**9. Bioavailability**

In human health risk assessment, ingestion of contaminated food is considered a major route of exposure to many contaminants either caused by industrial or environmental con‐ tamination or as result of production processes. The total amount of an ingested contami‐ nant (intake) does not always reflect the amount that is available to the body. Only a certain amount of the contaminant will be bioavailable (Versantvoort, Oomen, Van de Kamp, Rom‐ pelberg, Sips, 2005). Bioavailability is a term used to describe the proportion of the ingested contaminant in food that reaches the systemic circulation and then the organ or the appara‐ tus. Studies in animals and humans show that oral bioavailability of compounds from food can be significantly different depending on the food source (food product), food processing or food preparation (Wienk, Marx, Beynen, 1999; van het Hof, West, Weststrate, Hautvast, 2000). As a consequence, the intake of a contaminant in food matrix A can lead to toxicity whereas the intake of the same amount of contaminant in food matrix B will not exert a toxic effects. Thus, a better insight in the effect of the matrix on the oral bioavailability of a con‐ taminant will lead to a more accurate health risk assessment (Versantvoort, Oomen, Van de Kamp, Rompelberg, Sips, 2005).

Oral bioavailability of a compound can be seen as the resultant of three processes, namely the release of the compound from its matrix into digestive juice in the gastrointestinal tract (bioaccessibility); the transport across the intestinal epithelium into the vena Portae (intesti‐ nal transport); and the degradation of the compound in the liver and intestine (metabolism).

Release of the contaminant from the ingested product in the gastrointestinal tract is a pre‐ requisite for uptake and bioavailability of a contaminant in the body. The oral bioavailability of the contaminant can be reduced subsequently by partial transport of the contaminant across the intestinal epithelium, or by degradation of the contaminant. Thus, determination of the bioaccessibility of a contaminant from its matrix can be seen as an indicator for the maximal oral bioavailability of the contaminant. Quantification of bioavailability and bioac‐ cessibility of a compound from a certain matrix is difficult and often hampered by complex processes comprising digestion. The last decade there is an increasing interest in the use of *in vitro* methodologies to study the human oral bioavailability of compounds from the food chain (Minekus, Marteau, Havenaar, Huis, 1995; Glahn, Wien, Van Campen, Miller, 1996; Garrett, Failla, Sarama, 1999; Ruby, Schoof, Brattin, Goldade, Post, Harnois, Mosby, Casteel, Berti, Carpenter, Edwards, Cragin, Chappell, 1999; Oomen, Hack, Minekus, Zeijdner, Corne‐ lis, Schoeters, Verstraete, Wiele, Wragg, Rompelberg, Sips, Wijnen, 2002).

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

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‐

In human health risk assessment, ingestion of contaminated food is considered a major route of exposure to many contaminants either caused by industrial or environmental con‐ tamination or as result of production processes. The total amount of an ingested contami‐ nant (intake) does not always reflect the amount that is available to the body. Only a certain amount of the contaminant will be bioavailable (Versantvoort, Oomen, Van de Kamp, Rom‐ pelberg, Sips, 2005). Bioavailability is a term used to describe the proportion of the ingested contaminant in food that reaches the systemic circulation and then the organ or the appara‐ tus. Studies in animals and humans show that oral bioavailability of compounds from food can be significantly different depending on the food source (food product), food processing or food preparation (Wienk, Marx, Beynen, 1999; van het Hof, West, Weststrate, Hautvast, 2000). As a consequence, the intake of a contaminant in food matrix A can lead to toxicity whereas the intake of the same amount of contaminant in food matrix B will not exert a toxic effects. Thus, a better insight in the effect of the matrix on the oral bioavailability of a con‐ taminant will lead to a more accurate health risk assessment (Versantvoort, Oomen, Van de

Oral bioavailability of a compound can be seen as the resultant of three processes, namely the release of the compound from its matrix into digestive juice in the gastrointestinal tract (bioaccessibility); the transport across the intestinal epithelium into the vena Portae (intesti‐ nal transport); and the degradation of the compound in the liver and intestine (metabolism).

Release of the contaminant from the ingested product in the gastrointestinal tract is a pre‐ requisite for uptake and bioavailability of a contaminant in the body. The oral bioavailability of the contaminant can be reduced subsequently by partial transport of the contaminant across the intestinal epithelium, or by degradation of the contaminant. Thus, determination of the bioaccessibility of a contaminant from its matrix can be seen as an indicator for the maximal oral bioavailability of the contaminant. Quantification of bioavailability and bioac‐ cessibility of a compound from a certain matrix is difficult and often hampered by complex processes comprising digestion. The last decade there is an increasing interest in the use of *in vitro* methodologies to study the human oral bioavailability of compounds from the food chain (Minekus, Marteau, Havenaar, Huis, 1995; Glahn, Wien, Van Campen, Miller, 1996; Garrett, Failla, Sarama, 1999; Ruby, Schoof, Brattin, Goldade, Post, Harnois, Mosby, Casteel, Berti, Carpenter, Edwards, Cragin, Chappell, 1999; Oomen, Hack, Minekus, Zeijdner, Corne‐

lis, Schoeters, Verstraete, Wiele, Wragg, Rompelberg, Sips, Wijnen, 2002).

ing the market.

**9. Bioavailability**

352 Aflatoxins - Recent Advances and Future Prospects

Kamp, Rompelberg, Sips, 2005).

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

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‐ 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 B1 consumption may produce a significant decrease of serum leptin levels (Abdel-Wahhab, Ahmed, Hagazi, 2006). Leptin concentration is usually associated with the high levels of cor‐ tisol and interleukin-6, which act together to influence the feeding response (Barber, McMil‐ lan, Wallace, Ross, Preston, 2004). *Lactobacillus reuteri* might have contributed to reduce the aflatoxin B1 absorption in bacteria-treated rats and thus diminish its effect on leptin levels in blood serum.

reduction in excretion of aflatoxin metabolites in urine was found in presence of chlorophyl‐

Aflatoxins: Risk, Exposure and Remediation http://dx.doi.org/10.5772/52866 355

Furthermore, these results provide evidence for the hypothesis that chlorophyllin reduces

In risk assessment, a dose proportional relationship between contamination level and bioa‐ vailability is taken as basic assumption. This assumption simplifies risk assessment, since it can be assumed that regardless the level of contamination, a constant percentage of the con‐ taminant will be bioavailable. The extreme sensitivity of turkeys to the toxic effects of afla‐ toxin B1, a condition associated with a combination of efficient CYP-mediated activation and deficient GST-mediated detoxification of aflatoxin B1 (Klein, Buckner, Kelly, Coulombe, 2000), makes turkeys an excellent model in which to study various chemopreventives. We have recently shown that the observed chemopreventive properties of BHT in turkeys is due, at least in part, to its ability to inhibit hepatic conversion of aflatoxin B1 to the exo-afla‐

toxin B1-8,9-epoxide (AFBO) in vivo and in vitro (Guarisco, Hall, Coulombe, 2008).

Determining the outcome of inhibition of hepatic aflatoxin B1 bioactivation in whole animals is relevant to veterinary medicine and to food safety. Dietary butylated hydroxytoluene (BHT) can reduce aflatoxin B1 bioavailability, as demonstrated by serum concentrations of radiolabel which were reduced at every time interval after aflatoxin B1 administration. Among the possible explanations for reduced bioavailability is high first pass elimination prior to absorption into the blood, and/or an attenuation of mucosal aflatoxin B1 absorption. However, since no quantitative difference in the biliary elimination of aflatoxin B1 or its me‐ tabolites was identified, any change due to increased first pass effect would have to result in

The observed reduction in hepatic aflatoxin B1–DNA adducts in BHT fed animals is consis‐ tent with the fact that this antioxidant is a competitive inhibitor of hepatic in vitro CYP1A5 mediated aflatoxin B1 epoxidation to AFBO. Because of the critical role of AFBO and subsequent adduct formation in aflatoxicosis (as well as longer-term consequences such as tumor formation), a reduction in hepatic aflatoxin B1–DNA adducts would be expected to

Bioaccessibility has been defined as the fraction of a compound that is released from its ma‐ trix in the gastrointestinal tract and thus becomes available for intestinal absorption i.e. en‐ ters the blood stream (Benito, Miller, 1998). Bioaccessibility includes the entire sequence of events that take place during the digestive transformation of food into material that can be assimilated by the body, the absorption/assimilation into the cells of the intestinal epitheli‐ um, and lastly, the presystemic metabolism (both intestinal and hepatic). Bioaccessibility analyses can be approached using general experimental techniques (there are systematic techniques common to all types of foods) that can be adapted to all types of claims regard‐

have a positive effect on the overall health of aflatoxin B1-exposed turkeys.

lin (Versantvoort, Oomen, Van de Kamp, Rompelberg, Sips, 2005).

the absorption of aflatoxin B1 in humans.

increased elimination by non-biliary pathways.

**10. Bioaccessibility**

ing nutritional content.

The volume of the stomach is considered an important parameter for oral dosing in experi‐ mental animals. For rats, maximum oral dosage volume recommended is 10 mL kg–1 of body weight; for a 200 g rat this would mean a dosing volume of 2 mL (McConnell, Basit, Mur‐ dan, 2008). Therefore, it is possible that the volume supplied (every third day) by oral gav‐ age of aflatoxin and/or bacteria over the experiment, had partially met the basic water needs of the rats, which may explain the observed reduction in water consumption at the end of the experimental period (21 days).

A world-wide-accepted method for protecting animals against mycotoxicosis is the use of adsorbent materials. An effective adsorbent is one that tightly binds the mycotoxin in contaminated feed without dissociating in the gastrointestinal tract of the animal. The toxin–adsorbent complex passes then through the gastrointestinal tract without absorp‐ tion and is eliminated via the faeces. In other words, the bioavailability of the mycotox‐ in is reduced as less mycotoxin is absorbed because it is bound to the adsorbent, i.e. lower bioaccessibility. Therefore, these adsorbents can be used to evaluate the use of the in vitro digestion model as indicator for the in vivo bioavailability. The following mate‐ rials, representative for different classes of adsorbents, have been selected: an aluminosi‐ licate (HSCAS), which is a common anticaking additive in animal feeds to reduce mycotoxicosis in animals; activated charcoal, which is used in humans and animals as an antidote against poisoning; cholestyramine is an anion exchange resin and binds bile acids in the gastrointestinal tract and it has been used for over 20 years in the clinic for reduction of lowdensit y lipoproteins and cholesterol.

The effect of chlorophyllin on intestinal transport of aflatoxin B1 was studied by measure‐ ment of the transport of aflatoxin B1 with the intestinal Caco-2 cells. The rate at which com‐ pounds are transported across the Caco-2 cells, which is expressed as a permeability coefficient, is correlated with absorption in humans (Artursson, Karlsson, 1991).

Transport of 5ng/mL aflatoxin B1 across Caco-2 cells revealed that after 4h, 25±6% aflatoxin B1 was transported across Caco-2 cells into the basolateral compartment. Addition of chloro‐ phyllin (1 mg/mL) greatly reduced (>20-fold) the transport of aflatoxin B1 to only 1±1%. From this transport, a permeability coefficient can be calculated for aflatoxin B1 of 9x10-6 cm/s in absence, and 0.4x10-6 cm/s in presence of chlorophyllin. When we compare these transport rates with the S-shaped correlation found for absorption of compounds in hu‐ mans, the permeability coefficient of aflatoxin B1 alone (9x10-6 cm/s) corresponds with high absorption in humans whereas the permeability coefficient of aflatoxin B1 in presence of chlorophyllin (0.4x10-6 cm/s) indicates an intermediate absorption. Thus, these data are in ac‐ cordance with the human intervention study on chlorophyllin and aflatoxin B1, where a 50% reduction in excretion of aflatoxin metabolites in urine was found in presence of chlorophyl‐ lin (Versantvoort, Oomen, Van de Kamp, Rompelberg, Sips, 2005).

Furthermore, these results provide evidence for the hypothesis that chlorophyllin reduces the absorption of aflatoxin B1 in humans.

In risk assessment, a dose proportional relationship between contamination level and bioa‐ vailability is taken as basic assumption. This assumption simplifies risk assessment, since it can be assumed that regardless the level of contamination, a constant percentage of the con‐ taminant will be bioavailable. The extreme sensitivity of turkeys to the toxic effects of afla‐ toxin B1, a condition associated with a combination of efficient CYP-mediated activation and deficient GST-mediated detoxification of aflatoxin B1 (Klein, Buckner, Kelly, Coulombe, 2000), makes turkeys an excellent model in which to study various chemopreventives. We have recently shown that the observed chemopreventive properties of BHT in turkeys is due, at least in part, to its ability to inhibit hepatic conversion of aflatoxin B1 to the exo-afla‐ toxin B1-8,9-epoxide (AFBO) in vivo and in vitro (Guarisco, Hall, Coulombe, 2008).

Determining the outcome of inhibition of hepatic aflatoxin B1 bioactivation in whole animals is relevant to veterinary medicine and to food safety. Dietary butylated hydroxytoluene (BHT) can reduce aflatoxin B1 bioavailability, as demonstrated by serum concentrations of radiolabel which were reduced at every time interval after aflatoxin B1 administration. Among the possible explanations for reduced bioavailability is high first pass elimination prior to absorption into the blood, and/or an attenuation of mucosal aflatoxin B1 absorption. However, since no quantitative difference in the biliary elimination of aflatoxin B1 or its me‐ tabolites was identified, any change due to increased first pass effect would have to result in increased elimination by non-biliary pathways.

The observed reduction in hepatic aflatoxin B1–DNA adducts in BHT fed animals is consis‐ tent with the fact that this antioxidant is a competitive inhibitor of hepatic in vitro CYP1A5 mediated aflatoxin B1 epoxidation to AFBO. Because of the critical role of AFBO and subsequent adduct formation in aflatoxicosis (as well as longer-term consequences such as tumor formation), a reduction in hepatic aflatoxin B1–DNA adducts would be expected to have a positive effect on the overall health of aflatoxin B1-exposed turkeys.
