-We previously found that administration of ascorbic acid (AA) enhances the liver tumorpromoting activity of kojic acid (KA) in mice. To examine the reproducibility of these results in rats and the underlying mechanism of this effect, we employed a two-stage liver carcinogenesis model using male F344 rats. Two weeks after initiation with diethylnitrosamine (DEN), the animals received a diet containing 2% KA and drinking water with or without 5,000 ppm AA for a period of 7 weeks. A DENalone group was also established as a control. One week after the commencement of the administration, the animals were subjected to two-thirds partial hepatectomy. At the end of the experiment, the livers were analyzed immunohistochemically, and the mRNA expression level and extent of lipid peroxidation were measured. AA treatment enhanced the KA-induced tumor-promoting activity in terms of the number and area of liver cell foci that were positive for glutathione-S-transferase placental form. AA coadministration increased the number of hepatocytes positive for proliferating cell nuclear antigen and inversely decreased the number of TUNEL-positive cells. However, the increased level of thiobarbituric acid reactive substances resulting from KA treatment was suppressed by coadministration of AA. Gene expression analyses using low-density microarrays and real-time RT-PCR showed that coadministration of AA resulted in upregulation of genes related to cell proliferation and downregulation of those involved in apoptosis and/or cell cycle arrest. These results indicate that the concerted effects of AA on cell proliferation and apoptosis/cell cycle arrest probably through its antioxidant activity are involved in this enhancement.
To determine the threshold dose of piperonyl butoxide (PBO) that induces hepatocellular tumor-promoting effects, reactive oxygen species (ROS) generation, and drug-metabolizing enzymes that protect against ROS generation, partial hepatectomized rats were fed diets containing 0, 0.015, 0.03, 0.06, 0.125, 0.25, or 0.5% PBO after an i.p. injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Histopathologically, Glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in a dose-dependent manner in rats given 0.25% PBO or higher. The formation of microsomal ROS in the liver was significantly increased in 0.25 and 0.5% PBO. Real-time RT-PCR showed that the expression of the CYP1A1, UDPGTr-2, and Mrp3 genes was significantly upregulated in rats given 0.03% PBO or higher. These results suggest that 0.25% is the threshold dose of PBO that induces ROS-mediated hepatocarcinogenesis in rats, although the CYP1A1 gene that is related to ROS generation and the UDPGTr-2 and Mrp3 genes that are involved in protection against ROS were induced in the livers of rats even at a PBO dose of 0.03%.
ABSTRACT. We report a case of mixed epithelial and stromal tumor of the kidney (MESTK) in a 32-week-old heterozygous sphingosine 1-phosphate-2 (S1P2) receptor deficient female mouse. A white solid mass replacing the left kidney was observed at the left retroperitoneal wall. Histologically, the tumor mass consisted of dimorphic cellular components of epithelial and stromal cells. Epithelial cells formed various sized irregular-shaped tubular structures resembling renal tubules surrounded by stromal cells. Immunohistochemically, epithelial cells were positive for cytokeratin, while stromal cells showed positive immunoreactivity with α-smooth muscle actin as well as vimentin. Based on the morphological and immunohistochemical findings, this tumor was diagnosed as a MESTK. KEY WORDS: N-ethyl-N-nitrosourea, renal mixed epithelial and stromal tumor, S1P2 deficient mouse.J. Vet. Med. Sci. 70(5): 483-485, 2008 This report describes a case of mixed epithelial and stromal tumor of the kidney (MESTK) in a female heterozygous sphingosine 1-phosphate-2 (S1P2) deficient C57BL/6N mouse of 32 weeks old. S1P2 is a member of the G proteincoupled receptor family (formerly called the EDG family) that binds and mediates a variety of actions of sphingosine-1-phosphate, a plasma-derived pleiotropic sphingolipid mediator. A heterozygous S1P2-deficient female mouse which received a single subcutaneous injection of 120 mg/ kg body weight of N-ethyl-N-nitrosourea (ENU) at 6 weeks of age was subjected to complete necropsy on 32 weeks of age. Until necropsy, this mouse did not show any abnormal clinical signs. At necropsy, a white solid mass (15 mm × 10 mm × 10 mm) replacing the left kidney without involving the surrounding tissues was observed at the left retroperitoneal wall. While 3 groups of mice including wild type mice (n=14), heterozygous S1P2 deficient mice (n=9), and homozygous S1P2 deficient mice (n=8), all given ENU, were allocated, such a renal tumor was not observed in the other ENU-treated mice. On cut section, a mass was white, firm and mottled with hemorrhagic foci. No other particular pathologic abnormalities were observed in this mouse. The mass was fixed in 10% neutral buffered formalin and processed for routine histopathology with hematoxylin and eosin. Periodic acid-Schiff (PAS) staining was also applied on this tumor. As immunohistochemical analysis, avidinbiotin-peroxidase complex (ABC) technique was applied using antibodies against α-smooth muscle actin (monoclonal mouse antibody, clone 1A4; DAKO, Glostrup, Denmark), cytokeratin (monoclonal mouse antibody, clone MFN116; DAKO), desmin (monoclonal mouse antibody, clone D33; DAKO), estrogen receptor (monoclonal mouse antibody, clone 6F11; Novocastra Laboratories, Newcastle upon Tyne, UK), vimentin (polyclonal goat antibody, product no.C-20, Santa Cruz Biotechnology, Santa Cruz, CA) and WT-1 (polyclonal rabbit antibody, product no.C-19, Santa Cruz Biotechnology). These slides were lightly counterstained with hematoxylin. Appropriate positive and negative control tissu...
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