*3.2. Mycotoxins*

Mycotoxins are produced by fungi that can contaminate a variety of crops pre- and post-harvest, and which are associated with several diseases in animals and humans. Mycotoxins cannot be completely eliminated from food by food processing procedures, including thermal processing [272]. Of major concern are the mycotoxins aflatoxins, ochratoxin A and fumonisins [273,274].

**Figure 2.** Chemical structures of DNA-reactive carcinogenic mycotoxins and related chemicals present in foods. Asterisks indicate sites of activation.

#### 3.2.1. Aflatoxins

*Occurrence: Aflatoxins* (AFs) (Figure 2(1–6)) are mycotoxins formed by various strains of the fungus, *Aspergillus flavus*, and are present in contaminated foods, particularly corn and peanuts [68,275,276]. Food levels of AFs are often expressed as total AFs [276], which is useful for monitoring purposes. AFB1 has a tetrahydrocyclopenta[c]-furo [3- ,2- :4,5]-furo [2,3-h]chromene skeleton with oxygen functionality at positions 1, 4 and 11 (Figure 2(1)).

*Carcinogenicity*: AFB1 is the most highly carcinogenic AF [277] and one of the most potent carcinogens [278,279] (Table 2). Oral administration of AFB1, including as AFs mixtures, produced sufficient evidence for carcinogenicity in multiple species [275]. Specifically, AFB1-induced increases in the incidences of hepatocellular or cholangiocellular carcinomas were observed in rats, hamsters, marmosets, tree shrews, and monkeys; in addition, increase were observed in renal cell carcinomas and colon tumors in rats, lung adenomas in mice as well as osteogenic sarcoma, gallbladder tumors and adenocarcinoma of the pancreas in monkeys [68,275]. AFB2 (Figure 2(4)), AFG1 (Figure 2(2)), and AFM1 (Figure 2(3)) also produced liver tumors in experimental animals, but their potency was significantly lower compared to that of AFB1 [68,280]. No evidence for carcinogenicity of AFG2 have been reported [275].

*Genotoxicity/DNA Binding (Adducts)*: AFB1 is genotoxic in vitro and in vivo, producing mutagenic, aneugenic and clastogenic effects [275,281,282], as well as DNA adducts in multiple species [275,283–286], with the AFB1-*N*7-guanine adduct being assumed to be promutagenic and pro-carcinogenic [278,287–289]. The initial AFB1-DNA adduct is unstable in vivo; it either depurinates to give an AFB1-guanine residue which can be detected in the urine, or forms a more stable ring opened formamidopyrimidine derivative measurable in cellular DNA. AFB1-DNA adducts show high correlation with tumor incidence, but no threshold for hepatic DNA adduct formation was reported [25]. AFB1 also elicited DNA repair synthesis in cultured human hepatocytes [290] and γH2AX induction in human cell lines derived from hepatoblastoma, renal cell adenocarcinoma, and epithelial colorectal adenocarcinoma [291]. DNA adduct formation has been also reported after AFG1 and AFM1 exposures [292,293].

*Biotransformation*: The genotoxic and carcinogenic AF, AFB1, is metabolically activated predominantly by CYP3A4 oxidation at the 8–9 positions (Figure 2(1)) to form an AFB1- 8,9-epoxide, which is highly reactive and binds to the *N*7 position of guanine residues in DNA [287–289,294]. There is abundant evidence that in humans AFB1 is bioactivated by CYP1A2, 2B6, 3A4, 3A5, 3A7 and GSTM1 enzymes [281]. Ramsdell and Eaton [279] reported that mouse and monkey microsomes formed AFB1-8,9-epoxide at higher rates compared to rat and human; however, at lower substrate concentrations, conversion to AFB1-8,9-epoxide increased with rat and human microsomes, but not with mouse of monkey microsomes. Thus, the authors attributed interspecies differences in carcinogenic potency of AFB1 to differences in patterns of epoxide formation. AFG1 and AFM1, which also have a double bond at the 8,9-position (Figure 2(3,4)), can form epoxides; however, they are less DNAreactive compared to AFB1-8,9-epoxide [281]. Non- or weakly carcinogenic AFs, e.g., AFB2, AFG2, and AFM2, lack the double bond in the 8–9 position (Figure 2(4–6)) [276] and, except in the duck, are not metabolized to detectable levels of AFB1 [295]. CYP3A4 and CYP1A2 can also metabolize AFB1 to hydroxylated metabolites, AFM1 and AFQ1. Roebuck and Wogan [296] reported that AFQ1 was the principal metabolite produced by monkey, human, and rat liver, whereas duck liver produced mainly chloroform-insoluble derivatives. Monkey, human, and mouse liver also produced AFP1, which was not observed in duck and rat. The authors noticed that duck, monkey, and human livers were most active, each metabolizing approximately 80% of available substrate in half an hour. In comparison, activity of rat and mouse livers was lower, each metabolizing from 15 to 20% of substrate. No consistent pattern of metabolism that could explain interspecies differences in susceptibility to AFB1 carcinogenicity was detected. Detoxication of AFB1 occurs predominantly via conjugation with glutathione (GSH), and extent of this reaction

differs among species, with mouse showing the highest and humans having the lowest conjugation rates [281].

*MoA*: Covalent binding of AFB1-8,9-epoxide to *N*7 of guanine in DNA is considered to be the primary MoA of AFB1 carcinogenicity [275,278,281,289,297]. The adduct is believed to induce mutations of *TP53* gene in humans [275,276]. In addition, AFB1 epoxide reacts with serum proteins, including albumin. All have been used as biomarkers to assess AFB1 exposure [298,299]. Such studies have led to the clear association of AFB1 exposure and hepatocellular carcinoma, particularly in those infected with hepatitis B virus [278,281,300]. This is believed to be due to enhanced liver cell proliferation with hepatitis [300]. A strong correlation of urinary adducts indicative of AFB1 exposure, notably AFB1-*N*7-guanine, serological markers of hepatitis B infection, and liver cancer risk exists [301]. Induction of oxidative stress, immunomodulation and epigenetic modification also play a role in carcinogenicity of AFB1 [278,281].

*Human Exposure*: Overall, exposure to AFB1 results from ingestion of foods contaminated with *Aspergillus flavus*. Total EDI to AFs ranges from 0.0001 to 0.049 μg/kg bw/day in developing countries and is generally less than 0.001 μg/kg bw/day in developed countries [276]. In parts of the worlds where *Aspergillus* contamination of food is prevalent, AFB1 occurs in such foods at significant levels [278,302]. In the United States, consumption of food contaminated with up to 20 ppb AFB1, mainly corn and peanuts, is permitted [303], with the exception of milk, which is required to contain less than 0.5 ppb [304], corresponding to about 30 μg/day for a 70 kg adult. Obviously, high exposures are occurring in parts of the world where crop contaminations are not well controlled and accordingly, the cancer risk is much higher.

*Human Effects:* In humans, exposure to AFs is associated with increased risk of liver cancer, particularly in association with concurrent hepatitis B [68,275,281,299,300].

*Risk*: IARC [275] considers AFs to be carcinogenic to humans (Group 1) (Table 2). JECFA estimated the cancer potency for exposure to AFB1 per 100,000 population at 0.001 μg/kg bw/day, and recommended that efforts to reduce aflatoxin exposure continue [276]. The Committee also noticed that AFM1 will generally make a negligible (<1%) contribution to aflatoxin-induced cancer risk for the general population. EFSA estimated that MoEs, which range from 5000 to 29 for AFB1 and from 100,000 to 508 for AFM1 exposures, respectively, raise a concern for human health [281].
