2. Mechanisms of Carcinogenicity of DNA-Reactive Carcinogens

DNA-reactive carcinogens have structures that permit formation of electrophilic reactants that covalently bind (adduct) to nucleophilic sites in nuclear DNA, as well as in other macromolecules, including RNA and proteins, in the target tissue(s) of carcinogenicity ^[9][10][11][23,24,25]. In target tissue(s), a single DNA reactant can form different DNA adducts on various nucleophilic sites either on a single base or on different bases. Each adduct can undergo different rates of repair depending upon its location in the genome. For example, adducts in transcriptionally active regions are repaired by a transcription-coupled repair system whereas adducts in transcriptionally silent regions are repaired by a global repair system ^[12][26]. The levels of DNA adducts resulting from exposures are a function of several metrics including dose levels, the frequency of exposure, and rates of DNA repair for specific adducts. Each adduct has a characteristic efficiency with which it gives rise to mutations, with those at sites of base pairing being more mutagenic. Pro-mutagenic DNA alterations are converted to mutations during cell replication ^[13][14][15][27,28,29]. Mutations in critical growth control genes lead to neoplastic conversion, and subsequent neoplastic development ^[14][16][28,30]. DNA-reactive carcinogens can also exert other cellular effects, such as cytotoxicity, leading to enhanced cell proliferation, which can contribute to their carcinogenic activity ^[17][18][31,32]. DNA-reactive carcinogens can have additive effects with one another in their target organ(s). Some DNA adducts evidently do not lead to carcinogenicity, since some adducts can be found in tissues where no tumors are induced following administration of a carcinogen ^{[19][20][21][22]}[33,34,35,36]. For example, acrylamide, which is discussed below, forms adducts in target and non-target tissues ^[23][37]. It could also be the case that epigenetic effects are required to enable neoplastic conversion resulting from some adducts ^[24][25][38,39]. As a result of DNA interactions, DNA-reactive carcinogens are typically genotoxic in assay systems in which appropriate bioactivation is represented ^{[10][26][27][28][29]}[17,18,24,40,41]. Moreover, DNA-reactive carcinogens often produce tumors at multiple sites and with a short duration of exposure, even after administration of a single dose for some. This property underlies their activity in limited short-term bioassays ^[27][18]. Some DNA-reactive carcinogens have been demonstrated to exhibit no-observed-adverse-effect-levels (NOAELs) for carcinogenic effects in animal models ^{[11][17][30][31][32][33][34]}[25,31,42,43,44,45,46], although conflicting data have been reported. Based on the steps for tumorigenesis, it is evident that biological thresholds that may influence the likelihood of cancer progression for genotoxic carcinogens exist. Nevertheless, currently, thresholds are not generally accepted for DNA-reactive carcinogens from a risk assessment and management perspective ^[35][47]. It is acknowledged that the derivation of NOAELs can be dependent on the study design, and more research is needed in this space. It is outside the scope of this research paper to discuss thresholds for carcinogens in detail; however, this topic is reviewed elsewhere ^{[11][17][30][31][32][33][34]}[25,31,42,43,44,45,46].

3. Mechanisms of Carcinogenicity of Epigenetic Carcinogens

Epigenetic carcinogens do not chemically react with DNA ^{[26][36][37][38][39][40][41]}[17,20,48,49,50,51,52]. In the target tissue(s) of carcinogenicity, MoAs of these types of carcinogens involve molecular or cellular effects, which through secondary mechanisms, can either indirectly result in modification of DNA function or cell behavior ^[26][37][17,48]. For example, epigenetic carcinogens can induce oxidative stress, resulting in oxidative DNA damage ^[42][43][44][53,54,55], leading to either neoplastic conversion or stimulation of cell proliferation, thereby facilitating neoplastic development, often from cryptogenic pre-neoplastic cells. Epigenetic carcinogens can also affect gene expression ^[45][46][56,57], leading to neoplastic conversion. Such effects are often specific for rodents (e.g., d-limonene). Epigenetic carcinogens can enhance carcinogenicity of DNA-reactive carcinogens through interactive effects such as neoplasm promotion (e.g., butylated hydroxyanisole). Due to their lack of direct DNA reactivity, epigenetic carcinogens, in contrast to DNA-reactive agents, are typically negative in genotoxicity assays, even in the presence of bioactivation, unless some artifact, such as extreme cytotoxicity, mediates mutagenicity. To exert their carcinogenicity, epigenetic agents often require prolonged high-level exposures. Their MoA underlies the fact that in limited bioassays they are negative for initiating activity, but may be positive for promoting activity ^[27][18]. Epigenetic carcinogens are well established to exhibit NOAELs for the cellular effect underlying their carcinogenicity in animal models ^[26][47][17,19], as discussed for several of the food-borne carcinogens reviewed herein. Accordingly, thresholds are generally accepted for DNA-reactive carcinogens from a risk assessment perspective ^[35][47].

4. Risk Assessment of Food-Derived Carcinogens

4.1. Application of Carcinogenicity Data to Human Risk

Two types of carcinogenicity data are used in the assessment of risk: human epidemiologic data and tumor data obtained in testing in rodent models ^[48][58]. The former is considered more relevant for a variety of reasons ^{[49][50][51][52]}[59,60,61,62], although such data are often limited in human exposure information and can be poorly controlled ^[53][63]. Animal data are usually more robust, but frequently involve findings whose relevance to humans is uncertain ^[27][54][55][18,64,65], because the tumorigenic effect involves MoAs operational only in rodents. In addition, rodent studies do not mimic real life human exposures with respect to both the concentration and frequency of exposure. The human diet is also composed of mixture of components, which can both enhance and inhibit carcinogenicity. Thus, in assessing human risk, two considerations are critical, i.e., the MoA of carcinogenicity and human exposure dose ^[11][56][21,25]. Once a chemical has been identified in a food product and its structure determined, it is possible to undertake an in silico analysis to determine, based on structure-activity relationships, the potential for DNA reactivity ^[57][66]. While this works well for relatively simple compounds, with the complexity of many natural products, the subtleties of metabolic activation become increasingly difficult to predict. If sufficient material is available, direct testing for DNA reactivity is the preferred approach ^[27][18]. This research focuses primarily on chemicals present in food that have sufficient evidence of carcinogenicity in either humans or experimental animals and which were classified by the International Agency for Research on Cancer (IARC) as either carcinogenic to humans (Group 1), probably (Group 2A) or possibly (Group 2B) carcinogenic to humans ^[48][58][58,67]. IARC also recognizes a third group of substances (Group 3) which lack sufficient evidence to be classified as carcinogenic to humans but nonetheless can have the potential to cause carcinogenicity in animals. Moreover, a variety of chemicals has not yet been characterized as to their carcinogenic risk to humans. Where available, evaluations by other expert groups are cited. Data on classification of carcinogens by government agencies and their carcinogenic potencies (TD₅₀) calculated based on the tumorigenicity findings in rodents are provided in Table 2.