3.2.2. Ochratoxin A

*Occurrence: Ochratoxin A* (OTA) (*N*-[(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-3,4-dihydro-1H-isochromen-7-yl]carbonyl-L-phenylalanine) (Figure 2(7)), is a mycotoxin produced by a single *Penicillium* and several *Aspergillus* fungal species [305–307]. The ochratoxins are pentaketides, consisting of a dihydroisocoumarin coupled to 8-phenylalanine (Figure 2(7)) and, unusually for natural products, OTA is chlorinated. OTA is formed in improperly stored foods which have been produced mainly in Europe and Canada, including cereals, beans, ground nuts, oleaginous seeds, meat and wine [306].

*Carcinogenicity*: In several strains of mice, OTA fed in the diet induced kidney neoplasms, including carcinomas, at a concentration of 40 mg/kg bw, and liver neoplasms at 1 mg/kg bw. When administered by gavage to rats it induced renal tumors starting at 70 μg/kg bw, [305,308]. Male rats were considerably more susceptible than females. A feature of the renal toxicity of OTA is formation of karyomegalic nuclei in the tubular epithelia, predominantly in the corticomedullary zone [309].

*Genotoxicity/DNA Binding (Adducts)*: OTA was consistently negative in studies assessing mutagenicity in *Salmonella typhimurium*, both with and without exogenous metabolic activation. In contrast to bacteria, however, overall results from genotoxicity tests in mammalian cell systems provide some evidence for a weak genotoxic activity of OTA [305,307,310–312]. It elicited DNA repair synthesis in cultured rat and mouse hepatocytes at cytotoxin doses, increased DNA strand breaks levels and mutagenicity in target tissue, kidney, in rodents [307,312]. Controversy exits over whether OTA reacts directly

with DNA. OTA did not form DNA adducts in the kidneys of male rats when measured using radiolabeled OTA and accelerator mass spectrometry [313], while others obtained positive results in isolated DNA and cell culture by dehalogenation and redox reactions analyzed by nucleotide 32P-postlabeling (NPL) [314]. Mantle et al. [315] was able to identify a small amount (20–70 adducts per 109 normal nucleotides) of a single DNA adduct in the kidneys of rats using refined NPL methodology. These conflicting data have been reviewed [306,310,312]. EFSA concluded that while formation of covalent OTA-DNA adducts remains controversial, OTA mutagenicity cannot not simply be a consequence of oxidative DNA damage [307].

*Biotransformation*: OTA is characterized by rapid absorption and distribution, but slow elimination and excretion [307]. The major metabolite of OTA forms as a result of hydrolyses of amide bond between phenylalanine and dihydroisocoumaric acid. OTA also undergoes oxidative dichlorination in the presence of CYPs, generating electrophilic quinone, which can be further reduced to hydroquinone metabolite and excreted in urine, as has been shown in rats and humans [307,312]. In addition, peroxidase enzymes are involved in oxidation of OTA to electrophilic phenolic radical, which is believed to cause oxidative stress. Radical and benzoquinone intermediates formed during metabolism of OTA can covalently bind to DNA, generating C-bound C8-dG adducts [306].

*MoA:* The definitive MoA for carcinogenicity of OTA remains unclear, and most likely involves a combination of mutagenicity and increased reactive oxygen species (ROS) level leading to oxidative DNA damage [306,310,312,316,317]. Alternatively, an epigenetic MoA for renal carcinogenicity has been postulated to be a combination of inhibition of histone acetyltransferase, producing mitotic disruption leading to increased cell proliferation and genetic instability [310]. The mitotic disruption may be the basis for karyomegaly observed in rodent kidneys [318]. Thus, MoA other than DNA reactivity are possible for OTA. The pathogenesis of the renal tumors in male rats probably does not involve an α2u-globulin MoA [319]. Moreover, sex and strain differences are suggestive of biotransformation being important [320].

*Human Exposure:* In European Union, dietary exposures range between 0.00064 to 0.0178 μg/kg bw/day across all age groups [307]. The EDI values for OTA calculated from food products range from 1 × <sup>10</sup>−<sup>7</sup> to 0.0252 <sup>μ</sup>g/kg bw/day [306]. In areas where contamination occurs, biomarkers of OTA exposure are measurable in human blood, urine and milk [306].

*Human Effects:* OTA is suspected to be the main etiologic agent for human Balkan endemic nephropathy and the associated urinary tract tumors [311,321].

*Risk:* IARC [305] classified OTA as possibly carcinogenic to humans (Group 2B) based on sufficient evidence for carcinogenicity in experimental animals (Table 2). JECFA concluded that maximum levels of at 5 or 20 μg/kg in contaminated cereal grains would be unlikely to have an impact on dietary exposure to OTA, and established a provisional tolerable weekly intake of 0.112 μg/kg bw [322]. EFSA estimated that MoE for chronic neoplastic effects ranged from 22,615 to 815, indicating possible health concern for high-level consumers and breastfed infants [307].
