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Cisplatin is a chemotherapy drug used to treat a variety of cancers. Muscle loss in cancer patients is associated with increased cancer-related mortality. Previously, we suggested that cisplatin administration increases the atrophic gene expressions of ubiquitin E3 ligases, such as atrogin-1 and muscle RING finger-1 (MuRF1), which may lead to muscle atrophy. In this study, C57BL/6J mice were treated with cisplatin (3 mg/kg, intraperitoneally) or saline for 4 consecutive days. Twenty-four hours after the final injection of cisplatin, quadriceps muscles were removed from the mice. The gene expression of Psma and Psmb, which comprise the 20S proteasome, was upregulated by cisplatin administration in the quadriceps muscle of mouse. Systemic administration of cisplatin significantly reduced not only the quadriceps muscle mass but also the diameter of the myofibers. In addition, bortezomib (0.125 mg/kg, intraperitoneally) was administered 30 min before each cisplatin treatment. The co-administration of bortezomib, a proteasome inhibitor, significantly recovered the reductions in the mass of quadriceps and myofiber diameter, although it did not recover the decline in the forelimb and forepaw strength induced by cisplatin. Increased 20S proteasome abundance may play a significant role in the development of cisplatin-induced muscle atrophy. During cisplatin-induced skeletal muscle atrophy, different mechanisms may be involved between loss of muscle mass and strength. In addition, it is suggested that bortezomib has essentially no effect on cisplatin-induced muscle atrophy.

Background A reduction in the skeletal muscle mass worsens the prognosis of patients with various cancers. Our previous studies indicated that cisplatin administration to mice caused muscle atrophy. This is a concern for human patients receiving cisplatin. The insulin‐like growth factor 1 (IGF‐1)/phosphoinositide 3‐kinase (PI3K)/Akt pathway stimulates the rate of protein synthesis in skeletal muscle. Thus, IGF‐I can be a central therapeutic target for preventing the loss of skeletal muscle mass in muscle atrophy, although it remains unclear whether pharmacological activation of the IGF‐1/PI3K/Akt pathway attenuates muscle atrophy induced by cisplatin. In this study, we examined whether exogenous recombinant human IGF‐1 attenuated cisplatin‐induced muscle atrophy. Methods Male C57BL/6J mice (8–9 weeks old) were injected with cisplatin or saline for four consecutive days. On Day 5, quadriceps muscles were isolated. Mecasermin (recombinant human IGF‐1) or the vehicle control was subcutaneously administered 30 min prior to cisplatin administration. A dietary restriction group achieving weight loss equivalent to that caused by cisplatin administration was used as a second control. C2C12 myotubes were treated with cisplatin with/without recombinant mouse IGF‐1. The skeletal muscle protein synthesis/degradation pathway was analysed by histological and biochemical methods. Results Cisplatin reduced protein level of IGF‐1 by about 85% compared with the vehicle group and also reduced IGF‐1/PI3K/Akt signalling in skeletal muscle. Under this condition, the protein levels of muscle ring finger protein 1 (MuRF1) and atrophy gene 1 (atrogin‐1) were increased in quadriceps muscles (MuRF1; 3.0 ± 0.1 folds, atrogin‐1; 3.0 ± 0.3 folds, P

We have previously reported that swellings caused by haptens, such as 2,4,6-trinitrochlorobenzene (TNCB), may be associated with the extracellular signal-regulated kinase (ERK)-induced proliferation pathway. However, the involvement of the Spred/Sprouty family as critical negative regulators of the Ras/Raf/ERK signaling pathway at disease sites is not well-established. Thus, in the present study, the effects of hapten-challenge on the expression levels of genes and proteins associated with the Spred/Sprouty family in the ear of mice were investigated. The activation of ERK and epidermal growth factor receptor (EGFR) tyrosine kinase was inhibited by their selective inhibitors, namely, U0126 and PD168393, respectively. Twenty-four hours after the final challenge by the haptens TNCB, 2,4-dinitrofluorobenzene, or oxazolone, ear thickness was augmented by challenge with all haptens and the gene expression levels of Spred1, Spred2, Sprouty1, and Sprouty2 in swelling induced by all haptens were significantly decreased. Furthermore, Spred2, Sprouty1, and Sprouty2 genes were decreased in the epidermis and dermis of the TNCB-challenged ear. In conclusion, it is possible that the mechanism of hapten-challenge-induced skin thickening involves not only the enhancement of cell proliferative functions via the activation of ERK by EGFR tyrosine kinase activation but also the decreases expression of Spred/Sprouty family members.

It has recently been exhibited that Rac1 expression is increased in the bronchial tissue of a murine model with repeated antigen-challenged airway hyperresponsiveness (AHR). In the present study, the role of Rac1 in endothelin-1 (ET-1)-induced bronchial contraction and myosin light chain (MLC) phosphorylation was examined in AHR mice. Enhanced reactions in AHR mice were prevented by the Rac1 inhibitor NSC23766. These findings suggest that increased activation of Rac1 might be responsible for the enhancement of the bronchial contraction induced by ET-1 in AHR. Keywords: Airway hyperresponsiveness, Bronchial contraction, Rac1 signaling

We recently reported that swelling resulting from 2,4,6-trinitrochlorobenzene (TNCB) challenge might be associated with recruitment of neutrophils. However, it is not known whether neutrophil recruitment is affected by scratching at inflamed sites or not. Therefore, the effects of an Elizabethan collar on the TNCB-induced upregulation of ELR-positive chemokines (CXCL1, CXCL2, and CXCL5) and neutrophil recruitment were investigated. Mice were sensitized by the application of TNCB on abdominal skin. Then, the mice were challenged three times with TNCB to auricle of the ear. To prevent scratching at inflamed sites, an Elizabethan collar was placed on the mice from just before the first challenge until the end of the experiment. The effects of the Elizabethan collar on the TNCB-induced upregulation of CXCLs chemokines and recruitment of neutrophil were investigated. The increase of ear swelling by TNCB challenge was inhibited by the Elizabethan collar. TNCB-challenge-induced upregulation of TNF-α, IL-1β, IL-6, ELR+ chemokines, MPO, and ELA2 was also attenuated by the Elizabethan collar. The gene expression of CXCL1, CXCL2, and CXCL5 human homolog IL-8 was enhanced by TNF-α and IL-1β in human dermal fibroblasts and epidermal keratinocytes. We here suggest that scratching the site of inflammation leads to neutrophil accumulation mediated by TNF-α and IL-1β/ELR+ chemokines in TNCB-challenge-induced contact dermatitis in mice.

Previously it was shown that cisplatin causes muscle atrophy. Under this condition, cisplatin increased the expression of atorogenes, such as muscle ring finger 1 and atrogin-1 (also known as muscle atrophy F-box protein), in mouse skeletal muscle. It was reported recently that ubiquitin (Ub) and ubiquitinated protein levels in skeletal muscle were also up-regulated in cisplatin-induced muscle atrophy, and cisplatin-induced ubiquitinated proteins were degraded by the 26S proteasome pathway. Eicosapentaenoic acid (EPA) is effective against skeletal muscle atrophy in mice. However, it is unclear how EPA suppresses the Ub-proteasome pathway. In this study, the effect of EPA on cisplatin-induced muscle atrophy in mice was examined. Mice were intraperitoneally injected with cisplatin or vehicle control once daily for 4 d. EPA or its vehicle was orally administered 30 min before cisplatin administration. Cisplatin systemic administration induced decrease in muscle mass, myofiber diameter, and increase in Ub genes and ubiquitinated proteins in mouse skeletal muscle were recovered by co-treatment with EPA. However, weight loss and up-regulated atrogenes induced by cisplatin were not changed by co-treatment with EPA in skeletal muscle. In this study, EPA attenuated cisplatin-induced muscle atrophy via down-regulation of up-regulated Ub gene expression. Although further clinical studies are needed, EPA administration can be effective in the development of muscle atrophy in cisplatin-treated patients.

We previously demonstrated that cisplatin administration in mice induces muscle atrophy and an increase in the expression of two muscle-specific ubiquitin E3 ligase genes, muscle ring finger protein 1 (MuRF1), and atrophy gene-1 (atrogin-1), in skeletal muscle. Ubiquitination serves as a degradation signal in both the ubiquitin-proteasome and selective autophagy pathways. In the present study, we investigated changes in the expression of ubiquitin and ubiquitinated proteins and their degradation pathways. Ubiquitin and ubiquitinated protein levels were increased by cisplatin compared with those in the vehicle and dietary restriction (DR) groups. To quantify the levels of ubiquitin and ubiquitinated proteins, we conducted a dot blot assay using an anti-ubiquitin antibody. The expression of ubiquitin was also significantly increased by cisplatin compared with that in the vehicle and DR groups. Since the ubiquitin proteins were upregulated by cisplatin, we measured the mRNA levels of the ubiquitin genes: Ubb, Ubc, Rps27a, and Uba52. All these four genes were increased by cisplatin administration compared with those in both the vehicle-treated and DR groups in quadriceps muscle tissue. The anti-ubiquitin antibody-sensitive bands increased when C2C12 myotubes were treated with cisplatin. Furthermore, MG-132 (26 s proteasome inhibitor), but not bafilomycin A1 (autophagy inhibitor), caused a further increase in expression. In conclusion, ubiquitin and ubiquitinated proteins are upregulated in cisplatin-induced muscle atrophy. Cisplatin-induced ubiquitinated proteins are degraded by the 26 s proteasome pathway.

We previously revealed that Choreito, a traditional Kampo medicine, strongly inhibits bladder carcinogenesis promotion. We have also shown that Polyporus sclerotium, which is one of the crude drugs in Choreito, has the strongest bladder carcinogenesis inhibitory effect and that the ergosterol contained in Polyporus sclerotium is the main active component. In this study, we analyzed the mechanism by which ergosterol inhibits bladder carcinogenesis. Rats were given an N-butyl-N-(4-hydroxybutyl) nitrosamine (BHBN) solution ad libitum, and then a promoter [saccharin sodium (SS), DL-tryptophan, or BHBN] was administered together with ergosterol or its metabolite, brassicasterol. The bladders were removed from rats, and the inhibitory effect on carcinogenesis promotion was evaluated by an agglutination assay with concanavalin A (Con A). Although the oral administration of ergosterol inhibited the promotion of bladder carcinogenesis with SS, the intraperitoneal administration of brassicasterol showed a stronger effect. The effect of brassicasterol on carcinogenesis promotion was observed regardless of the type of promoter. Administration of testosterone to castrated rats increased the number of cell aggregates caused by Con A. In contrast, intraperitoneal administration of brassicasterol to castrated rats treated with testosterone significantly decreased the number of cell aggregates, confirming the inhibition of bladder carcinogenesis promotion. The inhibitory effect of ergosterol on bladder carcinogenesis is due to brassicasterol, a metabolite of ergosterol. The action of brassicasterol via androgen signaling may play a role in the inhibitory effect on bladder carcinogenesis promotion.

Dexamethasone for antiemetic therapy is typically administered with anticancer drugs such as cisplatin. We previously reported that cisplatin upregulates the muscle-specific E3 ubiquitin ligase genes, namely muscle ring-finger protein 1 (MuRF1) and atrophy gene-1 (atrogin-1), and promotes muscle atrophy in mice. It is well known that dexamethasone causes upregulation of MuRF1 and Atrogin-1 expression in skeletal muscles. Although it is speculated that a combination of dexamethasone and cisplatin worsens muscle atrophy, there are no reports based on research. We thereby investigated the effects of cisplatin and dexamethasone, alone or in combination, on the expression of MuRF1 and Atrogin-1 in murine skeletal muscles and C2C12 myotubes. Mice were intraperitoneally injected with cisplatin or the vehicle control once daily for 4 days. Dexamethasone or the vehicle control was subcutaneously administered 30 minutes prior to the administration of cisplatin. Dexamethasone enhanced MuRF1 and Atrogin-1 gene expression upregulated by cisplatin in murine quadriceps muscles and C2C12 myotubes. Cisplatin-caused upregulation of myostatin and downregulation of IGF-1 gene expression were also enhanced by co-administration of dexamethasone in murine quadriceps muscles and C2C12 myotubes. This study shows that the combination treatment of cisplatin and dexamethasone exacerbated muscle atrophy in mice. Therefore, this treatment regimen might exacerbate muscle atrophy in cancer patients.

Recently, we demonstrated that Rac1 upregulation is involved in augmented bronchial smooth muscle (BSM) contractions of antigen-challenged mice. However, change in G protein-coupled receptor (GPCR)-induced Rac1 activation remains unknown in BSMs of repeatedly antigen-challenged (Chal.) mice. We here examined carbachol (CCh)-induced Rac1 activation in BSMs of Chal. mice. Gene expression levels of both Rac1 and Rac-guanine nucleotide exchange factors (GEFs), such as Tiam1 and Trio, were increased in BSMs of Chal. mice. Furthermore, CCh-induced Rac1 activation was inhibited by pretreatment with Rac1-GEF inhibitor NSC23766 and Rac1 inhibitor EHT1864 in BSMs of sensitized-control (S.C.) and Chal. mice. Compared with S.C. mice, CCh-induced Rac1 activation was increased in BSMs of Chal. mice. In conclusion, we reported that increased CCh-induced Rac1 activation via Tiam1 and Trio upregulation, in addition to upregulate Rac1, may be involved in increased CCh-induced BSM contractions in Chal. mice.

Daiokanzoto (DKT) exerts its laxative effect via colonic inflammation caused by sennoside A in Daio (rhubarb). Previously, we showed that the laxative effect of sennoside A is related to decreased aquaporin-3 (AQP3) expression in mucosal epithelial cells due to colonic inflammation. We also found that a combination of glycyrrhizin, an ingredient in Kanzo (glycyrrhiza), and sennoside A attenuates the inflammatory response induced by sennoside A and reduces its laxative effect. These findings indicate that DKT may be a long-term treatment for chronic constipation, but there is no evidence supporting this hypothesis. In this study, we analyzed the laxative effect of repeated DKT administration, focusing on AQP3 expression in the colon. After rats were treated for 7 days, decreased AQP3 expression and the onset of diarrhea were observed in the DKT group, but were not seen in the Daio group either. Although the relative abundance of gut microbiota after repeated DKT administration was similar to that after control treatment, Daio reduced Lactobacillaceae, Bifidobacteriaceae, and Bacteroidaceae levels and markedly increased Lachnospiraceae levels. In this study, we show that DKT has a sustained laxative effect, even upon repeated use, probably because it maintains decreased AQP3 expression and gut microbiota homeostasis. This outcome therefore indicates that DKT can be used as a long-term treatment for chronic constipation.

We previously demonstrated that the expression levels of drug-metabolizing enzymes, cytochrome P450 (CYP) enzymes, in the liver are significantly decreased in a murine model of ulcerative colitis (UC). In this study, we investigated changes in the pharmacokinetics of phenytoin, a CYP2C substrate drug, in the presence of UC. Colitis was induced by feeding male mice 3.5% dextran sulfate sodium (DSS) dissolved in drinking water for 10 days. The messenger RNA (mRNA) expression of CYP2C29 and CYP2C37 and the protein expression of CYP2C in the liver were evaluated via real-time reverse transcription–polymerase chain reaction and Western blotting, respectively. In DSS-treated animals, both mRNA and protein expression levels of CYP2C in the liver were significantly reduced relative to those in control animals (by 20%-40%). Phenytoin (30 mg/kg) was administered orally in a single dose to mice, and plasma concentrations were measured. Plasma concentrations of phenytoin were higher in the DSS-treated group than in the control group at 12, 24, and 36 hours after administration. Animals given DSS also exhibited a higher area under the plasma concentration–time curve extrapolated to infinity (AUCinf, 315 μg·h/mL), a delayed elimination half-life ( T1/2, 8.1 hours), and a decreased body clearance (CL/F, 3.52 mL/h) compared with that of control animals (AUCinf, 215 μg·h/mL; T1/2, 3.6 h; CL/F, 5.58 mL/h). This study indicated that the presence of UC decreases CYP2C expression levels in the liver, thereby delaying the metabolism of CYP2C substrates, including phenytoin, and increasing blood concentrations of these substrates.

Xeroderma is a frequent complication in diabetic patients. In this study, we investigated the mechanism underlying the onset of diabetic xeroderma, focusing on aquaporin-3 (AQP3), which plays an important role in water transport in the skin. Dermal water content in diabetic mice was significantly lower than that in control mice. The expression level of AQP3 in the skin was significantly lower in diabetic mice than in control mice. One week after streptozotocin (STZ) treatment, despite their increased blood glucose levels, mice showed no changes in the expression levels of AQP3, Bmal1, Clock, and D site-binding protein (Dbp) in the skin and 8-hydroxydeoxyguanosine (8-OHdG) in the urine. In contrast, two weeks after STZ treatment, mice showed increases in the blood glucose level, decreases in AQP3, Bmal1, Clock, and Dbp levels, and increases in the urinary levels of 8-OHdG. The results of this study suggest that skin AQP3 expression decreases in diabetes, which may limit water transport from the vessel side to the corneum side, causing dry skin. In addition, in diabetic mice, increased oxidative stress triggered decreases in the expression levels of Bmal1 and Clock in the skin, thereby inhibiting the transcription of Aqp3 by Dbp, which resulted in decreased AQP3 expression.

In previous studies, we showed that a high-dose intake of green tea polyphenol (GP) induced a hepatospecific decrease in the expression and activity of the drug-metabolizing enzyme cytochrome P450 3A (CYP3A). In this study, we examined whether this decrease in CYP3A expression is induced by epigallocatechin gallate (EGCG), which is the main component of GP. After a diet containing 1.5% EGCG was given to mice, the hepatic CYP3A expression was measured. The level of intestinal bacteria of Clostridium spp., the concentration of lithocholic acid (LCA) in the feces, and the level of the translocation of pregnane X receptor (PXR) to the nucleus in the liver were examined. A decrease in the CYP3A expression level was observed beginning on the second day of the treatment with EGCG. The level of translocation of PXR to the nucleus was significantly lower in the EGCG group. The fecal level of LCA was clearly decreased by the EGCG treatment. The level of intestinal bacteria of Clostridium spp. was also decreased by the EGCG treatment. It is clear that the hepatospecific decrease in the CYP3A expression level observed after a high-dose intake of GP was caused by EGCG. Because EGCG, which is not absorbed from the intestine, causes a decrease in the level of LCA-producing bacteria in the colon, the level of LCA in the liver decreases, resulting in a decrease in the nuclear translocation of PXR, which in turn leads to the observed decrease in the expression level of CYP3A.

The chronic administration of morphine to patients with neuropathic pain results in the development of a gradual tolerance to morphine. Although the detailed mechanism of this effect has not yet been elucidated, one of the known causes is a decrease in μ-opioid receptor function with regard to the active metabolite of morphine, M-6-G(morphine-6-glucuronide), in the ventrotegmental area of the midbrain. In this study, the relationship between the concentration of morphine in the brain and its analgesic effect was examined after the administration of morphine in the presence of neuropathic pain. Morphine was orally administered to mice with neuropathic pain, and the relationship between morphine's analgesic effect and its concentration in the brain was analysed. In addition, the expression levels of the conjugation enzyme, UGT2B (uridine diphosphate glucuronosyltransferase), which has morphine as its substrate, and P-gp, which is a transporter involved in morphine excretion, were examined. In mice with neuropathic pain, the concentration of morphine in the brain was significantly decreased, and a correlation was found between this decrease and the decrease in the analgesic effect. It was considered possible that this decrease in the brain morphine concentration may be due to an increase in the expression level of P-gp in the small intestine and to an increase in the expression level and binding activity of UGT2B in the liver. The results of this study suggest the possibility that a sufficient analgesic effect may not be obtained when morphine is administered in the presence of neuropathic pain due to a decrease in the total amount of morphine and M-6-G that reach the brain.

In recent years, the intake of functional foods containing high-doses of green tea polyphenols (GP) has been increasing. In this study, the long-term safety of high-dose GP was assessed from a pharmacokinetic point of view by focusing on the drug-metabolizing enzyme, cytochrome P450 (CYP). Mice were fed a diet containing 3% GP for 4weeks, and the CYP expression levels and activity were determined. The GP-treated group showed a significant decrease in the hepatic CYP3A and an increase in the hepatic CYP2C expression compared with the control group. CYP1A, CYP2D, and CYP2E expression were not different between the GP-treated and the control groups. In the small intestine, there were no differences in the CYP3A protein levels between the groups. The increase in the plasma triazolam concentration in the GP-treated group was observed. Although no changes were found in the hepatic CYP3A levels in mice receiving a diet containing 0.1% GP for 4weeks, a significant decrease was seen in the hepatic CYP3A level in mice receiving a diet containing 3% GP for only 1week. This study revealed that the intake of a high-dose GP results in a liver-specific decrease in the CYP3A expression level. The results also indicated that the effects of GP on CYP3A were not observed following the intake of a low-dose GP. In the future, caution should be taken in cases when functional foods containing a high-dose GP are concomitantly consumed with a CYP3A substrate drug.

The drug-metabolizing enzyme CYP is mainly involved in the metabolism of various substances in the liver, such as drugs, endogenous substances, and carcinogens. Recent reports have also revealed that CYP1B1 plays a major role in the developmental process. Because the level of CYP expression is markedly high in the liver, we hypothesize that CYP plays a role in the developmental process of the liver. To verify this hypothesis, we analyzed the expression patterns of various CYP molecular species and their functions during the differentiation of embryonic stem cells (ES cells) into hepatocytes and the developmental process in mice. The results demonstrated that CYP2R1 and CYP26A1 are expressed at an earlier stage of the differentiation of ES cells into hepatocytes than hepatoblast-specific markers. Additionally, during the development of the mouse liver, CYP2R1 and CYP26A1 were mostly up-regulated during the stage when hepatoblasts appeared. In addition, when CYP2R1 and CYP26A1 expressions were forced in ES cells and liver of adult mice, they differentiated into hepatoblast marker positive cells. These results suggest that CYP2R1 and CYP26A1 may play a major role in hepatoblast cell differentiation during the development of the liver.

Approximately 30% of patients with cancer pain experience concurrent neuropathic pain. Since these patients are not sufficiently responsive to morphine, the development of an effective method of pain relief is urgently needed. Decreased function of the μ opioid receptor, which binds to the active metabolite of morphine M-6-G in the brain, has been proposed as a mechanism for morphine resistance. Previously, we pharmacokinetically examined morphine resistance in mice with neuropathic pain, and demonstrated that the brain morphine concentration was decreased, expression level of P-glycoprotein (P-gp) in the small intestine was increased, and expression level and activity of uridine diphosphate glucuronosyltransferase (UGT)2B in the liver were increased. In order to clarify the mechanism of the increased expression of UGT2B, we examined the phase of neuropathic pain during which UGT2B expression in the liver begins to increase, and whether this increased expression is nuclear receptor-mediated. The results of this study revealed that the increased expression of UGT2B in the liver occurred during the maintenance phase of neuropathic pain, suggesting that it may be caused by transcriptional regulation which was not accompanied by increased nuclear import of pregnane X receptor (PXR).

While irinotecan (CPT-11) has a potent anti-cancer effect, it also causes serious diarrhea as an adverse reaction. In this study, we analyzed the pathogenic mechanism of CPT-11-induced delayed diarrhea by focusing on water channel aquaporin-3 (AQP3) in the colon. When rats received CPT-11, the expression level of AQP3 was reduced during severe diarrhea. It was found that the expression levels of inflammatory cytokines and the loss of crypt cells were increased in the colon when CPT-11 was administered. When celecoxib, an anti-inflammatory drug, was concomitantly administered, both the diarrhea and the reduced expression of AQP3 induced by CPT-11 were suppressed. The inflammation in the rat colon during diarrhea was caused via activated macrophage by CPT-11. These results showed that when CPT-11 is administered, the expression level of AQP3 in the colon is reduced, resulting in delayed diarrhea by preventing water transport from the intestinal tract. It was also suggested that the reduced expression of AQP3 might be due to the inflammation that occurs following the loss of colonic crypt cells and to the damage caused by the direct activation of macrophages by CPT-11. Therefore, it was considered that anti-inflammatory drugs that suppress the reduction of AQP3 expression could prevent CPT-11-induced delayed diarrhea.

Aquaporin-3 (AQP3) is a water channel that is predominantly expressed in the colon, where it plays a critical role in the regulation of fecal water content. This study investigated the role of AQP3 in the colon in morphine-induced constipation. AQP3 expression levels in the colon were analyzed after oral morphine administration to rats. The degree of constipation was analyzed after the combined administration of HgCl(2) (AQP3 inhibitor) or fluoxetine (5-HT reuptake transporter [SERT] inhibitor) and morphine. The mechanism by which morphine increased AQP3 expression was examined in HT-29 cells. AQP3 expression levels in rat colon were increased during morphine-induced constipation. The combination of HgCl(2) and morphine improved morphine-induced constipation. Treatment with morphine in HT-29 cells did not change AQP3 expression. However, 5-HT treatment significantly increased the AQP3 expression level and the nuclear translocation of peroxisome proliferator-activated receptor gamma (PPARγ) 1 h after treatment. Pretreatment with fluoxetine significantly suppressed these increases. Fluoxetine pretreatment suppressed the development of morphine-induced constipation and the associated increase in AQP3 expression in the colon. The results suggest that morphine increases the AQP3 expression level in the colon, which promotes water absorption from the luminal side to the vascular side and causes constipation. This study also showed that morphine-induced 5-HT secreted from the colon was taken into cells by SERT and activated PPARγ, which subsequently increased AQP3 expression levels.

We have previously shown that menthol attenuates the anticoagulant effect of warfarin by increasing the expression levels of CYP3A and CYP2C in the liver. This study evaluated the effects of menthol on the pharmacokinetics of the CYP3A substrate triazolam and the CYP2C substrate phenytoin. Menthol was orally administered to mice for 7 d. Twenty-four hours after the administration of menthol, triazolam was orally administered, and the plasma concentration was measured. In addition, the CYP3A metabolic activity for triazolam and the CYP3A expression level in the liver were determined. The effects of menthol on the pharmacokinetics of phenytoin were assessed in the same manner. In the menthol-treated group, the area under the blood concentration-time curve (AUC) of triazolam was lower and its clearance was higher compared with the control group. The CYP3A metabolic activity and CYP3A expression level in the liver were significantly increased in the menthol-treated group compared with the control group. Similarly, the AUC of phenytoin was lower and the hepatic CYP2C expression level was higher in the menthol-treated group. Thus, menthol lowered the plasma concentrations of triazolam and phenytoin when concurrently administered. These effects may be attributed to an increased metabolic activity for these drugs due to the increased expression of CYP3A and CYP2C in the liver.

Skin function deteriorates with aging, and the dermal water content decreases. In this study, we have analyzed the mechanism of aging-related skin dryness focusing on aquaporins (AQPs), which are the water channels. Mice aged 3 and 20 months were designated as young and aged mice, respectively, to be used in the experiments. No differences were observed in transepidermal water loss between the young mice and aged mice. However, the dermal water content in aged mice was significantly lower than that in young mice, thus showing skin dryness. The expression of AQP1, AQP3, AQP4, AQP7, and AQP9 was observed in the skin. All the mRNA expression levels of these AQPs were significantly lower in aged mice. For AQP3, which was expressed dominantly in the skin, the protein level was lower in aged mice than in young mice. The results of the study showed that the expression level of AQPs in the skin decreased with aging, suggesting the possibility that this was one of the causes of skin dryness. New targets for the prevention and treatment of aging-related skin dryness are expected to be proposed when the substance that increases the expression of AQP3 is found.

Changes in the expression level and activity of cytochrome P450 (CYP) in the liver are caused by various factors and affect the pharmacokinetics of drugs. The purpose of this study was to determine whether the expression of CYP3A is affected by a high-fat diet. In addition, we examined whether the type of diet given to mice could produce changes in the expression level and activity of CYP3A. Mice were fed a purified diet containing 10 kcal% lard (control group) or 60 kcal% lard (HF group) or regular mouse chow containing 13 kcal% of fat (MF group) for 4 weeks. No significant differences were observed in the hepatic CYP3A protein expression level between the HF group and the control group. The CYP3A protein expression in the MF group was significantly higher than that observed in the control group. In the MF group, the area under the curve (AUC) of intraperitoneally administered triazolam was lower. Because lithocholic acid (LCA) is known to increase hepatic CYP3A expression, the levels of Clostridium sordellii and LCA in the feces were measured. In the MF group, the levels of Clostridium sordellii and LCA were higher. It has been demonstrated that a high-fat diet does not cause any changes in hepatic CYP3A expression. In addition, the different diets caused alterations in the enteric environment, which triggered changes in CYP3A expression. Therefore, it is necessary to carefully consider the type of feed while performing animal experiments to evaluate the pharmacokinetics of drugs.

Recent studies have reported that a high-fat diet during pregnancy exerts various effects on the foetus and newborn. The purpose of this study was to clarify the effects of a high-fat diet during pregnancy on the activity of hepatic cytochrome P450 (Cyp) 3a in offspring in mice. The protein expression level and activity of Cyp3a in the livers of 6-week-old mice born to mothers that were given a high-fat diet during pregnancy (HF group) decreased significantly compared with the Control group. Triazolam, which is a substrate of Cyp3a, was intraperitoneally administered to the mice in the HF group. Compared with the Control group, an increase in the area under the plasma concentration–time curve and a decrease in total clearance were observed in the HF group. The hepatic constitutive androstane receptor (CAR) mRNA expression level in the HF group was significantly lower than that in the Control group. An increase in phosphorylation of extracellular signal-regulated kinase (ERK) was also observed in the HF group. The results of this study revealed that a high-fat diet during pregnancy causes an increase in ERK phosphorylation and a decrease in the expression level of CAR in the livers of offspring, which leads to decreased Cyp3a expression and activity. The results suggest that individual differences in pharmacokinetics may not only be expressed by genetic predisposition but also by a mother’s living environment during pregnancy.

Living organisms eliminate foreign low-antigenic substances, such as drugs and environmental pollutants, by detoxification mediated by metabolizing cytochrome P450 (CYP). We have examined the possible regulation of CYP expression by enteric bacteria. Cyp mRNA expression levels, Cyp3a protein expression level, and the activity of Cyp3a in hepatic microsomal fractions were compared in germ-free (GF) and specific pathogen-free (SPF) mice. We evaluated hepatic Cyp3a11 mRNA expression levels and Cyp3a metabolic activity in GF and SPF mice after five days of antibiotic administration. The fecal levels of lithocholic acid (LCA)-producing bacteria and hepatic taurolithocholic acid (TLCA) were also measured. Cyp mRNA expression levels, Cyp3a protein expression level, and the activity of Cyp3a in SPF mice were higher than those in GF mice, indicating that enteric bacteria increases hepatic Cyp3a expression. The effects of enteric bacteria-reducing antibiotics on Cyp3a expression were examined. We observed that decreasing enteric bacteria with antibiotics in SPF mice caused a significant decrease in the hepatic Cyp3a11 mRNA expression, TLCA, and fecal LCA-producing bacteria compared to the group that did not receive antibiotics. No change in Cyp3a11 expression was observed in GF mice that were treated with antibiotics. Administration of LCA to GF mice showed an increase in Cyp3a11 expression similar to that of SPF mice. The enzymes of the enteric bacteria are believed to metabolize and detoxify drugs by either reduction or hydrolysis. The results of this study indicate that changes in enteric bacteria may alter the expression and activity of hepatic drug metabolizing enzymes and pharmacokinetics. Therefore, enteric bacteria should be closely monitored to ensure the safe use of drugs.

Patients with severe constipation are treated with combinations of several different laxatives. The purpose of this study is to examine whether the concomitant use of different laxatives enhances the laxative effect, using an osmotic laxative, magnesium sulphate (MgSO₄), and a stimulant laxative, bisacodyl. The faecal water content of rats, to which MgSO₄ and bisacodyl were coadministered, was lower than that in the MgSO₄ group, while the change in the faecal water content over time was very similar to that in the bisacodyl group. The mRNA expression of the osmotic pressure marker, sodium/myo-inositol transporter, in the coadministration group 5h after the administration was significantly higher than that in the control group and almost equal to that in the MgSO₄ group. The protein expression level of aquaporin-3 (AQP3), which plays an important role in water transfer, in the coadministration group decreased compared to the control group, as was the case in the bisacodyl group. The results of this study indicates that the coadministration of MgSO₄ and bisacodyl does not enhance the laxative effect because the expression level of AQP3 in the colon in the coadministration group was almost equal to that in the bisacodyl group.

Ethnopharmacological relevance We have previously reported that Byakkokaninjinto improves cutaneous pruritus by increasing the expression level of aquaporin-3 (AQP3). In this study, we examined the effect of Gypsum fibrosum (main component: CaSO 4 ), which is the main component of Byakkokaninjinto, on the cutaneous AQP3 expression level. Materials and methods KKAy mice were given a diet containing 0.3% Gypsum fibrosum extract, or a diet containing 0.3% CaSO 4 for 4 weeks. The urine volume, plasma glucose levels, cutaneous AQP3 protein expression, and the Ca 2+ content were measured. Results The 24-h urine volumes and the plasma glucose levels in the Gypsum fibrosum extract group were not significantly different from those in the control group. In the Gypsum fibrosum extract group, the cutaneous AQP3 protein levels increased significantly, by approximately 3.2-fold, compared to the control group. The cutaneous Ca 2+ content in the control group was approximately 35 μg/g. In the Gypsum fibrosum extract group, the Ca 2+ content increased to approximately 51 μg/g, which was significant compared to the control group. In the CaSO 4 group, an increase in the AQP3 protein expression levels and Ca 2+ content were observed; the extent of these increases were similar to those in the Gypsum fibrosum extract group. Conclusions The results of this study suggest that Gypsum fibrosum plays an important role in the increased levels of cutaneous AQP3 expression enhanced by Byakkokaninjinto. The results also indicate that the increase in AQP3 caused by Gypsum fibrosum is attributable to an increase in the cutaneous Ca 2+ content from its main component, CaSO 4 .

The purpose of this study was to clarify the anti-fatigue effect of Conclevan, which is mainly composed of liver hydrolysate, via a forced swimming test using mice. Conclevan was administered to mice for 6 weeks, and a forced swimming test was conducted to measure swimming time. After six weeks, the blood ammonia and glutamine concentrations were measured. In the Conclevan administration group, swimming time increased significantly compared to the swimming control group. In the swimming control group, an increase in blood ammonia and a decrease in blood glutamine were observed, relative to the non-swimming control group. In the Conclevan administration group, the increased blood ammonia and decreased blood glutamine induced by swimming were significantly reduced, compared to the swimming control group. The mRNA expression levels of the hepatic enzymes of the urea cycle (carbamoyl-phosphate synthetase, argininosuccinate synthetase, and arginase) and glutamine synthesis (glutamate dehydrogenase and glutamine synthetase) were significantly increased in the Conclevan administration group, compared to the swimming control group. The results of this study demonstrated the anti-fatigue effects of Conclevan. This product may inhibit an increase in the fatigue-inducing ammonia concentration in the blood by increasing the expression of hepatic enzymes, which convert ammonia to urea, leading to increased swimming time. In addition, Conclevan may prolong swimming time by increasing the hepatic synthesis of glutamine, which is an important amino acid for supplying energy in muscles.

In humans, digoxin is mainly eliminated through the kidneys unchanged, and renal clearance represents approximately 70% of the total clearance. In this study, we used the mouse models to examine digoxin pharmacokinetics in polyuria induced by diabetes mellitus and lithium carbonate (Li(2)CO(3)) administration, including mechanistic evaluation of the contribution of glomerular filtration, tubular secretion, and tubular reabsorption. After digoxin administration to streptozotocin (STZ)-induced diabetic mice, digoxin CL/F increased to approximately 2.2 times that in normal mice. After treatment with Li(2)CO(3) (0.2%) for 10 days, the CL/F increased approximately 1.1 times for normal mice and 1.6 times for STZ mice. Creatinine clearance (CLcr) and the renal mRNA expression levels of mdr1a did not differ significantly between the normal, STZ, and Li(2)CO(3)-treated mice. The urine volume of STZ mice was approximately 26 mL/day, 22 times that of normal mice. The urine volume of Li(2)CO(3)-treated mice increased approximately 7.3 times for normal mice and 2.3 times for STZ mice. These results suggest that the therapeutic effect of digoxin may be significantly reduced in the presence of polyuria either induced by diabetes mellitus or manifested as an adverse effect of Li(2)CO(3) in diabetic patients, along with increased urine volume.

Aquaporin (AQP) 3 plays an important role in regulating faecal water content in the colon. We investigated the role of AQP3 in the colon in the laxative effect of magnesium sulphate (MgSO(4)), a widely used osmotic laxative. Rats were administered MgSO(4), after which faecal water content, the colon mRNA expression levels of sodium myo-inositol transporter (SMIT) and taurine transporter (TauT), the colon protein expression levels of AQP3 were examined. Faecal water content increased over time after MgSO(4) administration, and severe diarrhoea was observed between 4 and 8 h after administration. The mRNA expression levels of SMIT and TauT, which are indicators of variations in osmotic pressure, were highest at 2 h after the administration of MgSO(4) and were still elevated at 8 h after administration when compared to immediately after the administration. The immunostaining analysis showed that AQP3 is a dominant AQP in the rat colon. The protein expression levels of AQP3 in the colon increased over time following the administration of MgSO(4) and at 8 h after administration were approximately 8 times higher than baseline levels. Previously, osmotic laxatives were believed to induce diarrhoea by elevating the osmotic pressure in the intestinal tract. The results of the present study suggest that the laxative effect of MgSO(4) is not simply caused by a change in the osmotic pressure in the intestinal tract, but could be a response to increased expression of AQP3.

We previously demonstrated that ciprofloxacin (CPX), a new quinolone antibiotic, suppresses Cyp3a in the mouse liver by reducing the hepatic level of lithocholic acid (LCA) produced by intestinal flora. The present study investigated the possibility that other antibiotics with antibacterial activity against LCA-producing bacteria also cause a decrease in the LCA level in the liver, leading to reduced expression of Cyp3a11. While the mRNA expression of Cyp3a11 in the liver was significantly reduced when SPF mice were administered antibiotics such as ampicillin, CPX, levofloxacin, or a combination of vancomycin and imipenem, no significant changes were observed after antibiotic treatment of GF mice lacking intestinal flora. LCA-producing bacteria in the feces as well as the hepatic level of the taurine conjugate of LCA were significantly reduced in the antibiotic-treated SPF mice, suggesting that the decrease in Cyp3a11 expression can be attributed to the reduction in LCA-producing intestinal flora following antibiotic administration. These results suggest that the administration of antibiotics with activity against LCA-producing bacteria can also cause a decrease in the LCA level in humans, which may lower CYP3A4 expression. The intestinal flora are reported to be altered not only by drugs, such as antibiotics, but also by stress, disease, and age. The findings of the present study suggest that these changes in intestinal flora could modify CYP expression and contribute to the individual differences in pharmacokinetics.

Recently, a new compound IT-M-07000 was designed as a prodrug of tamibarotene, one of the therapeutic agents for acute promyelocytic leukemia. In the present study, IT-M-07000 was administered to mice to investigate whether it is actually metabolized to tamibarotene. Its metabolic pathway and the utility as a tamibarotene prodrug were also evaluated. After oral administration of IT-M-07000, IT-M-07000, tamibarotene and two compounds that were supposed to be metabolic intermediates in a beta-oxidation pathway of IT-M-07000 to tamibarotene were detected in mouse plasma. It was thus shown that IT-M-07000 is probably beta-oxidized to tamibarotene in mice. Comparison of tamibarotene concentration profiles after oral administration of IT-M-07000 or tamibarotene showed that the plasma tamibarotene concentration increased slower and was retained stable, and the area under the plasma concentration-time curve (AUC) of tamibarotene was larger in mice administered IT-M-07000 than tamibarotene. These results indicate that IT-M-07000 is possibly useful as a prodrug of tamibarotene.

The injectable form of oxycodone contains hydrocotarnine that is supposed to potentiate the analgesic effect of oxycodone with unknown mechanism(s). In this study, the effects of hydrocotarnine on the cytochrome P450 (CYP) and P-glycoprotein (P-gp) were investigated. Hydrocotarnine did not induce a significant change in the metabolic activities of CYP2C9, 2C19, and 2E1 in an in vitro study using human CYP recombinants. Although weak inhibitory effects were observed on CYP3A4 and 2D6, these interactions did not seem to be clinically relevant. Hydrocotarnine also did not cause a significant change in the ATPase activity of human P-gp membranes, suggesting that it is not an inhibitor of P-gp. Furthermore, mice were intraperitoneally injected with hydrocotarnine for 14 days and the mRNA levels of major CYP isozymes and P-gp in the liver and small intestine were determined by real-time RT-PCR. As a result, none of the mRNAs investigated showed a significant change in their levels by hydrocotarnine treatment. In conclusion, it is unlikely that the potentiation of oxycodone effect by hydrocotarnine involves its effect on CYP and P-gp. The findings also demonstrate that hydrocotarnine is unlikely to cause drug interactions via CYP or P-gp.

The expression levels of cytochrome P450 (CYP) in the liver were analyzed over time in dextran sulfate sodium (DSS)-induced ulcerative colitis mouse model, from the initial active stage to the remission stage, to investigate the relationship between the changes in pathological conditions and CYP expression levels.DSS solution was given to mice for 10 days, after which water without DSS was provided for 40 days. Pathological conditions and CYP expression levels were examined over time. The mechanism for variation in CYP expression was also analyzed.The mRNA expression levels of CYP (CYP3A11, CYP1A2, CYP2C29, CYP2D9, and CYP2E1) decreased as pathological conditions worsened and reached their lowest levels on day 10 of DSS treatment. Pathological conditions improved following the discontinuation of DSS, and CYP expression levels normalized by day 50. Blood lipopolysaccharide levels, the hepatic expression of inflammatory cytokines, and the nuclear translocation of pregnane X receptor and constitutive androstane receptor in the liver exhibited patterns similar to the observed variations in CYP expression levels.The capacity for metabolizing drugs that are substrates of CYP decreases during the active stage of ulcerative colitis but subsequently improves during the remission stage. This decrease in CYP expression was likely caused by the observed reduction in the levels of nuclearly localized pregnane X receptor and constitutive androstane receptor, and the increase in the production of inflammatory cytokines triggered by lipopolysaccharides.

We have previously demonstrated that acacia polyphenol (AP) exerts strong anti-obesity, anti-diabetic, and anti-atopic dermatitis effects. In the present study, we investigated the anti-hypertensive effects of AP. Spontaneously hypertensive rats (SHR) with hypertension and control Wistar Kyoto rats (WKY) were used. WKY and SHR were fed AP-containing food or AP-free food (control group) ad libitum for 4 weeks, and their blood pressures were measured. After AP administration, both systolic and diastolic blood pressures were significantly lower in the SHR group than in the control group. There were no differences in the systolic or diastolic blood pressure of WKY between the AP group and the control group. Angiotensin-converting enzyme (ACE) activity, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression, and superoxide dismutase (SOD) activity in SHR kidneys were not altered by AP administration. Blood SOD activity in SHR was significantly higher in the AP group than in the control group. AP exerts anti-hypertensive effects on hypertension but has almost no effect on normal blood pressure. The anti-hypertensive effects of AP may be related to the anti-oxidative effects of increased blood SOD activity.

We had previously revealed that drug metabolism, as well as the expression level of hepatic CYP3A, a drug-metabolizing enzyme, increase 12 weeks after gastrectomy in mice. In this study, we elucidated the mechanism of the increased CYP3A expression. The levels of lithocholic acid (LCA)-producing bacteria (Bacteroides fragilis) and LCA in the colon did not show a significant increase up to 4 weeks after gastrectomy compared to the sham operation group. In contrast, at 12 and 24 weeks post-gastrectomy, the levels of Bacteroides fragilis and LCA were significantly higher in the gastrectomy group than in the sham operation group. At 12 and 24 weeks after gastrectomy, the hepatic nuclear translocation of pregnane X receptor (PXR) had also increased. The hepatic CYP3A11 mRNA expression and nuclear translocation of PXR after intraperitoneal administration of LCA to normal mice was significantly higher than those of the control group. The intraperitoneal administration of taurolithocholic acid (TLCA), a taurine conjugate of LCA, caused no change in the expression level of CYP3A11. We suggest that the increase in the expression level of CYP3A after gastrectomy is caused by an increase in the nuclear translocation of PXR, which is triggered by an increase in LCA-producing bacteria.

Recently, it was reported that the anticoagulant effect of warfarin was reduced when patients receiving warfarin also took menthol. The purpose of this study is to reveal the mechanism of this reduced anticoagulant effect of warfarin from the pharmacokinetic point of view. Warfarin was orally administered to mice 24h after the administration of menthol for 2 days, and the plasma warfarin concentration was measured. In the menthol administration group, the area under the blood concentration time curve of warfarin was decreased by approximately 25%, while total clearance was increased to 1.3-fold compared to the control group. The hepatic cytochrome P450 (CYP) 2C protein expression level in the menthol administration group was significantly increased compared to that in the control group. An increase in the nuclear translocation of constitutive androstane receptor (CAR) was also observed. The addition of menthol to human hepatic cells, HepaRG cells, caused an increase in the mRNA expression level of CYP2C9. The results of this study revealed that menthol causes an increase in CYP2C expression levels in the liver, which leads to an enhancement of warfarin metabolism, resulting in a decreased anticoagulant effect of warfarin. It was also suggested that menthol acted directly on the liver and increased the expression level of CYP2C by enhancing the nuclear translocation of CAR.

Ethnopharmacological relevance Aquaporin-3 (AQP3) is expressed in mucosal epithelial cells in the colon and is important for regulating fecal water content. We examined the role of AQP3 in the laxative effect of rhubarb extract. Methods After orally administering rhubarb extract or its major component (sennoside A) to rats, the fecal water content, AQP3 expression and prostaglandin E 2 (PGE 2 ) concentrations in the colon were examined. The mechanism by which sennoside A decreases the expression of AQP3 was examined using the human colon cancer HT-29 cells and macrophage-derived Raw264.7 cells. Results During diarrhea by rhubarb extract administration, the PGE 2 levels in the colon increased while the AQP3 expression significantly decreased. Similar changes were also observed when sennoside A was administered. When sennoside A or its metabolites, rheinanthrone and rhein were added to Raw264.7 cells, a significant increase in the PGE 2 concentration was observed only in cells treated with rheinanthrone. Fifteen minutes after adding PGE 2 to the HT-29 cells, the AQP3 expression decreased to approximately 40% of the control. When pretreated with indomethacin, sennoside A neither decreased the AQP3 expression nor induced diarrhea. Conclusions Sennoside A may decrease AQP3 expression in the colon to inhibit water transport from the luminal to the vascular side, leading to a laxative effect. The decreases in the levels of AQP3 are caused by rheinanthrone, which is a metabolite of sennoside A, this metabolite activates the macrophages in the colon and increases the secretion of PGE 2 ; PGE 2 acts as a paracrine factor and decreases AQP3 expression in colon mucosal epithelial cells.

Ulcerative colitis (UC) patients may have increased concentrations of drugs in their blood. We hypothesized that this response is mainly due to a decrease in the expression and activity of the drug-metabolizing enzyme, cytochrome P450 (CYP), in the liver. In this study, we have tried to demonstrate the hypothesis. UC was induced in mice by treatment with dextran sulfate sodium (DSS) solution. The mRNA and protein expression levels of CYP, inflammatory cytokine levels, and the metabolic activity of CYP3A in the liver were measured. The nuclear translocations of nuclear factor kappa B (NF-κB), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) were analyzed. The levels of hepatic inflammatory cytokines increased in the DSS-treated group. The hepatic mRNA and protein expression of CYP (CYP1A, CYP2C, CYP2D, CYP2E, and CYP3A) and the CYP3A metabolic activity significantly decreased compared to the control group. Hepatic NF-κB nuclear translocation significantly increased in the DSS-treated group. In contrast, the nuclear translocations of PXR and CAR were decreased. Lipopolysaccharides from inflammatory sites in the colon induce hepatic inflammation in DSS-induced murine colitis. This inflammation then causes an increase in the nuclear translocation of hepatic NF-κB and a decrease in the nuclear translocation of PXR and CAR, resulting in the decreased expression and activities of CYP. The results of this study indicated that at the onset of UC, the decreased activity of hepatic CYP causes an increase in the concentrations of drugs in the blood, leading to an increase in the incidence of adverse reactions.

Aquaporins (AQPs) are membrane channels that transport water within the human body and are therefore important for the regulation of water homeostasis. However, little is known regarding the details of the physiological role of AQP3, which is predominantly expressed in the colon. Thus, we investigated the role of AQP3 in the colon using laxative agents (magnesium sulfate and bisacodyl). The results suggest that the laxative effect produced by magnesium sulfate, which is classified as an osmotic laxative, is not simply a result of the changes in osmotic pressure but is also associated with the increased expression of AQP3 in the mucosal epithelial cells of the colon. In addition, magnesium sulfate increased colonic AQP3 expression through adenylate cyclase activation, which is caused by an increase in the intracellular Mg(2+) concentration. This effect may trigger CREB phosphorylation through PKA activation and promote AQP3 gene transcription. Meanwhile, bisacodyl, which is classified as a stimulant laxative, decreases the expression level of AQP3 in the mucosal epithelial cells of the colon, resulting in the inhibition of water transfer from the intestinal tract to the vascular side of the epithelium, eventually leading to the development of diarrhea. It was also observed that the direct activation of colon macrophages by bisacodyl increases the secretion of PGE2, which acts as a paracrine factor and decreases AQP3 expression in colon mucosal epithelial cells. Future studies of the enteric AQP3 expression level and water transport may aid in the development of new laxative and antidiarrheal agents that target AQP3.

In patients with gastrectomy, it is possible that drug effectiveness is reduced compared to healthy subjects due to the increased of the drug-metabolizing enzyme, Cytochrome P450 (CYP). The purpose of this study is to verify this possibility. Gastrectomy model mice were prepared to evaluate the expression level of various CYPs in the liver from 2 to 24 weeks post-operation. No significant differences were observed in the protein expression levels of CYP3A, CYP1A, CYP2C, and CYP2D between the sham operation group and the gastrectomy group up to 4 weeks after the gastrectomy. On the other hand, significant increases in the protein expression levels of any CYPs were observed in the gastrectomy group compared to the sham operation group from 12 weeks after the gastrectomy onward. These increases in expression levels were maintained until 24 weeks after the gastrectomy. The examination of metabolic activity in the liver in the gastrectomy group using triazolam revealed that the metabolic activity at 12 weeks after the gastrectomy was significantly increased in the gastrectomy group. The administration of the anticancer drug imatinib, which is a substrate of CYP3A, to mice at 12weeks after gastrectomy resulted in an increase in the metabolic rate, suggesting a possible decrease in drug effectiveness. It has been revealed that drug effectiveness may be reduced after gastrectomy because the expression levels of various CYPs in the liver were increased over a prolonged period. The results of this study can serve as valuable fundamental knowledge for drug therapy in patients with gastrectomy.

It has been recently reported that the consumption of a high-fat diet during pregnancy exerts various effects on fetuses and newborn mice. The purpose of this study was to determine the effects of a high-fat diet during pregnancy on the expression of cytochrome P450 (CYP) in the livers of offspring. Mouse dams were fed a high-fat diet during pregnancy from the time of conception. After their birth, the newborn mice were fed a normal diet until 12 weeks of age. In the livers of the infant male mice that consumed a high-fat diet, the protein expression of CYP3A and CYP2C was decreased, and the protein expression of CYP1A and CYP2E was increased at 6 and 12 weeks of age. However, almost no changes were observed in the CYP proteins at 6 and 12 weeks of age in the livers of the infant female mice that consumed a high-fat diet. The amount of pregnane X receptor (PXR) translocated into the nucleus was reduced in the livers of infant male mice that consumed a high-fat diet. However, there was neither an increase in tumor necrosis factor-α or interleukin-1β nor a decrease in lithocholic acid. These data suggested that CYP3A and CYP2C might decrease as a result of the decrease in the amount of nuclear PXR in infant male mice that consumed a high-fat diet. The results of this study suggested that the consumption of a high-fat diet by pregnant mothers may be one explanation for individual differences in pharmacokinetics.

Aquaporin (AQP) 3, which is predominantly expressed in the colon, is considered to play an important role in regulating the fecal water content in the colon. In this study, the role of AQP3 in the colon was examined using HgCl(2) and CuSO(4), which are known to inhibit AQP3 function. The fecal water content was measured up to 1 h after the rectal administration of HgCl(2) or CuSO(4) to rats. The results showed that the fecal water content in the HgCl(2) administration group increased significantly to approximately 4 times that in the control group, and severe diarrhea was observed. However, no changes were observed in the mRNA expression level of the osmoregulatory genes (sodium myo-inositol transporter and taurine transporter) and the level and distribution of AQP3 protein expression, as determined 1 h after the administration of HgCl(2). Comparable results were observed in the CuSO(4) administration group. The results of this study indicated that the inhibition of AQP3 function in the colon caused diarrhea. Therefore, it has been revealed that the fecal water content in the colon is controlled by the transport of water from the luminal side to the vascular side, which is mediated by AQP3. Our findings suggest that a drug that modulates the function or expression of AQP3 in the colon may represent a new target for the development of laxatives.

The purpose of this study was to investigate the role of aquaporin3 (AQP3) in the colon in the laxative effect of bisacodyl. After oral administration of bisacodyl to rats, AQP3, macrophages, cyclooxygenase 2 (COX2), and prostaglandin E2(PGE2) were examined in the colon. The mechanism by which bisacodyl decreases the expression of AQP3 was examined using HT-29 and Raw264.7 cells. When diarrhea occurred, a significant increase in the expression of PGE2and a decrease in AQP3 expression were observed. Immunostaining showed COX2 expression only in macrophages. The PGE2concentration increased significantly 30 min after the addition of bisacodyl to Raw264.7 cells. Thirty minutes after PGE2addition to HT-29 cells, the AQP3 expression level decreased to 40% of the control. When pretreated with indomethacin, bisacodyl did not induce an increase in the colon PGE2level, a decrease in the AQP3 expression level, or diarrhea. The results suggest that bisacodyl may decrease the expression of AQP3 in the colon, which inhibits water transfer from the luminal to the vascular side and leads to a laxative effect. This study also showed that direct activation of colon macrophages by bisacodyl increases the secretion of PGE2, which acts as a paracrine factor and decreases AQP3 expression in colon mucosal epithelial cells.

Enhanced expression of renal aquaporin-2 (AQP2) has been reported when polyuria occurs in diabetic animal models. The purpose of this study was to clarify the possibility that increased AQP2 expression in the kidneys play a role as a compensatory mechanism to alleviate diabetic dehydration. Lithium carbonate (Li₂CO₃), which decreases the renal expression of AQPs, was administered to streptozotocin (STZ)-induced model mice of type I diabetes mellitus (STZ mice), to investigate the relationship between urine volume and renal AQP expression. Plasma glucose and urine glucose levels were similar between STZ mice given feed containing Li₂CO₃ for 10 d and un-treated STZ mice. Urine volume increased to 70 ml/d for the Li₂CO₃-treated STZ mice, compared to 36 ml/d for un-treated STZ mice. No changes were observed in creatinine clearance or the mRNA expression levels of sodium myo-inositol transporter and taurine transporter, which are genes associated with the regulation of osmotic pressure in the kidney, in the Li₂CO₃-treated STZ mice relative to un-treated STZ mice. Protein expression levels of AQP2 and aquaporin-3 (AQP3) of the renal inner medulla were significantly decreased in the Li₂CO₃-treated STZ mice, compared to levels in the STZ group. This study revealed that the decreased expression levels of AQP2 and AQP3 in the kidney increased the urine volume in mice without a change in urinary osmotic pressure. The results of this study suggest that the increased renal AQP2 and AQP3 expression, in the setting of polyuria, physiologically serves as a compensatory mechanism to alleviate dehydration in diabetes mellitus.

Byakkokaninjinto (BKN) is an herbal medicine used for the relief of diuresis, thirst and dermal pruritus that are associated with diabetes. The effects of BKN on the expression of aquaporins (AQPs) in the kidney, salivary gland and skin were investigated in order to clarify the mechanism of drug action. Seven-week-old KKAy mice were given feed containing 4.5% BKN for 4 weeks. Compared with the control group, BKN administration did not affect the blood glucose and insulin concentration. However, water intake and urine volume were significantly reduced. AQP2 protein expression in the kidney inner medullary was significantly increased after BKN administration. AQP3 mRNA and protein expression in skin tissue was significantly increased after BKN administration. However, BKN administration did not affect AQP5 mRNA expression in the salivary gland. These results suggest that BKN treatment relieves diuresis, thirst, and dermal pruritis by increasing kidney AQP2 expression and skin AQP3 expression.

Aims Polyuria is a symptom that appears in association with diabetes mellitus. Because sustained polyuria causes serious dehydration, it is believed that the body has a compensating mechanism to alleviate dehydration. In the present study, the role of renal aquaporin 2 (AQP2) in the compensating mechanism was investigated in KKAy mice, a type 2 diabetes model. Main methods The renal AQP2 expression levels in KKAy mice aged between 5 and 24 weeks were determined using Western blotting. The hypothalamic vasopressin mRNA expression levels also were measured by real-time RT-PCR. Insulin was subcutaneously administered to 11-week-old KKAy mice twice a day for 7 days. After insulin treatment, the renal AQP2 protein expression and the hypothalamic vasopressin mRNA expression were measured. Key findings The urinary volumes of 5- and 12-week-old KKAy mice were 1.5 ± 0.3 mL and 9.5 ± 1.2 mL, respectively. The inner medullary AQP2 protein expression of 12-week-old KKAy mice was approximately 2.5-fold higher than that of 5-week-old KKAy mice. The hypothalamic vasopressin mRNA expression of 12-week-old KKAy mice was approximately twice that of 5-week-old KKAy mice. Insulin treatment in KKAy mice resulted in a significant reduction in the plasma glucose level, urinary volume, and inner medullary AQP2 protein and hypothalamic vasopressin mRNA expression. Significance The present study demonstrated that AQP2 is a renal functional molecule of vasopressin that controls urinary volume and that AQP2 in the kidney increases to alleviate dehydration due to type 2 diabetes with polyuria.

The pharmacokinetics of drugs can change in diabetes mellitus and even among diabetics. They may differ between type I diabetes (T1DM) and type 2 diabetes (T2DM). As triazolam was administered orally to Tsumura, Suzuki, obese, diabetes (TSOD) mice and streptozotocin (STZ) mice, clearance per body (CL/F) in TSOD mice did not differ compared with Tsumura, Suzuki, non-obesity (TSNO) mice. In STZ mice, CL/F was greater than in control mice. Small intestinal cytochrome P450 (Cyp) 3a expression in TSOD mice was significantly lower than in TSNO mice. No significant difference existed in small intestinal Cyp3a expression between STZ mice and control mice. In insulin-treated mice, small intestinal Cyp3a expression was significantly lower than in control mice. These results suggested that the differences in changes in small intestinal Cyp3a expression between T1DM and T2DM may be due to differences in plasma insulin concentrations. This may be a factor in the difference in the drug pharmacokinetics between T2DM and T1DM patients.

To investigate the pharmacokinetic characteristics in TSOD (Tsumura, Suzuki, obese, diabetes) mice, a model of type 2 diabetes and obesity, the expressions of major hepatic CYP enzymes in TSOD and TSNO (Tsumura, Suzuki, non-obesity; control) mice were compared. The 7-month-old TSOD mice, which represented severe obesity/diabetes-related pathophysiology, showed higher expressions of Cyp2c and Cyp3a compared with TSNO mice, while those of Cyp1a and Cyp2e were lower. Cyp3a metabolic activity was also higher in TSOD mice. In the 7-month-old liver, pregnane X receptor (PXR) (nuclear receptor) and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) (cofactor) mRNA expression were higher in TSOD mice, possibly playing a role in the altered expression of Cyp3a. This specifically altered CYP expression in TSOD mice suggests that the biotransformation of drugs metabolized by these CYP enzymes differs from that in normal animals. Based on these findings, further investigation on the relationship between altered CYP expression and pathophysiology may be useful in elucidating changes in pharmacokinetics in obese/diabetic patients.

Ciprofloxacin (CPX), a new quinolone antibiotic, is reported to reduce CYP3A expression in the liver when administered to rats. The present study investigates whether the reduction in intestinal flora is involved in this reduction of CYP3A. While hepatic Cyp3a11 expression and triazolam metabolic activity were significantly reduced by CPX treatment of SPF mice, no significant changes were seen by CPX treatment of germ-free (GF) mice. Lithocholic acid (LCA)-producing bacteria in the feces as well as hepatic level of taurine conjugate of LCA were significantly reduced in CPX-treated SPF mice. Cyp3a11 expression in GF mice was significantly elevated when treated with LCA, known as an activator of fernesoid X receptor and pregnane X receptor. These results indicate that antibiotics such as CPX, having antimicrobial spectrums against LCA-producing bacteria, possibly cause decrease in LCA in the liver, resulting in lower CYP3A expression. The intestinal flora is reported to be altered also by stress, disease and age etc. The findings of the present study suggest that these changes in intestinal flora may modify CYP expression and contribute to individual differences in pharmacokinetics.