Jun-ichi Onose

Evaluation of toxicity of green tea catechins with 90-day dietary administration to F344 rats

Green tea catechins (GTC), polyphenols extracted from the stalks and leaves of Camellia sinensis, are found in the different types of tea beverages and as antioxidant additives to many foods, snacks, fats and fatty oils. As a part of their safety assessment, subchronic toxicity was investigated in male and female F344 rats with dietary administration at concentrations of 0 (control), 0.3%, 1.25% and 5.0% for 90 days. The average daily intakes of GTC in each group were 180, 764 and 3525mg/kg body weight/day, respectively for males, and 189, 820 and 3542mg/kg body weight/day, respectively for females. No mortality or obvious clinical signs were observed throughout the experimental period but body weights were reduced from week 1 to the end of the experiment in 5.0% males. In serum biochemistry, alanine transaminase and alkaline phosphatase in 5.0% males and females and aspartate transaminase in 5.0% females were increased, together with the relative liver weights in both sexes receiving 5.0%. Although decreases were evident for total cholesterol in 0.3-5.0% males and triglycerides in 1.25% and 5.0% males and 5.0% females, these changes were not considered to be adverse. Hematology and histopathological observation revealed no GTC-related toxicological changes. Based on above findings, the no observed adverse effect level (NOAEL) of GTC was estimated to be 1.25% (764mg/kg body weight/day for males and 820mg/kg body weight/day for females).

Expeditious Synthesis of Vialinin B, an Extremely Potent Inhibitor of TNF-α Production

A first total synthesis of vialinin B, a powerful inhibitor (IC(50) 20 pM) of TNF-alpha production, is described. The key reactions include a double Suzuki-Miyaura coupling of electron-rich aryl bromide with a couple of phenylboronic acids, a Cu-mediated Ullmann reaction, and a LHMDS-promoted phenylacetylation. This synthesis proceeded in 11 steps with 18% overall yield from a known sesamol derivative.

Sequential analysis of development of invasive thyroid follicular cell carcinomas in inflamed capsular regions of rats treated with sulfadimethoxine after N-bis (2-hydroxypropyl) nitrosamine-initiation

A 2-stage thyroid follicular carcinogenesis model in rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN) is widely used to detect modifying effects of chemicals on thyroid carcinogenesis. A number of goitrogens are known to strongly promote carcinogenesis, and the carcinomas often originate adjacent to the thyroid capsule and show invasive growth into the capsule or adjacent tissues. To clarify mechanisms of progression to invasive carcinomas, we sequentially evaluated histopathological and immunohistochemical characteristics of thyroids in male F344 rats treated with sulfadimethoxine (SDM, 0.1% in drinking water) for 0—10 weeks beginning 1 week after DHPN initiation (2800 mg/kg body weight, single s.c. injection). In DHPN-SDM-treated rats, multiple focal hyperplasias and adenomas developed in thyroid follicular parenchyma at weeks 4 to 6. Apart from the proliferative lesions, capsular thickening with inflammatory cell infiltration, mainly consisting of macrophages, and migration of follicular epithelium into the capsule were also observed. Focal hyperplasias/adenomas adjacent to the capsule progressively developed to invasive carcinomas at weeks 6 to 10. In thyroid parenchyma, malignant lesions were seldom observed. With SDM-treatment alone, although no neoplastic lesions were observed, capsular thickening with inflammation and epithelial migration resulted in intracapsular residual follicles. Intracapsular residual follicular cells as well as invasive and intrathyroidal carcinoma cells generally showed increased cell proliferative activity, coincidental with cytoplasmic/nuclear positivity for β-catenin. These results suggested that β -catenin activation related to capsular inflammation may play a role in development of invasive carcinomas but is insufficient for tumor formation by itself. Whether this is associated with mutations in the β-catenin gene remains to be clarified.

Rapid induction of colorectal tumors in rats initiated with 1,2-dimethylhydrazine followed by dextran sodium sulfate treatment.

To establish a rapid bioassay system with neoplastic end-points for detection of colorectal carcinogenesis modifiers, we evaluated the effects of dextran sodium sulfate (DSS) treatment on the different stages of carcinogenesis in rats initiated with 1,2-dimethylhydrazine (DMH). F344 male rats were given three subcutaneous injections of DMH (40 mg/kg body weight) in a week, and were administered drinking water containing 1.0% DSS ad libitum either during or after the initiation period for a week, or both during and after initiation periods for 2 weeks. At the 10th week of the experiment, although the numbers of aberrant crypt foci were significantly decreased in all groups treated with DSS and given DMH-initiation as compared with DMH alone, dysplastic foci/adenomas/adenocarcinomas were increased. The incidences and multiplicities of these lesions were highest in rats treated with DSS after DMH-initiation period. At the 26th week, the incidences of adenocarcinomas (100 vs. 20% in DMH alone) and their multiplicities (6.6 +/- 0.8/rat vs. 0.2 +/- 0.4/rat in DMH alone) were also highest in this group. These results indicate that short-term DSS-treatment in the post-initiation period significantly accelerates DMH-induced colorectal tumor development in rats, so that this protocol may effective for establishment of a rapid bioassay system with neoplastic end-points.

Structural Revision of Thelephantin G by Total Synthesis and the Inhibitory Activity against TNF-α Production

This paper describes the total synthesis of thelephantin G, thus revising the proposed structure 1 to 2. The key steps involved a double Suzuki-Miyaura coupling and an esterification reaction. By a similar strategy, ganbajunins D and E (3 and 4) were also prepared. Compound 2 strongly inhibited TNF (tumor necrosis factor)-alpha production in rat basophilic leukemia (RBL-2H3) cells: IC(50) = 3.5 nM, while a mixture of 1 and its regioisomer 15 showed no such activity.

Effects of Peanut-Skin Procyanidin A1 on Degranulation of RBL-2H3 Cells

Peanut skin contains large amounts of polyphenols having antiallergic effects. We found that a peanut-skin extract (PSE) inhibits the degranulation induced by antigen stimulation of rat basophilic leukemia (RBL-2H3) cells. A low-molecular-weight fraction from PSE, PSEL, also had inhibitory activity against allergic degranulation. A main polyphenol in PSEL was purified by gel chromatography and fractionated by YMC-gel ODS-AQ 120S50 column. Electrospray ionization mass spectrometry (ESI-MS) analysis of the purified polyphenol gave m⁄z 599 [M+Na]+. Based on the results of 1H-NMR, 13C-NMR spectra, and optical rotation analysis, the polyphenol was identified as procyanidin A1. It inhibited the degranulation caused by antigen stimulation at the IC50 of 20.3 μM. Phorbol-12-myristate-13-acetate (PMA) and 2,5,-di(tert-butyl)-1,4-hydroquinone (DTBHQ)-induced processes of degranulation were also inhibited by procyanidin A1. These results indicate that peanut-skin procyanidin A1 inhibits degranulation downstream of protein kinase C activation or Ca2+ influx from an internal store in RBL-2H3 cells.

Development of invasive follicular cell carcinomas in a rat thyroid carcinogenesis model : Biological impact of capsular inflammation and reduced cyclooxygenase-2 expression

We have previously reported that thyroid capsular inflammation induced by sulfadimethoxine (SDM), a goitrogen, might play a role in development of invasive follicular cell adenocarcinomas in rats initiated with N‐bis(2‐hydroxypropyl)nitrosamine (DHPN). The present study was designed to examine the role of cyclooxygenase (COX)‐2, widely known to be up‐regulated in inflammatory states, during chemically induced rat thyroid carcinogenesis. Male F344 rats received a subcutaneous DHPN (2800 mg/kg) injection, and 1 week later were allowed free access to drinking water containing antithyroidal propylthiouracil (PTU, 0.003%) or SDM (0.1%) for 4 or 10 weeks. Control groups receiving goitrogen alone and no treatment were also included. At week 4, diffuse follicular cell hyperplasia was induced in all PTU‐ and SDM‐treated groups, along with fibrous capsular thickening and capsular thickening with inflammation, respectively. Additionally, multiple focal follicular cell hyperplasias and adenomas were observed in the DHPN + PTU and DHPN + SDM cases. At week 10, adenocarcinomas invasive to the capsule and restricted to the capsular adjacent region, were frequent in the DHPN + SDM group, but not observed in the animals given DHPN + PTU. Western blots and immunohistochemistry revealed constitutive COX‐2 expression in non‐neoplastic follicular cells of the control and all of the PTU‐ and SDM‐treated rats. However, COX‐2 reactivity was significantly reduced or negative in the preneoplastic/neoplastic lesions in the DHPN‐treated groups. In fibrous or inflamed thickened capsules, only a few component cells with inflammatory elements were positive for COX‐2, and there was no significant difference in this regard between the PTU and SDM treatments. The present results suggest that capsular inflammation could play a role in development of invasive carcinomas, but COX‐2 expression does not make a major contribution. (Cancer Sci 2005; 96: 31–38)

Vialinin A is a ubiquitin-specific peptidase inhibitor.

Vialinin A, a small compound isolated from the Chinese mushroom Thelephora vialis, exhibits more effective anti-inflammatory activity than the widely used immunosuppressive drug tacrolimus (FK506). Here, we show that ubiquitin-specific peptidase 5/isopeptidase T (USP5/IsoT) is a target molecule of vialinin A, identified by using a beads-probe method. Vialinin A inhibited the peptidase activity of USP5/IsoT and also inhibited the enzymatic activities of USP4 among deubiquitinating enzymes tested. Although USPs are a member of thiol protease family, vialinin A exhibited no inhibitions for other thiol proteases, such as calpain and cathepsin.

Structural elucidation and synthesis of vialinin C, a new inhibitor of TNF-α production.

A new inhibitor of TNF-α production (IC50=0.89 μM) named vialinin C (1) was isolated from dry fruiting bodies of an edible Chinese mushroom, Thelephora vialis. The structure of 1 was determined by high-resolution MS, NMR spectroscopic analysis, and confirmed by synthesis. Synthesis of ganbajunin B (5) obtained from the same origin was also described.

Evaluation of subchronic toxicity of dietary administered Cry1Ab protein from Bacillus thuringiensis var. Kurustaki HD-1 in F344 male rats with chemically induced gastrointestinal impairment

Bacillus thuringiensis (Bt) proteins are developed for genetically modified crops and the Bt proteins demonstrate no evidence of toxicity by the oral route in traditional animal models. However, the possible toxicity of Bt proteins under conditions of reduced gastric acid secretion and/or small intestinal damage has not been investigated. In the present study, we therefore evaluated following four F344 rat groups with a purified Bt protein Cry1Ab from B. thuringiensis var. Kurustaki HD-1. Gastrointestinal impairment (GI) alone and GI+Bt protein fed (GI+Bt) groups were given i.p. injections of famotidine to reduce gastric acid secretion twice a day at 30mg/kg body weight in weeks 2 and 4. GI and GI+Bt groups were additionally fed diets containing 80ppm indomethacin for induction of intestinal damage during weeks 1 and 3. Bt alone and GI+Bt groups were also fed diet containing Bt protein Cry1Ab at a concentration of 10ppm in weeks 2 and 4. A no treatment control group was also included. At the end of week 4, all animals were euthanized under ether anesthesia, blood samples were collected for hematology and serum biochemistry and a complete necropsy was performed. No significant changes indicative of toxicity of the Bt protein Cry1Ab used here were noted with any of the parameters investigated. In conclusion, no significant toxicological effects were detected in this subchronic gastrointestinal impairment rat model.