AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)

• Ted Simon

Revision dates for related pages

This AOP is applicable to all life stages, to many vertebrate taxons, and to both genders.

Support for Essentiality of KEs

Chemoprotection

A number of substances have been shown to act as chemoprotectant or chemopreventive agents against AFB1-induced liver tumors [1-3]. These compounds may act at several points in the pathway beginning with exposure to AFB1 and ending with the occurrence of tumors; such data address the essentiality of key events. Below we consider a framework for the action of chemoprotectants based on the KEs in the AOP and then attempt a classification of various chemoprotectants based on this framework with consideration of cellular signaling pathways that likely underlie their chemoprotective action [4,5]. Framework for Chemoprotectant Action

Chemoprotective agents may act in the three distinct ways: • protection against the formation of pro-mutagenic adducts • protection against mutations in tumor-critical locations • protection against the formation of pre-neoplastic lesions and tumors through appropriate cell cycle checkpoints or cytotoxicity

First, chemoprotectants could enhance detoxification metabolic pathways (e.g., GST conjugation) for AFB1 or decrease formation of the pro-mutagenic exo-epoxide metabolite. Second, AFB1 pro-mutagenic adducts may form in locations that lead to critical (pro-tumorigenic) mutations or in locations that are much less likely to lead to critical (pro-tumorigenic) mutations. Chemoprotectants could decrease the likelihood of adducts occurring in tumor-critical locations or enhance the fidelity and efficacy of DNA repair processes, thus reducing the effective numbers and impact of any pro-mutagenic adducts.

Third, chemoprotectants may act to block or lessen the progression from AFB1-induced critical mutations to pre-neoplastic lesions and tumors. Alternatively, chemoprotectants may enhance the ability of cell cycle checkpoint systems to interfere with cell replication (critical to render a mutation permanent and thus heritable), or may produce apoptotic cell death. In addition, chemoprotectants may produce cytotoxicity in cells with a sufficient number of adducts such that the cells die before any mutations can be fixed at the next cell division. Induction of cellular defense against electrophilic and oxidative stress is mediated through a signaling pathway via the transcription factor called nuclear factor erythroid 2-related factor 2 (Nrf2) and the Kelch-like erythroid cell-derived protein 1 (Keap1). Nrf2 turns over rapidly within cells via ubiquitination unless critical cysteine thiols of these two molecules are modified by oxidants or electrophiles. Reduced ubiquitination of Nrf2-Keap1 will increase cellular levels of free Nrf2 and lead to binding of Nrf2 dimers to anti-oxidant response elements (AREs) on DNA, resulting in the induction of anti-oxidant genes. Anti-oxidants such as the food additive butylated hydroxyanisole (BHA), phytochemicals such as genistein, quercetin and curcumin, redox-active bisphenols and quinones, thiocarbamates, dithiolethiones, polyphenols and triterpenoids are all capable of modifying the cysteine residues on Nrf2 and Keap1, and thus they can all serve as ARE inducers.

Nrf2 can induce a large number of ARE-dependent genes including the mixed function oxidase CYP2A5, the Phase 2 enzymes UGT1A1 and SULT3A1, superoxide dismutase, peroxiredoxin, glutathione peroxidase, heme oxygenase 1 (HO-1) and others [6]. In fact, the constellation of ARE-dependent enzymes likely act in all of the ways discussed in the framework above.

The majority of chemoprotective agents appear to work via activation of Keap1 due to cysteine modification.

Actions of Some Specific Chemoprotectants

In this section, the actions of specific chemoprotectants will be considered.

Selenium

Dietary selenium reduced the volume fraction of altered hepatic foci (AHF) occurring in the livers of rats fed a high fat diet ten weeks after initiation with 10 days of intragastric administration of 4 mg/kg AFB1. In rats fed diets containing 0.15 ppm Se or more, glutathione peroxidase was elevated compared with animals fed diets containing <0.02 ppm Se [7]. Se may modify cysteine residues on Nrf2-Keap1.

Oltipraz

The dithiolthione oltipraz was administered to rats at 0.01 to 0.1% in the diet for 1 week prior to administration of 250 μg/kg AFB1 by gavage 5 days per week for two weeks. The rats continued a diet containing oltipraz for one week following cessation of AFB1 administration. The animals were then restored to a control diet and sacrificed twelve weeks later. Oltipraz increased GST activity and reduced the volume of GGT-positive foci in a dose-dependent manner, but did not eliminate their formation completely. In rats administered a single gavage dose of 250 μg/kg (14C)-AFB1 and sacrificed two hours later, binding of the labeled AFB1 to DNA was reduced in a dose-dependent fashion, thus AFB1-induced DNA adduct formation is reduced, probably due to the induction of GST activity. In rats maintained on diets containing 0.01 and 0.1% oltipraz, a number of ARE-associated enzymes were induced in a dose-dependent fashion [8]. In a separate study, 0.075% oltipraz in the diet reduced the volume fraction of AFB1-induced GGT+ foci at 3 months and completely prevented the occurrence of tumors for times up to two years [9]. Oltipraz given following AFB1 exposure also reduced the volume percent of GST-P+ foci [10].

It is not entirely clear whether oltipraz acts by increasing DNA repair. A cell-free system was used to examine nucleotide excision repair in human cells in vitro and in livers from rats exposed to AFB1 and oltipraz; NER DNA repair enzymes were not induced by oltipraz and are not known to be associated with AREs [11]. However, some studies suggest that eukaryotic NER expression is induced by exposure to phytochemicals (Gross-Steinmeyer et al. 2010), and alteration of DNA repair has been suggested as a pathway of chemoprevention for AFB1 carcinogenesis (Gross-Steinmeyer and Eaton, 2012). Oltipraz likely acts by activating the Nrf2-Keap1 pathway [12]. Oltipraz has been shown to be successful as a chemoprotectant in humans exposed to AFB1, presumably by similar mechanisms to those elucidated in rats [3,13,14].

Chlorophyllin

In Rainbow trout, chlorophyllin interferes with the formation of AFB1 adducts by forming a non-covalent complex with AFB1 [15]. This complex is not subject to metabolism by Phase 1 enzymes and thus the pro-mutagenic exo-epoxide metabolite cannot be formed. Chlorophyllin also induces ARE-associated enzymes HO-1 and NQO1 [16]; however, the primary mechanism for cancer prevention appears to be reduction in the uptake and bioavailability of AFB1 and its exo-epoxide [17,18]. In rats, administration of chlorophyllin after treatment with AFB1 had no effect on reducing the occurrence or volume of AHF in rats [19].

In human volunteers administered 14C-AFB1, co-administration of chlorophyllin increased the level of AFB1 equivalents in the urine [20]. This finding indicates that chlorophyllin works in humans in a similar fashion—by binding AFB1 prior to metabolism and thus limiting uptake/bioavailability.

Sulforaphane

Sulforaphane also appears to be a potent activator of the Nrf2-Keap1 pathway leading to increased expression of carcinogen detoxifying enzymes [12,21].

Other Phytochemicals

Green tea polyphenols and Korean red ginseng act as chemoprotectants. The effect is based on their anti-oxidant and anti-inflammatory properties, and likely mediated through the Nrf2-Keap1 anti-oxidant pathway, similar to what was described previously [22,23].

CDDO-Im and other Triterpenoids

Very recently, a landmark paper by Johnson et al. (2014) demonstrated that administration of the triterpenoid CDDO-Im prior to and during 28 days of dosing rats with AFB1 provided complete protection against the formation of both pre-neoplastic lesions and liver tumors. [24]. In addition, the rats treated with CDDO-Im maintained a reduced yet still significant burden of AFB1 adducts in their liver. Triterpenoids are potent inducers of the Nrf2-Keap1 ARE pathway. Commentaries on this paper noted both that Nrf2 activation introduced a nonlinearity into the AFB1 adduct-to-tumor relationship and also might provide significant protection in human populations exposed to AFB1 [2,25].

1. Yates MS, Kensler TW (2007) Keap1 eye on the target: chemoprevention of liver cancer. Acta Pharmacol Sin 28: 1331-1342.

2. Olden K, Vulimiri SV (2014) Laboratory to community: chemoprevention is the answer. Cancer Prev Res (Phila) 7: 648-652.

3. Kensler TW, Egner PA, Wang J-B, Zhu Y-R, Zhang B-C, et al (2004) Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas. Gastroenterology 127: S310-S318.

4. Ma Q, He X (2012) Molecular basis of electrophilic and oxidative defense: promises and perils of Nrf2. Pharmacol Rev 64: 1055-1081.

5. Jaramillo MC, Zhang DD (2013) The emerging role of the Nrf2-Keap1 signaling pathway in cancer. Genes Dev 27: 2179-2191.

6. Kwak MK, Itoh K, Yamamoto M, Sutter TR, Kensler TW (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: 135-145.

7. Baldwin S, Parker RS (1987) Influence of dietary fat and selenium in initiation and promotion of aflatoxin B1-induced preneoplastic foci in rat liver. Carcinogenesis 8: 101-107.

8. Kensler TW, Egner PA, Dolan PM, Groopman JD, Roebuck BD (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: 4271-4277.

9. Roebuck BD, Liu YL, Rogers AE, Groopman JD, Kensler TW (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: 5501-5506.

10. Maxuitenko YY, MacMillan DL, Kensler TW, Roebuck BD (1993) Evaluation of the post-initiation effects of oltipraz on aflatoxin B1-induced preneoplastic foci in a rat model of hepatic tumorigenesis. Carcinogenesis 14: 2423-2425.

11. 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: 745-749.

12. Kensler TW, Roebuck BD, Wogan GN, Groopman JD (2011) Aflatoxin: a 50-year odyssey of mechanistic and translational toxicology. Toxicol Sci 120 Suppl 1: S28-S48.

13. Gross-Steinmeyer K, Eaton DL (2012) Dietary modulation of the biotransformation and genotoxicity of aflatoxin B(1). Toxicology 299: 69-79.

14. Sudakin DL (2003) Dietary aflatoxin exposure and chemoprevention of cancer: a clinical review. J Toxicol Clin Toxicol 41: 195-204.

15. Breinholt V, Schimerlik M, Dashwood R, Bailey G (1995) Mechanisms of chlorophyllin anticarcinogenesis against aflatoxin B1: complex formation with the carcinogen. Chem Res Toxicol 8: 506-514.

16. 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: 362-371.

17. Hayashi T, Schimerlik M, Bailey G (1999) Mechanisms of chlorophyllin anticarcinogenesis: dose-responsive inhibition of aflatoxin uptake and biodistribution following oral co-administration in rainbow trout. Toxicol Appl Pharmacol 158: 132-140.

18. Breinholt V, Arbogast D, Loveland P, Pereira C, Dashwood R, et al (1999) Chlorophyllin chemoprevention in trout initiated by aflatoxin B(1) bath treatment: An evaluation of reduced bioavailability vs. target organ protective mechanisms. Toxicol Appl Pharmacol 158: 141-151.

19. Orner GA, Roebuck BD, Dashwood RH, Bailey GS (2006) Post-initiation chlorophyllin exposure does not modulate aflatoxin-induced foci in the liver and colon of rats. J Carcinog 5: 6. 20. Jubert C, Mata J, Bench G, Dashwood R, Pereira C, et al (2009) Effects of chlorophyll and chlorophyllin on low-dose aflatoxin B(1) pharmacokinetics in human volunteers. Cancer Prev Res (Phila) 2: 1015-1022. 21. Keum Y-S, Yu S, Chang PP-J, Yuan X, Kim J-H, et al (2006) Mechanism of action of sulforaphane: inhibition of p38 mitogen-activated protein kinase isoforms contributing to the induction of antioxidant response element-mediated heme oxygenase-1 in human hepatoma HepG2 cells. Cancer Res 66: 8804-8813.

22. Qin G, Ning Y, Lotlikar PD (2000) Chemoprevention of aflatoxin B1-initiated and carbon tetrachloride-promoted hepatocarcinogenesis in the rat by green tea. Nutr Cancer 38: 215-222.

23. Kim Y-S, Kim Y-H, Noh J-R, Cho E-S, Park J-H, Son H-Y (2011) Protective Effect of Korean Red Ginseng against Aflatoxin B1-Induced Hepatotoxicity in Rat. J Ginseng Res 35: 243-249.

24. Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, et al (2014) Complete protection against aflatoxin B1-induced liver cancer with triterpenoid: DNA adduct dosimetry, molecular signature and genotoxicity threshold. Cancer Prev Res (Phila) .

25. Eaton DL, Schaupp CM (2014) Of mice, rats, and men: could Nrf2 activation protect against aflatoxin heptocarcinogenesis in humans? Cancer Prev Res (Phila) 7: 653-657.

Support for Biological Plausibility of KERs

Dose-Time Concordance Table Based on Available Data

AFB1 is a data-rich chemical, and it is clear that it induces hepatocellular carcinoma (HCC) in rats, fish, and in humans, among other species. The vast majority of experiments, however, were not designed with the intention of arraying the information in a dose- and time-concordant manner. As indicated in this table, there are no quantitative data (aside from the induction of pre-neoplastic lesions and HCCs) that can be used to develop the quantitative relationships between the KEs. The KEs that have been identified for AFB1-induced HCC begin with metabolism to reactive moieties and a key metabolite forming pro-mutagenic DNA adducts. If these adducts are not repaired, are mis-repaired, or are insufficiently repaired, this can lead to the induction of mutations (primarily G:C to T:A transversions) across the genome (Dycaico et al., 1996; Chen et al. 2010 ). There are sufficient data to indicate that at least one hot-spot for mutation induction occurs in codon 249 (G:C to T:A transversion) of the p53 gene. This conclusion is based on the fact that this mutation has been observed in up to 75% of liver cancers in humans residing in the high AFB1 incidence areas of China and East Africa, but the same mutation is rarely seen in no or low AFB1 areas (Hsu et al., 1991 and Bressac et al., 1991). Because of a lack of practicable methods, there are no studies conclusively demonstrating that AFB1 exposure induces these specific mutations in vivo. To be conclusive, demonstration of specific cancer gene mutation would have to be shown following relatively short exposure to AFB1 and long before the observation of any pre-neoplastic AHF lesions or HCCs.

There have, however, been some studies in which rodents have been exposed to AFB1 and either pro-mutagenic DNA adducts or increased mutant frequency in a surrogate gene (Lac I or cII) of a transgenic rodent have been observed. These data are shown in the table. There is some dose-response information for the AFB1 DNA adduct formation, but the rodent studies were conducted with only a single mutagenic dose. It should be noted that the neonatal mouse is very sensitive to the induction of mutations (and also susceptible to the induction of tumors), but the adult mouse exposed to AFB1 shows neither mutation nor tumors; this difference in sensitivity is believed to be due to differential ontological expression patterns in glutathione-S-transferase (GST) activity.

Data from studies with chemoprevention agents add considerably to the WOE and understanding of the KE as necessary but not sufficient. In particular, two datasets are shown in the Data Concordance Table: Roebuck et al. (1991) with oltipraz, and Johnson et al. (2014) with CDDO-Im. Both datasets demonstrated a significant reduction in tumor burden with a concomitant decrease in altered hepatic foci (AHF) (Johnson et al., 2014), that was correlated with reduction but not elimination of the pro-mutagenic DNA adducts. The reduction in DNA adducts was likely due in part to induction of GST activity. In any case, both datasets demonstrate that the presence of pro-mutagenic adducts, alone, does not result in AHF or HCC; that is, adduct formation is necessary but not sufficient.

Data from same study (Wogan et al., 1974, Table 1)

• Wogan et al., 1974, Table 1. [Wogan G.N., Paglialunga S., Newberne P.M. 1974. Carcinogenic effects of low dietary levels of aflatoxin B1 in rats, Food. Cosmet. Toxicol. 12, 681-68].

Table of Reversibility of KEs

Table References Chen T., Heflich R.H., Moore M.M., Mei N.2010. Differential mutagenicity of aflatoxin B1 in the liver of neonatal and adult mice.. Environ Mol Mutagen. 51(2), 156-63

Dycaico M.J., Stuart G.R., Tobal G.M., de Boer J.G., Glickman B.W., Provost G.S.1996. Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/lacI transgenic rats and mice following exposure to aflatoxin B1.. Carcinogenesis. 17(11), 2347-5

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).

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), 5501-6

Hypothetical and Ideal Data: Dose-Time Concordance Table

Given the importance of evaluating AOP information using a dose- and time-concordance table, and the fact that the appropriate data are not available for AFB1, we constructed a table to indicate what would be expected in a hypothetical ideal situation. It is expected that each of the KE will show a dose-response relationship with upstream KE observed at lower exposure levels than the downstream KE, and the AO observed only at the higher doses. Likewise the upstream KE would be expected to be observed at earlier times than the downstream KE or the apical outcome. Thus metabolism to reactive AFB1 forms must occur prior to the observation of DNA adducts. These adducts must be insufficiently repaired, not repaired, or mis-repaired prior to the observation of mutation in the critical cancer gene. For a mutagenic AOP, it is important that the mutation is induced prior to the clonal expansion of cells. Furthermore in this AOP a critical gene mutation leads to the formation of pre-neoplastic lesions, which must precede the formation of tumors. For chronic exposure, one would expect that the frequency of mutant cells would increase with the increased length of exposure; that is, the early KE will continue to occur with time leading to more adducts being formed and more mutations being induced.

While the studies presented in the concordance did not specifically measure DNA repair or induction of mutations, they do provide data on KE#3 (AHF frequency) and the AO (HCC tumor incidence). Treatment with chemoprotective agents decreased AHF formation significantly for oltipraz (Roebuck et al., 1991), while for CDDO-Im, AHF incidence was reduced to background/control levels (Johnson et al., 2014). In these studies, the AO HCC tumor incidence was either significantly reduced (oltipraz) or completely eliminated (CDDO-Im; 0% HCC incidence).

Support for Biological Plausibility of KERs

Uncertainties and Conflicting Evidence for KERs and AOP

Support for Biological Plausibility of KERs