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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Role of tumor necrosis factor-alpha in down-regulation of hepatic cytochrome P450 and P-glycoprotein by endotoxin.

We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in the down-regulation of hepatic P-glycoprotein and cytochrome P450 (CYP) by endotoxin, using TNF-alpha gene-deficient (TNF-alpha-/-) mice. In the case of P-glycoprotein, endotoxin (10 mg/kg) significantly decreased the expression of hepatic P-glycoprotein in wild-type mice 6 h, but not 24 h, after intraperitoneal injection, with no significant differences in the constitutional expression of P-glycoprotein between wild-type mice and TNF-alpha-/- mice. However, endotoxin had no effect on the expression of P-glycoprotein in TNF-alpha-/- mice either 6 or 24 h after injection. When doxorubicin was administered intravenously to TNF-alpha-/- mice treated 6 h earlier with and without endotoxin, no significant differences in the plasma concentrations of doxorubicin 3 h after injection were observed between endotoxin-treated and untreated TNF-alpha-/- mice. These results suggest that TNF-alpha plays a pivotal role in the down-regulation of P-glycoprotein by endotoxin. In the case of CYP, the constitutive expression of hepatic CYP3A2 and CYP2C11 had a tendency to decline in TNF-alpha-/- mice compared with that in wild-type mice. Endotoxin significantly decreased the expression of hepatic CYP3A2 and CYP2C11 in wild-type mice 24 h after injection, and that decreased expression was significantly greater in TNF-alpha-/- mice than wild-type mice. When antipyrine was administered intravenously to wild-type mice and TNF-alpha-/- mice treated 24 h earlier with endotoxin, the plasma concentrations of antipyrine in TNF-alpha-/- mice 3 h after injection were significantly higher than those in wild-type mice. These findings suggest that TNF-alpha plays a key role in endotoxin-induced down-regulation of hepatic P-glycoprotein, as well as plays a protective role in the regulation of hepatic CYP3A2 and CYP2C11 against endotoxin-induced acute inflammatory response. In TNF-alpha-/- mice, other cytokines appear to function as compensation for the lack of endogenous TNF-alpha.

Endotoxin from various gram-negative bacteria has differential effects on function of hepatic cytochrome P450 and drug transporters.

The differential effects of endotoxin derived from Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli on hepatic cytochrome P450 (CYP)-dependent drug-metabolizing enzyme activity and on the expression of hepatic CYP3A2, CYP2C11, P-glycoprotein and multidrug resistance-associated protein 2 (Mrp2) was investigated in rats. Endotoxin from all three different pathogens significantly decreased the systemic clearance of antipyrine, reflecting reduced hepatic drug-metabolizing enzyme activity 24 h after intravenous injection (0.5 mg/kg). The degree of the decreased systemic clearance by P. aeruginosa endotoxin was smaller than that by both K. pneumoniae and E. coli endotoxin. Western blot analysis revealed that the down-regulation of CYP3A2 by K. pneumoniae and E. coli endotoxin was greater than that by P. aeruginosa endotoxin. However, the down-regulation of CYP2C11 by all three different endotoxin was almost the same. Both K. pneumoniae and P. aeruginosa endotoxin significantly down-regulated P-glycoprotein, but did not down-regulate Mrp2. E. coli endotoxin had no effect on the expression of either P-glycoprotein or Mrp2, probably due to the low dose used. The down-regulation of CYP3A2 by endotoxin was parallel to the decreased systemic clearance of antipyrine. These results suggest that endotoxin has a differential effect on the hepatic CYP-mediated drug-metabolizing enzyme activity, and on the protein levels of hepatic CYP3A2 and P-glycoprotein, probably due to bacterial source-differences in the production of some proinflammatory mediators. Endotoxin appears to regulate coordinately CYP3A2, CYP2C11 and P-glycoprotein, but not Mrp2.

Behavioral changes following antisense oligonucleotide-induced reduction of organic cation transporter-3 in mice.

The organic cation transporter-3 (OCT3) can transport monoamines, similar to neuronal monoamine transporters. Due to the lack of selective ligands, however, the functional role of OCT3 is still unknown. Thus, we investigated behavioral effects of antisense against OCT3 (AS) in mice. AS (0.075-0.25 microg/0.25 microl/h, for 7 days) dose-dependently decreased immobility time. Moreover, although neither AS (0.075 microg/0.25 microl/h, for 7 days) or imipramine (4 mg/kg, i.p.) were effective, imipramine (4 mg/kg, i.p.) significantly decreased immobility time in mice treated with AS (0.075 microg/0.25 microl/h, for 7 days). Additionally, AS (0.25 microg/0.25 microl/h, for 7 days) significantly increased locomotor activity induced by methamphetamine (1 mg/kg, s.c.), but did not affect spontaneous locomotor activity. These results suggest that OCT3 might become a novel molecular target to treat depression and other diseases related to monoaminergic neuronal systems.

Down-regulation of cytochrome P450 proteins and its activities by Shiga-like toxin II from Escherichia coli O157:H7.

Escherichia coli O157:H7 infection frequently induces clinical complications such as hemolytic uremic syndromes and intestinal dysfunctions. These changes could alter the disposition of drugs, consequently changing their efficacy. However, the possible changes of drug-metabolizing activities by E. coli O157:H7 infection have not been addressed. Thus, we have investigated the effect of Shiga-like toxin type II (SLT-II), derived from E. coli O157:H7, on the hepatic cytochrome P450 (CYP) content and its activity in rats. SLT-II (2microg per animal, i.v.) time-dependently decreased total CYP content and the contents of CYP2C11 and CYP3A2 in hepatic microsomal preparations up to 24hr following injection. Consistently, SLT-II time-dependently decreased CYP activity in vivo, as represented by systemic clearance of antipyrine. An inhibitor of inducible nitric oxide synthase, S-methylisothiourea, restored the decreased systemic clearance of antipyrine by SLT-II, suggesting the involvement of the overproduction of nitric oxide by SLT-II. Moreover, dexamethasone restored the decreased systemic clearance of antipyrine by SLT-II. In the hepatic microsomal preparation, dexamethasone restored the SLT-II-induced decrease of CYP3A2 whereas S-methylisothiourea did not affect both CYP subtypes. Taken together, these results suggest that SLT-II might alter hepatic drug-metabolizing function during E. coli O157 infection and that more than one cytokines induced by SLT-II, including nitric oxide, might make a critical contribution to the decrease of CYP content and its activity.

The altered disposition of methamphetamine in the model of methamphetamine-induced neurotoxicity.

Methamphetamine (METH) is a drug of abuse, causing neurotoxic effects in mammals. Many hypotheses have been proposed to explain the underlying mechanisms of METH-induced toxicity, based on neurochemical/neuroanatomical changes. However, the pharmacokinetic properties of METH in the METH-induced neurotoxic model have not yet been evaluated. Thus, we investigated plasma and tissue levels of METH in the METH-induced neurotoxic model. As a result, when METH is administered multiply (5 mg/kg 4 times at 2-h intervals) in male Wistar rats, plasma METH levels at the third and forth injections were significantly higher than those at the first. The tissue distributions of METH in the brain as well as in the kidney were significantly decreased in the third injections, suggesting the importance of decreased transport of METH into tissues. Alternatively, one week after the establishment of METH-induced neurotoxicity, plasma levels of METH were back to normal, although METH levels in brain microdialysates were significantly higher than those in normal animals. These results suggest that the altered pharmacokinetic properties of METH, due to the abnormal membrane transport/disposition of METH into both central and peripheral tissues, might partially affect the emergence of METH-induced neurotoxicity.

Platelet factor 4 fragment induces histamine release from rat peritoneal mast cells.

Platelet factor 4 (PF-4) belongs to a superfamily of low-molecular weight proteins known as chemokines. However, its function has not been fully evaluated. In the present study, we investigated the effect of PF-4 on histamine release from rat peritoneal mast cells by employing its biologically-active carboxyl-terminal fragment, PF-4 (58-70). PF-4 (58-70) stimulated histamine release from mast cells in a dose-dependent manner (10(-8) to 10(-5)M). Histamine release induced by PF-4 (58-70) occurred rapidly (<30s) and was inhibited by extracellular Ca(2+). These results suggest that PF-4 might play a crucial role at the site of inflammation and/or immune response.

Shiga-like toxin II modifies brain distribution of a P-glycoprotein substrate, doxorubicin, and P-glycoprotein expression in mice.

The effect of Shiga-like toxin II (SLT-II), which was derived from Escherichia coli O157:H7, on doxorubicin transport across the blood-brain barrier (BBB) and P-glycoprotein function, was investigated in ddY mice. Doxorubicin (30 mg kg(-1)) was administered intravenously or fluorescein isothiocyanate labeled dextran (FD-4) was infused (20 microg min(-1)) to the mice, who had received an intravenous injection of SLT-II (0.2 microg/animal) 6 or 24 h earlier. Blood and brain were removed 4 h after injection of doxorubicin or 60 min after infusion of FD-4. SLT-II significantly elevated the brain concentration and brain-to-plasma concentration ratio (K(p)) of doxorubicin and FD-4 24 h after injection, but did not alter 6 h after. Cyclosporin A (200 mg kg(-1)) significantly increased the K(p) value of doxorubicin in the control mice, but did not alter it in mice treated 24 h earlier with SLT-II. Pentoxifylline (100 mg kg(-1)) a TNF-alpha production inhibitor, ameliorated SLT-II-induced increases in the brain concentrations of both drugs and the K(p) value of FD-4, suggesting that TNF-alpha, at least in part, causes damage to the brain capillaries. Western blot analysis revealed that SLT-II increased the protein level of P-glycoprotein in the brain of mice 6 h after injection and the increased level remained unchanged for 24 h. SLT-II did not change ATP content in the brain of mice. These results suggest that the increased P-glycoprotein level cannot explain SLT-II-induced increase in the doxorubicin accumulation in brain. The present findings indicate that SLT-II impairs the BBB function and doxorubicin transport across the BBB, while it overexpresses P-glycoprotein.