Donepezil, the most widely used drug for the treatment of Alzheimer's disease (AD), is an acetylcholinesterase (AChE) inhibitor and is thought to improve cognition by stimulating cholinergic neurotransmission. However, no correlation has yet been established between the inhibitory role of AChE inhibitors and their therapeutic effects when used in AD patients. The cleavage pathway of amyloid precursor protein (APP) includes amyloidgenic (β, γ-cleavage) and non-amyloidgenic (α-cleavage) pathways. The intracellular transportation of APP is important in determining these cleavage pathways. It has been suggested that sorting nexin (SNX) family proteins regulates the intracellular transport of APP, thereby enhancing α-cleavage. In this study, we examined the effects of donepezil on SNX33 expression changes and APP processing in primary cultures of fetal rat cortical neurons. While donepezil treatment increased the levels of SNX33 expression and soluble APPα (sAPPα) in culture media, no changes were observed regarding full-length APP expression in the cell lysate. Donepezil also reduced the release of amyloid β (Aβ) into culture media in a concentration- and time-dependent manner. This reduction was not affected by acetylcholine receptor antagonists. The membrane surface expression of APP was elevated by donepezil. Furthermore, SNX knockdown by antisense morpholino oligos prevented the effects of donepezil. These results indicated that donepezil increased APP expression at the surface of the plasma membrane by decreasing APP endocytosis through upregulation of SNX33, suggesting donepezil might stimulate the non-amyloidogenic pathway. This new mechanism of action for the currently used anti-AD drug may provide a valuable basis for future drug discovery.

Management of chemotherapy-induced adverse effects and the associated pharmaceutical interventions as well as supportive care evidence creation are the most important responsibilities of oncology pharmacists. We have evaluated the (1) efficacy of long-term and successive pharmaceutical care in outpatient chemotherapy and (2) nephroprotective effects of magnesium (Mg) against cisplatin-induced nephrotoxicity (CIN). The results revealed that the adoption rate of pharmaceutical proposals was 98%, and that approximately 70% of the proposals attenuated painful symptoms. Moreover, approximately 60% of pharmaceutical interventions were established after the third visit; in particular, approximately 20% were suggested after the tenth visit. These results have shown that long-term and successive pharmaceutical care by oncology pharmacists in outpatient chemotherapy contributes to a safe and less onerous chemotherapy implementation. CIN frequency and serum creatinine elevation were significantly attenuated by Mg premedication during the cisplatin, docetaxel, and fluorouracil regimen, without changes in adverse effects and response rate. Mg premedication has been suggested to exert a protective effect against CIN without influencing on adverse effects and anti-tumor efficacy. The nephroprotective effect of Mg against CIN was evaluated using Wistar rats. Cisplatin (2.5 mg/kg) was administered once or three times weekly with or without 40 mg/kg MgSO4. The results revealed that Mg regulates the expression of organic cation transporter 2, multidrug and toxin extrusion protein 1, and copper transporter 1, leading to reduced renal platinum accumulation, which results in CIN attenuation. In conclusion, evaluation of pharmaceutical care and supportive care by oncology pharmacists is necessary for advanced care of cancer patients.

Epigenetics is the study of heritable changes in gene expression that occur without alterations in the DNA sequence. Several studies have shown that environmental chemicals can alter epigenetic modifications, including histone modifications and DNA methylation. Environmental chemicals may show toxic effects via epigenetic mechanism-regulated changes in gene expression. Previously, we reported that zinc treatment rapidly decreased Lys(4)-trimethylated and Lys(9)-acetylated histone H3 in the metallothionein (MT) promoter, and also decreased total histone H3. The chromatin structure in the MT promoter may be locally disrupted by zinc-induced nucleosome removal. We also showed that chromium (VI) inhibited MT gene transcription by modifying the transcription potential of the co-activator p300. MT is a small cysteine-rich protein that is active in zinc homeostasis, cadmium detoxification, and protection against reactive oxygen species. Epigenetic changes might influence the cytoprotective function of the MT gene. In this review, I briefly summarize the results of previous studies and discuss the mechanisms and toxicological significance of metal-mediated epigenetic modifications.

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and fatal disease of unidentified pathogenesis. IPAH is pathologically characterized as sustained vasoconstriction and vascular remodeling of the pulmonary artery. In pulmonary arterial smooth muscle cells (PASMCs), an increase in cytosolic Ca2+ concentration ([Ca2+]cyt) triggers vasoconstriction and stimulates cell proliferation leading to vascular remodeling. However, dihydropyridine-type voltage-dependent Ca2+ channel blockers are only effective in very few patients with IPAH (<10%). It is unclear why dihydropyridine Ca2+ channel blockers are not therapeutically effective in a majority of IPAH patients. We have previously shown that extracellular Ca2+-sensing receptor (CaSR) is upregulated in PASMCs from IPAH patients, and it contributes to enhanced [Ca2+]cyt responses and augmented cell proliferation. In this study, the effects of dihydropyridine Ca2+ channel blockers on [Ca2+]cyt responses mediated by CaSR were examined in IPAH-PASMCs. Nifedipine (dihydropyridines) enhanced the CaSR-mediated increase in [Ca2+]cyt in IPAH-PASMCs, but not in PASMCs from normal subjects. Nicardipine (dihydropyridines) and Bay K 8644 (a dihydropyridine Ca2+ channel activator) also augmented the CaSR-mediated [Ca2+]cyt increase in IPAH-PASMCs. In contrast, non-dihydropyridine Ca2+ channel blockers such as diltiazem (benzothiazepines) and verapamil (phenylalkylamines) had no effect on the [Ca2+]cyt response in IPAH-PASMCs. Finally, in monocrotaline-induced pulmonary hypertensive rats, nifedipine caused further increase in right ventricular systolic pressure and thus right ventricular hypertrophy. In conclusion, dihydropyridine Ca2+ channel blockers could exacerbate symptoms of pulmonary hypertension in IPAH patients with upregulated CaSR in PASMCs.

Taste disorder is one of the adverse effects of cancer chemotherapy resulting in a loss of appetite, leading to malnutrition and a decrease in the quality of life of the patient. Oxaliplatin, a platinum anticancer drug, has a critical role in colon cancer chemotherapy and is known to induce taste disorder. Here, we evaluated the taste functions in oxaliplatin-administered rats. Among the taste receptors, expression levels of T1R2, one of the sweet receptor subunits, increased in the circumvallate papillae of the oxaliplatin-administered rats. In a brief-access test, i.e., behavioral analysis of the taste response, oxaliplatin-administered rats showed a decreased response to sweet taste. However, we did not detect any differences in the plasma levels of zinc, number of taste cells, or morphology of taste buds between control and oxaliplatin-administered rats. In conclusion, the decreased response to sweet taste by oxaliplatin administration may be due to the upregulation of T1R2 expression.

The use of nanotechnology has increased over the past 10 years, and various nanomaterials with a wide range of applications have been developed. Carbon nanotubes (CNTs), which are cylindrical molecules consisting of hexagonally arranged carbon atoms, are nanomaterials with high utility. Recently, applications of single-walled CNT (SWCNT) in the medical field for drug-delivery and as gene-delivery agents have been proposed. Due to its structural characteristics and physicochemical properties, the inhalation of SWCNT could be considered as one route for targeted drug delivery into the lungs. Therefore, it is necessary to investigate the effects of SWCNT on the physiological state and response of the cells upon delivery into the lung. We clarified the different response of two carcinoma cell lines to SWCNT exposure, and determined these differences may be due to different cell functions. Furthermore, SWCNT exposure resulted in a global downregulation of stress-responsive genes in normal human bronchial epithelial cells, thereby indicating that the factors involved in the stress responses were not activated by SWCNT. We then tried to ascertain the possible effect of SWCNT on the fate of drugs delivered with SWCNT. Exposure to SWCNT down-regulated the mRNA expression and enzymatic activity of CYP1A1 and CYP1B1 by preventing the binding of activated aryl hydrocarbon receptors to the enhancer region of these genes. This review provides basic information for the prediction of human responses to SWCNT exposure by inhalation, and in its use as a drug delivery carrier.

Naturally occurring inorganic arsenic is known to increase the risk of cancers of the skin and several other organs, including the urinary bladder, lung, and liver. Epidemiological studies have also indicated that gestational arsenic exposure is associated with increased incidences of cancers in several organs, including the bladder and liver, in adulthood. Previous studies have shown that epigenetic changes are involved in arsenic-induced carcinogenesis. Among epigenetic changes, DNA methylation changes that are specific to arsenic-induced tumors would be useful for distinguishing such tumors from tumors induced by other factors and for clarifying arsenic carcinogenesis. It has been reported that gestational arsenic exposure of C3H mice, whose males tend to spontaneously develop liver tumors, increases the incidence of tumors in the male offspring. Using the same experimental protocol, we found a number of regions where the DNA methylation status was altered in the liver tumors compared with the normal liver tissues by the methylated DNA immunoprecipitation (MeDIP)-CpG island microarray method. Among such regions, we demonstrated using real-time methylation-specific PCR and bisulfite sequencing that a gene body region of the oncogene Fosb underwent alteration in DNA methylation following gestational arsenic exposure. We also showed that the Fosb expression level significantly increased following gestational arsenic exposure. These findings suggest that the DNA methylation status of the Fosb region is implicated in tumor augmentation and can also be utilized for characterizing tumors induced by gestational arsenic exposure.

Collagen peptides, including glucosamine, are well-known functional food materials that act on bone and cartilage tissues. Collagen peptides are used at a volume of about 6,000 tons per year as a raw material in Japan. Evidence for the action of collagen peptides for both bone and cartilage tissues has accumulated both at basic research level (in vivo and in vitro ) and clinically. To confirm the clinical effect of these peptides and clearly identify their mechanism of action at the molecular level, further clinical studies comprising disease-free subjects, alongside basic research studies, are required.

Monosodium glutamate (MSG) is known to provide the umami taste in the food. We have recently reported that glutamate has the potential to protect the small intestine against non-steroidal anti-inflammatory drugs (NSAIDs)-induced lesions in rats. In this paper, we examined this protective effect using sodium loxoprofen, one of the NSAIDs frequently used in Asian countries, to determine whether MSG accelerates the healing of loxoprofen-induced small intestinal lesions in rats. Loxoprofen at 60 mg/kg caused hemorrhagic lesions in the small intestine, mainly in the jejunum and ileum. These lesions spontaneously healed within 7 days, but this healing process was delayed by repeated administration of loxoprofen at low doses (10, 30 mg/kg) for 5 d after lesion induction. The healing-impairment action of loxoprofen was accompanied by the down-regulation of vascular endothelium-derived growth factor (VEGF) expression and an angiogenic response. The impaired healing caused by loxoprofen was significantly restored by co-treatment with a diet containing 5% MSG for 5 d, accompanied by the enhancement of VEGF expression and angiogenesis. We suggest that daily intake of MSG not only protects the small intestine against NSAIDs-induced damage but also exerts healing-promoting effects on these lesions.

Ethanol has a variety of action on neuronal functions, though its mechanism of action remains uncertain. Previous investigations have demonstrated functional alteration of neurotransmitter receptors and ion channels by ethanol at its concentration observed in the blood of alcoholics. Our recent studies have shown that chronic ethanol treatment up-regulates high voltage-gated L-type calcium channels and ryanodine receptors, both of which regulate intracellular Ca2+ concentration, and that the up-regulation of these calcium channels participates in behavioral changes including the rewarding effect. Among inositol 1,4,5-trisphosphate receptors (IP3Rs) classified into three different subtypes (type 1 (IP3R-1), type 2, and type 3 IP3Rs) with distinct physiological properties, IP3R-1 is the major neuronal member in the central nervous system, predominantly enriched in cerebellar Purkinje cells and abundant in neurons in the cerebral cortex. Although the most important result of IP3R channel functions is the change in intracellular Ca2+ concentration and phosphorylation of IP3Rs, there are few available data on ethanol effect on IP3Rs. In this report, we demonstrate the functional relationship between ethanol-induced rewarding effect and IP3R-1 expression and regulatory mechanisms of IP3R-1 expression after chronic ethanol exposure, especially focusing on Ca(2+)-related signal transduction pathways via dopamine D1 receptors using mouse cerebral cortical neurons.

Inorganic arsenic is a natural environmental contaminant and known to be a human carcinogen. Although rodent models are pivotal in elucidating the mode of action of arsenic, it has been difficult to verify the carcinogenicity of arsenic in rodents until recently. Waalkes et al. (Toxicol Appl Pharmacol 2003;
186:7-17) reported that maternal exposure to arsenite increases the incidence of hepatic tumors in the male pups of C3H mice in adulthood. This finding indicated that the gestational period is vulnerable to arsenic. Using the same experimental model, we found that maternal arsenite exposure increases the incidence of hepatic tumors caused by a somatic mutation of the C61A Ha-ras gene, which encodes an activated oncogenic Ha-ras protein. The G:C to T:A transversion is attributable to oxidative stress. Our further studies of gpt delta transgenic mice, which enable detection of in vivo mutation, and genome-wide analysis of DNA methylation levels using the methylated DNA immunoprecipitation-CpG island microarray method suggest that oxidative-stress-induced mutation and DNA methylation changes are involved in the tumor augmentation in the pups maternally exposed to arsenic. Our recent study has also suggested that maternal arsenic exposure increases the incidence of hepatic tumors even in the grandchildren (the F2 generation). Consideration should be given to multigenerational and transgenerational effects of maternal exposure in future studies.

To date, 16 Gla-containing proteins have been discovered in humans, 7 and 9 of which are involved or not-involved in the blood coagulation cascade, respectively. They have a common feature that the carbon at the γ-position of glutamic acid in the specific amino acid sequence of the protein molecule is carboxylated. γ-Glutamyl carboxylase catalyzes this reaction in the presence of vitamin K as a cofactor. Similar to phosphorylation and glycosylation, γ-carboxylation of the glutamic acid has been thought to be one of the post-translational modification for the activation of proteins. However, undercarboxylated, but not highly carboxylated osteocalcin has been found to exhibit regulatory activities of glucose metabolism and insulin sensitivity in mice, suggesting that there would be more comprehensive mechanisms in the regulation of protein functions by the carboxylation of glutamic acid and the decarboxylation of γ-glutamic acid.

No effective treatment was available for myotonic dystrophy, even in animal model. We have established a new antisense oligonucleotide delivery to skeletal muscle of mice with bubble liposomes, and led to increased expression of chloride channel (CLCN1) protein and the amelioration of myotonia. In other experiments, we also identified small molecule compounds that correct aberrant splicing of Clcn1 gene. Manumycin A corrected aberrant splicing of Clcn1 in mouse model.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a mechanism against ER stress, wherein unfolded proteins accumulated in the ER are transported to the cytosol for degradation by the ubiquitin-proteasome system. We identified the novel ubiquitin ligase HRD1 involved in ERAD. HRD1 is expressed in brain neurons and protects against ER stress-induced apoptosis. In familial Parkinson's disease, accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R), a substrate of ubiquitin ligase Parkin involved in ERAD, leads to ER stress and apoptosis. We have demonstrated that HRD1 promotes ubiquitination and degradation of Pael-R and suppresses ER stress and apoptosis induced by Pael-R. Amyloid precursor protein (APP) is processed into amyloid β (Aβ) in Alzheimer's disease. We showed that HRD1 promotes APP ubiquitination and degradation, resulting in decreased generation of Aβ. Furthermore, suppression of HRD1 expression causes APP accumulation and Aβ generation associated with ER stress and apoptosis. Interestingly, HRD1 levels significantly decreased in the cerebral cortex of Alzheimer's disease patients, possibly because of its insolubilization. 4-phenylbutyrate (4-PBA) has been demonstrated to restore normal trafficking and activity of mutant proteins by acting as a chemical chaperone. We demonstrated that 4-PBA possesses chaperone activity in vitro, and this prevents protein aggregation. Furthermore, we revealed that 4-PBA attenuates the activation of ER stress responses and neuronal cell death, suggesting that HRD1 decreases unfolded protein accumulation in the ER. In addition, 4-PBA restores the normal expression of Pael-R protein and suppresses Pael-R-induced ER stress. Therefore, 4-PBA is a potential candidate for use in the pharmacotherapy of several neurodegenerative diseases linked to ER stress.

Replication-incompetent adenovirus (Ad) vectors are widely used in gene therapy studies because they are beneficial as a gene delivery vehicle enabling high-titer production and highly efficient gene transfer into a wide spectrum of dividing and non-dividing cells in vitro and in vivo. Theoretically, Ad genes should not be expressed following transduction with a replication-incompetent Ad vector. However, leaky expression of viral genes is known to occur following transduction with a conventional Ad vector, which leads to a cellular immunity against Ad proteins as well as Ad protein-induced toxicity. Such Ad protein-induced cellular immunity and toxicity frequently cause both an elimination of Ad vector-transduced cells and tissue damage, leading to short-lived transgene expression. To date, no detailed analysis of the leaky expression profile of Ad genes has been performed. First, we systematically examined the expression profiles of Ad genes in cells using real-time RT-PCR following transduction with a conventional Ad vector. The results revealed that significant expression was found for E2A, E4, and pIX genes. Next, in order to suppress the leaky expression of Ad genes, complementary sequences for microRNA (miRNA) were inserted into the 3'-untranslated region of the E2A, E4, or pIX genes. miRNAs are an approximately 22-nt length non-coding RNA, and bind to imperfectly complementary sequences in the 3'-untranslated region of target mRNA, leading to suppression of gene expression via post-transcriptional regulation. Incorporation of the miRNA-targeted sequences significantly suppressed the leaky expression of Ad genes in an miRNA-dependent manner.

Cadmium (Cd), a harmful metal, exerts severe toxic effects on various tissues such as those in the kidney, liver, lung, and bone. In particular, renal toxicity with damage to proximal tubule cells is caused by chronic exposure to Cd. However, the molecular mechanism underlying chronic Cd renal toxicity remains to be understood. In this review, we present our recent findings since we examined to search for the target molecules involved in the renal toxicity of Cd using toxicogenomics. In NRK-52E rat renal tubular epithelial cells, we found using DNA microarrays that Cd suppressed the expression of the gene encoding Ube2d4, a member of the Ube2d family. The Ube2d family consists of selective ubiquitin-conjugating enzymes associated with p53 degradation. Moreover, Cd suppressed the expressions of genes encoding all Ube2d family members (Ube2d1/2/3/4) prior to the appearance of cytotoxicity in NRK-52E cells. Cd markedly increased p53 protein level and induced p53 phosphorylation and apoptosis in the cells. In vivo studies showed that chronic Cd exposure also suppressed Ube2d family gene expression and induced p53 accumulation and apoptosis in the renal tubules of the mouse kidney. These findings suggest that Cd causes p53-dependent apoptosis due to the inhibition of p53 degradation through the down-regulation of Ube2d family genes in NRK-52E cells and mouse kidney. Thus, the Ube2d family genes may be one of the key targets of renal toxicity caused by Cd.