Expression of inducible nitric oxide (NO) synthase but not prevention by its gene ablation of hepatocarcinogenesis with fibrosis caused by a choline-deficient, L-amino acid-defined diet in rats and mice.

Expression of inducible nitric oxide synthase (iNOS) and effects of iNOS gene ablation on the hepatocarcinogenesis associated with fibrosis caused by a choline-deficient, L-amino acid-defined (CDAA) diet, were examined in male F344 rats and C57BL/6J wild-type and iNOS-/- mice. Western blot, RT-PCR and immunohistochemical analyses revealed increased expression of iNOS protein and mRNA in the livers of rats and wild-type mice fed a CDAA diet for 12-80 weeks, associated with elevated serum NO(x) and liver nitrotyrosine levels. iNOS-/- mice demonstrated greater liver injury and fibrosis in the early stage than their wild-type counterparts, but this did not significantly affect the incidence and multiplicity of altered foci, adenomas and hepatocellular carcinomas in spite of immunohistochemical iNOS expression in these lesions. Results suggested no major determinant roles of the expressed iNOS in the development of liver tumors caused by the CDAA diet.

Expression of receptor for advanced glycation end products during rat tongue carcinogenesis by 4-nitroquinoline 1-oxide and effect of a selective cyclooxygenase-2 inhibitor, etodolac.

OBJECTIVES: The expression of a receptor for advanced glycation end products (RAGE) in lesions developed during rat tongue carcinogenesis by 4-nitroquinoline 1-oxide (4-NQO) and the effect of a selective cyclooxygenase-2 inhibitor, etodolac, were investigated. METHODS: The tongue lesions were induced in Fischer 344 rats given 20-30 ppm 4-NQO in their drinking water for 12 weeks and then fed the basal diet containing 150 and 300 ppm of etodolac for 16 weeks, respectively. RESULTS: The incidence of carcinomas in the 4-NQO-alone group was 100%. Etodolac significantly reduced the incidences of carcinomas to 66.7% (p < 0.05) and 50% (p < 0.01) at doses of 150 and 300 ppm, respectively. RAGE protein was immunohistochemically expressed in dysplastic cells and cancer cells of dysplasias and carcinomas. Etodolac significantly decreased the stainability of RAGE protein in dysplasias (p < 0.02) and carcinomas (p < 0.01). The expression of RAGE mRNA analyzed by RT-PCR was clearly detected in carcinomas developing in the 4-NQO-alone group. In carcinomas developing in the etodolac-treated group, RAGE mRNA expression significantly decreased (p < 0.03). CONCLUSIONS: These results indicate that RAGE is involved in rat tongue carcinogenesis by 4-NQO and suggest that the chemopreventive effect exerted by etodolac is partly related to the inhibition of RAGE expression.

Inhibitory effects of selective cyclooxygenase-2 inhibitors, nimesulide and etodolac, on the development of squamous cell dysplasias and carcinomas of the tongue in rats initiated with 4-nitroquinoline 1-oxide.

The present study was conducted to examine the effects of selective cyclooxygenase (COX)-2 inhibitors, nimesulide and etodolac, on early stages of tongue carcinogenesis due to 4-nitroquinoline 1-oxide(4-NQO). Fischer 344 rats, 6 weeks old, were given 15 ppm of 4-NQO in their drinking water for 8 weeks followed by diet containing either nimesulide or etodolac at the doses of 150 and 300 ppm for 16 weeks. Rats were sacrificed at 24 weeks and tongue lesions were histologically examined. Nimesulide dose-dependently reduced the incidence and multiplicity of squamous cell dysplasias and carcinomas (SCCs), with significance at the 300 ppm dose. This suppression was associated with an increased incidence and multiplicity of hyperplasias. Etodolac exhibited similar but less extensive suppressive effects. The results suggest that COX-2 is involved in the progression of hyperplasia to dysplasia and from dysplasia to SCCs.

Increased expression of cyclooxygenase-2 protein during rat hepatocarcinogenesis caused by a choline-deficient, L-amino acid-defined diet and chemopreventive efficacy of a specific inhibitor, nimesulide.

Expression of cyclooxygenase (COX)-2 protein during rat hepatocarcinogenesis associated with fatty change, fibrosis, cirrhosis and oxidative DNA damage, caused by a choline-deficient, L-amino acid-defined (CDAA) diet were investigated in F344 male rats, along with the chemopreventive efficacy of the specific COX-2 inhibitor, nimesulide (NIM). Nimesulide, which was administered in the diet at concentrations of 200, 400, 600 and 800 p.p.m. for 12 weeks, decreased the number and size of preneoplastic enzyme-altered liver foci, levels of oxidative DNA damage, and the grade and incidence of fibrosis in a dose-dependent manner. A preliminary long-term study of 65 weeks also revealed that 800 p.p.m. NIM decreased the multiplicity of neoplastic nodules and hepatocellular carcinomas and prevented the development of cirrhosis. Western blot analysis revealed that COX-2 protein was barely expressed in control livers and increased approximately 2.9-fold in the livers of rats fed on a CDAA diet for 12 weeks and approximately 4.5-5.4-fold in tumors, with a diameter larger than 5 mm, at 80 weeks. Immunohistochemically, COX-2 protein was positive in sinusoidal and stromal cells in fibrotic septa, which were identified by immunoelectron microscopy as Kupffer cells, macrophages, either activated Ito cells or fibroblasts, after exposure to the CDAA diet for 12 weeks, whereas it was only occasionally weakly positive in sinusoidal, probably Kupffer, cells in control livers. In neoplastic nodules in rats fed on a CDAA diet for 30 and 80 weeks, sinusoidal cells and cells with relatively large round nuclei and scanty cytoplasm were strongly positive for COX-2 protein, with the neoplastic hepatocytes in the minority of the nodules, but not the cancer cells, being moderately positive. These results clearly indicate that rat hepatocarcinogenesis, along with fatty change, fibrosis and cirrhosis, is associated with increased expression of COX-2 protein, and point to the chemopreventive efficacy of a selective COX-2 inhibitor against, at least, the early stages of hepatocarcinogenesis.

Development of hepatocellular adenomas and carcinomas associated with fibrosis in C57BL/6J male mice given a choline-deficient, L-amino acid-defined diet.

Development of hepatocellular carcinomas in rats caused by a choline-deficient, L-amino acid-defined (CDAA) diet, usually associated with fatty liver, fibrosis, cirrhosis and oxidative DNA damage, has been recognized as a useful model of hepatocarcinogenesis caused by endogenous factors. In the present study, in order to further explore involved factors and genes, we established an equivalent model in spontaneous liver tumor-resistant C57BL/6J mice. Six-week-old males and females were continuously fed the CDAA diet and histological liver lesions and oxidative DNA damage due to 8-hydroxydeoxyguanosine (8-OHdG) were examined after 22, 65 and 84 weeks. In male mice, fatty change and fibrosis were evident at 22 weeks, and preneoplastic foci of altered hepatocytes were seen at an incidence of 8/8 (100%) and a multiplicity of 6.6 +/- 4.0 per mouse at 65 weeks. Hepatocellular adenomas and carcinomas developed at incidences of 16/24 (66.7%) and 5/24 (20.8%), and multiplicities of 1.42 +/- 1.32 and 0.29 +/- 0.62, respectively, at 84 weeks. The female mice exhibited resistance to development of these lesions. The CDAA diet also increased 8-OHdG levels in male but not female mice. These results indicate that a CDAA diet causes hepatocellular preneoplastic foci, adenomas and carcinomas associated with fibrosis and oxidative DNA damage in mice, as in rats, providing a hepatocarcinogenesis model caused by endogenous factors in mice.