Abstract
Chronic feeding, for up to 2 years, of a choline-deficient, L-amino acid-defined (CDAA) diet results in a high incidence of hepatocellular carcinomas (HCCs) in male rats through endogenous mechanisms. Its morphological course consists of sequentially developed (pre)neoplastic and nonneoplastic liver lesions, and its underlying mechanisms are associated with oxidative stress, multiple signaling alterations, and genetic and epigenetic changes of specific genes. These characteristics are closely similar to those seen in human hepatocarcinogenic cases including those due to an infection of hepatitis viruses. Furthermore, this has recently been recognized as a good animal model for human nonalcoholic steatohepatitis (famous as NASH). The present study was conducted as a part of our work to explore details of molecular events occurring in this model with our aim to obtain information contributable to the control of human cancers, especially HCC, a deadly human neoplasm. Male Fischer 344 rats (6 weeks old) were fed the CDAA diet or a control diet for up to 70 weeks and serially sacrificed to obtain livers. Using the livers obtained up to the end of week 12, mRNA and protein expressions of 18 cytokines were assessed by a ribonuclease protection assay and an immunohistochemical technique, respectively. Using the livers obtained at the end of week 70, comprehensive gene expression profiles were assessed for HCCs, their surrounding tissues and the normal tissues by an oligonucleotide microarray technique for 3757 genes. All assessments were conducted using at least 5 samples per group from individual animals. mRNAs of the cytokines including IL-1-alfa, IL-1-beta, TNF-alfa, TGF-beta-1, TGF-beta-2, and TGF-beta-3 were overexpressed in differentially time-dependent manners in the early phase within the first 12 weeks. At the end of week 12, proteins of such cytokines were also overexpressed. IL-1-alfa and TNF-alfa proteins were overexpressed more remarkably in preneoplastic liver lesions than in their surroundings. IL-1-beta and TGF-beta-1 proteins behaved similarly, but the differences between inside and outside of preneoplastic lesions were less evident. TGF-beta-2 protein was overexpressed less remarkably in preneoplastic liver lesions than in their surroundings. TGF-beta-3 protein behaved similarly, but the difference between inside and outside of preneoplastic lesions was less evident. Comparing profiles among HCCs, their surroundings and normal liver tissues, 147 genes were differentially expressed, which were classified according to the expression patterns into 4 identical clusters by different methods. These contained conspicuous changes for notable genes interacting each other within a bio-signaling complex. Such genes included transcription factors (e.g., hepatic nuclear factor-1 [Tcf1], hepatic nuclear factor-4 and Rheb), signaling factors (e.g., IL-1 receptor, IL-2 receptor, PDGF receptor, beta-, NGF, FGF-5, FGF-9, and COX-1), apoptosis and cell proliferation regulatory factors (e.g., caspase 3, inhibin, lifeguard, c-fos, src-related tyrosine kinase, and Tsc2), pre-mRNA processing factors (e.g., cyclin L and zinc finger protein 265), metabolic enzymes (e.g., CYP1B1) and anti-oxidant defense system members (e.g., phospholipase C and superoxide dismutase). The present results indicate that altered expression of numerous gene products is involved as a complex in the endogenous hepatocarcinogenesis of rats fed the CDAA diet.