**1. Introduction**

Based on the number of new cases of cancer in humans each year, hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide [20], the fifth in males and the seventh in females [37]. In the most recently reported year, 748,000 new cases of the tumour were recorded, constituting 9.2% of all new cancers [20]. Furthermore, the number of new cases of the tumour continues to increase year by year. Not only is HCC common, it also carries an especially grave prognosis, ranking third in annual cancer mortality rates. In the year men‐ tioned, the total death rate from the tumour was 695,900. Of the patients who died, 93% did so within 12 months of the onset of symptoms. This 12 month fatality ratio in HCC (0.93 - 0.96) is the highest of any human tumour.

HCC does not have a uniform geographical distribution. Rather, of all the new cases of the cancer recorded during recent years, approximately 84% occurred in resource-constrained (developing) countries [20], particularly in sub-Saharan Africa and the Asia Pacific region. In these regions the dominant cause of HCC is chronic hepatitis B virus (HBV) infection. This infection is almost invariably acquired very early in life, either as a result of perinatal transmission of the virus or of horizontal transmission in infancy or early childhood, times at which the infection very often becomes chronic [42]. The tumour resulting from the HBV infection frequently occurs at a young or relatively young age, and it carries a particularly grave prognosis.

In addition to chronic HBV infection, the other major cause of HCC in these high-risk re‐ gions is dietary exposure to aflatoxins, the toxic secondary metabolites of the fungi, *Aspergil‐ lus flavus* and *Aspergillus parasiticus.* These viral and fungal risk factors are largely responsible for the striking geographical variation in incidence of HCC. Both aflatoxin expo‐ sure and chronic HBV infection are more common in rural than in urban dwellers in re‐

© 2013 Kew; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Kew; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

source-constrained regions [42, 63]. In these regions, the association between aflatoxin exposure and the development of HCC is closest in sub-Saharan Africa [51].

ed that by reducing dietary AFB1 levels to below detectable limits in Asia and sub-Saharan Africa, between 72,800 and 98,800 new cases of HCC could be prevented each year [49].

The major human cytochrome P450 (CYP) enzymes involved in aflatoxin metabolism are CYP3A4, 3A5, 3A7 and 1A2, and the predominant site of metabolism is the liver [87, 39]. AFB1 is metabolized to an AFB1-8,9-*exo*-epoxide and, to a lesser extent, an AFB1-8,9-*endo*-ep‐ oxide. The *exo*-epoxide binds to DNA to form the predominant promutagenic 8,9-dihydro-8-

secondary forms, an apurinic site and a more stable ring-opened AFB1-formamidopyrimi‐ dine (AFB1-FABY) adduct, which is far more persistent *in vivo.* This adduct causes G to T transversion mutations [28, 4, 87], the most prevalent of which are targeted to the site of the original adduct. AFB1-FABY exists as a mixture of two rotameric forms. In *Escherichia coli*

mutations also occurring adjacent to the site of adduct formation [82]. AFB1-FABY also re‐ sulted in blocked replication. Subsequent studies showed that the form of AFB1-FABY nor‐ mally present in double-stranded DNA is mutagenic, whereas the dominant species in

Chronic liver injury and regenerative hyperplasia are critical to the development of HCC [30]. AFB1-induced DNA adducts may therefore be fixed as mutations consequent to an HBV-related increase in cell proliferation and hyperplasia. Inflammation and oxidative stress associated with chronic active hepatitis and aflatoxin exposure may also result direct‐

The 'DNA damage checkpoint response' acts as an anti-tumour mechanism against genotox‐ ic agents. By playing a central role in co-ordinating DNA repair and cell cycle progression, 'DNA damage checkpoint response' proteins play a key role in preventing mutations [66]. Genotoxic doses of AFB1 induce an incomplete and inefficient 'DNA damage checkpoint re‐

AFB1 has a geographical distribution similar to that of chronic HBV infection, colonizing a variety of foodstuffs in the same Far Eastern and sub-Saharan African countries. According‐ ly, a synergistic interaction between the hepatocarcinogenic effects of HBV and AFB1 would offer a plausible explanation for the very high incidence of HCC, and perhaps also the

**2. Evidence for a synergistic hepatocarcinogenic interaction between**

Although a study in Guanxi, China published in the mid-1980s showed that HCC occurring in individuals infected with HBV who lived in villages with a "high" consumption of afla‐ toxins had a mortality rate that was 10 times higher than that in individuals living in vil‐ lages with a "low" consumption [93], other early studies of the consequences of exposure to aflatoxins did not include data on the HBV status of the populations studied. All of these

sponse', which may contribute to the carcinogenic properties of the toxin [27].

AFB1-FABY induced a six-fold higher G to T mutation frequency than AFB1-N7


Synergistic Interaction Between Aflatoxin and Hepatitis B Virus in Hepatocarcinogenesis


http://dx.doi.org/10.5772/51396

225


(N7


single-stranded DNA blocks replication [5].

ly in DNA damage and mutations [52].

young age of the patients, in these regions.

**aflatoxins and hepatitis B virus**

Aflatoxins are structurally-related difuranocoumarin derivatives, some of which are muta‐ genic and carcinogenic in humans and animals [89, 87]. These toxins are widely distributed in nature. Because atmospheric humidity and moisture content of plants are important fac‐ tors in determining growth of, and toxin production by, these moulds, contamination of crops occurs mainly in tropical and sub-tropical climates with high humidity and tempera‐ ture. These conditions exist in sub-Saharan Africa, the Asia Pacific region, and parts of South America. Contamination is particularly likely to occur in subsistence farming com‐ munities in regions with these climates and where regulations to control exposure to the fungi are either non-existent or unenforceable in practice.

In these regions, the moulds contaminate a variety of staple foods, especially maize and groundnuts [89, 34, 87]. Because most rural dwellers can afford only limited food variation, these staples make up a significant portion of their diets. Contamination of crops with afla‐ toxins occurs either during their growth or as a result of their storage under conditions that promote fungal growth and toxin production [31, 32, 33]. Exposure begins *in utero* as a result of trans-placental transmission of the toxins [86] and in the postnatal period as a result of breast-feeding [85], and continues throughout life. Exposure increases with increasing age for example, in Malaysia evidence of exposure was more common in the population aged 31 to 50 years than that aged 18 to 30 years [46].

Approximately 4.5 billion of the world's population are believed to be exposed to aflatoxins [88]. Between 25,200 new cases of HCC each year (or 4.6% of all cases of the tumour world‐ wide) and 155,000 new cases each year (or 28.2% of all cases of the tumour world wide) may be attributed to this exposure [51]. It has been estimated that aflatoxins play a causative role in at least 4.6% and at most 28.2% of all cases of HCC worldwide [51]. These large ranges stem from the considerable uncertainty and variability in data on cancer potency factors, HBV prevalence, aflatoxin exposure, and other risk factors [51].

Although the parent aflatoxin molecule is harmless, it is converted by members of the cyto‐ chrome 450 superfamily into electrophilic intermediates that are mutagenic and carcinogen‐ ic [90, 87, 40, 71]. Of the four naturally occurring aflatoxins, aflatoxin B1 (AFB1), B2, G1 and G2, toxigenic strains of *A. flavus* typically produce only aflatoxins B1 and B2, whereas most strains of *A. parasiticus* produce all of the aflatoxins [18]. AFB1 is the most potent experimen‐ tal hepatocarcinogen known to man -- no animal model exposed to the toxin thus far has failed to develop HCC. AFM1, the hydroxylation product of AFB1, is found in milk and milk products when animals intended for dairy production consume aflatoxin-contaminated feed [70]. In rodents exposed to AFB1 in equivalent doses to those occurring in humans, levels of aflatoxin adduct in the serum have correlated with levels of hepatic DNA damage and with development of HCC [83].

AFB1 is the aflatoxin most often found in contaminated human foodstuffs [78], and exposure to AFB1 is causally related to the development of HCC in humans [34]. The correlation be‐ tween the degree of exposure to AFB1 and the incidence of HCC is direct. It has been estimat‐ ed that by reducing dietary AFB1 levels to below detectable limits in Asia and sub-Saharan Africa, between 72,800 and 98,800 new cases of HCC could be prevented each year [49].

source-constrained regions [42, 63]. In these regions, the association between aflatoxin

Aflatoxins are structurally-related difuranocoumarin derivatives, some of which are muta‐ genic and carcinogenic in humans and animals [89, 87]. These toxins are widely distributed in nature. Because atmospheric humidity and moisture content of plants are important fac‐ tors in determining growth of, and toxin production by, these moulds, contamination of crops occurs mainly in tropical and sub-tropical climates with high humidity and tempera‐ ture. These conditions exist in sub-Saharan Africa, the Asia Pacific region, and parts of South America. Contamination is particularly likely to occur in subsistence farming com‐ munities in regions with these climates and where regulations to control exposure to the

In these regions, the moulds contaminate a variety of staple foods, especially maize and groundnuts [89, 34, 87]. Because most rural dwellers can afford only limited food variation, these staples make up a significant portion of their diets. Contamination of crops with afla‐ toxins occurs either during their growth or as a result of their storage under conditions that promote fungal growth and toxin production [31, 32, 33]. Exposure begins *in utero* as a result of trans-placental transmission of the toxins [86] and in the postnatal period as a result of breast-feeding [85], and continues throughout life. Exposure increases with increasing age for example, in Malaysia evidence of exposure was more common in the population aged 31

Approximately 4.5 billion of the world's population are believed to be exposed to aflatoxins [88]. Between 25,200 new cases of HCC each year (or 4.6% of all cases of the tumour world‐ wide) and 155,000 new cases each year (or 28.2% of all cases of the tumour world wide) may be attributed to this exposure [51]. It has been estimated that aflatoxins play a causative role in at least 4.6% and at most 28.2% of all cases of HCC worldwide [51]. These large ranges stem from the considerable uncertainty and variability in data on cancer potency factors,

Although the parent aflatoxin molecule is harmless, it is converted by members of the cyto‐ chrome 450 superfamily into electrophilic intermediates that are mutagenic and carcinogen‐ ic [90, 87, 40, 71]. Of the four naturally occurring aflatoxins, aflatoxin B1 (AFB1), B2, G1 and G2, toxigenic strains of *A. flavus* typically produce only aflatoxins B1 and B2, whereas most strains of *A. parasiticus* produce all of the aflatoxins [18]. AFB1 is the most potent experimen‐ tal hepatocarcinogen known to man -- no animal model exposed to the toxin thus far has failed to develop HCC. AFM1, the hydroxylation product of AFB1, is found in milk and milk products when animals intended for dairy production consume aflatoxin-contaminated feed [70]. In rodents exposed to AFB1 in equivalent doses to those occurring in humans, levels of aflatoxin adduct in the serum have correlated with levels of hepatic DNA damage and with

AFB1 is the aflatoxin most often found in contaminated human foodstuffs [78], and exposure to AFB1 is causally related to the development of HCC in humans [34]. The correlation be‐ tween the degree of exposure to AFB1 and the incidence of HCC is direct. It has been estimat‐

exposure and the development of HCC is closest in sub-Saharan Africa [51].

fungi are either non-existent or unenforceable in practice.

HBV prevalence, aflatoxin exposure, and other risk factors [51].

to 50 years than that aged 18 to 30 years [46].

224 Aflatoxins - Recent Advances and Future Prospects

development of HCC [83].

The major human cytochrome P450 (CYP) enzymes involved in aflatoxin metabolism are CYP3A4, 3A5, 3A7 and 1A2, and the predominant site of metabolism is the liver [87, 39]. AFB1 is metabolized to an AFB1-8,9-*exo*-epoxide and, to a lesser extent, an AFB1-8,9-*endo*-ep‐ oxide. The *exo*-epoxide binds to DNA to form the predominant promutagenic 8,9-dihydro-8- (N7 -guanyl)-9-hydroxy AFB1 (AFB1-N7 -Gua) adduct. AFB1-N7 -Gua can result in two secondary forms, an apurinic site and a more stable ring-opened AFB1-formamidopyrimi‐ dine (AFB1-FABY) adduct, which is far more persistent *in vivo.* This adduct causes G to T transversion mutations [28, 4, 87], the most prevalent of which are targeted to the site of the original adduct. AFB1-FABY exists as a mixture of two rotameric forms. In *Escherichia coli* AFB1-FABY induced a six-fold higher G to T mutation frequency than AFB1-N7 -Gua, with mutations also occurring adjacent to the site of adduct formation [82]. AFB1-FABY also re‐ sulted in blocked replication. Subsequent studies showed that the form of AFB1-FABY nor‐ mally present in double-stranded DNA is mutagenic, whereas the dominant species in single-stranded DNA blocks replication [5].

Chronic liver injury and regenerative hyperplasia are critical to the development of HCC [30]. AFB1-induced DNA adducts may therefore be fixed as mutations consequent to an HBV-related increase in cell proliferation and hyperplasia. Inflammation and oxidative stress associated with chronic active hepatitis and aflatoxin exposure may also result direct‐ ly in DNA damage and mutations [52].

The 'DNA damage checkpoint response' acts as an anti-tumour mechanism against genotox‐ ic agents. By playing a central role in co-ordinating DNA repair and cell cycle progression, 'DNA damage checkpoint response' proteins play a key role in preventing mutations [66]. Genotoxic doses of AFB1 induce an incomplete and inefficient 'DNA damage checkpoint re‐ sponse', which may contribute to the carcinogenic properties of the toxin [27].

AFB1 has a geographical distribution similar to that of chronic HBV infection, colonizing a variety of foodstuffs in the same Far Eastern and sub-Saharan African countries. According‐ ly, a synergistic interaction between the hepatocarcinogenic effects of HBV and AFB1 would offer a plausible explanation for the very high incidence of HCC, and perhaps also the young age of the patients, in these regions.
