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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....

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The expression patterns of the liver-enriched transcription factors CCAAT/enhancer-binding protein (C/EBP) alpha and beta and hepatocyte nuclear factor (HNF)-1 and HNF-4 were studied in liver nodules and hepatocellular carcinomas from male rats treated according to the resistant hepatocyte (RH) model. C/EBP alpha expression was lower at the transcriptional, mRNA, and protein levels in persistent nodules than in the respective surrounding livers. Expression was further decreased in the tumors. Transcriptional downregulation of C/EBP alpha gene expression was observed already in very early nodules, isolated 3 wk after partial hepatectomy in the RH model. However, no detectable changes were observed in preneoplastic nodules in the transcription or in steady-state mRNA levels of C/EBP beta, HNF-1, and HNF-4. A slight decrease in C/EBP beta protein and a more pronounced attenuation of HNF-1 and HNF-4 levels was observed in nodules, being 67%, 37%, and 46% of the levels in the corresponding surrounding livers, respectively. In conclusion, differential regulation of several transcription factors that are associated with the maintenance of the differentiated state of the hepatocytes was observed in preneoplastic and neoplastic liver lesions. This could have an impact on the regulation of a wide array of genes during liver carcinogenesis. Furthermore, the attenuation of C/EBP alpha expression, regarded as a negative growth regulator, could contribute to the proliferative advantage of nodules during liver carcinogenesis.

Sewage effluents contain pharmaceuticals, personal care products and industrial chemicals, exposing aquatic organisms to complex mixtures. The consequences of exposure to combinations of different classes of drugs in fish are largely unknown. In this study, we exposed adult zebrafish (Danio rerio) males and females for two weeks to low, environmentally relevant concentrations of the endocrine disrupting chemical 17α-etinylestradiol (EE) and the selective serotonin re-uptake inhibitor (SSRI) citalopram, alone and in combination, and analyzed behaviors of importance for population fitness, scototaxis (light/dark preference), the novel tank test and shoal cohesion. Control water contained 0.4ng/L EE and the measured exposure concentrations were 0.9ng/L EE (nominal 0.1) and 1ng/L EE (nominal 0.5). The measured concentrations of citalopram were 0.1 (nominal 0.1) and 0.4μg/L (nominal 0.5). Both EE exposures increased anxiety in males in the scototaxis test, with significantly longer latency periods before entering and fewer visits to the white zone of the tank. The combined exposures (0.9ng/L EE+0.1μg/L citalopram and 1ng/L EE+0.4μg/L citalopram) resulted in abolishment of effects of EE, with shorter latency period and more transitions to white than for fish exposed to EE alone. In the novel tank test, the results surprisingly indicated lower anxiety after both EE and citalopram exposure. Significantly more transitions to the upper half of the tank observed in males exposed to 0.1μg/L citalopram alone compared to control males. Males exposed to EE (0.9ng/L) had shorter latency period to the upper half. Combination exposure resulted in a longer latency and fewer transitions to the upper half compared to both control, EE- and citalopram-exposed males. Males exposed to the combination spent significantly less time in the upper half than males EE or citalopram-exposed males. Females exposed to 1ng/L EE had fewer transitions to the upper half than the control group and females exposed to 0.4μg/L citalopram. In the shoaling test, males exposed to 0.1μg/L citalopram+0.9ng/L EE showed more transitions away from peers than males exposed to 0.1μg/L citalopram alone. In conclusion, low concentrations of EE, closely above the predicted no effect concentration (NOEC) of 0.1ng/L, created anxiety-like behavior in zebrafish males. Citalopram showed marginal effects at these low concentrations but in the combination exposure the behavioral effects of EE were abolished. This is an initial effort to understand the effects of cocktails of anthropogenic substances contaminating aquatic environments.