**5. Aflatoxins in food and commodities**

From the mycological perspective, there are great qualitative and quantitative differences in the toxigenic abilities displayed by different strains within each aflatoxigenic species. For ex‐ ample, only about half of *Aspergillus flavus* strains produce aflatoxins (Klich, Pitt, 1988), while those that do may produce more than 106 µg/kg (Cotty, Bayman, Egel, Elias, 1994). Many substrates support growth and aflatoxin production by aflatoxigenic molds. Natural contamination of cereals, figs, oilseeds, nuts, tobacco, and a long list of other commodities is a common occurrence (Detroy, Lillehoj, Ciegler, 1971; Diener, Cole, Sanders, Payne, Lee, Klich, 1987).

Crops can be contaminated with aflatoxins in the field before harvest (Diener, Cole, Sanders, Payne, Lee, Klich, 1987; Klich, 1987). Even more problematic is the fate of crops stored under conditions that favor mold growth. The most relevant variables to keep under control dur‐ ing the storage are considered the moisture content of the substrate and the relative humidi‐ ty of the surroundings (Detroy, Lillehoj, Ciegler, 1971; Wilson, Payne, 1994). There are many side implications of aflatoxins contamination. Aflatoxin contamination has been linked to in‐ creased mortality in farm animals and thus significantly lowers the value of grains as an ani‐ mal feed and as an export commodity (Smith, Moss, 1985). Milk products can also be an indirect source of information on aflatoxins presence in the diet, and considering the broad diffusion of these products mainly addressed to infants, children, and people affected by health conditions, the risk associated to aflatoxins M1 and M2 is relevant. When cows assume aflatoxin-contaminated feed, they metabolically biotransform aflatoxin B1 into a hydroxylat‐ ed form, namely aflatoxin M1, as a detoxification way for animal exposed to aflatoxins B1 or B2 (Van Egmond, 1989).

ganism (Newberne, Butler, 1969; Shank, Bhamarapravati, Gordon, Wogan, 1972; Peers, Lin‐ sell, 1973; Eaton, Groopman, 1994). The diseases caused by aflatoxin consumption are loosely called aflatoxicoses. Acute aflatoxicosis results in death; chronic aflatoxicosis results in cancer, immune suppression, and other "slow" pathological conditions (Hsieh, 1988). The liver is the primary target organ, with liver damage occurring when poultry, fish, rodents, and non human primates are fed aflatoxin B1 contaminated foodstuff. This data is not unex‐ pected because the liver is a lipophilic organ and all compounds carried by blood stream, i.e. drugs, contaminants, mycotoxins etc., are stored and concentrated in the hepatocytes that, with a long exposure time, may transform themselves in a cancer cell line. There are sub‐ stantial differences in species susceptibility. Moreover, within a given species, the magni‐ tude of the response is influenced by age, sex, weight, diet, exposure to infectious agents, and the presence of other mycotoxins and pharmacologically active substances. Thousands of studies on aflatoxin toxicity have been conducted, mostly on laboratory models or agri‐ cultural important species (Cullen, Newberne, 1994; Eaton, Groopman, 1994; Newberne,

Aflatoxins: Risk, Exposure and Remediation http://dx.doi.org/10.5772/52866 349

Cytochrome P450 enzymes convert aflatoxins to the reactive 8,9-epoxide form (also known as aflatoxin-2,3 epoxide), which is capable of binding to both DNA and proteins (Eaton, Groopman, 1994). The reactive aflatoxin epoxide binds to the N7 position of gua‐ nines. Moreover, aflatoxin B1-DNA adducts can result in GC to TA transversions. A re‐ active glutathione *S*-transferase system found in the cytosol and microsomes catalyzes the conjugation of activated aflatoxins with reduced glutathione, leading to the excretion of aflatoxins (Raj, Prasanna, Mage, Lotlikar, 1986). Variation in the level of the gluta‐ thione transferase system as well as variations in the cytochrome P450 system are con‐ sidered contributor to the differences observed in interspecies aflatoxin susceptibility

Considering the differences exhisting in aflatoxin susceptibility in test animals, it has been proven not easy to extrapolate the possible effects of aflatoxins to humans. Acute toxicity of

In 1974 it has been reported in India an outbreak of hepatitis and 100 cases of death attribut‐ ed to the consumption is heavily aflatoxins contaminated maize, causing an aflatoxins in‐ take of 2 to 6 mg per day (Krishnamachari, Bhat, Nagarajan, Tilnak, 1975). Based on these data, it has been estimated that the acute lethal dose (LD) for adults is approximately 10 to 20 mg of aflatoxins (Pitt, 2000). Aflatoxins have been in years associated to various health conditions and are considered a poison. For example it has been associated kwashiorkor, a severe malnutrition disease, to a form of pediatric aflatoxicosis (Hendrickse, 1997). Aflatox‐ ins, according to reported studies non completely assessed, could be involved in Reye's syn‐ drome, an encephalopathy, and to fatty degeneration of some target organs in children and

Exposure to aflatoxins in the diet is considered an important risk factor for the development of primary hepatocellular carcinoma, particularly in individuals already exposed to hepatitis B. There are also observed nonhepatic effects of aflatoxin B1 as reported by Coulombe (Cou‐ lombe, 1994). Several epidemiological studies have linked liver cancer incidence to estimated

Butler, 1969).

(Eaton, Ramsdel, 1992; Eaton, Groopman, 1994).

adolescents (Hayes, 1980).

aflatoxins in humans however represent a serious threat.

#### **6. Occurence**

Aflatoxins often occur in crops in the field before harvest so frequently that they are consid‐ ered mycotoxins originating from the field compared to other mycotoxins that are common‐ ly found in post-harvesting of field crops. Postharvest contamination can occur if crop drying is delayed and during crop storage if water is present in the amount required for the mold growth. Insect or rodent presence can facilitate mold onset on stored commodities. Aflatoxins have been also detected in milk, cheese, corn, peanuts, cottonseed, nuts, almonds, figs, grape berries, spices, and a variety of other foods and feeds. Milk, eggs and meat prod‐ ucts are contaminated sometimes due to the consumption by the animal of aflatoxin conta‐ minated feed, and are a clear example of carry-over. A few years after the discovery of mycotoxins, scientific understanding of the carry-over phenomenon raised immediately the interest of scientists and put focus on the risk related to food contaminated by molds. The commodities with the highest risk of aflatoxin contamination are corn, peanuts, and cotton‐ seed. Corn is probably the commodity of greatest worldwide concern, because it is grown in climates that are likely to have perennial contamination with aflatoxins. Corn is the staple food of many countries, and, also for some population corn represents the main ingredient of the diet. It is usually named as single-food with all nutritional and unwanted contami‐ nants related to its consumption. Corn can be used to produce flour and starch products and this links back to the problem statement such as aflatoxins is a likely toxin to be found in foodstuff. However, procedures used in the processing of corn help to reduce contamination of the resulting food product. This is because although aflatoxins are stable to moderately stable in most food processes, they are unstable in processes such as those used in making tortillas that employ alkaline conditions or oxidizing steps. Aflatoxin-contaminated corn and cottonseed meal in dairy rations have resulted in aflatoxin M1 contaminated milk and milk products, including non-fat dry milk, cheese, ice creams and yogurts. Even in the case of the butter, during its production due to its chemical lipid rich compostion, the accumula‐ tion and concentration of any aflatoxin M1 present in milk is usually involved.

#### **7. Aflatoxins toxicity**

Aflatoxins, and especially aflatoxin B1, are associated with both toxicity and carcinogenicity in human and animal populations. The International Agency for Research on Cancer has classi‐ fied aflatoxin B1 as a group I carcinogen (International Agency for Research on Cancer, 1982).

In particular, aflatoxin B1 is considered by medicine doctors and toxicologists as the most hepatocarcinogenic compound not produced by human activites but produced by a life or‐ ganism (Newberne, Butler, 1969; Shank, Bhamarapravati, Gordon, Wogan, 1972; Peers, Lin‐ sell, 1973; Eaton, Groopman, 1994). The diseases caused by aflatoxin consumption are loosely called aflatoxicoses. Acute aflatoxicosis results in death; chronic aflatoxicosis results in cancer, immune suppression, and other "slow" pathological conditions (Hsieh, 1988). The liver is the primary target organ, with liver damage occurring when poultry, fish, rodents, and non human primates are fed aflatoxin B1 contaminated foodstuff. This data is not unex‐ pected because the liver is a lipophilic organ and all compounds carried by blood stream, i.e. drugs, contaminants, mycotoxins etc., are stored and concentrated in the hepatocytes that, with a long exposure time, may transform themselves in a cancer cell line. There are sub‐ stantial differences in species susceptibility. Moreover, within a given species, the magni‐ tude of the response is influenced by age, sex, weight, diet, exposure to infectious agents, and the presence of other mycotoxins and pharmacologically active substances. Thousands of studies on aflatoxin toxicity have been conducted, mostly on laboratory models or agri‐ cultural important species (Cullen, Newberne, 1994; Eaton, Groopman, 1994; Newberne, Butler, 1969).

aflatoxin-contaminated feed, they metabolically biotransform aflatoxin B1 into a hydroxylat‐ ed form, namely aflatoxin M1, as a detoxification way for animal exposed to aflatoxins B1 or

Aflatoxins often occur in crops in the field before harvest so frequently that they are consid‐ ered mycotoxins originating from the field compared to other mycotoxins that are common‐ ly found in post-harvesting of field crops. Postharvest contamination can occur if crop drying is delayed and during crop storage if water is present in the amount required for the mold growth. Insect or rodent presence can facilitate mold onset on stored commodities. Aflatoxins have been also detected in milk, cheese, corn, peanuts, cottonseed, nuts, almonds, figs, grape berries, spices, and a variety of other foods and feeds. Milk, eggs and meat prod‐ ucts are contaminated sometimes due to the consumption by the animal of aflatoxin conta‐ minated feed, and are a clear example of carry-over. A few years after the discovery of mycotoxins, scientific understanding of the carry-over phenomenon raised immediately the interest of scientists and put focus on the risk related to food contaminated by molds. The commodities with the highest risk of aflatoxin contamination are corn, peanuts, and cotton‐ seed. Corn is probably the commodity of greatest worldwide concern, because it is grown in climates that are likely to have perennial contamination with aflatoxins. Corn is the staple food of many countries, and, also for some population corn represents the main ingredient of the diet. It is usually named as single-food with all nutritional and unwanted contami‐ nants related to its consumption. Corn can be used to produce flour and starch products and this links back to the problem statement such as aflatoxins is a likely toxin to be found in foodstuff. However, procedures used in the processing of corn help to reduce contamination of the resulting food product. This is because although aflatoxins are stable to moderately stable in most food processes, they are unstable in processes such as those used in making tortillas that employ alkaline conditions or oxidizing steps. Aflatoxin-contaminated corn and cottonseed meal in dairy rations have resulted in aflatoxin M1 contaminated milk and milk products, including non-fat dry milk, cheese, ice creams and yogurts. Even in the case of the butter, during its production due to its chemical lipid rich compostion, the accumula‐

tion and concentration of any aflatoxin M1 present in milk is usually involved.

Aflatoxins, and especially aflatoxin B1, are associated with both toxicity and carcinogenicity in human and animal populations. The International Agency for Research on Cancer has classi‐ fied aflatoxin B1 as a group I carcinogen (International Agency for Research on Cancer, 1982).

In particular, aflatoxin B1 is considered by medicine doctors and toxicologists as the most hepatocarcinogenic compound not produced by human activites but produced by a life or‐

B2 (Van Egmond, 1989).

348 Aflatoxins - Recent Advances and Future Prospects

**7. Aflatoxins toxicity**

**6. Occurence**

Cytochrome P450 enzymes convert aflatoxins to the reactive 8,9-epoxide form (also known as aflatoxin-2,3 epoxide), which is capable of binding to both DNA and proteins (Eaton, Groopman, 1994). The reactive aflatoxin epoxide binds to the N7 position of gua‐ nines. Moreover, aflatoxin B1-DNA adducts can result in GC to TA transversions. A re‐ active glutathione *S*-transferase system found in the cytosol and microsomes catalyzes the conjugation of activated aflatoxins with reduced glutathione, leading to the excretion of aflatoxins (Raj, Prasanna, Mage, Lotlikar, 1986). Variation in the level of the gluta‐ thione transferase system as well as variations in the cytochrome P450 system are con‐ sidered contributor to the differences observed in interspecies aflatoxin susceptibility (Eaton, Ramsdel, 1992; Eaton, Groopman, 1994).

Considering the differences exhisting in aflatoxin susceptibility in test animals, it has been proven not easy to extrapolate the possible effects of aflatoxins to humans. Acute toxicity of aflatoxins in humans however represent a serious threat.

In 1974 it has been reported in India an outbreak of hepatitis and 100 cases of death attribut‐ ed to the consumption is heavily aflatoxins contaminated maize, causing an aflatoxins in‐ take of 2 to 6 mg per day (Krishnamachari, Bhat, Nagarajan, Tilnak, 1975). Based on these data, it has been estimated that the acute lethal dose (LD) for adults is approximately 10 to 20 mg of aflatoxins (Pitt, 2000). Aflatoxins have been in years associated to various health conditions and are considered a poison. For example it has been associated kwashiorkor, a severe malnutrition disease, to a form of pediatric aflatoxicosis (Hendrickse, 1997). Aflatox‐ ins, according to reported studies non completely assessed, could be involved in Reye's syn‐ drome, an encephalopathy, and to fatty degeneration of some target organs in children and adolescents (Hayes, 1980).

Exposure to aflatoxins in the diet is considered an important risk factor for the development of primary hepatocellular carcinoma, particularly in individuals already exposed to hepatitis B. There are also observed nonhepatic effects of aflatoxin B1 as reported by Coulombe (Cou‐ lombe, 1994). Several epidemiological studies have linked liver cancer incidence to estimated aflatoxin consumption in the diet (Peers, Linsell, 1973; Van Rensburg, Cook-Mazaffari, van Schalkwyk, van der Watt, Vincent, Purchase, 1985; Li, Yoshizawa, Kawamura, Luo, Li, 2001) even if the long term quantification of individual exposure to aflatoxins is difficult. The inci‐ dence of liver cancer varies widely from country to country, but it is one of the most common occurring in China, the Philippines, Thailand, and many African countries. The presence of hepatitis B virus infection, an important risk factor for primary liver cancer, complicates many of the epidemiological studies. In one case-control study involving more than 18.000 urine samples collected over 3.5 years in Shanghai, China, aflatoxin exposure alone yielded a relative risk of about 2; hepatitis B virus antigen alone yielded a relative risk of about 5; combined expo‐ sure to aflatoxin and hepatitis B yielded a relative risk of about 60 (Ross, Yuan, Yu, Wogan, Qian, Tu, Groopman, Gao, Henderson, 1992).

ganization/World Health Organization/United Nations Environment Programme Conference report stated that "in developing countries, where food supplies are already limited, drastic le‐ gal measure may lead to lack of food and to excessive prices. It must be remembered that peo‐ ple living in these countries cannot exercise the option of starving to death today in order to live

Aflatoxins: Risk, Exposure and Remediation http://dx.doi.org/10.5772/52866 351

**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‐

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‐

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‐

a better life tomorrow" (Henry, Bosch,. Troxell, Bolger, 1999).

ids (Wild, Umbenhauer, Chapot, Montesano, 1986).

drolysed by b-glucuronidase and sulfatase (Wei, Marshall, Hsieh, 1985).

Using molecular epidemiology, it is possible to asses a link exhisting between putative carci‐ nogens and specific cancers. Biomonitoring of aflatoxins can be done by analyzing for the presence of aflatoxin metabolites in blood, milk, and urine. In addition, excreted DNA ad‐ ducts and blood protein adducts can also be monitored (Sabbioni, Sepai, 1994). The aflatoxin B1-N7 -guanine adduct is considered a reliable urinary biomarker for aflatoxin exposure but reflects only recent exposure. Many studies have shown that carcinogenic potency is highly correlated with the extent of total DNA adducts formed *in vivo* (Eaton, Gallagher, 1994; Ea‐ ton, Groopman, 1994).

Inactivation of the p53 tumor suppressor gene may be important in the development of pri‐ mary hepatocellular carcinoma. Studies of liver cancer patients in Africa and China have shown that a mutation in the p53 tumor suppressor gene at codon 249 is associated with a G-to-T transversion (Bressac, Kew, Wands, Ozturk, 1991; Hsu, Metcalf, Sun, Welsh, Wang, Harris, 1991). It is known that the reactive aflatoxin epoxide binds to the N7 position of gua‐ nines. Moreover, aflatoxin B1-DNA adducts can result in GC to TA inversion. The specific mutation in codon 249 of the p53 gene has been called the first example of a "carcinogenspecific" biomarker that remains fixed in the tumor tissue (Eaton, Gallagher, 1994).

There is also considerable evidence associating aflatoxin with neoplasms in extrahepatic tis‐ sues, particularly the lungs. For example, one early epidemiological study of Dutch peanut processing workers exposed to dust contaminated with aflatoxin B1 showed a correlation be‐ tween both respiratory cancer and total cancer in the exposed group compared with unex‐ posed cohorts (Hayes, van Nienwenhuise, Raatgever, Ten Kate, 1984). Exposition even indirect to aflatoxins can result in a severe health issue: Deger (Deger, 1976) reported for ex‐ ample that dust from scrapings of chromatographic plates from aflatoxin analyses contribut‐ ed to causing cancer in two young adults.

In developed countries, sufficient amounts of food combined with regulations that monitor aflatoxin levels protect human populations from significant aflatoxins ingestion. However, in countries where populations are facing starvation or where regulations are either not enforced or nonexistent, routine ingestion of aflatoxin may occur (Cotty, Bayman, Egel, Elias, 1994). Worldwide, liver cancer incidence rates are 2 to 10 times higher in developing countries than in developed countries (Henry, Bosch, Troxell, Bolger, 1999). A joint Food and Agriculture Or‐ ganization/World Health Organization/United Nations Environment Programme Conference report stated that "in developing countries, where food supplies are already limited, drastic le‐ gal measure may lead to lack of food and to excessive prices. It must be remembered that peo‐ ple living in these countries cannot exercise the option of starving to death today in order to live a better life tomorrow" (Henry, Bosch,. Troxell, Bolger, 1999).

aflatoxin consumption in the diet (Peers, Linsell, 1973; Van Rensburg, Cook-Mazaffari, van Schalkwyk, van der Watt, Vincent, Purchase, 1985; Li, Yoshizawa, Kawamura, Luo, Li, 2001) even if the long term quantification of individual exposure to aflatoxins is difficult. The inci‐ dence of liver cancer varies widely from country to country, but it is one of the most common occurring in China, the Philippines, Thailand, and many African countries. The presence of hepatitis B virus infection, an important risk factor for primary liver cancer, complicates many of the epidemiological studies. In one case-control study involving more than 18.000 urine samples collected over 3.5 years in Shanghai, China, aflatoxin exposure alone yielded a relative risk of about 2; hepatitis B virus antigen alone yielded a relative risk of about 5; combined expo‐ sure to aflatoxin and hepatitis B yielded a relative risk of about 60 (Ross, Yuan, Yu, Wogan,

Using molecular epidemiology, it is possible to asses a link exhisting between putative carci‐ nogens and specific cancers. Biomonitoring of aflatoxins can be done by analyzing for the presence of aflatoxin metabolites in blood, milk, and urine. In addition, excreted DNA ad‐ ducts and blood protein adducts can also be monitored (Sabbioni, Sepai, 1994). The aflatoxin

Inactivation of the p53 tumor suppressor gene may be important in the development of pri‐ mary hepatocellular carcinoma. Studies of liver cancer patients in Africa and China have shown that a mutation in the p53 tumor suppressor gene at codon 249 is associated with a G-to-T transversion (Bressac, Kew, Wands, Ozturk, 1991; Hsu, Metcalf, Sun, Welsh, Wang, Harris, 1991). It is known that the reactive aflatoxin epoxide binds to the N7 position of gua‐ nines. Moreover, aflatoxin B1-DNA adducts can result in GC to TA inversion. The specific mutation in codon 249 of the p53 gene has been called the first example of a "carcinogen-

There is also considerable evidence associating aflatoxin with neoplasms in extrahepatic tis‐ sues, particularly the lungs. For example, one early epidemiological study of Dutch peanut processing workers exposed to dust contaminated with aflatoxin B1 showed a correlation be‐ tween both respiratory cancer and total cancer in the exposed group compared with unex‐ posed cohorts (Hayes, van Nienwenhuise, Raatgever, Ten Kate, 1984). Exposition even indirect to aflatoxins can result in a severe health issue: Deger (Deger, 1976) reported for ex‐ ample that dust from scrapings of chromatographic plates from aflatoxin analyses contribut‐

In developed countries, sufficient amounts of food combined with regulations that monitor aflatoxin levels protect human populations from significant aflatoxins ingestion. However, in countries where populations are facing starvation or where regulations are either not enforced or nonexistent, routine ingestion of aflatoxin may occur (Cotty, Bayman, Egel, Elias, 1994). Worldwide, liver cancer incidence rates are 2 to 10 times higher in developing countries than in developed countries (Henry, Bosch, Troxell, Bolger, 1999). A joint Food and Agriculture Or‐

specific" biomarker that remains fixed in the tumor tissue (Eaton, Gallagher, 1994).


Qian, Tu, Groopman, Gao, Henderson, 1992).

350 Aflatoxins - Recent Advances and Future Prospects

ed to causing cancer in two young adults.

B1-N7

ton, Groopman, 1994).
