Carcinogenesis Advance Access originally published online on January 19, 2008
Carcinogenesis 2008 29(3):638-646; doi:10.1093/carcin/bgm303
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Genetic and epigenetic changes in rat preneoplastic liver tissue induced by 2-acetylaminofluorene
Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA
1 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
2 Toxicologic Pathology Associates, National Center for Toxicological Research, Jefferson, AR 72079, USA
* To whom correspondence should be addressed. Tel: +1 870 543 7096; Fax: +1 870 543 7720;Email: igor.pogribny{at}fda.hhs.gov
Genotoxic carcinogens, including 2-acetylaminofluorene (2-AAF), in addition to exerting their genotoxic effects, often cause a variety of non-genotoxic alterations in cells. It is believed that these non-genotoxic effects may be indispensable events in tumorigenesis; however, there is insufficient knowledge to clarify the role of carcinogens in both the genetic and epigenetic changes in premalignant tissues and a lack of conclusive information on the link between epigenetic alterations and carcinogenic exposure. In the current study, we investigated whether or not the mechanism of 2-AAF-induced hepatocarcinogenesis consists of both genotoxic (genetic) and non-genotoxic (epigenetic) alterations. Male and female Sprague–Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 6, 12, 18 or 24 weeks. The levels of DNA adducts obtained from 2-AAF in liver and kidney tissues were assessed by high-performance liquid chromatography combined with electrospray tandem mass spectrometry (HPLC-ES-MS/MS). N-(Deoxyguanosine-8-yl)-2-aminofluorene was the major adduct detected at all time points in both tissues. Global DNA methylation in the livers and kidneys, as determined by an HpaII-based cytosine extension assay and by HPLC-ES-MS/MS, did not change over the 24-week period. In the livers of male rats, there was a progressive decrease of global and long interspersed nucleotide element-1-associated histone H4 lysine 20 trimethylation, as well as hypermethylation of the p16INK4A gene. These epigenetic changes were not observed in the livers of female rats or the kidneys of both sexes. Importantly, morphological evidence of formation and progression of neoplastic process was observed in the liver of male rats only. In conclusion, we have demonstrated that exposure of rats to genotoxic hepatocarcinogen 2-AAF, in addition to formation of 2-AAF-specific DNA lesions, resulted in substantial alterations in cellular epigenetic status.
Abbreviations: 2-AAF, 2-acetylaminofluorene; dG-C8-AF, N-(deoxyguanosine-8-yl)-2-aminofluorene; DNMT1, DNA methyltransferase 1; GST-P, glutathione-S-transferase placental form; HPLC-ES-MS/MS, high-performance liquid chromatography combined with electrospray tandem mass spectrometry; H4K20me3, histone H4 lysine 20 trimethylation; H3K9me3, histone H3 lysine 9 trimethylation; LINE-1, long interspersed nucleotide element-1; ORF1, open reading frame 1; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; ROP dG-C8-AF, oxidation products of N-(deoxyguanosine-8-yl)-2-aminofluorene
These authors contributed equally to this work. Received October 10, 2007; revised December 13, 2007; accepted December 20, 2007.
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