Event:373

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Event Title

Pro-mutagenic DNA Adducts, Formation

Molecular Initiating Event

Action - how the event is affected

Alkylation, binding

Biological Process/Object

The Molecular Initiating Event for this AOP Formation of AFB1-induced pro-mutagenic DNA adducts: N7-AFB1-G and/or AFB1-FAPy

Key Event Overview

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AOPs Including This Key Event

AOP Name Event Type Essentiality
AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC) MIE Strong

Chemical Initiators

The following are chemical initiators that operate directly through this Event:

  1. Aflatoxin B1

Taxonomic Applicability

Name Scientific Name Evidence Links
rainbow trout Oncorhynchus mykiss Strong NCBI
Human, rat, mouse Strong
chickens, ducks, turkeys Strong

Level of Biological Organization

Biological Organization
Molecular

Evidence Supporting Essentiality

Strong

Evidence supporting the formation of an AFB1-induced pro-mutagenic DNA adduct as the molecular initiating event (MIE) is strong and stems from many datasets in different biological systems. The formation of N7-AFB1-G DNA adducts after AFB1 exposure has been demonstrated across phyla, from bacteria through yeast, fish, birds, and including many mammalian systems up through non-human primates and humans (Croy et al., 1978; IARC, 1993; Cupid et al., 2004).

The reactive metabolite AFB1 exo-epoxide intercalates into DNA and then binds to the nucleophilic N7-G residue via an SN2 reaction. This N7-G DNA adduct can then spontaneously ring-open to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido-9-hydroxyaflatoxin B1, or formamidopyrimidine adduct, AFB1-FAPy (Brown et al., 2006).

The essentiality of this MIE is demonstrated by the effects of modulation of metabolism to reactive forms. Inhibition of activation results in reduced formation of the critical exo-epoxide. Likewise, increased GST activity results in increased metabolism of the exo-epoxide to less toxic forms. In both cases, less reactive metabolite is available to form DNA adducts, resulting in fewer adducts (Guengerich et al., 1996). Pre-treatment of rats with oltipraz provides a specific example, wherein a 65-70% reduction in AFB1-induced DNA adducts was demonstrated due to increased GST activity; this corresponds with a subsequent 100% reduction in liver tumors (Roebuck et al., 1991; Kensler et al., 1998).


Another line of evidence for essentiality of the MIE is the recognized species difference in sensitivity to AFB1-induced liver tumors between mice and rats. Mice, with considerably increased metabolic activation of AFB1 to the exo-epoxide compared with rats, are nonetheless much less sensitive to AFB1-induced liver tumors (Degen and Neumann, 1981). This difference is believed to be due to the constitutive presence of GST-alpha activity in mice vs. rats, where this activity is not found (Monroe and Eaton, 1987).

Taxonomic Applicabilty

AFB1-induced DNA adduct measurements have focused mainly on mammalian species, including rats, mice, non-human primates, and humans; however, all species capable of metabolic activation of AFB1 to the exo-epoxide—including yeast, birds, and fish--will form the pro-mutagenic N7-AFB1-G DNA and AFB1-FAPy adducts described above (IARC, 1993).

Level of Biological Organization : Molecular

Formation of DNA adducts is at the sub-cellular level, as the target is nuclear DNA, leading to a potential outcome of heritable mutation. For AFB1, the cross-species critical target is hepatocyte nuclear DNA, as the apical outcome is hepatocellular carcinoma (HCC).

How this Key Event works

The initial AFB1-induced pro-mutagenic DNA adduct is the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, or N7-AFB1-G (Croy et al., 1971). Once the exo-epoxide is bound to the N7-guanine, it can then ring-open to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido)-9-hydroxyaflatoxin B1, or formamidopyrimidine adduct, AFB1-FAPy (Brown et al., 2006).

The N7-AFB1-G adduct has a short half-life; it can spontaneously depurinate, leaving an apurinic (AP) site, a DNA lesion that typically is rapidly repaired (Denissenko et al., 1998). AP sites are the predominant background or endogenous lesion identified to date in DNA from control rats, with about 30,000 AP sites/cell present ubiquitously and continually (Swenberg et al., 2011). Thus, although the N7-AFB1-G is considered to be a pro-mutagenic lesion due to its capability to intercalate in DNA and its bulkiness (Bailey et al., 1996), it may not be the most important DNA adduct in the process of AFB1-induced tumorigenesis.

The AFB1-FAPy adduct has a longer half-life and demonstrates higher mutagenic efficiency or potency than the N7-AFB1-G (Brown et al., 2006). Data indicate that about 20% of the N7-AFB1-G adducts undergo opening of the ring to become AFB1-FAPy adducts (Bedard et al., 2005; Croy and Wogan, 1981a); others report that by about 24 post-exposure, AFB1-FAPy adducts predominate (Boysen et al., 2009; Croy and Wogan, 1981a). These adducts do not spontaneously depurinate, thus can accumulate over time, which likely contributes to their increased mutagenic efficacy (Smela et al., 2002).

The pro-mutagenicity of these two adducts was demonstrated by assessing their mutant frequencies (MF) in non-human primate-derived cell line COS-7; these cells employ an error-prone replication bypass repair system. The N7-AFB1-G adducts demonstrated a MF of 45% in COS-7 cells (Lin et al., 2014a), while the N7-AFB1-FAPy adduct MF was 97% (Lin et al., 2014b).

How it is Measured or Detected

Sensitive analytical techniques are available for structural quantification of the AFB1-specific DNA adducts, including the N7-AFB1-G and AFB1-FAPy adducts (Himmelstein et al., 2009). DNA is isolated from tissues or cells and the isolated DNA subjected to neutral thermal or enzyme or acid hydrolysis. This releases the adducted bases, which are then further analyzed with specialized approaches. Techniques include high pressure liquid chromatography (HPLC) or liquid chromatography (LC) separation coupled with tandem mass spectrometry (HPLC-MS/MS or LC-MS/MS). These techniques allow for definitive identification of the AFB1-related adducts using authentic standards. These capabilities can be further enhanced by the use of stable isotope-labelled test materials, e.g., with 13C, 15N, or D3. More sensitivity is reported with accelerated mass spectrometry (AMS) approaches; these require the use of radiolabelled (14C) test material but can detect adducts down into the attomolar range. Demonstration of dose-responses of adduct formation and temporal-response relationships are possible with administration of a variety of dose regimens, including repeated doses

Evidence Supporting Taxonomic Applicability

AFB1-induced DNA adduct measurements have focused mainly on mammalian species, including rats, mice, non-human primates, and humans; however, all species seem capable of metabolic activation of AFB1 to the exo-epoxide, including yeast, birds, and fish. These will form the pro-mutagenic N7-AFB1-G DNA and AFB1-FAPy adducts described above (Croy et al., 1978; IARC, 1993; Cupid et al., 2004; Lin et al., 2014b; Smela et al., 2002).

Evidence for Chemical Initiation of this Molecular Initiating Event

An extensive database demonstrates the formation of AFB1-specific DNA adducts in many different systems and from several laboratories. In particular, Groopman’s lab and Essigman’s group, among others, have provided pivotal data to demonstrate the formation of these pro-mutagenic AFB1-induced DNA adducts (Croy and Wogan, 1981a,b; Croy et al., 1978; Groopman et al., 1992; Smela et al., 2002; Egner et al., 2006). Lutz (1987) summarized data from a thesis that measured tritiated DNA in liver following p.o. administration of tritiated AFB1 to male F344 rats over a range of doses, from 1 ng AFB1/kg bw to 104 ng AFB1/kg bw and the dose-response was reported to be linear; only limited experimental details are available for this dataset, which relied on less sophisticated and less specific analytical methods than are currently available.

References

Bailey EA, Iyer RS, Stone MP, et al. (1996). Mutational properties of the primary aflatoxin B1-DNA adduct. Proc Natl Acad Sci USA, 93, 1535–1539.

Bedard L, Alessi, M, Davey SK, Massey TE (2005). Susceptibility to aflatoxin B1-induced carcinogenesis correlates with tissue-specific differences in DNA repair activity in mouse and in rat. Cancer Res 65:1265-1270.

Boysen G, Pachkowski BF, Nakamura J, Swenberg JA. (2009). The formation and biological significance of N7-guanine adducts. Mutat Res, 678, 76–94.

Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199.

Croy RG, Wogan GN (1981a). Temporal patterns of covalent DNA adducts in rat liver after single and multiple doses of aflatoxin B1. Cancer Res 41:197-203.

Croy RG, Wogan GN (1981b). Quantitative comparison of covalent aflatoxin-DNA adducts formed in rat and mouse livers and kidneys. J Natl Cancer Inst 66:761-768.

Croy RG, Essigman JM, Reinhold VN, Wogan GN (1978). Identification of the principal aflatoxin N1-DNA adduct formed in vivo in rat liver. Proc Natl Acad Sci USA 75:1745-1749.

Cupid BC, Lightfoot TJ, Russell D, Grant SJ, Turner PC, Dingley KH, Curtis KD, Leveson SH, Turteltaub KW, Garner RC (2004). The formation of AFB1-macromolecular adducts in rats and humans at dietary levels of exposure. Food Chem Toxicol 42:559-569.

Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306.

Denissenko MF, Koudriakova TB, Smith L, O'Connor TR, Riggs AD, and Pfeifer GP. The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. Oncogene. 1998, Dec 10;17(23):3007-14.

Egner PA, Groopman JD, Wang J-S, Kensler TW, Friesen MD (2006). Quantification of aflatoxin-B1-N7-Guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry. Chem Res Toxicol 19:1191-1195.

Groopman JD, Roebuck BD, Kensler TW. (1992). Molecular dosimetry of aflatoxin DNA adducts in humans and experimental rat models. Prog Clin Biol Res. 374:139-155.

Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect. 104(Suppl 3):557-562.

Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.

IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395.

Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–34.

Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506.

Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468.

Lutz, W. (1987). Quantitative evaluation of DNA-binding data in vivo for low-dose extrapolations. Arch.Toxicol, Suppl. 11: 66-74.

Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409.

Roebuck BD, Liu Y-L, Rogers AE, et al. (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.

Smela ME, Hamm ML, Henderson PT, Harris CM, Harris TM, Essigmann JM (2002). The aflatoxin B(1) formamidopyrimidine adduct plays a major role in causing the types of mutations observed in human hepatocellular carcinoma. Proc Natl Acad Sci USA 99:6655-6660.

Swenberg JA, Lu K, Moeller BC, et al. (2011). Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment. Tox Sci, 120, S130–45.