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Key Event Title
Increased, Insufficient repair or mis-repair of pro-mutagenic DNA adducts
|Level of Biological Organization|
Key Event Components
|DNA repair||Nuclear deoxyribonucleic acid||functional change|
Key Event Overview
AOPs Including This Key Event
Key Event Description
The AFB1-induced pro-mutagenic DNA adducts, N7-AFB1-G and /or AFB1-FAPy, are recognized by DNA repair proteins or systems, which then initiate their repair processes (Bedard and Massey, 2006).
Although there are no data specific to AFB1 for this key event of insufficient/mis-repair of DNA, it is a process known to occur and known to result in mutations. The system that recognizes the damage depends in part upon which stage of the cell cycle is in progress when the pro-mutagenic DNA adducts are recognized. Initial steps can include recruitment of a series of proteins specific to that repair system and can also include blocking of progress of the DNA replication fork. This latter step ensures that DNA replication waits for repair to occur before proceeding, preventing replication of the damaged DNA, thus avoiding a mutation when the repair might otherwise faithfully copy the error.
Mis-repair: For many of the DNA repair systems the repair process is quite faithful; that is, damage is correctly repaired with the correct or original base substituted for the adducted base. This is not always the case, however, and erroneous repair is one source of mutations, when an incorrect base inserted opposite a correct or an adducted base (mis-repair).
Insufficient repair: An alternative route to mutation can occur when on-going DNA replication is not stopped, and DNA replication occurs across an adducted (typically non-informational lesion) base that has not undergone any repair (insufficient repair). This situation can result in the incorporation of an incorrect base into the nascent DNA strand.
Insufficient repair or mis-repair results in an incorrect base incorporated into the nascent DNA strand, an error which is then eventually replicated, resulting in a so-called “fixed” change in the primary DNA sequence; that is, a mutation. This mutation may be silent (no impact on protein product structure and/or function) or expressed (non-functional or differently functional protein product). Depending on the location or gene of the expressed mutation, it may have a substantial or slight biological impact, or can have no impact. For example, if the mutation is located in codon 249 of the p53 gene and is expressed, there is potential for substantial biological impact, including steps on a pathway to carcinogenesis. There is less likelihood of a substantial biological impact when a mutation results in no change in the coded amino acid due to lack of stringency in the genetic code. Likewise a change in amino acid sequence may not result in any change in the activity or function of the protein product.
Level of Biological Organization
Repair of DNA adducts occurs at the sub-cellular level, as the target is nuclear DNA. For AFB1, the cross-species critical target is repair of hepatocyte nuclear DNA, as the eventual AFB1-induced tumor is hepatocellular carcinoma (HCC), which is initiated in hepatocytes.
Evidence Supporting Essentiality
Evidence supporting the insufficient repair or mis-repair of pro-mutagenic DNA adducts is strong, but mainly indirect. This evidence comes from different biological systems and datasets, mostly mammalian, and is based in large part on the biological understanding of a DNA-reactive mode of action requiring a change in primary DNA sequence (mutation). Such a change occurs when DNA lesions are not repaired prior to cell replication (Preston and Williams, 2005; Jarabek et al, 2009; Pottenger et al., 2014). AFB1 forms two types of pro-mutagenic adducts: N7-AFB1-G and AFB1-FAPy DNA adducts, and resulting mutations are predominantly G:C to T:A transversions. These changes are the expected result of such adducts not being repaired or undergoing mis-repair (Bedard and Massey, 2006; Lin et al., 2014a,b).
Data from diverse cell types and systems demonstrate that AFB1-induced DNA adducts are repaired by a variety of DNA repair processes, including SOS repair, nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (Bedard and Massey, 2006). The work by Guo et al. (2005), conducted in transgenic yeast modified to express human CYP1A2, is particularly useful as several DNA repair systems were evaluated for efficacy towards the AFB1-induced DNA adducts. Mutations were more likely to be induced in strains deficient in certain repair systems. Mutations were also induced in strains with active secondary repair pathways; these include pathways for error-prone post-replication repair and those relying on apurinic endonucleases. However, NER appears to be most important for repair of AFB1-induced adducts in mammalian systems (Bedard and Massey, 2006).
The codon 249 of the p53 gene has been identified as a particular target of AFB1-induced adduction and subsequent mutation. Using DNA polymerase fingerprint analysis, Puiseux et al. (1991) showed a specificity of AFB1 epoxide binding for this codon. This specificity was not found with benzo[a]pyrene (B[a]P), although B[a]P also forms bulky, intercalating epoxides, similar to AFB1 exo-epoxide, which result in bulky N7-B[a]P-G adducts.
Essentiality of this key event of insufficient or mis-repair is supported in part by datasets demonstrating that a reduction in AFB1 adduct burden (e.g., 65-70% reduction) results in a significantly reduced or even eliminated altered hepatic foci (AHF) and liver tumor burden in experimental animals (Roebuck et al., 1991; Elegbede and Gould, 2002; Yates et al., 2006; Johnson et al., 2014). Data from studies in human populations show that treatment with known modulators of AFB1 metabolism (e.g., with oltipraz or chlorophyllin) resulted in reduced urinary levels of N7-AFB1-G, derived from AFB1-induced DNA adducts (Egner et al., 2006). These treatments are also observed to decrease levels of a human biomarker of exposure, AFB1-induced albumin adducts (Kensler et al., 1998). A similar dataset in rats pre-treated with the triterpenoid, ODDC-Im, provides an even more convincing picture, wherein a chronic bioassay demonstrated a ~60% reduction in AFB1 DNA adducts, complete elimination of liver tumors, and nearly complete elimination of altered hepatic foci (Johnson et al., 2014).
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).
How It Is Measured or Detected
One approach would be quantitation of chemical-specific DNA adducts, such as the N7-AFB1-G and AFB1-FAPy DNA adducts, before and after DNA repair occurs combined with DNA sequence data to determine whether the decrease in adducts were correctly repaired or not. While this would be a very satisfying dataset, it is not a very practical approach. Measurement of mutations following AFB1 treatment of yeast with a variety of DNA repair deficiencies has been used to elucidate the role of DNA repair (Guo et al., 2005) by comparing mutant frequency (MF) in wild type vs. DNA repair deficient strains. In this instance MF serves as a marker for the insufficient or mis-repair of the AFB1 DNA adducts. The exquisitely sensitive analytical techniques available for structural quantification of these chemical-specific DNA adducts require specialized analytical chemistry techniques conducted on DNA isolated from tissues or cells and subjected to neutral thermal or enzyme or acid hydrolysis; this releases the adducted bases, which are then further analyzed with specialized approaches (Himmelstein et al., 2009). Demonstration of adduct dose-responses and temporal relationships are possible with administration of a variety of doses, including repeated doses.
Domain of Applicability
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction by AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014), It is likely that all species which are capable of DNA repair following binding of AFB1 exo-epoxide to DNA are subject to insufficient or mis-repair. These species include bacteria, yeast, birds, mammals, and fish.
Bedard, L.L., and Massey, T.T. (2006). Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 241(2):174-183.
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.
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.
Gross-Steinmeyer K, and Eaton DL. (2012). Dietary modulation of the biotransformation and genetoxicity of aflatoxin B1. Toxicology, 299: 69–79.
Guo Y, Breeden LL, Zarbl H, et al. (2005). Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage. Mol Cell Biol, 25, 5823–5833.
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.
Jarabek, AM, Pottenger, LH, Andrews, LS, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization. Crit. Rev. Toxicol. 39: 659-678.
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.
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, 127–134.
Leadon SA, Tyrrell RM, Cerutti PA. (1981). Excision repair of aflatoxin B1-DNA adducts in human fibroblasts. Cancer Res. 41: 5125-5129.
Leung MC, Goldstone JV, Boyd WA, Freedman JH, and Meyer JN. (2010). Caenorhabditis elegans generates biologically relevant levels of genotoxic metabolites from aflatoxin B1 but not benzo[a]pyrene in vivo. Toxicol Sci.118(2):444-453.
Levy DD, Groopman JD, Lim SE, et al. (1992). Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells. Cancer Res. 52: 5668–5673. 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
Meier B, Cooke SL, Weiss J, Bailly AP, Alexandrov LB, Marshall J, et al. (2014). C. elegans whole genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency. Genome Res. 24(10):1624-1636.
Oleykowski CA, Mayernick JA, Lim SE, et al. (1993). Repair of aflatoxin B1 DNA adducts by the UvrABC endonuclease of Escherichia coli. J Biol.Chem. 268: 7900–8002.
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.
Preston RJ, Williams GM. (2005). DNA-reactive carcinogens: mode of action and human cancer hazard. Crit Rev Toxicol, 35, 673–83
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):6185-6189.
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.
Sarasin AR, Smith CA Hanawalt PC. (1977). Repair of DNA in human cells after treatment with activated aflatoxin B1. Cancer Res. 37: 1786–1793.
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.