**8. Monitoring techniques for assessing human exposure to aflatoxins**

In the last few years, new technologies have been developed that more accurately monitor individual exposures to aflatoxins. Particular attention has been paid to the analysis of afla‐ toxin DNA adducts and albumin adducts as surrogates for genotoxicity in people. Autrup et al. (Autrup, Bradly, Shamsuddin, Wakhisi, Wasunna, 1983) proposed for the first time the use of synchronous fluorescence spectroscopy for the measurement of aflatoxin DNA ad‐ ducts in urine. Urine samples collected after exposure to alfatoxins were found to contain 2,3-dihydroxy-2-(N7-guanyl)-3-hydroxyaflatoxin B1, trivially known as aflatoxin B-Gual. Wild et al. used highly sensitive immunoassays to quantitate aflatoxins in human body flu‐ ids (Wild, Umbenhauer, Chapot, Montesano, 1986).

An enzyme linked immunosorbent assay (ELISA) was used to quantitate aflatoxin B1 in a range from 0.01 ng/mL to 10 ng/mL, and was validated in human urine samples. Using this method, aflatoxin-DNA adduct excretion into urine was found to be positively correlated with dietary intake, and the major aflatoxin B1-DNA adduct excreted in urine was shown to be an appropri‐ ate dosimeter for monitoring aflatoxin dietary exposure. Several epidemiological studies have found positive association between aflatoxin B1 dietary exposure and an increased risk of hu‐ man liver cancer (Sudakin, 2003; Zhu, Zhang, Hu, Xiao, Chen, Xu, Fremy, Chu, 1987; Groop‐ man, Donahue,1988; Bean, Yourtee, 1989). Cytochrome P-450 enzymes further convert aflatoxins to different metabolites (Eaton, Ramsdell, Neal, 1994), e.g. aflatoxin B1 is converted to metabolites like aflatoxin B1-epoxide and the hydroxylated aflatoxins M1, P1 and Q1. The hy‐ droxylated metabolites form glucuronide and sulfate conjugates that can be enzymatically hy‐ drolysed by b-glucuronidase and sulfatase (Wei, Marshall, Hsieh, 1985).

The European Union (EU) introduced measures to minimise the presence of aflatoxins in different foodstuffs. Maximum levels of aflatoxins are laid down in Commission Regulation (EC) No. 1881/2006. In an opinion adopted in January 2007, the European Food Safety Au‐ thority (EFSA) scientific Panel on contaminants in the food chain (CONTAM), concluded that increasing the current EU maximum levels of 4 µg/kg total aflatoxins in nuts to 8 or 10 µg/kg total afatoxins would have had minor effects on the estimated dietary exposure, can‐ cer risk and calculated margin of exposure. The Panel also concluded that exposure to afla‐ toxins from all food sources should be kept as low as reasonably achievable because aflatoxins are genotoxic and carcinogenic. In June 2009 the European Commission asked EF‐ SA to assess the effect on public health of an increase of the maximum level for total aflatox‐ ins from 4 µg/kg to 10 µg/kg allowed for tree nuts other than almonds, hazelnuts and pistachios (e.g. Brazil nuts and cashews). This would facilitate the enforcement of the maxi‐ mum levels, in particular regarding commercially available mixtures of nuts. The Panel con‐ cluded that public health would not be adversely affected by increasing the levels for total aflatoxins from 4 µg/kg to 8 or 10 µg/kg. However, the Panel reiterated its previous conclu‐ sions regarding the importance of reducing the number of highly contaminated foods reach‐ ing the market.

Most of the *in vitro* digestion models simulate in a simplified manner the digestion processes in mouth, stomach and small intestine, in order to enable investigation of the bioaccessibility

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Extensive studies involving animal models have indicated that the primary site for absorp‐ tion of aflatoxin is the small intestine, in particular the duodenum (Wogan, Edwards, Shank, 1967; Ramos, Hernandez, 1996). *Lactobacillus spp.* has previously proven to be capable to sur‐ vive at the gastrointestinal tract after oral intake (Taranto, Medici, Perdigon, Ruiz-Holgado, Valdez, 2000; Valeur, Engel, Carbajal, Connolly, Ladefoged, 2004); therefore, it is probable that mycotoxins were in contact with bacteria in the intestinal lumen, which then favored

It has been reported that the binding process might be dependent on the environmental pH (Bolognani, Rumney, Rowland, 1997) and that the presence of bile salts could produce sig‐ nificant effects in the aflatoxin B1 binding ability of the bacteria (Hernandez-Mendoza, Gar‐ cia, Steele, 2009). These two factors are closely related during the normal digestive process and its relationship varies along the small intestine (Low, 1990). Hence, the difference on aflatoxin binding ability of *Lactobacillus spp.* observed at the different portions of the intes‐ tine could be influenced by conditions prevailing in each region of the gastrointestinal tract. Once the aflatoxin B1 has been absorbed at intestinal level, it proceeds to the bloodstream and binds with plasma proteins especially albumin to form aflatoxin B1-albumin adduct (Verma, 2004). The average half-life of albumin (approximately 20 days in humans) allows accumulation of adducts after chronic exposure to the toxin (Chapot, Wild, 1991). According to this, the amount of adducts present in blood samples of rats treated only with aflatoxin B1 represent the cumulative dose of aflatoxin intake over the experimental period, which indi‐ cates that the reduction of aflatoxin B1-Lys adduct observed in animals treated with aflatoxin plus bacteria was originated by the ability of *Lactobacillus spp.*to bind aflatoxin B1 inside the intestinal lumen, thus avoiding its passage into the bloodstream. In a related work (Gratz, Täubel, Juvonen, Viluksela, Turner, 2006) no significant differences were found in the amounts of aflatoxin B1-Lys adduct present in animals receiving *Lactobacillus rhamnosus* GG daily for 3 d before and 3 d after a single oral dose of aflatoxin B1 compared with those re‐ ceiving only the mycotoxin. Other reports suggested that probiotics are less capable of bind‐ ing aflatoxin B1 in the presence of mucus and are more susceptible to interfere factors in the intestinal tract, which may explain the behavior observed in the levels of adduct (Gratz, Mykkänen, Ouwehand, Juvonen, Salminen, 2004; Gratz, Täubel, Juvonen, Viluksela, Turner, 2006). This effect could have been surmounted by the numbers of bacteria implanted before oral dose of aflatoxin B1, and the constant administration of probiotic bacteria during the ex‐

of compounds from their matrix during transit in the gastrointestinal tract.

aflatoxin B1 binding by bacteria prior to its natu ral process of absorption.

perimental period (Gratz, Mykkänen, Ouwehand, Juvonen, Salminen, 2004).

In agreement with earlier reports (Ward, Sontag, Weisburger, Brown, 1975; Maurice, Bodine, Rehrer, 1983), body weight gain was not adversely affected. However, there was a reduction in feed intake in rats receiving only aflatoxin B1. This effect could be induced by the dose of aflatoxin received, since it has been reported that aflatoxin B1 induces reduction of food in‐ take in some animal species, including rats and birds, in a dose-dependent manner (Maur‐ ice, Bodine, Rehrer, 1983). In addition, toxicological studies in rats have shown that aflatoxin
