Key Event Title
Key Event Component
|DNA alkylation||Nuclear deoxyribonucleic acid||abnormal|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)||MolecularInitiatingEvent|
Level of Biological Organization
|rainbow trout||Oncorhynchus mykiss||Strong||NCBI|
|Human, rat, mouse||Human, rat, mouse||Strong||NCBI|
|chickens, ducks, turkeys||chickens, ducks, turkeys||Strong||NCBI|
How This Key Event Works
Evidence Supporting Essentiality
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).
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).
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 Perturbation by Stressor
Overview for 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.
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