Mechanisms of the pH- and Oxygen-Dependent Oxidation Activities of Artesunate.

Artemisinin was discovered in 1971 as a constituent of the wormwood genus plant (Artemisia annua). This plant has been used as an herbal medicine to treat malaria since ancient times. The compound artemisinin has a sesquiterpene lactone bearing a peroxide group that offers its biological activity. In addition to anti-malarial activity, artemisinin derivatives have been reported to exert antitumor activity in cancer cells, and have attracted attention as potential anti-cancer drugs. Mechanisms that might explain the antitumor activities of artemisinin derivatives reportedly induction of apoptosis, angiogenesis inhibitory effects, inhibition of hypoxia-inducible factor-1α (HIF-1α) activation, and direct DNA injury. Reactive oxygen species (ROS) generation is involved in many cases. However, little is known about the mechanism of ROS formation from artemisinin derivatives and what types of ROS are produced. Therefore, we investigated the iron-induced ROS formation mechanism by using artesunate, a water-soluble artemisinin derivative, which is thought to be the underlying mechanism involved in artesunate-mediated cell death. The ROS generated by the coexistence of iron(II), artesunate, and molecular oxygen was a hydroxyl radical or hydroxyl radical-like ROS. Artesunate can reduce iron(III) to iron(II), which enables generation of ROS irrespective of the iron valence. We found that reduction from iron(III) to iron(II) was activated in the acidic rather than the neutral region and was proportional to the hydrogen ion concentration.

Spermatogenesis in the Microminipig.

The microminipig has considerable potential as an animal model to evaluate general toxicity; however, there are few studies on the male reproductive system, particularly regarding spermatogenesis. The objectives of the present study were to clarify the stages of the seminiferous epithelium cycle on the basis of spermiogenesis and to determine the duration of spermatogenesis in the microminipig. Eleven microminipigs from 6 to 9 months of age were used for histological analyses. Spermiogenesis and stages of the seminiferous epithelium cycle were classified according to the degree of acrosomal development as shown by the periodic acid-Schiff reaction. Three of the animals were intravenously injected with 5-bromo-2-deoxyuridine to determine the duration of spermatogenesis by immunohistochemistry. Spermiogenesis was classified into 15 steps according to the morphological development of the acrosome, nucleus, and flagellum. The seminiferous epithelium cycle was classified into 11 stages based on the steps of spermatid development and germ cell associations. The length of the seminiferous epithelium cycle and the overall spermatogenesis process in the microminipig were estimated to be approximately 9.1 and 40.9 days, respectively. The results indicate the potential application of the microminipig in the evaluation of testicular toxicity, such as spermatogenesis disruption, in general toxicity studies.

Mechanism of action of efinaconazole, a novel triazole antifungal agent.

The mechanism of action of efinaconazole, a new triazole antifungal, was investigated with Trichophyton mentagrophytes and Candida albicans. Efinaconazole dose-dependently decreased ergosterol production and accumulated 4,4-dimethylsterols and 4α-methylsterols at concentrations below its MICs. Efinaconazole induced morphological and ultrastructural changes in T. mentagrophytes hyphae that became more prominent with increasing drug concentrations. In conclusion, the primary mechanism of action of efinaconazole is blockage of ergosterol biosynthesis, presumably through sterol 14α-demethylase inhibition, leading to secondary degenerative changes.

An adjustment in BMP4 function represents a treatment for diabetic nephropathy and podocyte injury.

Podocyte injury has been proposed to play an important role in diabetic nephropathy; however, its pathological mechanism remains unclear. We have shown that bone morphogenetic protein 4 (BMP4) signaling leads to the glomerular changes characteristic of this disorder. To analyze the molecular mechanism of podocyte injury, the effect of BMP4 was investigated using streptozotocin (STZ)-induced, Bmp4 heterozygous knockout (Bmp4+/-) and podocyte-specific Bmp4 knockout mice. Mice with STZ-induced diabetes exhibited glomerular matrix hyperplasia and decreased numbers of podocyte nucleus-specific WT1-positive cells. The number of podocytes and proteinuria were improved in both diabetic Bmp4 knockout mouse models compared to the effects observed in the control mice. The effect of BMP4 overexpression on Bmp4-induced or podocyte-specific transgenic mice was examined. Tamoxifen-induced Bmp4-overexpressing mice exhibited mesangial matrix expansion and decreased numbers of WT1-positive cells. Podocyte-specific Bmp4-overexpressing mice displayed increased kidney BMP4 expression and mesangial matrix expansion but decreased nephrin expression and numbers of WT1-positive cells. Both lines of Bmp4-overexpressing mice exhibited increased albuminuria. In cultured podocytes, BMP4 increased phospho-p38 levels. BMP4 decreased nephrin expression but increased cleaved caspase-3 levels. p38 suppression inhibited caspase-3 activation. Apoptosis was confirmed in STZ-diabetic glomeruli and Bmp4-overexpressing mice. Bmp4 +/- mice with diabetes displayed reduced apoptosis. Based on these data, the BMP4 signaling pathway plays important roles in the development of both podocyte injury and mesangial matrix expansion in diabetic nephropathy.

Suppression of epithelial restitution using an inhibitor against Rho-associated coiled-coil containing protein kinase aggravates colitis through reduced epithelial expression of A-kinase anchor protein 13.

In the gastrointestinal tract, the immediate healing response to mucosal damage is critical to sustain mucosal homeostasis. The migration of surrounding epithelial cells to cover the denuded area without proliferation is termed restitution, followed by early reparation of the damage. In this study, we determined the role of A-kinase anchor protein 13 (AKAP13) in mice with dextran sulphate sodium (DSS)-induced colitis upon mucosal injury and restitution, and investigated whether inhibition of Rho-associated coiled-coil containing protein kinase (ROCK), downstream effector of AKAP13, affects these mucosal responses. BALB/c mice were challenged with 4% or 2% DSS in their drinking water for up to 8 or 16days, respectively. During this period, mice received subcutaneous injections of fasudil hydrochloride hydrate (FH, 10mg/kg, twice per day), an inhibitor of phosphorylation of ROCK. In immunohistochemistry, AKAP13 was highly expressed in the mucosal epithelium prior to DSS-induced mucosal injury, and also expressed in ulcer-covering non-proliferative epithelium, which corresponded to restituted epithelial cells. Coadministration of FH increased serum amyloid A levels and histopathological scores for mucosal injury, as compared with the DSS group. The effects were associated with a decrease in gene expression of Akap13 in the mucosal tissue and the inhibition of restitution rata (the length of restituted epithelial cells per ulcer). These results suggested that AKAP13 and ROCK are involved in mucosal response at early injury and restitution during healing in DSS-induced colitis in mice.

Expression of A-kinase anchor protein 13 and Rho-associated coiled-coil containing protein kinase in restituted and regenerated mucosal epithelial cells following mucosal injury and colorectal cancer cells in mouse models.

We demonstrate the expression patterns of A-kinase anchor protein 13 (AKAP13), a scaffold protein that acts upstream of Rho signaling, and Rho-associated coiled-coil containing protein kinase (ROCK) 1/2 in mouse colorectal cancer and during the healing stage of mouse colitis. BALB/c mice received an intraperitoneal injection of azoxymethane at 10mg/kg, followed by two 7-day cycles of 3% dextran sulfate sodium (DSS) administered through their drinking water to induce colon cancer, or a 7-day administration of 4% DSS to induce colitis. The colorectal tissue was then analyzed for gene expression, histopathology, and immunohistochemistry. In the colorectal cancer, AKAP13 and ROCK1/2 were highly expressed in adenocarcinoma compared to the control tissue and low-grade dysplasia. In colitis, AKAP13 and ROCK1 were highly expressed in the restituted and regenerated mucosa but were only moderately expressed in the injured mucosal epithelium, compared to the normal epithelium that exhibited weak expression levels. ROCK2 was weakly expressed in these cells, consistent with the expression of AKAP13 and ROCK1. Furthermore, we found several clumps of epithelial cells expressing AKAP13 and ROCK1/2 in the lamina propria during the mucosal healing process, and these cells also expressed interleukin-6, which is a multipotential cytokine for both inflammation and healing. These data suggest that AKAP13 was expressed in relation with ROCK1/2, which probably play an overall role in both mucosal healing and tumorigenesis.

Anti-inflammatory effects of the selective phosphodiesterase 3 inhibitor, cilostazol, and antioxidants, enzymatically-modified isoquercitrin and α-lipoic acid, reduce dextran sulphate sodium-induced colorectal mucosal injury in mice.

Developing effective treatments and preventing inflammatory bowel disease (IBD) are urgent challenges in improving patients' health. It has been suggested that platelet activation and reactive oxidative species generation are involved in the pathogenesis of IBD. We examined the inhibitory effects of a selective phosphodiesterase-3 inhibitor, cilostazol (CZ), and two antioxidants, enzymatically modified isoquercitrin (EMIQ) and α-lipoic acid (ALA), against dextran sulphate sodium (DSS)-induced colitis. BALB/c mice were treated with 0.3% CZ, 1.5% EMIQ, and 0.2% ALA in their feed. Colitis was induced by administering 5% DSS in drinking water for 8days. The inhibitory effects of these substances were evaluated by measuring relevant clinical symptoms (faecal blood, diarrhoea, and body weight loss), colon length, plasma cytokine and chemokine levels, whole genome gene expression, and histopathology. Diarrhoea was suppressed by each treatment, while CZ prevented shortening of the colon length. All treatment groups exhibited decreased plasma levels of interleukin (IL)-6 and tumour necrosis factor (TNF)-α compared with the DSS group. Microarray analysis showed that cell adhesion, cytoskeleton regulation, cell proliferation, and apoptosis, which might be related to inflammatory cell infiltration and mucosal healing, were affected in all the groups. DSS-induced mucosal injuries such as mucosal loss, submucosal oedema, and inflammatory cell infiltration in the distal colon were prevented by CZ or antioxidant treatment. These results suggest that anti-inflammatory effects of these agents reduced DSS-induced mucosal injuries in mice and, therefore, may provide therapeutic benefits in IBD.

Cilostazol and enzymatically modified isoquercitrin attenuate experimental colitis and colon cancer in mice by inhibiting cell proliferation and inflammation.

We previously reported the anti-inflammatory effects of cilostazol, a selective inhibitor of phosphodiesterase 3, and two antioxidants, enzymatically modified isoquercitrin and α-lipoic acid in a dextran sodium sulphate-induced colitis mouse model. We further examined the chemopreventive effects of these substances in a murine azoxymethane/dextran sodium sulphate -induced colorectal carcinoma model and compared the effects with those of the well-known anticancer natural plant pigment, anthocyanin. In addition, the effects on cell proliferation activity were evaluated in colon cancer cell lines and mucosal epithelial cells in a model of acute dextran sodium sulphate-induced colitis. Cilostazol and enzymatically modified isoquercitrin improved the outcome of azoxymethane/dextran sodium sulphate-induced colorectal cancer along with anthocyanin though inhibiting inflammation and cell proliferation, but the effect of α-lipoic acid was minimal. Inhibition of cell proliferation by cilostazol was confirmed in vitro. In the acute dextran sodium sulphate-induced colitis model, cilostazol and enzymatically modified isoquercitrin prevented the decrease in epithelial proliferative cells. These results indicate that cilostazol and enzymatically modified isoquercitrin first exhibited an anti-dextran sodium sulphate effect at the initial stage of colitis and then showed antitumour effects throughout subsequent inflammation-related cancer developmental stages.

Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats.

We determined effects of the NADPH oxidase (NOX) inhibitor apocynin (APO) or the antioxidant enzymatically modified isoquercitrin (EMIQ) on an early stage of hepatocarcinogenesis in the liver with steatosis. Male rats were given a single intraperitoneal injection of N-diethylnitrosamine (DEN) and fed a high-fat diet (HFD) to subject to a two-stage hepatocarcinogenesis model. Two weeks later, rats were fed a HFD containing the lipogenic substance malachite green (MG), which were co-administered with EMIQ or APO in drinking water for 6 weeks. Three after DEN initiation, rats were subjected to a two-third partial hepatectomy to enhance cell proliferation. The HFD increased total cholesterol and alkaline phosphatase levels, which were reduced by EMIQ co-administration. APO co-administration reduced MG-increased preneoplastic liver lesions, glutathione S-transferase placental form (GST-P)-positive, adipophilin-negative liver foci, and tended to decrease MG-increased Ki-67-positive or active caspase-3-positive cells in the liver foci. EMIQ or APO co-administration reduced the expression of a NOX subunit p22phox in the liver foci, but did not alter the numbers of LC3a-positive cells, an autophagy marker. We identified no treatment-related effects on p47phox and NOX4 expression in the liver foci. The results indicated that APO or EMIQ had the potential to suppress hyperlipidaemia and steatosis-preneoplastic liver lesions, through suppression of NOX subunit expression in rats.

Maternal exposure to ochratoxin A targets intermediate progenitor cells of hippocampal neurogenesis in rat offspring via cholinergic signal downregulation and oxidative stress responses.

To elucidate the developmental exposure effects of ochratoxin A (OTA) on postnatal hippocampal neurogenesis, pregnant SD rats were provided a diet containing 0, 0.12, 0.6, or 3.0ppm OTA from gestation day 6 to day 21 on weaning after delivery. Offspring were maintained through postnatal day (PND) 77 without OTA exposure. At 3.0ppm, offspring of both sexes showed a transient body weight decrease after weaning. Changes in hippocampal neurogenesis-related parameters as measured in male PND 21 offspring were observed at 3.0ppm. In the subgranular zone (SGZ) of the dentate gyrus, PAX6+ or TBR2+ cells were decreased, while GFAP+ or DCX+ cells did not fluctuate in number, suggesting decreased numbers of type-2 progenitor cells. On the other hand, SGZ cells accumulating malondialdehyde increased. In the hilus of the dentate gyrus, SST+ or CHRNB2+ γ-aminobutyric acid (GABA)-ergic interneurons decreased, accompanied with transcript downregulation of Chrnb2 in the dentate gyrus. These results suggest that maternal exposure to OTA decreased type-2 progenitor cells by reducing hilar GABAergic interneurons innervating type-2 progenitor cells via cholinergic signal downregulation and also by increasing oxidative stress in the SGZ. Transcript levels of neurotrophin (Bdnf), glutamatergic receptors (Gria1, Gria2, and Grin2a), serotonin-synthesizing enzyme, and serotonergic receptors (Tph2, Htr1a, and Htr4) increased in the dentate gyrus, suggesting multiple neuroprotective actions against OTA-induced aberrant neurogenesis. All observed fluctuations were reversed by PND 77. The no-observed-adverse-effect level for offspring neurogenesis was determined to be 0.6ppm, corresponding to 39.3-76.0µg/kg body weight/day.

Induction of duodenal mucosal tumors of intestinal epithelial cell origin showing frequent nuclear ß-catenin accumulation similar to the concurrently induced colorectal tumors in rats after treatment with azoxymethane.

Azoxymethane (AOM) is a potent carcinogen used for induction of colon tumors in rats and mice. It is also known that AOM treatment induces small bowel tumors in addition to colorectal tumors in rats. The present study examined the histogenesis of AOM-induced rat duodenal tumors in comparison with concurrently induced colorectal tumors by histochemical and immunohistochemical approaches. Duodenal and colorectal tumors were positive for both periodic acid-Schiff reaction and Alcian blue staining. Immunohistochemically, duodenal tumors were positive for intestinal epithelial markers such as cytokeratin (CK) 20 (100%) and mucin (MUC) 2 (91.7%) but negative for pancreaticobiliary markers such as CK7 (100%) and MUC1 (100%). All colorectal tumors were also negative for CK7 and MUC1 but positive for CK20. Eighty percent of colorectal tumors were positive for MUC2. In addition, nuclear accumulation of ß-catenin was found in duodenal tumors (70.8%), which was similar to colorectal tumors (90.0%). These results indicate that duodenal tumors induced by AOM treatment of rats were derived from intestinal epithelium. Similar to colorectal tumors, nuclear accumulation of ß-catenin indicates activation of Wnt signaling as a driving force for tumor progression in AOM-induced duodenal tumors.

Spontaneous Mammary Adenocarcinoma in a Twelve-week-old Female Sprague-Dawley Rat.

Spontaneous mammary adenocarcinoma was observed in a 12-week-old female SD rat. A movable mass in the right cervical region was found at 11 weeks of age, and the rat was sacrificed the following week. The mass was located in the vicinity of the right salivary gland and measured 38 mm × 26 mm × 16 mm in gross size. It was a firm whitish mass, with a cut surface that was also whitish in appearance. Histopathologically, neoplastic cells formed glandular structures that contained secreted eosinophilic material. Ultrastructurally, similar secreted material and lipid droplets were in the cytoplasm of the neoplastic cells. Immunohistochemically, the neoplastic cells were positive for cytokeratin 8, cytokeratin 18 and estrogen receptor α. Based on these findings, the tumor was diagnosed as a mammary gland adenocarcinoma, and we therefore conclude that this tumor type can occur spontaneously in female SD rats as young as 12 weeks of age.