The effects of dietary 2-acetylaminofluorene (2-AAF) on cell cycle-related proteins was studied in regenerating livers from male Wistar rats. The levels of cyclins, cyclin dependent kinases (cdks), and related proteins were studied at different times during the first cell cycle after partial hepatectomy (PH). The frequency of proliferation cell nuclear antigen (PCNA)-positive nuclei, a marker of S phase progression, was almost zero during the first 27 hours after PH in the mitoinhibited 2-AAF-treated rats, while about 50% of the nuclei were labeled 24 hours after PH in control animals. Accordingly, Western blot tests showed markedly elevated PCNA protein levels from 18 hours to the end of S phase in untreated animals but no upregulation in response to 2-AAF. Compared with control animals, animals treated with 2-AAF showed increased levels of cdk 4 and cyclin D 3 from 12 and 15 hours after PH, respectively, and altered cyclicity in cyclin D 3 expression. No effects on cyclin E were observed, while the increase in cdk 2 levels in control animals during late G 1 /S (15-27 hours) was abolished by 2-AAF. p53 was induced by 2-AAF treatment during the same period, with a peak at 24 hours. The protein detected with p21 antibodies was highly expressed in unstimulated hepatocytes in control animals, and further increased by 2-AAF. The expression was sustained until 15 hours after PH in control rats while 2-AAF-treated animals lacked detectable protein during this period; however, a transient increase was observed at 21 hours. Thus, 2-AAF affects several parameters of cell cycle regulation of possible relevance for its inhibitory effects on hepatocyte proliferation in vivo. (HEPATOLOGY 1998;27:691-696.)Many liver tumor promoters such as 2-acetylaminofluorene (2-AAF), phenobarbital, orotic acid, clofibrate and ethinylestradiol have inhibitory effects on proliferation of hepatocytes in vivo during long-term treatment, 1-5 which is believed to be an important aspect of the promotion mechanism. In the classical resistant hepatocyte model (RH-model) for rat liver carcinogenesis, where promotion is performed by dietary 2-AAF treatment combined with a partial hepatectomy (PH), the selective growth advantage of preneoplastic foci during treatment has been suggested to be caused by the inhibited proliferation of hepatocytes surrounding the lesions, combined with focal resistance to 2-AAF-induced inhibition. 1 This makes the putatively preneoplastic foci, in contrast to the surrounding tissue, able to respond to the proliferative stimulus provided by the PH. The mitoinhibitory effects of 2-AAF are caused by the formation of sulfated metabolites of N-hydroxyacetylaminofluorene, 6 which is low in the lesions compared with the surrounding hepatocytes. 7 These sulfated metabolites give rise to two different Nacetylated adducts causing major distortion of the DNA structure, and lead to an efficient DNA replication block both in vitro and in vivo. 6,[8][9] The molecular mediators of the inhibitory signals generated by 2-AAF are not known....

Previous studies have indicated that isolated preneoplastic rat hepatocytes in vitro fail to induce nuclear p53 protein and fail to block replication in response to genotoxic compounds. This suggests that defects in the protection of genomic integrity are part of their premalignant character. In the present study, we have investigated if similar defects occur in vivo. Preneoplastic glutathione-S-transferase (GST) 7-7-positive foci were induced in male Wistar rats by diethylnitrosamine (DEN) initiation and promotion with 2-acetylaminofluorene (2-AAF)/partial hepatectomy (PH). The response to genotoxic damage was studied by X-irradiation. p53 protein was moderately expressed in nuclei in surrounding hepatocytes. This nuclear p53 staining had decreased 2 weeks after 2-AAF treatment. In foci, the protein was detected in the cytoplasm whereas the nuclei were negative. Levels of p21 waf1/cip1 protein were high in nuclei and cytoplasm of surrounding hepatocytes, whereas the expression in foci was low. A low level of Mdm2 in nuclei was observed in surrounding liver, while both Mdm2 and Bcl-2 protein were strongly expressed in the cytoplasm in foci. X-ray exposure further induced nuclear expression of p53, p21 waf1/cip1 , and Mdm2 in surrounding hepatocytes, but focal nuclei were still negative. DNA replication was strongly reduced by X-irradiation in surrounding hepatocytes, but only partially reduced in the foci. These results indicate that the p53 pathway of response to genomic stress is impaired in preneoplastic Over the last decades the process of experimental hepatocarcinogenesis in rats has been intensively studied (reviewed in Schulte-Hermann 1 and Pitot 2 ). Early in the process, preneoplastic cells emerge, which are able to proliferate during exposure to cytotoxic/genotoxic tumor promoters 3 while phenotypically normal cells are blocked in their growth. 4 Changes in expression of several proteins have been related to the premalignant nature of the preneoplastic cells, such as increased expression of glutathione-S-transferase (GST) 7-7, p-glycoprotein, and ␥-glutamyltranspeptidase, and decreased expression of a sulfotransferase and alterations in cytochrome P450s and c-myc expression. [5][6][7][8][9][10][11][12] The molecular mechanisms of their escape from growth inhibition as normally seen in hepatocytes after exposure to genotoxic compounds are, however, not clear.The tumor suppressor p53 plays an important role in maintaining the integrity of the genome. 13 Wild-type p53 protein blocks the cell cycle in G1 after DNA damage and may stimulate DNA repair. [13][14][15][16] The p53 protein is mutated in over 50% of the human tumors indicating that loss of p53 is an important event in tumor development. 13,14 We have shown earlier that compounds with hepatic tumor promoting activity are able to induce p53 in rat liver in vivo, 17,18 and that cells from preneoplastic foci in vitro and in vivo express less nuclear p53 compared with surrounding hepatocytes during treatment with tumor promoters 19 (Ohlson et al., subm...

Synthetic estrogens act as tumor promoters in rat liver. Because estrogen treatment markedly increases the secretion of pituitary prolactin, also shown to be a tumor promoter in rat liver, the possibility of a pituitary influence in estrogen promotion was investigated in Wistar rats. In diethylnitrosamine (DEN)-initiated hypophysectomized (hx) female rats, 24 weeks of ethinyl estradiol (EE) administration (500 microg/kg/d, intraperitoneally) did not increase the number of hepatocyte nodules and did not induce hepatocellular carcinoma (HCC) in a 2-year study. Very few placental forms of glutathione-S-transferase (GST-P)-positive foci were observed at the end of EE administration. Estrogen receptor (ER) messenger RNA (mRNA) levels in hx females were 20% of the levels in intact females. EE administration (range, 160-210 microg/kg/d, subcutaneous release pellets) to DEN-initiated intact males and females increased the number and size of hepatocyte foci. A significant increase in HCC frequency was observed in EE-treated females compared with females receiving sham-release pellets, and the latency period for HCC induction was decreased by EE in both males and females. Inhibition of prolactin (PRL) secretion by bromocriptine (Brc) (ParlodelLAR, slow intramuscular release vehicles) during EE treatment decreased the number of foci without affecting their size and markedly prolonged the latency period in both sexes. EE treatment also significantly increased the expression of c-myc, and c-jun, enhanced the levels of growth hormone receptor (GHr) mRNA in females and the levels of ER mRNA in males and "feminized" the expression of the GH-regulated genes cytochrome P450 (CYP), 2C11, CYP 2C12, and GHr in male liver. Brc administration decreased the mRNA levels of the female-predominant CYP 2C12 in EE-treated males but otherwise had no effects. In conclusion, a decreased promotive effect of EE was obtained by decreasing the PRL levels, indicating that estrogens exert at least part of their promotion effects indirectly, by increasing the levels of pituitary PRL.

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