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).

Promotion of thyroid carcinogenesis by para-aminobenzoic acid in rats initiated with N-bis(2-hydroxypropyl)nitrosamine.

Sulfonamide analogues of para-aminobenzoic acid (PABA), a precursor of folate synthesis, have beneficial effects as antifolate, but thyroid peroxidase inhibition has been reported as a side effect that results in promotion of rat thyroid carcinogenesis. In the present study, effects of PABA itself on F344 rat thyroid carcinogenesis after initiation with N-bis(2-hydroxypropyl)nitrosamine (DHPN) were evaluated. In experiment 1, rats in groups 1-4 received a single subcutaneous injection of DHPN at 2800 mg/kg, and groups 5 and 6 received vehicle saline alone. From 1 week after DHPN initiation, rats in groups 2, 3, 4, and 6 were fed basal diet containing 0.25%, 0.5%, 1.0%, and 1.0% PABA, respectively, for 40 weeks. Rats in groups 1 and 5 received basal diet alone throughout the experiment. The final incidence of thyroid follicular cell adenomas and adenocarcinomas was significantly (p < 0.05 or 0.01) increased in groups 3 and 4 as compared to group 1. No thyroid tumors were found in groups 5 and 6. In experiment 2, animals in group 1 were fed basal diet alone, while groups 2 and 3 were given 0.5% and 1.0% PABA in the diet, respectively, for 2 weeks. Thyroid weights in group 3, and serum thyroid stimulating hormone level and proliferative activity of follicular cells in groups 2 and 3 were significantly (p < 0.05 or 0.01) elevated. In addition, the serum thyroxine level in group 3 was significantly (p < 0.05) depressed. These results clearly indicate that PABA exerts promotion/progression effects on rat thyroid carcinogenesis as a result of hypothyroidism followed by negative-feedback via the thyroid-pituitary axis.

Ca2+-ATPase inhibitor induces IL-4 and MCP-1 production in RBL-2H3 cells.

The effects of a Ca2(+)-ATPase inhibitor, cyclopiazonic acid (CPA), and two hydroquinone-antioxidants, 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ) and 2,5-di-(tert-amyl)-1,4-hydroquinone (DTAHQ) on the release of IL-4 and MCP-1 from RBL-2H3 cells were investigated. CPA, DTBHQ and DTAHQ, all of which induce intracellular free Ca2+ concentration ([Ca2+]i) increase, induced IL-4 and MCP-1 release in a dose-dependent manner. The release of TNF-alpha required both a Ca2(+)-ATPase inhibitor and 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the Ca2(+)-ATPase inhibitors induced IL-4 and MCP-1 production without TPA. The release of IL-4 and MCP-1 reached a maximum at 9 and 6 h, respectively. IL-4 and MCP-I release was inhibited by treatment with the immunosuppressant FK-506 and actinomycin D. Therefore, in our system IL-4 and MCP-1 release involves Ca2(+)-dependent and FK-506-sensitive signaling pathways. This is the first report about Th-2 type cytokine and chemokine production in RBL-2H3 cells.

Casein kinase II-like ectokinase activity on RBL-2H3 cells.

We studied the properties of the ectokinase activity on the outer cell surfaces of RBL-2H3 cells and examined the phosphorylation of exogenous substrates to clarify the substrate specificity of the ectokinases on RBL-2H3 cells. Among the several protein substrates tested, casein was the most strongly phosphorylated with [gamma-32P]ATP, and the net incorporation of 32P into casein was 0.65 pmol P/50 microg/10(6) cells. Casein kinase II peptide was also phosphorylated with [gamma-32P]ATP. The phosphorylation of casein and casein kinase II peptide was almost completely inhibited by the addition of 3 microg/ml of cell-impermeable K252b. Phosphorylation of casein and casein kinase II peptide was also observed by [gamma-32P]GTP. Western blot analysis using anti-casein kinase II antibody revealed a 44-kDa casein kinase band in the membrane fraction and Fc epsilonRI complexes. The immunofluorescence microscopic analysis using anti-casein kinase II antibody showed the existence of casein kinase II on the surface of the cells. This is the first report about the existence of ectokinase on mast cells.

Quantitative changes in glycosaminoglycans in the lungs of rats exposed to diesel exhaust.

Exposure to diesel exhaust (DE) induces lesions in lung epithelium by generation of reactive oxygen species. Glycosaminoglycans (GAG), components of extracellar matrix, are thought to play important roles in cell proliferation and differentiation in the repair process of injured tissue. We investigated how GAG are related to the recovery of lung tissue from injury. Using high-performance liquid chromatography analysis, we determined the amounts of GAG, such as chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronan (HA) in the lungs of rats exposed to DE for 4 weeks at concentrations of 0.3 or 3 mg/m(3) as suspended particulate matter, or to filtered air. The contents of CS and HA in the surroundings of the bronchi were significantly increased after exposure to DE. In addition, immunohistochemical staining showed that the number of 8-hydroxydeoxyguanosine-positive cells as a marker of cell damage, and proliferating cell nuclear antigen-positive cells also increased in the same areas in which the levels of GAG were elevated in the lungs of rats exposed to 3 mg/m(3) DE. These results suggest that CS and HA in the lung contribute to cell proliferation and remodeling in the process of recovery from injury caused by exposure to DE.

Effect of Ca(2+) ATPase inhibitors on MCP-1 release from bone marrow-derived mast cells and the involvement of p38 MAP kinase activation.

The effect of two Ca(2+) ATPase inhibitors, cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ), on the release of MCP-1 from bone marrow-derived mast cells (BMMCs) were investigated. CPA and DTBHQ increased the intracellular free Ca(2+) concentration ([Ca(2+)](i)) and induced MCP-1 release in a dose-dependent manner. These Ca(2+) ATPase inhibitors induced MCP-1 release in the absence of phorbol ester, in contrast to their induction of TNF-alpha. MCP-1 release reached a maximum at 6-9 h. It was inhibited by treatment with actinomycin D, the immunosuppressant cyclosporin A, and the cytosolic Ca(2+) chelator BAPTA-AM. Furthermore, RT-PCR showed a time-dependent increase of MCP-1 mRNA. Thus MCP-1 release seems to depend on Ca(2+)-dependent transcriptional activation. MCP-1 release was dose-dependently inhibited by the p38 MAP kinase inhibitor SB202190, but not by the p44/42 MAP kinase inhibitor PD98059. Therefore, transcriptional activation of MCP-1 production and its release seem to be dependent on the nuclear factor of activated T cells and p38 MAP kinase activation. This is the first report to show the regulation of MCP-1 production in BMMCs.

Ca2+-ATPase inhibitors and PKC activation synergistically stimulate TNF-alpha production in RBL-2H3 cells.

OBJECTIVE AND DESIGN: To investigate the effect of Ca2+-ATPase inhibitors on the production of TNF-alpha in rat basophilic leukemia (RBL-2H3) cells. MATERIAL: Two Ca2+-ATPase inhibitors, thapsigargin (TG) and cyclopiazonic acid (CPA), and three hydroquinone-antioxidants, 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ), 2,5-di-(tert/amyl)-1,4-hydroquinone (DTAHQ), 2-(tertbutyl)-1,4-hydroquinone (MTBHQ) were used. TREATMENT: Cells were treated with TG, CPA, DTBHQ, DTAHQ and MTBHQ for 3 h in the presence of 12-Otetradecanoylphorbol-13-acetate (TPA) and released TNF-alpha from the cells was measured (n > or = 4). RESULTS: All Ca2--ATPase inhibitors (TG, CPA, DTBHQ and DTAHQ) induced TNF-alpha release in a dose-dependent manner. TNF-alpha release was inhibited by treatment with protein kinase C inhibitors (staurosporine, Ro31-8220, calophostin C) (p < or = 0.05). In contrast, MTBHQ, which does not induce increases in [Ca2+]i, did not induce the release of TNF-alpha. TNF-alpha release induced by DTBHQ and CPA was inhibited by treatment with actinomycin-D, the immunosuppressant FK506 and the glucocorticoid dexamethasone (p < or = 0.01). CONCLUSIONS: These results suggest 1) that [Ca2+]i increase and subsequent activation of protein kinase C is necessary for the release of TNF-alpha, and they work synergistically, 2) that the TNF-alpha release induced by Ca2+-ATPase inhibitors can be regulated at the transcriptional level.