of de novo L1 insertions in various tumors, but little is known about its mechanism. Here, we found that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), food-borne carcinogens that are present in broiled meats, induced L1-RTP. This induction was dependent on a cellular cascade comprising the aryl hydrocarbon receptor (AhR), a mitogen-activated protein kinase, and CCAAT/enhancer-binding protein ß. Notably, these compounds exhibited differential induction of L1-RTP. MeIQx-induced L1-RTP was dependent on AhR nuclear translocator 1 (ARNT1), a counterpart of AhR required for gene expression in response to environmental pollutants. By contrast, PhIP-induced L1-RTP did not require ARNT1 but was dependent on estrogen receptor α (ERα) and AhR repressor. In vivo studies using transgenic mice harboring the human L1 gene indicated that PhIP-induced L1-RTP was reproducibly detected in the mammary gland, which is a target organ of PhIP-induced carcinoma. Moreover, picomolar levels of each compound induced L1-RTP, which is comparable to the PhIP concentration detected in human breast milk. Data suggest that somatic cells possess machineries that induce L1-RTP in response to the carcinogenic compounds. Together with data showing that micromolar levels of heterocyclic amines (HCAs) were non-genotoxic, our observations indicate that L1-RTP by environmental compounds is a novel type of genomic instability, further suggesting that analysis of L1-RTP by HCAs is a novel approach to clarification of modes of carcinogenesis.

Prediction of fluoroquinolone-induced elevation in serum creatinine levels: a case of drug-endogenous substance interaction involving the inhibition of renal secretion.

The aim of this study was to examine the mechanism underlying the elevation in serum creatinine levels caused by a novel des-fluoro(6)-quinolone antibacterial agent, DX-619, in healthy subjects. hOCT2 showed a prominent uptake of creatinine (K(m) = 56.4 mmol/l) among renal organic ion transporters. DX-619 is a potent inhibitor of hOCT2 (K(i) = 0.94 micromol/l), hMATE1 (0.82 µmol/l), and hMATE2-K (0.10 micromol/l). The pharmacokinetic model involving the inhibition of hOCT2 (model 1), hOCT2, and MATE1 or MATE2-K (model 2) could predict the elevation in serum creatinine levels in individual subjects receiving DX-619. This assumes that a significant contribution of tubular secretion (59, 38, and 31%) and reabsorption ranged from 3-50, 4-30, and 5-21% in model 1, -2a (hOCT2/hMATE1), and -2b (hOCT2/hMATE2-K), respectively, for creatinine. In conclusion, DX-619, at its therapeutic dose, is able to inhibit hOCT2, hMATE1, and hMATE2-K, leading to a significant inhibition of tubular secretion of creatinine and consequently to elevation of serum creatinine levels.

Effects of organic solvents on the time-dependent inhibition of CYP3A4 by diazepam.

The effects of organic solvents, acetonitrile, dimethyl sulfoxide (DMSO), and methanol, which are used to dissolve lipophilic test compounds and cytochrome P450 (P450) substrates, and carried into pre-incubation at 1% (v/v), on time-dependent inhibition of CYP3A4 by diazepam, were evaluated using human liver microsomes (HLM) and recombinant human P450 expressed microsomes (rCYPs). The inactivation kinetics of CYP3A4 by diazepam dissolved in acetonitrile and methanol were almost equal with k(inact)/K(I) values, 0.095 and 0.15 min(-1) mM(-1) for HLM and 1.1 and 1.4 min(-1) mM(-1) for rCYP3A4, respectively. In contrast, the inactivation by diazepam dissolved in 1% DMSO significantly decreased and the kinetic parameter could not be calculated. The formation rate of nordiazepam and temazepam metabolized from diazepam dissolved in DMSO were approximately half of those using substrate dissolved in acetonitrile and methanol in both HLM and rCYP3A4. Dixon plots revealed that the metabolism of diazepam in rCYP3A4 were inhibited by DMSO in a competitive or mixed-type manner with K(i) (inhibition constant) values of 6 and 24 mM for nordiazepam and temazepam, respectively. In conclusion, the time-dependent inhibition of CYP3A4 by diazepam was attenuated by DMSO, while acetonitrile and methanol had no effect. The metabolite formation profile under the conditions tested suggested that DMSO competitively inhibit the formation of the reactive metabolites of diazepam by CYP3A4. The effect of organic solvents should be taken into consideration when evaluating the in vitro time-dependent inhibition of new chemical entities.

Comparison of mechanism-based inhibition of human cytochrome P450 2C19 by ticlopidine, clopidogrel, and prasugrel.

Mechanism-based inhibition of CYP2C19 in human liver microsomes by the thienopyridine antiplatelet agents clopidogrel, prasugrel and their thiolactone metabolites was investigated by determining the time- and concentration-dependent inhibition of the activity of S-mephenytoin 4'-hydroxylase as typical CYP2C19 activity and compared with ticlopidine and its metabolite. Clopidogrel was shown to be a mechanism-based inhibitor of CYP2C19 with the inactivation kinetic parameters, k(inact) and K(I), equal to 0.0557 min(-1) and 14.3 microM, respectively, as well as ticlopidine (0.0739 min(-1) and 3.32 microM, respectively). The thiolactone metabolite of ticlopidine and clopidogrel inhibited CYP2C19 only in a concentration-dependent manner. In contrast, neither prasugrel nor its thiolactone metabolite inhibited CYP2C19 at concentrations up to 100 microM. The oxidation of the thiophene moiety of clopidogrel to form their respective thiolactones was found to be the critical reaction that produces the chemically reactive metabolites which cause the mechanism-based inhibition of CYP2C19. Estimation of in vivo drug-drug interaction using in vitro parameters predicted clinically observed data. For clopidogrel, there was no increase in the area under the curve (AUC) at its clinical dose level as predicted by the in vitro parameters, and for ticlopidine the prediction agreed with the clinically observed AUC increase. In conclusion, clopidogrel is potent mechanism-based inhibitors of CYP2C19 as well as ticlopidine, whereas prasugrel did not inactivate CYP2C19. Administration of prasugrel would not cause a clinically relevant interaction with CYP2C19.

Up-regulation of carnitine transporters helps maintain tissue carnitine levels in carnitine deficiency induced by pivalic acid.

PURPOSE: Pivalic acid (PVA) forms conjugates with endogenous carnitine and enhances its excretion. The purpose of this study is to determine whether tissue carnitine levels decrease in parallel with plasma levels in carnitine deficiency induced by PVA. METHODS: PVA was orally administered to rats for 5 days. Carnitine levels in plasma, liver, kidney, muscle, and heart were monitored. The tissue uptake clearance (CLuptake) was determined in vivo by the integration plot method. Hepatocytes were prepared from control and PVA-treated rats, and the uptake of L-carnitine was determined. RESULTS: Plasma concentrations of L-carnitine decreased as a result of the enhanced carnitine elimination as pivaloylcarnitine (PCN) when rats were treated with PVA. However, L-carnitine concentrations in liver, muscle, and heart remained relatively constant during the study. period. CLuptake increased in liver and muscle and, thus, the rate of carnitine uptake from plasma into these tissues did not change even at low plasma concentrations. This helps maintain carnitine levels in these tissues. Up-regulation of carnitine transporters is suggested to be a mechanism for the increased CLuptake. CONCLUSIONS: In the carnitine deficiency state induced by PVA, increased CLuptake owing to up-regulation of carnitine transporters is suggested to help maintain carnitine levels in some tissues.

Polarized efflux of mono- and diacid metabolites of ME3229, an ester-type prodrug of a glycoprotein IIb/IIIa receptor antagonist, in rat small intestine.

ME3229 is an ester-type prodrug of a glycoprotein IIb/IIIa receptor antagonist ME3277. In our previous study, it was shown that only a small part of the drug taken up into the enterocytes reached the mesenteric vein, mainly due to transporter-mediated efflux of its hydrolyzed metabolites formed in the cells. To characterize the efflux transport system for the metabolites, the transport of the diacid metabolite ME3277 and the monoacid metabolites PM-10 and PM-11 were studied. ME3277 and PM-10 were preferentially transported in the serosal-to-mucosal direction across the rat small intestine in the presence of glucose. Permeability of ME3277 across monolayer of Caco-2 cells with P-glycoprotein (P-gp) and indomethacin-sensitive efflux pump expression did not show any directionality and verapamil, an inhibitor of P-gp, and indomethacin did not affect the permeability of ME3277 across rat intestinal tissue. Directional transport was not site specific and was observed in the Eisai hyperbilirubinemic rat whose canalicular multispecific organic anion transporter/multidrug resistance-associated protein (cMOAT/MRP2) is hereditarily defective as well as in normal rats. The efflux transport of ME3277 was inhibited by 1-naphthol, 1-choloro-2,4-dinitrobenzene, and sulfobromophthalein, and efflux of ME3277 and monoacid metabolites from intestinal tissue preloaded with ME3229 fell in the presence of 1-naphthol and sulfobromophthalein. These results demonstrate that mono- and diacid metabolites of ME3229 were pumped out into the gut lumen by an energy-dependent transport system located on the mucosal membrane of intestinal tissue and distinct from either P-gp, indomethacin-sensitive efflux pump or canalicular multispecific organic anion transporter/MRP2. An inhibition study suggested that this unknown transporter has a substrate specificity similar to that of MRP transporter families.

A study of the intestinal absorption of an ester-type prodrug, ME3229, in rats: active efflux transport as a cause of poor bioavailability of the active drug.

The intestinal absorption of a prodrug is affected by a number of factors, such as its membrane permeability, stability in the gut lumen, and conversion to the parent drug in enterocytes. We evaluated the absorption of ME3229, an ester-type prodrug of a hydrophilic glycoprotein IIb/IIIa antagonist. Although the octanol/water distribution coefficient and permeability across a Caco-2 cell monolayer of ME3229 was high enough for us to expect good oral absorption, less than 10% of the dose was absorbed in rats. To clarify this unexpected outcome, we evaluated the rate of its disappearance from the gut lumen (V1), its degradation in the gut lumen (V(deg)), uptake into enterocytes (V(uptake)), and appearance in the mesenteric vein (V2) by using a single-pass perfusion technique in combination with an in vitro metabolism study. Our data suggested that ME3229 crossed the apical membrane and was taken up into enterocytes at a rate compatible with its lipophilicity, but that only a small fraction of the metabolites formed in enterocytes reached the mesenteric vein, primarily attributable to efflux into the intestinal lumen. Transport of the main metabolite across rat intestinal tissue mounted on an Ussing chamber suggested that an active efflux system pumped out any ionic metabolite(s) present.

A survey of bedroom and bed climate of the elderly in a nursing home.

Bedroom and bed climate of the elderly in a nursing home were surveyed. Twenty-two elderly subjects were divided into four groups depending on their Activity of Daily Living (ADL) and gender. The four groups were: male and female subjects with almost no problems in daily life and an ADL score higher than 5 [H males, H females] and male and female subjects who normally stayed in bed all day with an ADL score lower than 2 [L males, L females]. The temperature and humidity of the bedroom and bed climate were measured continuously for 24 hours. Bedding and clothing condition, subjective sensational vote and subjective sleep evaluation were surveyed before and after sleep for five days continuously. In the daytime, bedroom and bed climate temperature was significantly lower in H females than in the other groups. At night, no significant effect of gender and ADL was observed in bed climate, which was maintained at 33-35 degrees C, RH50-60% in all the groups. Bedding under the body increased significantly in L males and L females compared with H males and H females. The number of underwear increased significantly in H females. Subjective sleep evaluation was significantly better in H females than the other groups. These results suggest that ADL and gender based differences should be taken into account with regard to the care of the elderly in a nursing home.

Effects of truss mattress upon sleep and bed climate.

The purpose of this study was to examine the effects of a truss mattress upon sleep and bed climate. The truss mattress which has been designed to decrease the pressure and bed climate humidity was tested. Six healthy female volunteers with a mean age of 23.3 years, served as subjects. The experiment was carried out under two conditions: a truss mattress (T) and a futon (F) (Japanese bedding). The ambient temperature and relative humidity were controlled at 19-20 degrees C, and RH 50-60% respectively. Sleep was monitored by an EEG machine and the rectal temperature, skin temperature and bed climate were also measured continuously. Subjective evaluations of bed and sleep were obtained before and after the recording sessions. No significant difference was observed in the sleep parameters and time spent in each sleep stage. Rectal temperature was significantly lower in T than F. Although there was no significant difference in bed climate over the T/F, the temperature under T/F was significantly higher in T. No significant difference was observed in subjective sleep evaluation. The subjective feeling of the mattress was significantly warmer in F than T before sleep. These results suggest that although T does not disturb the sleep parameters and the bed climate is maintained at the same level as with F, it may affect rectal temperature which can be due to low thermal insulation.

The effects of a newly designed air mattress upon sleep and bed climate.

In our previous study, an air mattress with three series of air cells with inflation pressure, that was increased/decreased in time interval of 20 min, did not affect sleep quality and quantity, although the relative and absolute humidity inside the bedding was kept significantly higher than that of a Futon. The purpose of this study was to confirm the effects of a newly designed air mattress upon sleep and bed climate. In this newly designed air mattress, the cell series and time interval was reduced. Six healthy female volunteers, aged 18-22, served as subjects. The experiment was carried out under two conditions: using a regular Futon (Futon), and a newly-designed air mattress with the timer and pump activated (Airmat). The room temperature and relative humidity were controlled at 22-23 degrees C and RH 50-60%, respectively. The subjects' sleep was monitored by using an EEG machine and their skin temperatures and bed climates were also measured continuously. Subjective evaluations of bed comfort and sleep were obtained before and after the recording sessions. Sleep onset latency, wake after sleep onset and the sleep efficiency index showed no significant differences between the two conditions. A significant difference was observed in the bed climate of the waist area. The temperature of the waist was lower overall under the Airmat than the Futon, while relative humidity was higher under the Airmat. Absolute humidity also tended to be higher in the Airmat. Sleep evaluation and comfort sensation were good under both conditions. Although sleep was not disturbed and subjective sleep evaluation tended to be better in Airmat, our results indicate that changing the time intervals and cell series until this air mattress level is not effective in decreasing the bed climate humidity.

The effects of air mattress upon sleep and bed climate.

The purpose of this study was to investigate the effects of an air mattress upon sleep and bed climate. This air mattress, which employs a pump and timer to increase or decrease the inflation pressure in order to cure and prevent decubitus was tested. Six healthy female volunteers, aged 18 to 23, served as subjects. The experiments were carried out under three conditions: using regular Futon (Futon), the air mattress with pump and timer activated (Air+) and the same mattress without pump and timer activated (Air-). Room temperature and relative humidity were controlled at 22-23 degrees C and RH 50-60% respectively. Subjects' sleep was monitored by using EEG machine throughout the night, and subject's body temperature and bed climate were also continuously checked. Subjective estimation of bed and sleep were obtained before and after the recording sessions. Sleep onset latency and wake after sleep onset tended to be reduced in Air+ compared to Futon and Air-. The time and percentage of Stage 3 was increased significantly in the middle one third of the night in Air+. A significant difference was observed in bed climate of the waist area. Temperature tended to be higher in Futon than in Air+ and Air-, while relative and absolute humidity were significantly higher in Air+ and Air-. Significant difference between Air+ and Air- was observed only during one hour after sleep recordings started. Thermal sensation in the morning was cooler and comfort sensation tended to be better in Air+ and Air-. Subjective sleep estimation was somewhat good under all conditions. These results suggest that although these air mattresses do not affect sleep, we have to be cautious in using these mattresses as relative and absolute humidity were kept higher than with Futon. Further study on materials and construction of these air mattresses to decrease the humidity is needed.

Flunitrazepam effects on human sleep EEG spectra: Differences in NREM, REM and individual responses.

Flunitrazepam (FNZ) (4mg), an intermediate type benzodiazepine (BDZ) hypnotic, was administered orally to five healthy male subjects (Ss) for seven consecutive nights. Sleep EEG from the baseline night (BLN), the initial drug night (IDN), the fourth and the seventh drug nights (4DN, 7DN) was subjected to fast Fourier transform (FFT) analysis. During NREM sleep of 4DN and 7DN the sigma band (11.0-12.5 Hz) activity was similarly enhanced in every S. In REM of 4DN and 7DN beta band (23.0-29.0 Hz) was enhanced, but with larger variations among Ss. High intra-individual consistency of the relative EEG power patterns on 4DN and 7DN was observed. These results suggest that 1) EEG responses to FNZ are different in sleep states; explorations of these differences may provide better understandings of sleep mechanisms, and 2) individual variations in EEG responses may reflect individual variations of the BDZ receptor system. These methods may be useful for exploring receptor changes in neuropsychiatric disorders.

Flunitrazepam effects on human sleep EEG spectra. II: Sigma and beta alterations during NREM sleep.

Flunitrazepam (FNZ) is known to enhance the higher EEG frequencies, including sigma (10-15 Hz) and beta (20-28 Hz). Both sigma and beta frequency bands show an inverse relationship with delta (0.3-3 Hz) during NREM periods, as we have previously reported. It is not known whether generation of these two EEG frequencies is mediated by the same or different neuronal mechanisms. In this report, we compare alterations of delta, sigma and beta EEG induced by FNZ (4 mg) orally administered to five healthy male subjects for seven consecutive nights. Sleep EEG on the baseline night (BLN), and the fourth and seventh drug nights (4DN, 7DN) was subjected to fast Fourier transform (FFT) analysis. On drug nights, sigma was enhanced without regard to delta amount, but beta was enhanced only during epochs containing low delta. Thus, sigma and beta EEG were altered differently by the same pharmacological agent. These results suggest that sigma and beta EEG are mediated by different neuronal mechanisms.

FK 506 metabolism in male and female rat liver microsomes.

The main metabolite of the immunosuppressant FK 506 in hepatic microsomes from male and female Sprague-Dawley rats was identified by mass spectrometry as an O-desmethyl derivative. The rate of formation of the metabolite exhibited saturation kinetics in the range of 0.6-40 microM with Vmax and KM equal to 0.66 +/- 0.47 nmol/min/mg protein and 24 +/- 18 microM, respectively, for microsomes from male rats, and 0.28 +/- 0.15 nmol/min/mg protein and 24 +/- 16 microM, respectively, for microsomes from female rats. CYP3A enzymes are thought to be responsible for metabolizing FK 506 in male rats. Because untreated female rats show no classical CYP3A activity, our work suggests that other CYP enzymes metabolize FK 506 in untreated female rats. O-Desmethyl FK 506 did not cross-react in the standard clinical ELISA assay for FK 506. This suggests that demethylation had occurred at the C13-methoxy group.

Sleep and respiration under acute hypobaric hypoxia.

The purpose of this study was to elucidate the effects of acute hypobaric hypoxia on nocturnal sleep architecture and respiratory responses in a hypobaric simulator. Five healthy young males (19-23 years old) were recruited to sleep for 8 h at sea level and at simulated altitudes of 1,500, 3,000, and 4,000 m in the simulator (61.2 m3, 20 degrees C, and 60% RH). Each experimental run was separated by at least 3 d. Standard polysomnograph, respiration, and arterial oxygen saturation (SaO2) during sleep were observed. 1) SaO2 decreased significantly with increasing altitude. At 4,000 m, SaO2 showed its lowest value during 1 to 3 h after sleep onset. 2) Sleep architecture below 3,000 m showed almost the same pattern. However, reduction in REM sleep and increased wakefulness were observed at 4,000 m, though such sleep disturbance was not observed in the first one-third of the night spent in bed. 3) Periodic breathing (PB) with apnea and/or hypopnea developed in all subjects above 3,000 m. PB tended to appear in light sleep, though sleep was not always disturbed by PB. It might be concluded that there was no sleep disturbance up to 3,000 m altitude. Nocturnal sleep at 4,000 m, however, was disturbed after a few hours from sleep onset by severe hypoxemia induced by multiplicative effects of hypoxia and hypoventilation during deep sleep. At high altitude, PB seems to not induce arousals consistently, which was different from sleep apnea syndrome at sea level.

Enhancing effect of bromovinyldeoxyuridine on antitumor activity of 5'-deoxy-5-fluorouridine against adenocarcinoma 755 in mice. Correlation with pharmacokinetics of plasma 5-fluorouracil levels.

5'-Deoxy-5-fluorouridine (DFUR), whether or not combined with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was pursued in BDF1 mice from both a pharmacokinetic viewpoint, following a single oral dose administration, and an anticancer viewpoint, following 5 daily oral doses in mice inoculated subcutaneously with adenocarcinoma 755 tumor cells. Half-life (t1/2) values for the elimination of DFUR and 5-fluorouracil (5-FU) from plasma following DFUR (100 mg/kg) administration were about 0.80 and 0.39 hr, respectively. Plasma 5-FU AUC (area under the curve) values following oral DFUR (100 mg/kg) was 0.224 micrograms.hr/ml. If DFUR (100 mg/kg) was combined with BVDU (10 mg/kg) the t1/2 and AUC values for 5-FU increased from 0.39 to 1.24 hr, and from 0.224 to 1.699 micrograms.hr/ml, respectively. Thus, BVDU significantly increased the plasma levels of 5-FU. It had no effect on the plasma levels of DFUR. At 100 mg/kg, DFUR did not show a significant antitumor activity. At 500 mg/kg it effected a 90% inhibition in tumor growth. When combined with BVDU (10 mg/kg), DFUR at 100, 200 and 300 mg/kg reduced tumor growth by 96, 100 and 100%, respectively. The antitumor activity achieved by DFUR, in the presence or absence of BVDU, correlated highly significantly with the AUC values for plasma 5-FU.

Effect of procainamide on renal tubular transport of cimetidine in the isolated perfused rat kidney.

The effect of procainamide on renal tubular transport of cimetidine was studied in isolated perfused rat kidney based on the multiple indicator dilution (MID) technique. T-1824-labeled albumin (a vascular reference), [14C]creatine (an extracellular reference), and [3H]cimetidine were rapidly injected into the renal artery of isolated perfused rat kidney in the presence or absence of procainamide (100 microM) in the perfusate, and normalized outflow-time patterns were secured from rapidly sampled renal perfusate. A distributed two-compartmental model was fitted to the dilution data by non-linear least-squares regression, and the influx, efflux and sequestration rate constants were estimated. Net transport and influx processes of cimetidine were competitively inhibited by procainamide (PA), while the efflux and sequestration processes were increased. The increase in the values of the efflux and sequestration rate constants by addition of procainamide may be explained by the increase in the tissue binding of cimetidine. However, these three processes were not significantly affected by p-aminohippurate (PAH). These results suggest that both cimetidine and procainamide are secreted into the lumen by an organic base transport mechanism in the perfused kidney, in which the spatial organization and cell polarity of the kidney are maintained.

No basic rest-activity cycle in head, wrist or ankle.

Kleitman's theory of a Basic Rest-Activity Cycle (BRAC) was tested by recording activity of the head, wrists, and left ankle from 10 healthy subjects. No 90-100 min rhythms in activity were found.

Theophylline delays human sleep phase.

Twelve healthy male volunteers were given theophylline 250 mg in order to test effects on 24-hr rhythms. Rhythms of sleep/wake and subjective sleepiness were delayed. Ingestion of xanthines such as theophylline in coffee, tea, colas and chocolate may contribute to some sleep disorders. Theophylline might likewise be useful in treating disorders of circadian oscillators.