3.1.3.1. Cycasin

*Occurrence: Cycasin* (methylazoxymethanol-D-glucoside) (Figure 1(9)) is a glucoside produced by the cycad nut, which grows in most tropical climates [89,163]. The amount of cycasin ranges from 0.02% to 2.3% [89].

*Carcinogenicity*: With oral administration, cycasin induced neoplasia in mice, rats, hamsters, guinea pigs, and monkeys mainly in liver, kidney and colon [89,164]. A metabolite of cycasin, methylazoxymethanol (MAM) (Figure 1(10)), has also been shown to induce hepatocellular carcinomas and tumors in other organs, including kidneys and intestinal tract, in nonhuman primates [164], and colon carcinogenesis in rodents [165–168].

*Genotoxicity/DNA Binding (Adducts)*: Cycasin was genotoxic after removal of a sugar residue to yield the aglycone, MAM, which is an alkylating agent [89,169–173]. MAM produced DNA adducts, specifically O6-methylguanine and *N*7-methylguanine, in vitro and in vivo in rats and guinea pigs [174–178].

*Biotransformation*: Bioactivation of cycasin to MAM occurs by hydroxylation of the methyl group, a reaction which is catalyzed by CYP2E1 [179]. Interspecies differences in metabolic bioactivation of cycasin to MAM was suggested to underly different susceptibility to its carcinogenicity [178].

*MoA:* The genotoxic metabolite MAM was shown to target cellular processes involved in carcinogenesis [180].

*Human Exposure*: Human exposure to cycasin is limited since cycad nuts are no longer used as a source of starch. Cycasin can, however, contaminate improperly prepared flour, as has occurred in Guam, where concentrations of cycasin ranged from 0.004 to 75.93 μg/g [175].

*Human Effects*: Human ingestion of cycad plant toxins has been associated with neurodegenerative disorders in inhabitants of Guam [181,182], but no appreciable increase in cancer mortality was evident at 2 to 7 years after heavy intake [89]. Cases of acute toxicity from high exposures have been reported but all with complete initial recovery [183].

*Risk*: IARC [89] classified both, cycasin and MAM, as possibly carcinogenic to humans (Group 2B) (Table 2).

#### 3.1.3.2. Ptaquiloside and Bracken Fern

*Occurrence*: *Ptaquiloside* (Figure 1(11)) is an unstable norsesquiterpene glucoside of the illudane type [184,185]. It is present in bracken fem (*Pteridium aquilinum*), in wild species and in products made from fronds at concentration ranges of 6300 ± 520 and 44 ± 3 μg/g, respectively [186]. High quantities of ptaquiloside, in various studies ranging from 0.0006 to 0.0058 μg/mL, were found in the milk from farm animals that consume diet containing bracken fern [187–190].

*Carcinogenicity*: Ingestion of bracken fern by cattle and sheep has been reported to cause cancers of the esophagus and urinary bladder [191,192]. Feeding of bracken fern to rats and mice induced intestinal and bladder cancers [193], which was initially attributed to the content of quercetin [194], but ptaquiloside was subsequently demonstrated to be the carcinogenic constituent [190,195]. With oral administration, ptaquiloside induced tumors of mammary glands, ileum and urinary bladder in female rats [196,197] and oral squamous cell carcinomas in HPV16-transgenic mice [198].

*Genotoxicity/DNA Binding (Adducts)*: Ptaquiloside was genotoxic in bacterial mutagenicity assays and in the rat hepatocyte primary culture DNA-repair assay [199–201]. In addition, it produced chromosomal aberrations in Chinese hamster lung cells and human mononuclear blood cells [202,203], and DNA damage in human gastric epithelial cells [204]. Formation of DNA adducts was reported in upper gastrointestinal tract of mice that were fed bracken fern [205,206] and in target tissue of carcinogenicity, ileum, in rats injected with ptaquiloside intravenously [207]. DNA adducts formed after exposure to bracken fern were distinctly different from the adducts formed by ptaquiloside [206].

*Biotransformation*: Bioactivation of ptaquiloside is not enzyme mediated, and involves conversion to aglycone, ptaquilosin, which, under alkaline conditions undergoes aromatization resulting in a reactive metabolite, bracken dienone [185]. Dienone has an ability to alkylate DNA, forming adducts primarily on *N*3 position of adenine and *N*7 position of guanine [185].

*MoA:* DNA alkylation of adenine bases with subsequent DNA depurination and breakage leading to induction of mutations, in particular to activation of *H-ras* protooncogenes and frameshift mutations of *p53* gene [192,204,208,209] is thought to be the main mechanism underlying ptaquiloside-related carcinogenicity. Other potential MoAs, including clastogenicity and aneugenicity, as well as alteration of monocyte function, TNFα expression and cell proliferation, cannot be excluded [202,207,210].

*Human Exposure:* Estimation of human consumption of ptaquiloside with cow's milk resulted in intake levels ranging from 1.75 to 13.4 mg/day [211]. Some populations in Japan, Brazil and Canada can also consume cooked or salted bracken crosiers [185,186].

*Human Effects*: No study on human carcinogenicity of ptaquiloside is available. However, in areas where bracken fern is consumed, there is a correlation between the consumption of ptaquiloside-contaminated milk and increased risk of esophageal or stomach cancer [184,212,213].

*Risk*: While IARC recognizes bracken fern as possibly carcinogenic to humans (Group 2B), it considers ptaquiloside to be unclassifiable as to its carcinogenicity (Group 3) (Table 2) based on limited evidence [193]. Nevertheless, genotoxicity and mutagenicity of ptaquiloside, as well as some epidemiological evidence of potential carcinogenicity raises concerns for human safety [184,190,202].

#### 3.1.4. Psoralens

*Occurrence*: *Psoralen* (7H-furo[3,2-g][1]benzopyran-7-one) (Figure 1(12)) is a furocoumarin which is naturally present in several plants, notably *Psoralea corylifolia*, celery, parsley and in all citrus fruits, including bergamot orange peel, whose oils are used as flavors [214–217]. In citrus-flavored beverages, the highest levels of psoralens, 29 and 24 mg/L, were found in bergamot juice and home-made limoncello, respectively [218]. Levels of psoralens in celery varied, depending on when it was harvested, from 26 to 84 μg/g fresh weight [216]. Psoralens are widely used in the photochemotherapy of various skin conditions in humans [219].

*Carcinogenicity*: Psoralen derivatives, 5- and 8-methoxypsoralen (methoxsalen) (Figure 1(13,14)), produced skin tumors in mice in the presence of UV A light, even with

oral administration [15,68,193,220,221]. In male rats, tumors of Zymbal glands and kidneys were also reported after oral gavage with methoxsalen [222].

*Genotoxicity/DNA Binding (Adducts)*: Psoralen can be photoactivated to DNA cross linking reactant which exhibit genotoxicity and photomutagenicity [15,193,222–224]. Intercalation occurs predominantly on pyrimidine bases of DNA, mainly with thymine, which leads to inhibition of DNA synthesis, in addition, psoralens have high affinity for uridine bases on RNA [219].

*Biotransformation*: Metabolism of psoralen involves hydroxylation of phenyl ring, hydrogenation and hydrolysis of the unsaturated lactone ester, and oxidation of the furan ring to generate epoxide or/and γ-ketoenal intermediates [225,226]. These reactions are catalyzed by CYP3A4, CYP1A1, CYP1A2 and CYP2B6 [226–230]. Incubation of psoralen with liver microsomes from different species, including humans, dogs, non-human primates and rodents, demonstrated similarity of metabolites produced by humans and dogs, while metabolic capabilities of rat and monkey microsomes were the closest to those of human microsomes [225].

*MoA*: Photochemical genotoxicity and mutagenicity are most likely responsible mechanism of psoralens carcinogenicity [15,68,193]. Other potential MoAs may involve oxidative damage [219].

*Human Exposure:* Dietary exposure to psoralens occurs mainly from either limes, with estimated per capita exposure of 1300 μg/day [231], or grapefruit juice, with dietary exposure re-estimated to be in the range of 548 to 2237 μg/day [232].

*Human Effects:* Human exposures have so far been mainly associated with photodermatitis due to occupational contact [216,233,234] with only one report of phototoxicity following ingestion [235]. Human carcinogenicity studies relating to oral psoralens have only been made with patients receiving photodynamic therapy (PUVA) [15,68,236,237] and no attempt to extrapolate these positive results to normal populations has been undertaken. One study identified an association between high citrus consumption and melanoma [238]. This study, however, did not specifically assess risk from psoralen consumption.

*Risk:* IARC classifies methoxsalen with UV A radiation as a human carcinogen (Group 1) and 5-methoxypsoralen as probable human carcinogen (Group 2A) [15,193] (Table 2). Further investigation to establish potential health risks of dietary intake of psoralens in humans is warranted [219].
