Ghrelin and obestatin promote the allergic action in rat peritoneal mast cells as basic secretagogues.

Ghrelin is an endogenous ligand of the type 1a growth hormone secretagogue receptor (GHSR1a) that regulates energy balance. Ghrelin and obestatin, derived from the post-translational processing of preproghrelin, are involved in a diverse range of biological activities, yet their effect on the immune system is not fully understood. In the present study, we investigated the roles of ghrelin and obestatin on mast cell degranulation and found that both ghrelin and obestatin induce the release of histamine from rat peritoneal mast cells. This induced histamine release was inhibited by the pertussis toxin, an inhibitor of Gα(i) protein, and extracellular Ca(2+). Rat peritoneal mast cells and rat basophilic leukemia (RBL-2H3) cells did not express the ghrelin receptor GHSR1a, suggesting that histamine release induced by ghrelin occurs via a receptor-independent mechanism. We report here that ghrelin and obestatin, belonging to the family of basic secretagogues, stimulate mast cells independent of a receptor, and this may play a crucial role at the site of allergy or inflammation.

Second-line weekly paclitaxel in resistant or relapsed non-small cell lung cancer treated with docetaxel and carboplatin: a multi-center phase II study.

We conducted a phase II trial to evaluate the safety and efficacy of weekly paclitaxel in patients with resistant or relapsed non-small cell lung cancer (NSCLC) treated with docetaxel and carboplatin. Thirty-two NSCLC patients at a median age of 58.0 years (range 33-75) were enrolled. The Eastern Cooperative Oncology Group performance status scores (0/1/2) were 18/9/5, respectively. The majority of patients had adenocarcinoma (84%) and stage IV disease (81%). The response rate for the first-line chemotherapy was 28%. Paclitaxel was administered at a dose of 80 mg/m(2) as an intravenous infusion 60 min weekly for 6 consecutive weeks of an 8-week cycle. All patients were assessable for response and toxicity. The median number of cycles administered was two (range 1-8), and the overall response rate was 15.6%. The median survival time (MST) was 10.6 months (95% CI=8.2-12.5), while the 1-year survival rate was 37.5%, and the median progression-free survival was 4.9 months (95% CI=3.0-7.1). Hematological toxicities (grade 3 or 4) were observed in 15 patients (46.9%) with leukopenia, and in 4 (12.5%) with anemia. Non-hematological toxicity was generally mild, though grade 3 anorexia was observed in 3 patients (9.3%). No treatment-related deaths were observed. In conclusion, second-line weekly paclitaxel is effective in NSCLC patients treated with docetaxel plus carboplatin and is associated with a tolerable toxicity profile.

Heme oxygenase-1 mediates the anti-allergic actions of quercetin in rodent mast cells.

OBJECTIVE AND DESIGN: We investigated the involvement of heme oxygenase (HO)-1 in the anti-allergic action of quercetin against degranulation of rat basophilic leukemia (RBL-2H3) cells, rat peritoneal mast cells, and mouse bone marrow-derived mast cells. METHODS: The strength of allergic reaction was evaluated by the extent of degranulation in mast cells sensitized with various stimulants. The levels of HO-1, HO-2, and nuclear factor erythroid 2-related factor 2 (Nrf2) expressions were determined by quantitative RT-PCR, western blotting, or immunocytochemistry. RESULTS: Heme oxygenase activity was upregulated after short exposure to quercetin, followed by the induction of HO-1 expression after long exposure to quercetin. The inhibition of degranulation by quercetin was reversed using tin protoporphyrin IX (SnPP), an HO-1 inhibitor. HO-1 metabolites, bilirubin and CO, led to inhibit degranulation, and quercetin translocated Nrf2 from cytoplasm into nucleus in RBL-2H3 cells. CONCLUSION: These results strongly suggest that quercetin exerted anti-allergic actions via activation of Nrf2-HO-1 pathway.

Involvement of heme oxygenase-1 in kaempferol-induced anti-allergic actions in RBL-2H3 cells.

Kaempferol is one of the most commonly found dietary flavonoids. The exposure to kaempferol is known to inhibit degranulation from mast cells, but the inhibitory mechanism of degranulation has not been clarified yet. In this study, we investigated the involvement of heme oxygenase (HO)-1 in the anti-allergic action of kaempferol against degranulation in rat basophilic leukemia (RBL-2H3) cells. Our results demonstrate upregulation of HO enzymatic activity after short (15 min) exposure to kaempferol, followed by the induction of HO-1 expression in protein. The involvement of HO-1 in the kaempferol-induced inhibition of degranulation was confirmed using tin protoporphyrin IX (SnPP), a HO-1 inhibitor. These findings strongly suggest that kaempferol exerts anti-allergic actions via activation of the HO-1.

Up-regulation of surfactant protein production in a mouse model of secondary pulmonary alveolar proteinosis.

Although Pneumocystis infection might be one of the causes of secondary pulmonary alveolar proteinosis (PAP), the mechanism of its pathogenesis is uncertain. We analyzed a mouse model of secondary PAP resulting from Pneumocystis infection using mice deficient in CD40 (CD40KO), and evaluated the mechanism of the pathogenesis of secondary PAP from the viewpoint of surfactant-associated protein (SP) homeostasis, the overproduction of SP by type II alveolar epithelial cells, and the phagocytic function of alveolar macrophages (AMs). The effect of CD40 on SP production was also investigated in vitro using the H441 cell line, which has a phenotype similar to type II alveolar epithelial cells and primary alveolar epithelial cells. After long-term exposure to ovalbumin, CD40KO mice showed Pneumocystis infection and accumulation of surfactants in the alveoli (ApCD40KO). The amounts of SP production were up-regulated in ApCD40KO mice compared with wild-type mice treated using the same procedure. On the other hand, AMs from ApCD40KO mice did not show either phagocytic dysfunction or down-regulation of PU.1 expression. Furthermore, the stimulation of CD40-CD40 ligand (CD154) pathway regulated the production of SPs in H441 cells or primary alveolar epithelial cells. These results suggested that CD40KO mice could be one of the models useful for developing secondary PAP resulting from Pneumocystis infection. Surfactant accumulation was due to the overproduction in our model of secondary PAP. The CD40-CD154 interaction plays an important role in the regulation of surfactant-associated protein production.

Effect of the organophosphorus pesticide diazinon on glucose tolerance in type 2 diabetic rats.

We have reported that the toxicity of the organophosphorus pesticide diazinon (DZN) and its metabolites is increased in streptozotocin-induced diabetic rats (type 1 diabetic rats). In the present study, we have investigated the effect of DZN on glucose tolerance in genetic type 2 diabetic rats, Goto-Kakizaki (GK) rats. Oral glucose tolerance test (OGTT) (2g/(5 ml kg)) was assessed before, and 1 and 2 weeks after intraperitoneal injection of DZN (6.5 mg/kg) in Wistar and GK rats. DZN significantly increased the levels of glucose in plasma at designated blood sampling points in GK rats. The activity of hepatic drug-metabolizing enzymes and expression of hepatic cytochrome P450 (CYP) 1A2, CYP3A2 and CYP2D1, which oxidize DZN to DZN-oxon, a potent ChE inhibitor, were measured before DZN injection. There were no significant differences in the activity and expression of CYPs between both rat groups, indicating that the ability of metabolic activation might be almost the same in Wistar and GK rats. DZN dramatically decreased the activity of cholinesterase (ChE) in plasma by approximately 40% in both Wistar and GK rats. However, no significant differences in the activity of ChE in plasma were observed between Wistar and GK rats for 5 days after DZN injection. No massive necrotic and apoptotic areas, leukocyte infiltration and immunoreactive insulin-positive cells (beta-cells) were observed in pancreas 2 weeks after DZN injection. Moreover, DZN might not affect plasma insulin levels in Wistar and GK rats. These results suggest that DZN deteriorates the glucose tolerance in GK rats. It is unlikely that this phenomenon is due to differences in ChE activity and/or DZN-oxon production levels between Wistar and GK rats.

Effect of thalidomide on endotoxin-induced decreases in activity and expression of hepatic cytochrome P450 3A2.

Thalidomide has been reported to inhibit the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) that are involved in the down-regulation of hepatic cytochrome P450 (CYP) induced by endotoxin. In the present study, we investigated the effects of thalidomide on endotoxin-induced decreases in the activity and expression of hepatic CYP3A2 in rats. Thalidomide (50 mg/kg) was administered orally 22 h and 2 h before intraperitoneal injection of endotoxin (1 mg/kg). Twenty-four hours after the injection of endotoxin, antipyrine clearance experiments were conducted, in which the rats were sacrificed and protein levels of hepatic CYP3A2 were measured. There were no significant differences in the histopathological changes in the liver between the endotoxin-treated and endotoxin plus thalidomide-treated rats. Thalidomide had no effect on the systemic clearance of antipyrine, which is a proper indicator for hepatic CYP3A2 activity, whereas it enhanced endotoxin-induced decrease in the systemic clearance of antipyrine. Western blot analysis revealed that thalidomide had no effect on the protein levels of hepatic CYP3A2, whereas it enhanced the down-regulation of hepatic CYP3A2 by endotoxin. However, there were no significant differences in the concentrations of TNF-alpha and NO in plasma between the endotoxin-treated and endotoxin plus thalidomide-treated rats. The present findings suggest that thalidomide enhances endotoxin-induced decreases in the activity and expression of hepatic CYP3A2.

TH1/TH2 immune response in lung fibroblasts in interstitial lung disease.

BACKGROUND: Inflammatory response in pulmonary fibrosis closely resembles a T-helper (Th) 2 immune response. For recruitment to an inflammatory lesion, the majority of Th1 cells express CXC chemokine receptor 3, recognizing monokine induced by interferon-gamma (Mig), interferon gamma-inducible protein of 10 kD (IP-10), and interferon-inducible T-cell alpha chemoattractant (I-TAC). Th2 cells express CC chemokine receptor 4, recognizing thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC). We investigated the Th1/Th2 chemokine production patterns by lung fibroblasts and their evaluation in bronchoalveolar lavage (BAL) fluid of interstitial lung disease. METHODS: The production pattern of Th1/Th2 chemokines by lung fibroblasts was examined in ELISA and quantitative reverse transcriptase polymerase chain reactions. Th1/Th2 chemokine levels in BAL fluid of idiopathic pulmonary fibrosis (IPF) and nonspecific interstitial pneumonia (NSIP) were examined to evaluate the clinical relevance of Th1/Th2 chemokines. RESULTS: The lung fibroblasts were polarized to produce Th1-type chemokines by the pro-inflammatory cytokine, tumor necrosis factor (TNF)-alpha and the anti-fibrotic cytokine, interferon (IFN)-gamma. However, the induction patterns of chemokines by these two cytokines were different, i.e., involving predominant induction of IP-10 and I-TAC by TNF-alpha and induction of Mig by IFN-gamma. Although Mig, IP-10, and I-TAC were produced within the BAL fluid of patients, TARC and MDC were at significantly low levels. CONCLUSIONS: Our results suggest that lung fibroblasts tend to induce a Th1-type immune response under normal conditions, and that a Th2-type immune response does not play a significant role in smoldering inflammation around the established lesions in IPF and NSIP.

Biological monitoring of pyrethroid exposure of pest control workers in Japan.

Synthetic pyrethroids such as cypermethrin, deltamethrin and permethrin, which are usually used in pest control operations, are metabolized to 3-phenoxybenzoic acid (3-PBA) and excreted in urine. Though 3-PBA can be used to assess exposure to pyrethroids, there are few reports describing urinary 3-PBA levels in Japan. This study aimed to investigate the seasonal variation of the exposure levels of pyrethroids and the concentration of urinary 3-PBA among pest control operators (PCOs) in Japan. The study subjects were 78 and 66 PCOs who underwent a health examination in December 2004 and in August 2005, respectively. 3-PBA was determined using gas chromatography-mass spectrometry. The geometric mean concentration of urinary 3-PBA in winter (3.9 microg/g creatinine) was significantly lower than in summer (12.2 microg/g creatinine) (p<0.05). Geometric mean concentrations of urinary 3-PBA in the spraying workers and the not-spraying workers within 2 d before the survey were 5.4 microg/g creatinine and 0.9 microg/g creatinine for winter with a significant difference between the groups (p<0.05), and 12.3 microg/g creatinine and 8.7 microg/g creatinine for summer (p>0.05), respectively. A significant association of 3-PBA levels and pyrethroid spraying was thus observed only in winter. In conclusion, the results of the present study show that the exposure level of pyrethroids among PCOs in Japan assessed by monitoring urinary 3-PBA was higher than that reported in the UK but comparable to that in Germany. Further research should be accumulated to establish an occupational reference value in Japan.

The role of organic cation transporter-3 in methamphetamine disposition and its behavioral response in rats.

Organic cation transporter-3 (OCT3) is expressed in several tissues including the brain. We have previously demonstrated that rats with behavioral sensitization to methamphetamine (METH) increased the brain penetration of METH with decreased expression of OCT3 in brain. Considering the earlier in vitro studies demonstrating that 1) OCT3 could transport dopamine (DA) and 2) the specific transport via OCT3 could be inhibited by METH, these results suggest that decreased OCT3 might decrease the efflux of METH and/or DA from brain, subsequently causing the development of behavioral sensitization. Thus, in the present study, behavioral task related to DA and pharmacokinetic experiment were performed using rats treated with antisense against OCT3 (OCT3-AS) since no specific ligands for OCT3 are still available. The continuous infusion of OCT3-AS into the third ventricle significantly decreased the expression of OCT3 in choroid plexus (CP) epithelial cells. Both METH-induced hyperlocomotion and METH-induced extracellular DA levels in nucleus accumbens and prefrontal cortex were significantly increased in OCT3-AS-treated rats. Moreover, the concentrations of METH were significantly increased in cerebrospinal fluid as well as extracellular areas at the nucleus accumbens in OCT3-AS-treated rats. These results suggested that decreased OCT3 elevated the concentration of METH and/or DA in brain, subsequently enhancing dopaminergic neuronal transmission and increasing METH-induced hyperlocomotion. In summary, OCT3 at the CP could regulate the effect of METH by controlling the levels of METH and/or DA in brain. Thus, these results suggest that OCT3 may be a new molecular target to treat METH-related disorders such as drug abuse and schizophrenia.

Macrophage-derived chemokine in malignant and tuberculous pleural effusions.

BACKGROUND AND OBJECTIVES: Macrophage-derived chemokine (MDC/CCL22) is recognized as a T-helper (Th) 2-type chemokine. Both malignant and tuberculous pleural effusions are typically lymphocytic pleural effusions. Tuberculous pleural effusions have a more polarized Th1 reaction than malignant effusions, which are predominantly Th2 in nature. The aim of this study was to compare the levels of MDC in malignant pleural effusions with those in tuberculous pleural effusions to help delineate the role of MDC in Th2 versus Th1 effusions. METHODS: Forty-three patients with pleural effusions (32 malignant, 11 tuberculous) were studied. The concentration of MDC in the pleural effusion was measured by ELISA. RESULTS: The median concentration of MDC was lower in malignant pleural effusions than in tuberculous pleural effusions (P < 0.005). CONCLUSIONS: MDC has been reported to both promote and suppress antitumour immunity. The low concentration of MDC in malignant effusions is likely to minimise its antitumour activity but the precise role of MDC in malignant and tuberculous effusions needs to be investigated further.

Toxicity of diazinon and its metabolites increases in diabetic rats.

The effect of diazinon (DZN) on the activities of cholinesterase (ChE) in plasma and acetylcholinesterase (AChE) in erythrocyte and brain was investigated in normal and streptozotocin-induced diabetic rats. Hepatic drug-metabolizing enzyme activity was also estimated by measuring the systemic clearance of antipyrine, and the expression of hepatic cytochrome P450 (CYP) 3A2 and CYP1A2, which is closely related to the metabolism from DZN to DZN-oxon, a strong inhibitor of both ChE and AChE. No significant differences in the activities of ChE in plasma and AChE in erythrocyte were observed between normal and diabetic rats. Treatment with DZN significantly decreased these activities in diabetic rats more than in normal rats 6h after injection (6.5 mg/kg). Treatment with DZN significantly decreased the activity of AChE in brain of diabetic rats than normal rats 3h after injection (65 mg/kg), although no significant difference in the activity was found between normal and diabetic rats. The urinary recovery of diethylphosphate (DEP), a metabolite of DZN-oxon, was significantly increased in diabetic rats, but that of diethylthiophosphate (DETP), a metabolite of DZN, was unchanged. Significant increases in the systemic clearance of antipyrine and protein levels of hepatic CYP1A2, not CYP3A2, were observed in diabetic rats. These results suggest the possibility that a metabolite of DZN, DZN-oxon, causes higher toxicity in diabetic rats due to the enhancement of hepatic CYP1A2-mediated metabolism of DZN.

The pharmacokinetic properties of methamphetamine in rats with previous repeated exposure to methamphetamine: the differences between Long-Evans and Wistar rats.

Repeated treatment with methamphetamine (METH) causes long-term behavioral changes, so-called behavioral sensitization (BS), in humans as well as experimental animals. However, there are no reports as to whether repeated METH treatment can establish BS in stress-sensitive Long-Evans (LE) rats. Thus, we investigated the effect of repeated METH treatment (5 mg/kg x 5 days) on the establishment of BS in LE rats. Wistar (WIS) rats were used as a reference. In LE rats, repeated METH treatment failed to cause BS although it did enhance METH-induced hyperlocomotion in WIS rats. The levels of METH in brain dialysate and the ratio of the area under the concentration-time curve area in plasma to that in brain dialysate was increased in repeated METH-treated WIS rats as reported previously, but not in repeated METH-treated LE rats. METH increases plasma corticosterone (CORT) in both strains. However, the intensity of increment of CORT by repeated METH was lower in LE rats than that in WIS rats. Repeated METH treatment decreased the expression of METH-transposable and CORT-sensitive transporter, organic cation transporter 3 (OCT3), in the brain of WIS rats. However, the intensity of the decrement of OCT3 with repeated METH treatment was similar between both strains. Taken together, these results suggest that the lack of establishment of BS in LE rats might have been caused by the unchanged brain penetration of METH after repeated METH administration, and that the differential CORT response to METH is an important strain difference.

Phase I/II trial of docetaxel and carboplatin as a first-line therapy in patients with stage IV non-small-cell lung cancer.

A phase I/II study was conducted to determine the maximum-tolerated dose, the safety and tolerability, and the clinical efficacy of carboplatin and docetaxel in combination in patients with stage IV non-small-cell lung cancer. Patients with measurable, previously untreated, good performance status, and stage IV non-small-cell lung cancer were eligible. Increasing doses of docetaxel were given in combination with a fixed dose of carboplatin except at level 5. Cycles were repeated every four weeks. Seventy-seven patients were registered. In phase I, 27 patients were entered at five different dose levels. A docetaxel dose of 60 mg/m(2) and carboplatin area under the concentration time curve 6 was recommended for phase II, and an additional 50 patients were entered at this level for a total of 56 patients. Grade 3/4 neutropenia was the most common adverse event and occurred in 70% of the patients. Two patients had febrile neutropenia. Fifty-six patients were assessable for response; 21 partial responses were observed for an overall response rate of 37.5%. The median time to tumor progression was 4.0 months (range, 1.0-21.0 months), and the median survival was 12.9 months (range, 0.4-51.3 months). The one-year survival rate was 46.4%. The combination of docetaxel 60 mg/m(2) and carboplatin area under the concentration time curve 6 is feasible and effective in patients with stage IV non-small-cell lung cancer.

Protein kinase Cbeta isoform down-regulates the expression of MDR3 P-glycoprotein in human Chang liver cells.

The MDR3 protein is a transporter of phosphatidylcholine on the canalicular membrane of human hepatocytes. Previously we showed that the expression of MDR3 mRNA was down-regulated by phorbol 12-myristate 13-acetate (PMA) in human Chang liver cells. In the present study, to elucidate the isoform of protein kinase C (PKC), which influences the level of MDR3 protein, we investigated the effects of PKC-specific inhibitors and antisense oligonucleotides. The level of protein decreased around 50% after treatment for 3-5 days using the dosage of PMA effective against the mRNA expression. The half-life of the MDR3 protein was estimated to be about 5 days. This decrease was antagonized by GF109203X, a non-selective inhibitor of PKCs, and Gö6976, a selective inhibitor for PKCalpha/beta. These inhibitors also suppressed the reduction in MDR3 protein. To specify the isoform of PKC, the cells were treated with antisense oligonucleotide of PKCalpha or PKCbeta. The suppressive effects on MDR3 mRNA of PMA were attenuated in antisense PKCbeta-treated cells, but those in antisense PKCalpha-treated cells were not attenuated. These suggested that PKCbeta plays a regulatory role in the expression of MDR3.

Gender-related differences in expression and function of hepatic P-glycoprotein and multidrug resistance-associated protein (Mrp2) in rats.

To clarify whether gender-related differences exist in the expression and function of hepatic P-glycoprotein- and/or multidrug resistance-associated protein (Mrp2), we measured the hepatobiliary excretion of doxorubicin and their protein levels in male and female Sprague-Dawley rats. When rats received a single intravenous injection of doxorubicin (5 mg/kg), a delay in the disappearance of doxorubicin from plasma was observed in male rats. When rats received a constant-rate infusion of doxorubicin, no significant gender-related differences in the apparent biliary clearance of doxorubicin based on the steady state plasma concentrations were observed between male and female rats. However, the net biliary clearance of doxorubicin based on the liver concentration, which represents the actual function of P-glycoprotein and/or Mrp2, was higher in female rats than in male rats. These results suggest that the actual function of the hepatobiliary transport of doxorubicin is greater in female than in male rats. Western blot analysis revealed that the expression of P-glycoprotein and Mrp2 in the liver of female rats was significantly higher than in male rats, similar to results of hepatobiliary excretion experiments. The expression of hepatic cytochrome P450 (CYP) 2B1, which is involved in the metabolism of doxorubicin, was significantly higher in male than in female rats. By pretreatment with testosterone (10 mg/day for 7 days), the actual biliary clearance of doxorubicin in female rats was nearly that of male rats. The protein levels of P-glycoprotein and Mrp2 in female rats were also lowered by treatment with testosterone so as to be nearer those in male rats. These results suggest that gender-related differences exist in P-glycoprotein- and Mrp2-mediated hepatobiliary transport and that these two transporters may be regulated by sex hormones.

Effect of a newly developed ketolide antibiotic, telithromycin, on metabolism of theophylline and expression of cytochrome P450 in rats.

The effects of a newly-developed ketolide antibiotic, telithromycin, on the metabolism of theophylline and the expression of hepatic cytochrome P450 (CYP) 1A2 and CYP3A2 were investigated in rats. Telithromycin at a high dose (100 mg/kg of body weight) was injected intraperitoneally once a day for 3 days. Twenty-four hours (day 4) after the final administration of telithromycin, theophylline (10 mg/kg) was administered intravenously. The presence of telithromycin significantly delayed the disappearance of theophylline from plasma. Parameters related to the pharmacokinetic interaction between theophylline and telithromycin were examined by noncompartmental methods. A significant decrease in the systemic clearance of theophylline was observed in the presence of telithromycin. Pretreatment with telithromycin significantly decreased the metabolic clearance of the major metabolites, 1-methyluric acid and 1,3-dimethyluric acid, with no change in the renal clearance of theophylline, suggesting that the decreased systemic clearance of theophylline by telithromycin is due to reduction of their metabolic clearance. Pretreatment with telithromycin significantly decreased the activity of 7-ethoxyresorufin O-deethylation and testosterone 6 beta-hydroxylation, suggesting that telithromycin decreases the activity of hepatic CYP1A2 and CYP3A2. Western blot analysis revealed that telithromycin significantly decreased the protein levels of CYP1A2 and CYP3A2 in the liver, which could explain the observed decreases in the systemic clearance of theophylline and metabolic clearance of 1-methyluric acid and 1,3-dimethyluric acid. The present study suggests that telithromycin at the dose used in this study alters the pharmacokinetics and metabolism of theophylline, due to reductions in the activity and expression of hepatic CYP1A2 and CYP3A2.

Simultaneous determination of urinary dialkylphosphate metabolites of organophosphorus pesticides using gas chromatography-mass spectrometry.

In this study, we developed a safe and sensitive method for the simultaneous determination of urinary dialkylphosphates (DAPs), metabolites of organophosphorus insecticides (OPs), including dimethylphosphate (DMP), diethylphosphate (DEP), dimethylthiophosphate (DMTP), and diethylthiophosphate (DETP), using a pentafluorobenzylbromide (PFBBr) derivatization and gas chromatography-mass spectrometry (GC-MS). Several parameters were investigated: pH on evaporation, reaction temperature and time for the derivatization, the use of an antioxidant for preventing oxidation, and a clean-up step. The pH was set at 6, adjusted with K2CO3, and the reaction temperature and time of derivatization were 80 degrees C and 30 min, respectively. Sodium disulfite was chosen as an antioxidant. The clean-up step was performed with a Florisil/PSE mini-column to remove the unreacted PFBBr and sample matrix. This established procedure markedly shortened the sample preparation time to only about 3 h, and completely inhibited the unwanted oxidization of dialkylthiophosphates. The limits of determination (LOD) were approximately 0.3 microg/L for DMP, and 0.1 microg/L for DEP, DMTP, and DETP in 5 mL of human urine. Within-series and between-day imprecision for the present method using pooled urine spiked with DAPs was less than 20.6% in the calibration range of 1-300 microg/L, and the mean recovery was 56.7-60.5% for DMP, 78.5-82.7% for DEP, 88.3-103.9% for DMTP, and 84.2-92.4% for DETP. This method detected geometric mean values of the urinary DAPs in Japanese with and without occupational exposure to OPs, 16.6 or 27.4 for DMP, 1.0 or 0.7 for DEP, 1.3 or 2.3 for DMTP, and 1.0 or 1.1 microg/L for DETP, respectively. The present method, which does not require special equipment except for GC-MS, is quick, safe, and sensitive enough to be adopted in routine biological monitoring of non-occupational as well as occupational exposure to OPs.

Effect of Shiga-like toxin II from Escherichia coli O157:H7 on intestinal clearance of norfloxacin in rats.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection causes severe clinical symptoms, due to its bacterial toxin, called Shiga-like toxin (SLT). However, little is known about the information to establish a safe and efficient prescription to treat for EHEC O157:H7 patients. Thus, we investigated the effect of SLT-II on intestinal function in rats by using the antibiotic norfloxacin (NFLX) as a model drug. The intestinal clearance (CLi) of NFLX, determined by loop method in the jejunum, was significantly decreased by SLT-II. In histopathological experiment, epithalaxia was observed in SLT-II-treated rats without structural changes of tight junction suggesting the deterioration of active transport systems by SLT-II. CLi of NFLX in normal rats was decreased by carnitine (CAR), suggesting the possible involvement of CAR-sensitive transporter in CLi of NFLX. Taken together, these results suggest that the EHEC O157:H7 infection might affect the intestinal disposition of NFLX due to the changing intestinal expression/function of drug transporters by SLT-II.

Involvement of the drug transporters p glycoprotein and multidrug resistance-associated protein Mrp2 in telithromycin transport.

The present study aims to investigate the role of P glycoprotein and multidrug resistance-associated protein (Mrp2) in the transport of telithromycin, a newly developed ketolide antibiotic, in vitro and in vivo. The in vitro experiments revealed that the intracellular accumulation of telithromycin in adriamycin-resistant human chronic myelogenous leukemia cells (K562/ADR) overexpressing P glycoprotein was significantly lower than that in human chronic myelogenous leukemia cells (K562/S) not expressing P glycoprotein. Cyclosporine significantly increased the intracellular accumulation of telithromycin in K562/ADR cells. When telithromycin was coadministered intravenously with cyclosporine in Sprague-Dawley (SD) rats, cyclosporine significantly delayed the disappearance of telithromycin from plasma and decreased its systemic clearance to 60% of the corresponding control values. Hepatobiliary excretion experiments revealed that cyclosporine almost completely inhibited the biliary clearance of telithromycin, suggesting that telithromycin is a substrate of P glycoprotein and a potential substrate of Mrp2. Moreover, the biliary clearance of telithromycin was significantly decreased by 80% in Eisai hyperbilirubinemic mutant rats with a hereditary deficiency in Mrp2, indicating that Mrp2, as well as P glycoprotein, plays an important role in the biliary excretion of telithromycin. When the effect of telithromycin on the biliary excretion of doxorubicin, a substrate of P glycoprotein and Mrp2, was examined in SD rats, telithromycin significantly decreased the biliary clearance of doxorubicin by 80%. Results obtained from this study indicate that telithromycin is a substrate of both P glycoprotein and Mrp2, and these transporters are involved in the hepatobiliary transport of telithromycin.