**3. Possible mechanisms of interaction between AFB1 and HBV in hepatocarcinogenesis**

A number of possible mechanisms for the interaction between HBV and AFB1 in causing HCC have been suggested. One is that HBV infection directly or indirectly sensitizes hepatocytes to the carcinogenic effects of AFB1. One way in which this may be accomplished is that the specific cytochrome P450s that metabolize AFB1 to AFB1-8,9-epoxide may be induced either by chronic hepatitis caused by HBV infection or by the presence of the virus itself. Induc‐ tion of these phase I enzymes has been described in HBV transgenic mice [21, 6], where this effect appeared to result from hepatocyte injury induced by the virus rather than the pres‐ ence of the virus *per se* [6]. The observation that Gambian and Taiwanese children and adoles‐ cents chronically infected with HBV have higher concentrations of AFB1 adducts than uninfected individuals [2, 76, 11] is consistent with this mechanism. But studies in adults in China, Taiwan and The Gambia have either failed to show a significant difference in serum AFB1 adduct levels between HBsAg-positive and -negative subjects [23, 79, 12] or showed only a margin‐ ally significant difference [74]. Moreover, a study in woodchucks with chronic woodchuck hepatitis virus infection did not show enhanced activation of AFB1 [75, 44].

those individuals also chronically infected with HBV [49]. Individuals infected with HBV alone have a population attributable risk of 8.8% [49]. If the one study in the meta-analysis that contributed most to heterogeneity in the analysis was excluded, the summarised odds ratio of HCC (with 95% confidence limits) was 73 (36 to 148.3) for the combined effects of AFB1 and HBV, 11.3 (6.75 to 18.9) for HBV alone, and 6.37 (3.74 to 10.86) for AFB1 alone [49]. The effect of a synergistic interaction between AFB1 and HBV on the age of onset of HCC was specifically addressed in a study of Taiwanese patients. HBV-infected patients in whom

Although they have had limitations, various animal models with natural hepatitis viral in‐ fections have been used to examine the interaction between hepadnaviruses and AFB1 [84]. In woodchucks and tree shrews, animal species with hepadnaviral-induced liver pathology similar to that observed in HBV-infected humans, the administration of AFB1 resulted in a higher incidence of liver tumours than in infected animals not receiving AFB1 [92, 3]. More‐ over, HBsAg transgenic mice over-expressing the large envelope protein of HBsAg in the liver developed HCC when exposed to aflatoxin, whereas their littermates not exposed to

In those human populations in which an interaction between the fungal toxin and HBV has been described, the infection is predominantly acquired in infancy or early childhood. Dur‐ ing the early years of HBV infection, a state of immune tolerance towards the virus exists and little if any cellular damage occurs. With loss of this tolerance, the ongoing infection re‐ sults in recurring cell damage. Exposure to AFB1 in contaminated foodstuffs also occurs in

Nevertheless, it is likely, certainly in China and Taiwan, where perinatal transmission of HBV is the predominant mode of infection, and also probably in Africa, where slightly later horizontal infection is the major route of infection, that the HBV carrier state is established,

A number of possible mechanisms for the interaction between HBV and AFB1 in causing HCC have been suggested. One is that HBV infection directly or indirectly sensitizes hepatocytes to the carcinogenic effects of AFB1. One way in which this may be accomplished is that the specific cytochrome P450s that metabolize AFB1 to AFB1-8,9-epoxide may be induced either by chronic hepatitis caused by HBV infection or by the presence of the virus itself. Induc‐ tion of these phase I enzymes has been described in HBV transgenic mice [21, 6], where this effect appeared to result from hepatocyte injury induced by the virus rather than the pres‐ ence of the virus *per se* [6]. The observation that Gambian and Taiwanese children and adoles‐ cents chronically infected with HBV have higher concentrations of AFB1 adducts than uninfected individuals [2, 76, 11] is consistent with this mechanism. But studies in adults in China, Taiwan

**3. Possible mechanisms of interaction between AFB1 and HBV in**

not before exposure to, but before heavy exposure to the toxin.


tumour tissue was shown by histochemical staining to be positive for AFB1-N7

carcinogens did not [67, 47].

228 Aflatoxins - Recent Advances and Future Prospects

young children [81].

**hepatocarcinogenesis**

were on average 10 years younger than those with adduct-negative tumours [10].

The generated aflatoxin-8,9-epoxide has been shown to bind to proteins, causing acute toxic‐ ity, or to DNA inducing changes that over time increase the risk of malignant transforma‐ tion [26]. DNA damage can also increase the chance of integration of the viral DNA into the host genome [16]. This effect could be exerted directly by AFB1 or indirectly by oxidative stress induced by chronic viral hepatitis.

A guanine to thymine transversion at the third base of codon 249 of the p53 tumour sup‐ pressor gene (arginine to serine substitution; 249ser, R249S) is present in between 40 and 66% of HCC patients in regions with heavy dietary exposure to AFB1 [28, 4, 45, 30]. The mutation is also detectable in circulating cell-free DNA from the plasma of HCC patients and healthy subjects from these regions [77]. The exact timing of the development of the 249ser mutation remains uncertain, although it has been shown to be an early event. The mutation abrogates the normal functions of p53, including those in cell cycle control, DNA repair, and apopto‐ sis, thereby contributing to the multistep process of hepatocarcinogenesis. This mutation is extremely uncommon in tumors other than HCC [58].

A specific and close association between this inactivating mutation, the presence of AFB1 biomarkers, and the development of HCC was recognised in epidemiological studies in re‐ gions with high or low AFB1 exposure rates [4, 60, 15, 19, 45, 30, 62, 22], and evidence that the mutation induced chromosomal instability was found [62]. Arising from the observation of the co-existence of the p53 mutation and AFB1 exposure, the presence of the 249ser muta‐ tion was believed to be a primary genetic event in hepatocarcinogenesis. It occurs early in the series of events leading to AFB1-associated HCC, and may thus provide an early bio‐ marker of exposure to the fungal toxin and AFB1-induced hepatocarcinogenesis [36].

But the findings have been inconsistent with support for an aetiological association being provided by some but not all studies. In an investigation of Taiwanese patients with HCC the mutation was present in 36.3% of HBV-infected patients with HCC, compared with 11.7% of those without HBV markers [79]. In a second analysis in Taiwan, all of the 249ser mutations occurred in patients positive for HBsAg, giving an odds ratio of 10.0 (95% confi‐ dence limits 1.6; 17.5) [54]. In a study in The Gambia patients positive for HBsAg alone had an increased relative risk of 10, those with 249ser mutation alone of13, and those with both an estimated risk of 399 [45]. Other studies, however, showed a similar, but not a statistically significant trend [68, 19], and in yet other analyses from a variety of countries no association could be found [listed in reference: 69]. Furthermore, in a meta-analysis of 49 published studies using a method that takes into account both within-study and study-to-study varia‐ bility, little evidence for HBV-AFB1 interaction in modulating the 249ser mutation was found [69]. In addition, the absence of the 249ser mutation from the serum of patients from coun‐ tries with a low incidence of HBV-induced HCC to date suggests that chronic HBV infection alone is insufficient to result in the development of the 249ser mutation [82].

Another suggested possibility is that the activity of phase II detoxification enzymes (gluta‐ thione S transferase (GST) and epoxide hydrolase(EPHX)) may play a role in the genesis of HCC induced jointly by AFB1 and HBV [94; 73; 56]. A multiplicative interaction in the gene‐ sis of HCC in West African and Chinese patients was demonstrated between HBV infection and mutations of EPHX [56]: patients with chronic HBV infection but with normal EPHX al‐ leles were at a 15-fold increase in risk, and those with both HBV infection and at least one EPHX mutant were at a 77-fold increased risk. In further studies in these patients a positive interaction between HBV and AFB1 seemed to depend on the presence of a polymorphism of the GST M1, GST T1, and EPHX genes that are normally responsible for converting the car‐ cinogenic AFB1 -8,9-epoxide to non-reactive metabolites [56, 8, 94, 73]. But again no consis‐ tent pattern has emerged. In one analysis in Taiwan the risk of HCC formation was greater in HBV carriers who had the GST M1 null genotype compared with the non-null genotype [94], in a second study the risk appeared to depend on the presence of a GST T1 null geno‐ type [73], and in a third the risk was considerably greater in those with null genotypes of both GST M1 and GST T1 [8].

complete, and there is clearly a need for further research to be undertaken into the pathoge‐ netic mechanisms involved in this interaction between the two common hepatocarcinogens

Synergistic Interaction Between Aflatoxin and Hepatitis B Virus in Hepatocarcinogenesis

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

231

1 Department of Medicine, Faculty of Health Sciences, University of Cape Town, Depart‐ ment of Medicine, Faculty of Health Sciences, University of the Witwatersrand, Johannes‐

[1] Ahn, J. I., Jung, E. Y., Kwun, H. J., et al. (2002). Dual effects of hepatitis B virus X pro‐ tein on the regulation of cell cycle depending on the status of cellular p53. *J Gen Virol*,

[2] Allen, S. J., Wild, C. P., Wheeler, J. G., et al. (1992). Aflatoxin exposure and hepatitis B infection in rural Gambian children. *Trans Roy Soc Trop Med Hyg*, 86, 426-430.

[3] Bannasch, P., Khoshkhou, N. I., Hacker, H. J., et al. (1995). Synergistic hepatocarcino‐ genic effect of hepadnaviral infection and dietary aflatoxin B1 in woodchucks. *Cancer*

[4] Bressac, B., Kew, M. C., Wands, J. R., et al. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. *Nature Lond*, 350, 429-430. [5] Brown, K. L., Deng, J. Z., Iver, R. S., et al. (2006). Unravelling the aflatoxin-FABY co‐ nundrum: structural basis for differential replicative processing of isomeric forms of the formaminopyrimidine DNA adduct of aflatoxin B1. *J Am Chem Soc*, 128,

[6] Chemin, I., Ohgaki, H., Chisari, F. V., et al. (1999). Altered expression of hepatic car‐ cinogen metabolizing enzymes with liver injury in HBV transgenic mice lineages ex‐

[7] Chen, C. J., Liang, K. Y., Chang, A. S., et al. (1991). Effect of hepatitis B virus, alcohol drinking, cigarette smoking and familial tendency on hepatocellular carcinoma. *Hep‐*

[8] Chen, C. J., Wang, L. Y., Lu, S. N., et al. (1996). Elevated aflatoxin exposure and in‐

pressing various amounts of hepatitis B surface antigen. *Liver* , 19, 81-87.

creased risk of hepatocellular carcinoma. *Hepatology*, 24, 38-42.

in resource-constrained geographical regions.

Address all correspondence to: michael.kew@uct.ac.za

**Author details**

Michael C. Kew1\*

burg, South Africa

83, 2765-2772.

*Res*, 55, 3318-3330.

15188-15199.

*atology*, 13, 398-406.

**References**

Another possible mechanism for a carcinogenic interaction between AFB1 and HBV is that increased hepatocyte necrosis and proliferation cause by chronic HBV infection increases the likelihood of both AFB1 mutations, including 249ser, and the subsequent clonal expansion of cells containing these mutations [13]. Chronic necroinflammatory hepatic disease, including that resulting from HBV infection, results in the generation of oxygen and nitrogen reactive species [50, 35]. Both of the latter are mutagenic, but, in addition, increased oxidative stress has been shown to induce 249ser mutations [29].

The HBV x gene is frequently included in sequences of the virus that are integrated into cel‐ lular DNA [43]. AFB1-DNA adducts are normally repaired by the nucleotide excision repair pathway. The HBV X protein interferes with the nuclear excision repair pathway [38; 43] and might, by this means, favour persistence of existing mutations or impaired DNA. DNA repair is also compromised by the rapid cell turnover rate in chronic hepatitis. In the pres‐ ence of dietary exposure to AFB1, the HB X protein may contribute to the uncontrolled cell proliferation in other ways. The transcription of p21 waf1/ cip1, which induces cell cycle arrest at the G1-S checkpoint, is activated by HB X protein in a dose-dependent manner in the pres‐ ence of functional p53. This transcription is, however, repressed by HB X protein when p53 is not functional or is functional at a low level [1]. The expression of HB X protein also corre‐ lates with an increase in the overall frequency of DNA mutations in transgenic mice and a 2 fold increase the incidence of the 249ser mutation in transgenic mice exposed to AFB1 [55].

Altered methylation of genes may play a role in hepatocarcinogenesis [43]. For example, the methylation status of the human *ras* association domain gene (RASSF1A) and the P16 gene has been incriminated in the pathogenesis of HCC [95]. No association was found between methylation status and P53 status {95]. A statistically significant association was, however, found between RASSF1A methylation status and the level of AFB1-DNA adducts in HCC tissues [95].

An understanding of the mechanisms responsible for the heightened risk of malignant transformation in patients chronically infected with HBV and exposed to AFB1 is far from complete, and there is clearly a need for further research to be undertaken into the pathoge‐ netic mechanisms involved in this interaction between the two common hepatocarcinogens in resource-constrained geographical regions.
