Recently the idea of hormesis, a biphasic dose-response relationship in which a chemical exerts opposite effects dependent on the dose, has attracted interest in the field of carcinogenesis. With non-genotoxic agents there is considerable experimental evidence in support of hormesis and the present review highlights current knowledge of dose-response effects. In particular, several in vivo studies have provided support for the idea that non-genotoxic carcinogens may inhibit hepatocarcinogenesis at low doses. Here, we survey the examples and discuss possible mechanisms of hormesis with cytochrome P450 inducers, such as phenobarbital, 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), α-benzene hexachloride (α-BHC), and other non-genotoxins. Epigenetic processes differentially can be affected by agents that impinge on oxidative stress, DNA repair, cell proliferation, apoptosis, intracellular communication and cell signaling. Non-genotoxic carcinogens may target nuclear receptors, cause aberrant DNA methylation at the genomic level and induce post-translational modifications at the protein level, thereby impacting on the stability or activity of key regulatory proteins, including oncoproteins and tumor suppressor proteins. Via multiple epigenetic lesions, non-genotoxic carcinogens can elicit a variety of changes contributing to cellular carcinogenesis.

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Cell proliferation activity and correlation between the LI (labeling index) of BrdU (5-bromo-2'-deoxy-uridine) and the expression of the Ki-67 antigen or PCNA (proliferating cell nuclear antigen) were evaluated by immunohistochemistry in 18 canine spontaneous neoplasms. Spearman's rank correlation coefficient and linear regression analysis revealed a significant correlation between the BrdU LI and Ki-67 LI, BrdU LI and PCNA LI, and BrdU LI and MI (mitotic index) in each case [r_s = 0.917, P < 0.01 (BrdU LI vs. Ki-67 LI), r_s = 0.736, P < 0.01 (BrdU LI vs. PCNA LI), r_s = 0.748, P < 0.01 (BrdU LI vs. MI) by Spearman's rank correlation coefficient; and r = 0.918, P < 0.01 (BrdU LI vs. Ki-67 LI), r = 0.626, P < 0.01 (BrdU LI vs. PCNA LI), r = 0.763, P < 0.01 (BrdU LI vs. MI) by linear regression analysis]. Ki-67 LIs were more closely correlated with BrdU LIs than PCNA LIs or MIs, and the Ki-67 antigen is useful as an endogenous proliferation marker in canine neoplasms.

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The present study was conducted as a preliminary evaluation of toxicologic and carcinogenic risks of copper gluconate and to determine its optimal dose for use in an upcoming study utilizing the medium-term multi-organ carcinogenesis protocol. Male BrlHan:WIST@Jcl (GALAS) rats were initially treated with N-nitrosodiethylamine, N-methylnitrosourea, 1,2-dimethylhydrazine, N-butyl-N-(4-hydroxybutyl)-nitrosamine and 2,2'-dihydroxy-di-n-propylnitrosamine for a total multiple initiation period of 4 weeks. In addition, rats were fed a diet containing copper gluconate at 0, 1000, 3000, 4800, 6000, or 12000 ppm from the commencement for 13 weeks. While no systemic toxicity was detected, hepatic granulomas were observed at copper gluconate doses of 6000 ppm, or greater. Hepatic copper accumulation and metallothionein induction were demonstrated at doses of 3000 ppm and 1000 ppm, respectively, or greater. Putative preneoplastic lesions in the liver appeared to increase at a dose of 12000 ppm, and 8-hydroxydeoxyguanosine formation was enhanced at doses of 6000 ppm, or greater. These results suggest that copper gluconate exerts effects on the liver at the relatively low dietary dose of 1000 ppm and causes hepatic injury at 6000 ppm. The hepatotoxicity and possible hepatocarcinogenicity of copper gluconate may be attributable to oxidative stress due to the impairment of hepatic copper metabolism.

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Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation and migration, and protects cells from apoptosis. It interacts with at least three specific G protein-coupled transmembrane receptors, LPA1, LPA2 and LPA3. In the present study, the involvement of LPA receptor genes in the development of rat tumors was evaluated by measuring mRNA expression of the LPA1, LPA2 and LPA3 genes in nitroso-compound induced lung adenocarcinomas and hepatocellular carcinomas (HCCs) in rats. Total RNA was extracted from 12 lung adenocarcinomas and 6 HCCs, and expression analysis was performed by real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR). In lung adenocarcinomas, the expressions of LPA2 and LPA3 were significantly increased (p < 0.001 and p < 0.005, respectively) compared with normal lung tissue. In contrast, LPA1 showed a lower level of expression, but the difference was not significant. In HCCs, the expression of LPA2 was significantly increased (p < 0.05) compared with normal liver tissue. In contrast, varying levels of LPA1 and LPA3 expression were detected. The ratio of LPA2/LPA1, which is important for understanding the effects of LPA on target cells than the levels of LPA1 and LPA2 separately, increased markedly in both lung (p<0.005) and liver tumors (p<0.005). These results suggest that aberrant expressions of LPA receptors may be involved in the development of lung and liver tumors in the rat.

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Pulmonary masses were observed in two aged Beagles maintained for a long period without any treatment. In the histopathology in both cases, the poorly demarked masses were composed of neoplastic cells proliferating in a sheet-like fashion within the alveolar lumen. These cells showed moderate to marked pleomorphism and atypia and had abundant eosinophilic to foamy cytoplasm. Phagocytosis and multinucleated giant cells were occasionally observed. Similar lesions with pulmonary infiltrates were not noted in any of the observed tissues in both cases. In the immunohistochemistry, the neoplastic cells were positive for vimentin, lysozyme and alpha-1-antitrypsin, but not for cytokeratin or lymphocytic markers such as CD3 or CD79a. The tumor cells also showed lectin binding affinities such as concanavalin A and ricinus communis agglutinin-1. These findings indicated that these neoplastic cells were derived from a histiocytic lineage. Therefore, we diagnosed them as pulmonary histiocytic sarcomas.

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A tumor was observed in the left mandible of a 3-year-old male Shih-Tzu dog. Histopathology revealed neoplastic alveolar proliferation of round cells, sarcomatoid proliferation of round to spindle-shaped cells and an existing parotid gland tissue around the tumor. A cancellous bone framework was also seen throughout the tumor. The tumor cells in the sarcomatoid area were positive and weakly positive for vimentin and cytokeratin, respectively, demonstrating their myoepithelial character. We diagnosed this tumor as a malignant mixed tumor of the salivary glands, because of the location of the tumor, its invasiveness into the surrounding tissue and the identification of both epithelial and myoepithelial tumor cells.

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Connexin32 (Cx32), one of the components of gap junction intercellular communication, is commonly detected by the fluorescent antibody method, although this method has some defects when conducting detailed histopathological investigations. In order to develop a new method to improve the fluorescent antibody method, the optimal immunohistochemical procedure on formalin-fixed paraffin-embedded sections was examined. According to the results, the localization of Cx32 was observed on sections prepared with the Dako catalyzed signal amplification (CSA) method after 0.2% protease digestion for 30 minutes after a 3-day or 6-month fixation. As the CSA method is highly sensitive and based on signal amplification by biotinylated tyramide, the localization of Cx32 was clearly shown on the cell membranes of hepatocytes. In fact, by this CSA method, a decrease in the signals of Cx32 was shown in the liver of rats treated with phenobarbital or in hepatocellular adenoma, which were consistent with previous reports using cryosections. In conclusion, the CSA method is considered an appropriate method for the detection of Cx32 in formalin-fixed paraffin-embedded sections.

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