Relationship: 342



Increased, Induced Mutations in Critical Genes leads to Increased, Proliferation/Clonal Expansion of Mutant Cells (Pre-Neoplastic Lesions/Altered H

Upstream event


Increased, Induced Mutations in Critical Genes

Downstream event


Increased, Proliferation/Clonal Expansion of Mutant Cells (Pre-Neoplastic Lesions/Altered H

Key Event Relationship Overview


AOPs Referencing Relationship


Taxonomic Applicability


Sex Applicability


Life Stage Applicability


Key Event Relationship Description


There is no direct evidence addressing AFB1 induced critical gene mutations and the subsequent induction of AHF. However, in general the progression from chemical exposure to pre-neoplastic lesions appears to include six general mechanisms: 1) decrease in the activity of the Keap1/Nrf2/ARE pathway; 2) decrease in p53 function leading to increased survival in the presence of genomic instability; 3) changes in the tumor microenvironment within cells; 4) alterations in apoptosis; 5) changes in Wnt/b-catenin signaling, and 6) gene expression changes that may be related to cancer promotion/progression (Caballero et al., 2004; Ma and He, 2012; Gross-Steinmeyer and Eaton, 2012; Liby et al., 2008; Yates and Kensler, 2007; Liby and Sporn, 2012; Ikeda et al., 2004; Harida et al., 2004; Kwak et al, 2001; Honda et al., 2011; Shelton and Jaiswal, 2013; Johnson et al., 2014; Fabregat, 2009). In addition to these six possible mechanisms, epigenetic changes in DNA methylation may occur and may be related to the progression of cancer (Zhang et al., 2003, 2006, 2012; Wu et al, 2013; Pogribney et al., 2011).

Evidence Supporting this KER


Biological Plausibility


AFB1 induces G:C to T:A transversions. The third nucleotide in codon 249 of the p53 gene is a guanine and is a preferred binding target for AFB1 metabolites. In humans, some evidence exists that this codon is a mutational hot spot in liver cancers in people exposed to AFB1. (Puisieux et al., 1991). Although there is a large literature describing mutations in this location in humans and the corresponding p53 codon 247 in rodents, whether these mutations by themselves actually lead to tumors is unknown. (Lee et al., 1998) In vitro data collected from two human cell lines suggest that doses of AFB1 sufficient to induce DNA damage also induce an incomplete cell cycle checkpoint response, likely involving p53; this suggests a potential mechanism that might ultimately lead to tumor formation in vivo.(Gursoy-Yuzugullu et al., 2011). In rats, Ki-ras codon 12 mutations observed after AFB1 dosing may also be associated with hyperplastic foci to a greater extent than in hepatocellular carcinomas (HCCs), suggesting some change in the mutational spectrum during tumor development. (Tokusashi et al., 1994)

In general, the exact origin of cells that form altered hepatic foci (AHF) is not known with certainty. (Sell, 1993, 2002; Sell and Leffert, 2008). Four types of cells in the liver are thought to be capable of becoming tumor cells: 1) mature hepatocytes, 2) bile duct progenitor cells, 3) ductular bipolar progenitor cells, and 4) periductular stem cells. Pre-neoplastic foci and HCCs likely form from the last two cell types.[1] In rats, pre-neoplastic foci expressing the placental form of GST appear to be descended from hepatocytes. (Bralet et al., 2002; Avril et al., 2004; Gournay et al., 2002).

However, Sell (2008) has criticized the hypothesis the HCC arise by de-differentiation of hepatocytes and suggested the cellular origin of HCC may be liver stem cells undergoing an arrest of maturation. Trosko (2014) has considered de-differentiation in light of one of the emerging “Hallmarks of Cancer”—alterations of cellular metabolism—and argues that the likelihood of cancer arising through de-differentiation is vanishingly small. (Hanahan and Weinberg, 2011; Trosko and Kang, 2012; Trosko, 2013, 2014). The questions of the origin of the progenitor cells that replace hepatocytes after liver injury and the origin of liver cancer stem cells are the subjects of intense research. (Gong et al., 2013; Jia et al., 2013; Miyajima et al., 2014; Rehman et al., 2014; Shin and Kaestner, 2014; Tanimizu and Mitaka, 2014). The cyotokine IL-6 may be involved in the transformation of hepatic progenitor cells into cancer stem cells. (Dang et al., 2014; He et al., 2013). In addition, Dragan et al. (1994) observed that the increase in BrdU labeling, indicating cell proliferation, occurred to a greater extent in altered hepatic foci (AHF).

There is no direct evidence addressing AFB1 induced critical gene mutations and the subsequent induction of AHF. However, in general the progression from chemical exposure to pre-neoplastic lesions appears to include six general mechanisms: 1) decrease in the activity of the Keap1/Nrf2/ARE pathway; 2) decrease in p53 function leading to increased survival in the presence of genomic instability; 3) changes in the tumor microenvironment within cells; 4) alterations in apoptosis; 5) changes in Wnt/β-catenin signaling, and 6) gene expression changes that may be related to cancer promotion/progression (Miyajima et al., 2014; Gong et al., 2013; Jia et al., 2013; Dang et al., 2014; He et al., 2013; Caballero et al., 2004; Ma and He, 2012; Gross-Steinmeyer and Eaton, 2012; Liby et al., 2008; Yates et al., 2007; Liby and Sporn, 2012; Ikeda et al., 2004; Haridas et al., 2004). In addition to these six possible mechanisms, epigenetic changes in DNA methylation may occur and may be related to the progression of cancer (Kwak et al., 2001; Honda et al., 2011; Shelton and Jaiswal, 2013; Fabregat, 2009; Zhang et al., 2003).

The following is an overview of these 6 general biological processes that appear to be involved in the progression of mutant cells (whether occurring as the result of direct chemical induction or increased due other events such as cell proliferation and clonal expansion of spontaneously occurring mutant cells) to AHF and pre-neoplastic lesions.

Decrease in p53 function A defective p53 gene may contribute to telomere dysfunction. Continuous hepatocyte renewal may be associated with telomere shortening and chromosomal instability [2-4]. In transgenic mice, telomere dysfunction enhanced tumor initiation but interfered with tumor progression/promotion. However, p53 mutation leading to inactivation of the gene, in combination with telomere dysfunction and increased hepatocyte turnover, may increase the risk of HCC (Zhang et al., 2006, 2012; Wu et al., 2013; Pogribny et al., 2011; Farazi et al., 2003).

The Tumor Microenvironment Intracellular signaling mechanisms are involved in the creation of tumor microenvironment. (Hanahan and Weinberg, 2011). These include alterations in cellular circuits governing proliferation, differentiation, motility, and viability. The x gene is one of the four genes in the Hepatitis B Virus (HBV) genome that is capable of trans-activating cellular genes to contribute to the development of a tumor microenvironment (Brechot et al., 2000, 2010). The x gene can be expressed through stable transfection into LE/6 cells, a cell line derived from hepatic oval cells harvested from rats fed a choline-deficient diet. When LE/6 cells that were transfected with HBx and injected into nude mice administered a single dose of 240 μg/kg AFB1 by IP injection, or in the drinking water continuously for 16 weeks, the mice developed HCC from these rat liver stem cells, indicative of the stem cell-origin of HCC and suggesting that HBV and x expression may foster a tumor microenvironment.

TGF-β has a dual effect on hepatocytes—inhibiting proliferation and inducing apoptosis. Experiments using TGF-β null mutant mice show that this signal is required for normal development and appears to be involved in destruction of damaged cells in the liver. TGF-β 1 also mediates interferon-induced apoptosis in AHF in rats. In rats, AFB1 altered biological processes in hepatic stem-like cells related to cell motion, cell adhesion, immune response, and signal transduction (Yang et al., 2014). Reduced levels of TGF-β produce an increase in E-cadherin and reduce cellular motility, one of the intracellular networks associated with the cancer hallmark of invasion and metastasis (Fransvea et al., 2008).

Effects on Apoptotic Signaling TGF-β normally inhibits growth and proliferation and induces apoptosis of damaged cells. In primary rat hepatocytes and a cell line (NRML) derived from these primary rat cells, TGF-β inhibited cell proliferation measured by DNA synthesis and the ability to form colonies. In vitro treatment of NRML cells with AFB1 blocked the TGF-β-mediated inhibition of cell growth and proliferation (McMahon et al., 1986). AFB1 transformed cells were also resistant to the growth-inhibiting effects of tumor necrosis factor-alpha (TNF-α) (Chapekar et al., 1989). However, BL9 cells, derived from rat hepatocytes transformed with reactive metabolites if activated AFB1, retained the sensitivity to the inhibitory effects of TGF-β on cell growth.

The product of the p53 gene mutated at codon 249 may itself impair the apoptotic process. In Hep3B human hepatoma cells, transfection with mutant p53 (p53mt249) increased transcription of insulin-like growth factor II (IGF-II). Wild-type p53 inhibited binding of the TATA-box binding protein (TBP) and the Sp1 transcription factors to the IGF-II promoter, possibly by binding to this promoter. However, p53mt249 increased the formation of transcription complexes, possibly due to lack of binding to the promoter. Chang liver cells show characteristics of hepatocytes; transfection with p53mt249 or addition of IGF-II blocked apoptosis mediated by TNF-α or the HBV x gene (Lee et al., 2000).

Adult tree shrews were administered AFB1 at 150 μg/kg/d five times weekly for 105 weeks. Interim sacrifices were performed every 30 weeks to observe liver histopathology, specifically immunohistochemical staining for p53, bcl-2, and bax, proteins in the p53 pathway that leads to apoptosis (Duan et al., 2005). Immunohistochemistry was also conducted to visualize survivin, a protein that acts directly on caspases to inhibit the activity of caspase-3 and caspase-7 to block the terminal pathway of apoptosis (Cheung et al., 2013). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to detect apoptotic cells.

Bcl-2 and bax were observed in the livers of less than 10% of treated animals at 30 weeks. This result seems somewhat paradoxical because these proteins are intermediates in the p53 apoptotic pathway; however, the determinant of apoptotic progression may be the bcl-2 / bax ratio (Korsmeyer et al., 1993). The actions of survivin occur much later in the apoptotic pathway and this protein was detected in 40% of livers. Also, the apoptotic index measured by TUNEL was increased significantly at 30 weeks (Duan et al., 2005).

Wnt/β-catenin Signaling Wnt/β-catenin signaling is a key determinant of liver cell proliferation and eventual differentiation to mature hepatocytes or cholangiocytes. This signaling cascade is generally inactive in healthy adult liver save for a small periportal region of a hepatic lobule. Hepatocytes in this region have constitutively active β-catenin that in turn regulates expression of genes associated with ammonia and xenobiotic metabolism (Monga, 2014). An increase in Wnt/β-catenin signaling appears to play a role in the development of hepatocellular cancers, and mutations in the β-catenin gene have been observed in HCCs from humans (Laurent-Puig and Zucman-Rossi, 2006; Devereux et al., 2001). In areas of high AFB1 exposure in China, β-catenin was overexpressed in a higher proportion of liver samples from HCC patients than in Malaysia where AFB1 exposure is lower (Chen Ban et al., 2004).

Paradoxically, AFB1 appears to increase microRNA-33a, which in turn down-regulates β-catenin (Fang et al., 2013). However, this study was conducted in human cancer cell lines and the signaling pathways in such cells may be considerably different than they are in vivo.

Genomic Changes in Response to AFB1 Administration of AFB1 to rat hepatic stem-like WB-F344 cells derived from oval cells produced changes in pathways related to TGF-β signaling, Wnt signaling, MAPK signaling, regulation of the cytoskeleton, cell adhesion, changes in energy metabolism, and ECM-receptor interaction (Yang et al., 2014). A number of these can be conceptually linked to the hallmarks of cancer (Hanahan and Weinberg, 2011). In another study, the AFB1-induced transcriptome, measured by RNA-Seq with increased resolution and sensitivity was compared to microarrays for pathways with differentially expressed genes, such as glutathione metabolism, cell cycle dysregulation, cancer pathways, Nrf2-mediated oxidative stress, and ten various intermediary and amino acid metabolism pathways (Merrick et al., 2013).

Empirical Evidence


In rats administered AFB1 then treated with oltipraz, decreases in the frequency of gamma-glutamyl transferase positive (GGT+) foci, the number of foci observed, and the size of the foci were all demonstrated, as well as decreased tumor incidence one to two years later (Roebuck et al., 1991). In rats treated with a single dose of 5 mg/kg AFB1, a progressive increase in the number and size of tigroid cell foci and neoplastic nodules was observed over a two-year period following dosing. Within these foci and nodules, the mitotic index was 100-fold higher than in the surrounding liver parenchyma (Bannasch et al., 1985).

Uncertainties and Inconsistencies


There is no direct information as to whether AFB1 induced mutations in critical genes leads to cellular proliferation and clonal expansion of mutant cells. It is known, however that the Hepatitis B virus plays a large role in the development of HCC in many endemic AFB1 regions, and viral infection is thought to interact with AFB1 exposure increasing the possibility that individual cells progress down the pathway to tumor formation. It seems reasonable that this infection may contribute to increased cell proliferation which could contribute to more clonal expansion (and, therefore, an increase in tumor formation) of AFB1-induced critical cancer gene mutant cells. It might also contribute to inflammation and increased cell proliferation which could result in an increased number of spontaneously mutant cells containing mutants in cancer critical genes (such as p53) thus also resulting in HCC (via a non-mutagenic MOA). This interaction between AFB1 and viral infection has motivated much research (Chittmittrapap et al., 2013; Farazi et al., 2006; Gouas et al., 2010; Kew 2003).

Quantitative Understanding of the Linkage


While studies have been conducted to quantitate clonal expansion and pre-neoplastic foci following exposure to AFB1, there are no studies that evaluate the quantitative relationship between cells carrying critical cancer gene mutations and clonal expansion/pre-neoplastic foci. This is primarily due to the lack of appropriate techniques. The chemoprevention data, however, indicate that the relationship between induced mutations in critical genes and AHF is likely not a linear one; additional data, such as with ACB-PCR techniques (Parsons et al., 2010), collected in a carefully designed study, could address this.

Response-response Relationship




Known modulating factors


Known Feedforward/Feedback loops influencing this KER


Domain of Applicability


In mammals, the identification of AHF as a pre-neoplastic lesion has been recognized for many years as part of the general etiology of HCC (Beer and Pitot, 1987; Dragan et al., 1995; Goldsworthy et al., 1986; Pitot et al., 1990; Sargent et al., 1989). In rats, the presence of AHF with altered growth characteristics has been observed in a number of studies (Bannasch et al., 1985; Fischer, 1986; Fischer et al., 1987; Gil et al., 1988; Godlewski et al., 1985; Manson et al., 1984; Newberne, 1976; Nishizumi et al., 1977; Roebuck et al., 1991; Youngman and Campbell, 1992). The mechanisms involved in the formation of AHF appear to be generalizable across animals and likely apply to fish and birds as well as mammals.



Andersen, M.E., Preston, R.J., Maier, A., Willis, A.M. & Patterson, J., 2014, Dose-response approaches for nuclear receptor-mediated modes of action for liver carcinogenicity: Results of a workshop, Crit Rev Toxicol, 44(1), pp. 50-63.

Ansher, S.S., Dolan, P. & Bueding, E., 1986, Biochemical effects of dithiolthiones, Food Chem Toxicol, 24(5), pp. 405-15.

Avril, A., Pichard, V., Bralet, M.P. & Ferry, N., 2004, Mature hepatocytes are the source of small hepatocyte-like progenitor cells in the retrorsine model of liver injury, J Hepatol, 41(5), pp. 737-43.

Bannasch, P., Benner, U., Enzmann, H. & Hacker, H.J., 1985, Tigroid cell foci and neoplastic nodules in the liver of rats treated with a single dose of aflatoxin B1, Carcinogenesis, 6(11), pp. 1641-8.

Beer, D.G. & Pitot, H.C., 1987, Biological markers characterizing the development of preneoplastic and neoplastic lesions in rodent liver, Arch Toxicol Suppl, 10, pp. 68-80.

Besaratinia, A., Kim, S.I., Hainaut, P. & Pfeifer, G.P., 2009, In vitro recapitulating of TP53 mutagenesis in hepatocellular carcinoma associated with dietary aflatoxin B1 exposure, Gastroenterology, 137(3), pp. 1127-37, 1137.e1-5.

Bralet, M.P., Pichard, V. & Ferry, N., 2002, Demonstration of direct lineage between hepatocytes and hepatocellular carcinoma in diethylnitrosamine-treated rats, Hepatology, 36(3), pp. 623-30.

Brechot, C., Kremsdorf, D., Soussan, P., Pineau, P., Dejean, A., Paterlini-Brechot, P. & Tiollais, P., 2010, Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC): molecular mechanisms and novel paradigms, Pathol Biol (Paris), 58(4), pp. 278-87.

Bréchot, C., Gozuacik, D., Murakami, Y. & Paterlini-Bréchot, P., 2000, Molecular bases for the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), Semin Cancer Biol, 10(3), pp. 211-31.

Budinsky, R.A., Schrenk, D., Simon, T., Van den Berg, M., Reichard, J.F., Silkworth, J.B., Aylward, L.L., Brix, A., Gasiewicz, T., Kaminski, N., Perdew, G., Starr, T.B., Walker, N.J. & Rowlands, J.C., 2014, Mode of action and dose-response framework analysis for receptor-mediated toxicity: The aryl hydrocarbon receptor as a case study, Crit Rev Toxicol, 44(1), pp. 83-119.

Caballero, F., Meiss, R., Gimenez, A., Batlle, A. & Vazquez, E., 2004, Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis, Int J Exp Pathol, 85(4), pp. 213-22.

Chao, H.K., Tsai, T.F., Lin, C.S. & Su, T.S., 1999, Evidence that mutational activation of the ras genes may not be involved in aflatoxin B(1)-induced human hepatocarcinogenesis, based on sequence analysis of the ras and p53 genes, Mol Carcinog, 26(2), pp. 69-73.

Chapekar, M.S., Huggett, A.C. & Thorgeirsson, S.S., 1989, Growth modulatory effects of a liver-derived growth inhibitor, transforming growth factor beta 1, and recombinant tumor necrosis factor alpha, in normal and neoplastic cells, Exp Cell Res, 185(1), pp. 247-57.

Chen Ban, K., Singh, H., Krishnan, R. & Fong Seow, H., 2004, Comparison of the expression of beta-catenin in hepatocellular carcinoma in areas with high and low levels of exposure to aflatoxin B1, J Surg Oncol, 86(3), pp. 157-63.

Cheung, C.H.A., Huang, C.-C., Tsai, F.-Y., Lee, J.Y.-C., Cheng, S.M., Chang, Y.-C., Huang, Y.-C., Chen, S.-H. & Chang, J.-Y., 2013, Survivin - biology and potential as a therapeutic target in oncology, Onco Targets Ther, 6, pp. 1453-62.

Chittmittrapap, S., Chieochansin, T., Chaiteerakij, R., Treeprasertsuk, S., Klaikaew, N., Tangkijvanich, P., Komolmit, P. & Poovorawan, Y., 2013, Prevalence of aflatoxin induced p53 mutation at codon 249 (R249s) in hepatocellular carcinoma patients with and without hepatitis B surface antigen (HBsAg), Asian Pac J Cancer Prev, 14(12), pp. 7675-9.

Corton, J.C., Cunningham, M.L., Hummer, B.T., Lau, C., Meek, B., Peters, J.M., Popp, J.A., Rhomberg, L., Seed, J. & Klaunig, J.E., 2014, Mode of action framework analysis for receptor-mediated toxicity: The peroxisome proliferator-activated receptor alpha (PPARα) as a case study, Crit Rev Toxicol, 44(1), pp. 1-49.

Dang, H.T., Budhu, A. & Wang, X.W., 2014, The origin of cancer stem cells, J Hepatol, 60(6), pp. 1304-5.

Davidson, N.E., Egner, P.A. & Kensler, T.W., 1990, Transcriptional control of glutathione S-transferase gene expression by the chemoprotective agent 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz) in rat liver, Cancer Res, 50(8), pp. 2251-5.

Denissenko, M.F., Koudriakova, T.B., Smith, L., O'Connor, T.R., Riggs, A.D. & Pfeifer, G.P., 1998, The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts, Oncogene, 17(23), pp. 3007-14.

Devereux, T.R., Stern, M.C., Flake, G.P., Yu, M.C., Zhang, Z.Q., London, S.J. & Taylor, J.A., 2001, CTNNB1 mutations and beta-catenin protein accumulation in human hepatocellular carcinomas associated with high exposure to aflatoxin B1, Mol Carcinog, 31(2), pp. 68-73. Dragan, Y., Teeguarden, J., Campbell, H., Hsia, S. & Pitot, H., 1995, The quantitation of altered hepatic foci during multistage hepatocarcinogenesis in the rat: transforming growth factor alpha expression as a marker for the stage of progression, Cancer Lett, 93(1), pp. 73-83.

Dragan, Y.P., Hully, J., Crow, R., Mass, M. & Pitot, H.C., 1994, Incorporation of bromodeoxyuridine in glutathione S-transferase-positive hepatocytes during rat multistage hepatocarcinogenesis, Carcinogenesis, 15(9), pp. 1939-47.

Duan, X.-X., Ou, J.-S., Li, Y., Su, J.-J., Ou, C., Yang, C., Yue, H.-F. & Ban, K.-C., 2005, Dynamic expression of apoptosis-related genes during development of laboratory hepatocellular carcinoma and its relation to apoptosis, World J Gastroenterol, 11(30), pp. 4740-4.

Elcombe, C.R., Peffer, R.C., Wolf, D.C., Bailey, J., Bars, R., Bell, D., Cattley, R.C., Ferguson, S.S., Geter, D., Goetz, A., Goodman, J.I., Hester, S., Jacobs, A., Omiecinski, C.J., Schoeny, R., Xie, W. & Lake, B.G., 2014, Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator, Crit Rev Toxicol, 44(1), pp. 64-82.

Fabregat, I., 2009, Dysregulation of apoptosis in hepatocellular carcinoma cells, World J Gastroenterol, 15(5), pp. 513-20.

Fang, Y., Feng, Y., Wu, T., Srinivas, S., Yang, W., Fan, J., Yang, C. & Wang, S., 2013, Aflatoxin B1 negatively regulates Wnt/β-catenin signaling pathway through activating miR-33a, PloS one, 8(8), p. e73004.

Farazi, P.A., Glickman, J., Horner, J. & Depinho, R.A., 2006, Cooperative interactions of p53 mutation, telomere dysfunction, and chronic liver damage in hepatocellular carcinoma progression, Cancer Res, 66(9), pp. 4766-73.

Farazi, P.A., Glickman, J., Jiang, S., Yu, A., Rudolph, K.L. & DePinho, R.A., 2003, Differential impact of telomere dysfunction on initiation and progression of hepatocellular carcinoma, Cancer Res, 63(16), pp. 5021-7.

Fischer, G., 1986, Increased UDP-glucuronyltransferase and gamma-glutamyltranspeptidase in enzyme-altered rat liver lesions produced by low doses of aflatoxin B1, Virchows Arch B Cell Pathol Incl Mol Pathol, 51(5), pp. 443-60.

Fischer, G., Domingo, M., Lodder, D., Katz, N., Reinacher, M. & Eigenbrodt, E., 1987, Immunohistochemical demonstration of decreased L-pyruvate kinase in enzyme altered rat liver lesions produced by different carcinogens, Virchows Arch B Cell Pathol Incl Mol Pathol, 53(6), pp. 359-64.

Fransvea, E., Angelotti, U., Antonaci, S. & Giannelli, G., 2008, Blocking transforming growth factor-beta up-regulates E-cadherin and reduces migration and invasion of hepatocellular carcinoma cells, Hepatology, 47(5), pp. 1557-66.

Ghebranious, N. & Sell, S., 1998, The mouse equivalent of the human p53ser249 mutation p53ser246 enhances aflatoxin hepatocarcinogenesis in hepatitis B surface antigen transgenic and p53 heterozygous null mice, Hepatology, 27(4), pp. 967-73.

Gil, R., Callaghan, R., Boix, J., Pellin, A. & Llombart-Bosch, A., 1988, Morphometric and cytophotometric nuclear analysis of altered hepatocyte foci induced by N-nitrosomorpholine (NNM) and aflatoxin B1 (AFB1) in liver of Wistar rats, Virchows Arch B Cell Pathol Incl Mol Pathol, 54(6), pp. 341-9.

Godlewski, C.E., Boyd, J.N., Sherman, W.K., Anderson, J.L. & Stoewsand, G.S., 1985, Hepatic glutathione S-transferase activity and aflatoxin B1-induced enzyme altered foci in rats fed fractions of brussels sprouts, Cancer Lett, 28(2), pp. 151-7.

Goldsworthy, T.L., Hanigan, M.H. & Pitot, H.C., 1986, Models of hepatocarcinogenesis in the rat--contrasts and comparisons, Crit Rev Toxicol, 17(1), pp. 61-89.

Golubovskaya, V.M., Filatov, L.V., Behe, C.I., Presnell, S.C., Hooth, M.J., Smith, G.J. & Kaufmann, W.K., 1999, Telomere shortening, telomerase expression, and chromosome instability in rat hepatic epithelial stem-like cells, Mol Carcinog, 24(3), pp. 209-17.

Gong, P., Wang, Y., Zhang, J. & Wang, Z., 2013, Differential hepatic stem cell proliferation and differentiation after partial hepatectomy in rats, Mol Med Rep, 8(4), pp. 1005-10.

Gouas, D.A., Shi, H., Hautefeuille, A.H., Ortiz-Cuaran, S.L., Legros, P.C., Szymanska, K.J., Galy, O., Egevad, L.A., Abedi-Ardekani, B., Wiman, K.G., Hantz, O., Caron de Fromentel, C., Chemin, I.A. & Hainaut, P.L., 2010, Effects of the TP53 p.R249S mutant on proliferation and clonogenic properties in human hepatocellular carcinoma cell lines: interaction with hepatitis B virus X protein, Carcinogenesis, 31(8), pp. 1475-82.

Gournay, J., Auvigne, I., Pichard, V., Ligeza, C., Bralet, M.P. & Ferry, N., 2002, In vivo cell lineage analysis during chemical hepatocarcinogenesis in rats using retroviral-mediated gene transfer: evidence for dedifferentiation of mature hepatocytes, Lab Invest, 82(6), pp. 781-8.

Gross-Steinmeyer, K. & Eaton, D.L., 2012, Dietary modulation of the biotransformation and genotoxicity of aflatoxin B(1), Toxicology, 299(2-3), pp. 69-79.

Gursoy-Yuzugullu, O., Yuzugullu, H., Yilmaz, M. & Ozturk, M., 2011, Aflatoxin genotoxicity is associated with a defective DNA damage response bypassing p53 activation, Liver Int, 31(4), pp. 561-71.

Hanahan, D. & Weinberg, R.A., 2011, Hallmarks of cancer: the next generation, Cell, 144(5), pp. 646-74.

Haridas, V., Hanausek, M., Nishimura, G., Soehnge, H., Gaikwad, A., Narog, M., Spears, E., Zoltaszek, R., Walaszek, Z. & Gutterman, J.U., 2004, Triterpenoid electrophiles (avicins) activate the innate stress response by redox regulation of a gene battery, J Clin Invest, 113(1), pp. 65-73.

He, G., Dhar, D., Nakagawa, H., Font-Burgada, J., Ogata, H., Jiang, Y., Shalapour, S., Seki, E., Yost, S.E., Jepsen, K., Frazer, K.A., Harismendy, O., Hatziapostolou, M., Iliopoulos, D., Suetsugu, A., Hoffman, R.M., Tateishi, R., Koike, K. & Karin, M., 2013, Identification of liver cancer progenitors whose malignant progression depends on autocrine IL-6 signaling, Cell, 155(2), pp. 384-96.

Hoare, M., Das, T. & Alexander, G., 2010, Ageing, telomeres, senescence, and liver injury, J Hepatol, 53(5), pp. 950-61.

Honda, T., Yoshizawa, H., Sundararajan, C., David, E., Lajoie, M.J., Favaloro, F.G., Janosik, T., Su, X., Honda, Y., Roebuck, B.D. & Gribble, G.W., 2011, Tricyclic compounds containing nonenolizable cyano enones. A novel class of highly potent anti-inflammatory and cytoprotective agents, J Med Chem, 54(6), pp. 1762-78.

Hulla, J.E., Chen, Z.Y. & Eaton, D.L., 1993, Aflatoxin B1-induced rat hepatic hyperplastic nodules do not exhibit a site-specific mutation within the p53 gene, Cancer Res, 53(1), pp. 9-11.

Hussain, S.P., Schwank, J., Staib, F., Wang, X.W. & Harris, C.C., 2007, TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer, Oncogene, 26(15), pp. 2166-76.

Ikeda, H., Nishi, S. & Sakai, M., 2004, Transcription factor Nrf2/MafK regulates rat placental glutathione S-transferase gene during hepatocarcinogenesis, Biochem J, 380(Pt 2), pp. 515-21.

Jia, S.Q., Ren, J.J., Dong, P.D. & Meng, X.K., 2013, Probing the hepatic progenitor cell in human hepatocellular carcinoma, Gastroenterol Res Pract, 2013, p. 145253.

Johnson, N.M., Egner, P.A., Baxter, V.K., Sporn, M.B., Wible, R.S., Sutter, T.R., Groopman, J.D., Kensler, T.W. & Roebuck, B.D., 2014, Complete protection against aflatoxin B1-induced liver cancer with triterpenoid: DNA adduct dosimetry, molecular signature and genotoxicity threshold, Cancer Prev Res (Phila).

Kensler, T.W., Egner, P.A., Dolan, P.M., Groopman, J.D. & Roebuck, B.D., 1987, Mechanism of protection against aflatoxin tumorigenicity in rats fed 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) and related 1,2-dithiol-3-thiones and 1,2-dithiol-3-ones, Cancer Res, 47(16), pp. 4271-7.

Kensler, T.W., Egner, P.A., Trush, M.A., Bueding, E. & Groopman, J.D., 1985, Modification of aflatoxin B1 binding to DNA in vivo in rats fed phenolic antioxidants, ethoxyquin and a dithiothione, Carcinogenesis, 6(5), pp. 759-63.

Kensler, T.W., Gange, S.J., Egner, P.A., Dolan, P.M., Muñoz, A., Groopman, J.D., Rogers, A.E. & Roebuck, B.D., 1997, Predictive value of molecular dosimetry: individual versus group effects of oltipraz on aflatoxin-albumin adducts and risk of liver cancer, Cancer Epidemiol Biomarkers Prev, 6(8), pp. 603-10.

Kew, M.C., 2003, Synergistic interaction between aflatoxin B1 and hepatitis B virus in hepatocarcinogenesis, Liver Int, 23(6), pp. 405-9.

Korsmeyer, S.J., Shutter, J.R., Veis, D.J., Merry, D.E. & Oltvai, Z.N., 1993, Bcl-2/Bax: a rheostat that regulates an anti-oxidant pathway and cell death, Semin Cancer Biol, 4(6), pp. 327-32.

Kwak, M.K., Itoh, K., Yamamoto, M., Sutter, T.R. & Kensler, T.W., 2001, Role of transcription factor Nrf2 in the induction of hepatic phase 2 and antioxidative enzymes in vivo by the cancer chemoprotective agent, 3H-1, 2-dimethiole-3-thione, Mol Med, 7(2), pp. 135-45.

Laurent-Puig, P. & Zucman-Rossi, J., 2006, Genetics of hepatocellular tumors, Oncogene, 25(27), pp. 3778-86.

Lee, C.C., Liu, J.Y., Lin, J.K., Chu, J.S. & Shew, J.Y., 1998, p53 point mutation enhanced by hepatic regeneration in aflatoxin B1-induced rat liver tumors and preneoplastic lesions, Cancer Lett, 125(1-2), pp. 1-7.

Lee, Y.I., Lee, S., Das, G.C., Park, U.S. & Park, S.M., 2000, Activation of the insulin-like growth factor II transcription by aflatoxin B1 induced p53 mutant 249 is caused by activation of transcription complexes; implications for a gain-of-function during the formation of hepatocellular carcinoma, Oncogene, 19(33), pp. 3717-26.

Liby, K., Yore, M.M., Roebuck, B.D., Baumgartner, K.J., Honda, T., Sundararajan, C., Yoshizawa, H., Gribble, G.W., Williams, C.R., Risingsong, R., Royce, D.B., Dinkova-Kostova, A.T., Stephenson, K.K., Egner, P.A., Yates, M.S., Groopman, J.D., Kensler, T.W. & Sporn, M.B., 2008, A novel acetylenic tricyclic bis-(cyano enone) potently induces phase 2 cytoprotective pathways and blocks liver carcinogenesis induced by aflatoxin, Cancer Res, 68(16), pp. 6727-33.

Liby, K.T. & Sporn, M.B., 2012, Synthetic oleanane triterpenoids: multifunctional drugs with a broad range of applications for prevention and treatment of chronic disease, Pharmacol Rev, 64(4), pp. 972-1003.

Liu, Y.L., Roebuck, B.D., Yager, J.D., Groopman, J.D. & Kensler, T.W., 1988, Protection by 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) against the hepatotoxicity of aflatoxin B1 in the rat, Toxicol Appl Pharmacol, 93(3), pp. 442-51.

Ma, Q. & He, X., 2012, Molecular basis of electrophilic and oxidative defense: promises and perils of Nrf2, Pharmacol Rev, 64(4), pp. 1055-81.

Manson, M.M. & Neal, G.E., 1984, The influence of partial hepatectomy on the biphasic response of gamma glutamyl transpeptidase to aflatoxin B1, Cancer Lett, 25(1), pp. 81-7.

McMahon, J.B., Richards, W.L., del Campo, A.A., Song, M.K. & Thorgeirsson, S.S., 1986, Differential effects of transforming growth factor-beta on proliferation of normal and malignant rat liver epithelial cells in culture, Cancer Res, 46(9), pp. 4665-71.

Merrick, B.A., Phadke, D.P., Auerbach, S.S., Mav, D., Stiegelmeyer, S.M., Shah, R.R. & Tice, R.R., 2013, RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats, PloS one, 8(4), p. e61768.

Miura, N., Horikawa, I., Nishimoto, A., Ohmura, H., Ito, H., Hirohashi, S., Shay, J.W. & Oshimura, M., 1997, Progressive telomere shortening and telomerase reactivation during hepatocellular carcinogenesis, Cancer Genet Cytogenet, 93(1), pp. 56-62.

Miyajima, A., Tanaka, M. & Itoh, T., 2014, Stem/progenitor cells in liver development, homeostasis, regeneration, and reprogramming, Cell Stem Cell, 14(5), pp. 561-74.

Monga, S.P.S., 2014, Role and regulation of β-catenin signaling during physiological liver growth, Gene Expr, 16(2), pp. 51-62.

Newberne, P.M., 1976, Experimental hepatocellular carcinogenesis, Cancer Res, 36(7 PT 2), pp. 2573-8.

Newberne, P.M. & Rogers, A.E., 1968, Carcinoma, thymidine uptake, and mitosis in the livers of rats exposed to aflatoxin, N Z Med J, 67(426), pp. Suppl:8-17.

Nishizumi, M., Albert, R.E., Burns, F.J. & Bilger, L., 1977, Hepatic cell loss and proliferation induced by N-2-fluorenylacetamide, diethylnitrosamine, and aflatoxin B1 in relation to hepatoma induction, Br J Cancer, 36(2), pp. 192-7.

Pitot, H.C., 1990, Altered hepatic foci: their role in murine hepatocarcinogenesis, Annu Rev Pharmacol Toxicol, 30, pp. 465-500. Pitot, H.C., Dragan, Y., Xu, Y.H., Pyron, M., Laufer, C. & Rizvi, T., 1990, Role of altered hepatic foci in the stages of carcinogenesis, Prog Clin Biol Res, 340D, pp. 81-95.

Pogribny, I.P., Muskhelishvili, L., Tryndyak, V.P. & Beland, F.A., 2011, The role of epigenetic events in genotoxic hepatocarcinogenesis induced by 2-acetylaminofluorene, Mutat Res, 722(2), pp. 106-13.

Puisieux, A., Lim, S., Groopman, J. & Ozturk, M., 1991, Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens, Cancer Res, 51(22), pp. 6185-9.

Rehman, K., Iqbal, M.J., Zahra, N. & Akash, M.S., 2014, Liver stem cells: from preface to advancements, Curr Stem Cell Res Ther, 9(1), pp. 10-21.

Roebuck, B.D., Liu, Y.L., Rogers, A.E., Groopman, J.D. & Kensler, T.W., 1991, Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short-term molecular dosimetry, Cancer Res, 51(20), pp. 5501-6.

Sargent, L., Xu, Y.H., Sattler, G.L., Meisner, L. & Pitot, H.C., 1989, Ploidy and karyotype of hepatocytes isolated from enzyme-altered foci in two different protocols of multistage hepatocarcinogenesis in the rat, Carcinogenesis, 10(2), pp. 387-91.

Scholl, P.F., McCoy, L., Kensler, T.W. & Groopman, J.D., 2006, Quantitative analysis and chronic dosimetry of the aflatoxin B1 plasma albumin adduct Lys-AFB1 in rats by isotope dilution mass spectrometry, Chem Res Toxicol, 19(1), pp. 44-9.

Sell, S., 1993, The role of determined stem-cells in the cellular lineage of hepatocellular carcinoma, Int J Dev Biol, 37(1), pp. 189-201.

Sell, S., 2002, Cellular origin of hepatocellular carcinomas, Semin Cell Dev Biol, 13(6), pp. 419-24.

Sell, S. & Leffert, H.L., 2008, Liver cancer stem cells, J Clin Oncol, 26(17), pp. 2800-5. Shelton, P. & Jaiswal, A.K., 2013, The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene? FASEB J, 27(2), pp. 414-23.

Shin, S. & Kaestner, K.H., 2014, The origin, biology, and therapeutic potential of facultative adult hepatic progenitor cells, Curr Top Dev Biol, 107, pp. 269-92.

Sparfel, L., Langouët, S., Fautrel, A., Salles, B. & Guillouzo, A., 2002, Investigations on the effects of oltipraz on the nucleotide excision repair in the liver, Biochem Pharmacol, 63(4), pp. 745-9.

Tanimizu, N. & Mitaka, T., 2014, Re-evaluation of liver stem/progenitor cells, Organogenesis, 10(2). Tokusashi, Y., Fukuda, I. & Ogawa, K., 1994, Absence of p53 mutations and various frequencies of Ki-ras exon 1 mutations in rat hepatic tumors induced by different carcinogens, Mol Carcinog, 10(1), pp. 45-51.

Trosko, J.E., 2013, Evolution of energy metabolism, stem cells and cancer stem cells: how the Warburg and Barker hypothesis might be linked, BMC Proc, 7(Suppl 2 Sao Paulo Advanced School of Comparative Oncology: AbstractsClaudia Aparecida Rainho, Patricia Pintor dos Reis and Silvia Regina RogattoPublication of this supplement was funded by the Sao Paulo Research Foundation (FAPESP)), p. K8.

Trosko, J.E., 2014, Induction of iPS cells and of cancer stem cells: the stem cell or reprogramming hypothesis of cancer? Anat Rec (Hoboken), 297(1), pp. 161-73.

Trosko, J.E. & Kang, K.S., 2012, Evolution of energy metabolism, stem cells and cancer stem cells: how the warburg and barker hypotheses might be linked, Int J Stem Cells, 5(1), pp. 39-56.

Wu, H.C., Wang, Q., Yang, H.I., Tsai, W.Y., Chen, C.J. & Santella, R.M., 2013, Global DNA methylation in a population with aflatoxin B1 exposure, Epigenetics, 8(9), pp. 962-9.

Yang, L., Ji, J., Chen, Z., Wang, H. & Li, J., 2014, Transcriptome profiling of malignant transformed rat hepatic stem-like cells by aflatoxin B1, Neoplasma, 61(2), pp. 193-204.

Yates, M.S. & Kensler, T.W., 2007, Keap1 eye on the target: chemoprevention of liver cancer, Acta Pharmacol Sin, 28(9), pp. 1331-42.

Youngman, L.D. & Campbell, T.C., 1992, Attenuation of preneoplastic lesion development by dietary protein intervention: apparent persistence and regression, Cancer Lett, 66(2), pp. 165-74.

Zhang, Y., Guan, L., Wang, X., Wen, T., Xing, J. & Zhao, J., 2008, Protection of chlorophyllin against oxidative damage by inducing HO-1 and NQO1 expression mediated by PI3K/Akt and Nrf2, Free Radic Res, 42(4), pp. 362-71.

Zhang, Y.J., Chen, Y., Ahsan, H., Lunn, R.M., Lee, P.H., Chen, C.J. & Santella, R.M., 2003, Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation and its relationship to aflatoxin B1-DNA adducts and p53 mutation in hepatocellular carcinoma, Int J Cancer, 103(4), pp. 440-4.

Zhang, Y.J., Rossner, P., Chen, Y., Agrawal, M., Wang, Q., Wang, L., Ahsan, H., Yu, M.W., Lee, P.H. & Santella, R.M., 2006, Aflatoxin B1 and polycyclic aromatic hydrocarbon adducts, p53 mutations and p16 methylation in liver tissue and plasma of hepatocellular carcinoma patients, Int J Cancer, 119(5), pp. 985-91.

Zhang, Y.J., Wu, H.C., Yazici, H., Yu, M.W., Lee, P.H. & Santella, R.M., 2012, Global hypomethylation in hepatocellular carcinoma and its relationship to aflatoxin B(1) exposure, World J Hepatol, 4(5), pp. 169-75.