**2. Evidence for a synergistic hepatocarcinogenic interaction between aflatoxins and hepatitis B virus**

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 studies showed a statistically significant increase in incidence of HCC in those individuals who were exposed to the fungal toxin [31, 53, 32]. But, given the high frequency of chronic HBV infection in this region at that time, the probability exists that at least some, and possi‐ bly a significant number, of the subjects included in these early studies were also chronically infected with HBV and that the virus, rather than the exposure to aflatoxins, could have caused the malignant transformation or, at least, contributed to it. In two studies, one earlier and the other later, the roles of AFB1 and HBV in explaining the varying frequencies of HCC in different areas of Swaziland in southern Africa [61] and in Guanxi Province in China [93] were assessed. Both analyses concluded that with simultaneous exposure to the two poten‐ tial carcinogenic agents, AFB1 exposure was the more important determinant of geographi‐ cal variation in the incidence of HCC than was HBV infection, at least in those regions. However, no attempt was made in either study to evaluate a possible interactive hepatocar‐ cinogenic effect between the two risk factors.

**HBV alone AFB1 alone HBV and AFB1 RR (95% CL)\* RR (95% CL) RR (5% CL)**

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0.3 (0 - 3.6) 70.0 (11.5 –


[65] 4.8 (1.2 -19.7) 1.9 (0.5 - 7.5) 60.1 (6.4 - 561.8) [64] 7.3 (2.2 - 24.4) 3.4 (1.1 - 10.0) 59.4 (15.6 - 212)

[54] 17.0 (2.8 - 103.9) 17.4 (3.4 - 90.3) 67.6 (12.2 - 373.2) [59] 3.3 (1.3 - 8.3) 32.0 (4.0 - 255.8) 40.7 (12.7- 130.9)

**Table 1.** Findings in five studies comparing the risk of HBV infection alone, dietary exposure to AFB1 alone, and the

A synergistic interaction between AFB1 exposure and chronic HBV infection in causing HCC was evident in each of the five studies - odds ratios ranged from 40.7 to 70.0 with a mean of 59.6 (Table 1). In three of these studies there was a striking multiplicative effect, and in the other two a sub-multiplicative effect between exposure to AFB1 alone and exposure to AFB1 in the presence of chronic HBV infection in inducing HCC, in comparison with each carcino‐ gen alone. The study which did not show an increased odds ratio with AFB1 alone had the highest odds ratios for both HBV infection alone and for co-existing AFB1 exposure and HBV infection [79]. The finding in this study that exposure to AFB1 alone did not increase the risk of HCC development [79] could conceivably be the source of the erroneous view held by some hepatologists and oncologists that AFB1 alone does not cause HCC and is im‐

In other investigations, also in countries with high rates of contamination of foodstuffs by AFB1, only individuals chronically infected with HBV were studied and the influence of AFB1 exposure in further increasing their risk of HCC development was analysed. In Qi‐ dong county, China, over a 10-year prospective follow-up period, the risk of HCC in male carriers of the virus was shown to be increased 3-fold (95% confidence limits 1.2, 8.7) in those with detectable urinary levels of AFB1 metabolites in comparison with those without these metabolites [72]. This result was later confirmed in a longer follow-up of the same co‐ hort of HBV carriers, when the risk of HCC was increased 3.5-fold (95% confidence limits 1.5, 8.1) [57]. A dose-response relationship between urinary AFB1 metabolites and the risk of HCC was shown in HBV carriers in Taiwan [94]. Comparing high and low urinary levels of the aflatoxin metabolite, AFM1, a multivariate-adjusted odds ratio of 6.0 (95% confidence limits1.2, 29) was calculated. The risk was greater (odds ratio 10.0: 95% confidence limits

the urine. In another study performed in chronic carriers of HBV in the same country, a stat‐ istically significant relationship was noted between detectable levels of AFB1 adducts in se‐ rum and the risk of HCC, with an age-adjusted odds ratio of 2.0 (95% confidence limits 1.1, 3.7) [73]. A recent meta-analysis has shown that the population attributable risk of develop‐ ing HCC in individuals exposed to dietary aflatoxins is 17%, with the risk being 21% in

[80] 17.4 (3.6 – 143.4)

portant only as a co-carcinogen with HBV.

1.6 ; 60.9) when both AFM1 and AFB1-N7

425.4)

two risk factors together in the genesis of HCC. \* Relative risk (95% confidence limits).

The first published evidence consistent with synergism between AFB1 and HBV in the gene‐ sis of HCC was provided by experiments in which transgenic mice over-expressing the large envelope polypeptide of HBV were fed AFB1. These mice produced more rapid and exten‐ sive hepatocyte dysplasia than did their unexposed littermates, and HCCs developed [67]. Shortly thereafter, further experimental evidence for a positive interaction between AFB1 and another member of the *Hepadnaviridae* family, the woodchuck hepatitis virus, in the de‐ velopment of HCC was presented [3]. Woodchucks infected with woodchuck hepatitis virus and exposed to AFB1 developed, after six to 26 months of exposure, a high incidence of pre‐ neoplastic foci of altered hepatocytes followed by hepatocellular adenomas and HCCs. Moreover, woodchucks infected with woodchuck hepatitis virus had earlier been shown to have enhanced activation of the biologically inactive AFB1 to AFB1-8,9-epoxide [17]. The de‐ velopment of liver tumours was also reported in ducks infected with duck hepatitis virus and exposed to AFB1 [14] and in tree shrews (*Tupaia belangeri chinensis*) infected with HBV and exposed to AFB1 [48].

Following the introduction of methods to measure aflatoxin metabolites and aflatoxin-DNA adducts in urine and aflatoxin-albumin adducts in serum, biomarkers that were a far more accurate and reliable indicator of AFB1 exposure than the hitherto used food sampling and dietary questionnaires, five large cohort studies were undertaken in Shanghai and Qidong county, China and in Taiwan to assess the carcinogenic effects of AFB1 and HBV alone and in combination. In four of the studies an hepatocarcinogenic effect of AFB1 alone was shown, with increased odds ratios ranging from 1.9 to 32.0 with a mean ratio of 13.7 [65, 64, 54, 59] (Table 1). These studies proved that exposure to AFB1 alone could cause malignant transformation of hepatocytes in humans. The fifth study failed to show an increased odds ratio of AFB1 exposure alone [79].

As expected, these studies (including the one that did not show an increased odds ratio for the development of HCC for AFB1 alone [79]) confirmed an hepatocarcinogenic effect of HBV alone - odds ratios ranged from 3.3 to 17.4, with a mean ratio of 10.0 (Table 1).


studies showed a statistically significant increase in incidence of HCC in those individuals who were exposed to the fungal toxin [31, 53, 32]. But, given the high frequency of chronic HBV infection in this region at that time, the probability exists that at least some, and possi‐ bly a significant number, of the subjects included in these early studies were also chronically infected with HBV and that the virus, rather than the exposure to aflatoxins, could have caused the malignant transformation or, at least, contributed to it. In two studies, one earlier and the other later, the roles of AFB1 and HBV in explaining the varying frequencies of HCC in different areas of Swaziland in southern Africa [61] and in Guanxi Province in China [93] were assessed. Both analyses concluded that with simultaneous exposure to the two poten‐ tial carcinogenic agents, AFB1 exposure was the more important determinant of geographi‐ cal variation in the incidence of HCC than was HBV infection, at least in those regions. However, no attempt was made in either study to evaluate a possible interactive hepatocar‐

The first published evidence consistent with synergism between AFB1 and HBV in the gene‐ sis of HCC was provided by experiments in which transgenic mice over-expressing the large envelope polypeptide of HBV were fed AFB1. These mice produced more rapid and exten‐ sive hepatocyte dysplasia than did their unexposed littermates, and HCCs developed [67]. Shortly thereafter, further experimental evidence for a positive interaction between AFB1 and another member of the *Hepadnaviridae* family, the woodchuck hepatitis virus, in the de‐ velopment of HCC was presented [3]. Woodchucks infected with woodchuck hepatitis virus and exposed to AFB1 developed, after six to 26 months of exposure, a high incidence of pre‐ neoplastic foci of altered hepatocytes followed by hepatocellular adenomas and HCCs. Moreover, woodchucks infected with woodchuck hepatitis virus had earlier been shown to have enhanced activation of the biologically inactive AFB1 to AFB1-8,9-epoxide [17]. The de‐ velopment of liver tumours was also reported in ducks infected with duck hepatitis virus and exposed to AFB1 [14] and in tree shrews (*Tupaia belangeri chinensis*) infected with HBV

Following the introduction of methods to measure aflatoxin metabolites and aflatoxin-DNA adducts in urine and aflatoxin-albumin adducts in serum, biomarkers that were a far more accurate and reliable indicator of AFB1 exposure than the hitherto used food sampling and dietary questionnaires, five large cohort studies were undertaken in Shanghai and Qidong county, China and in Taiwan to assess the carcinogenic effects of AFB1 and HBV alone and in combination. In four of the studies an hepatocarcinogenic effect of AFB1 alone was shown, with increased odds ratios ranging from 1.9 to 32.0 with a mean ratio of 13.7 [65, 64, 54, 59] (Table 1). These studies proved that exposure to AFB1 alone could cause malignant transformation of hepatocytes in humans. The fifth study failed to show an increased odds

As expected, these studies (including the one that did not show an increased odds ratio for the development of HCC for AFB1 alone [79]) confirmed an hepatocarcinogenic effect of

HBV alone - odds ratios ranged from 3.3 to 17.4, with a mean ratio of 10.0 (Table 1).

cinogenic effect between the two risk factors.

226 Aflatoxins - Recent Advances and Future Prospects

and exposed to AFB1 [48].

ratio of AFB1 exposure alone [79].

**Table 1.** Findings in five studies comparing the risk of HBV infection alone, dietary exposure to AFB1 alone, and the two risk factors together in the genesis of HCC. \* Relative risk (95% confidence limits).

A synergistic interaction between AFB1 exposure and chronic HBV infection in causing HCC was evident in each of the five studies - odds ratios ranged from 40.7 to 70.0 with a mean of 59.6 (Table 1). In three of these studies there was a striking multiplicative effect, and in the other two a sub-multiplicative effect between exposure to AFB1 alone and exposure to AFB1 in the presence of chronic HBV infection in inducing HCC, in comparison with each carcino‐ gen alone. The study which did not show an increased odds ratio with AFB1 alone had the highest odds ratios for both HBV infection alone and for co-existing AFB1 exposure and HBV infection [79]. The finding in this study that exposure to AFB1 alone did not increase the risk of HCC development [79] could conceivably be the source of the erroneous view held by some hepatologists and oncologists that AFB1 alone does not cause HCC and is im‐ portant only as a co-carcinogen with HBV.

In other investigations, also in countries with high rates of contamination of foodstuffs by AFB1, only individuals chronically infected with HBV were studied and the influence of AFB1 exposure in further increasing their risk of HCC development was analysed. In Qi‐ dong county, China, over a 10-year prospective follow-up period, the risk of HCC in male carriers of the virus was shown to be increased 3-fold (95% confidence limits 1.2, 8.7) in those with detectable urinary levels of AFB1 metabolites in comparison with those without these metabolites [72]. This result was later confirmed in a longer follow-up of the same co‐ hort of HBV carriers, when the risk of HCC was increased 3.5-fold (95% confidence limits 1.5, 8.1) [57]. A dose-response relationship between urinary AFB1 metabolites and the risk of HCC was shown in HBV carriers in Taiwan [94]. Comparing high and low urinary levels of the aflatoxin metabolite, AFM1, a multivariate-adjusted odds ratio of 6.0 (95% confidence limits1.2, 29) was calculated. The risk was greater (odds ratio 10.0: 95% confidence limits 1.6 ; 60.9) when both AFM1 and AFB1-N7 - gua metabolites were tested for and detected in the urine. In another study performed in chronic carriers of HBV in the same country, a stat‐ istically significant relationship was noted between detectable levels of AFB1 adducts in se‐ rum and the risk of HCC, with an age-adjusted odds ratio of 2.0 (95% confidence limits 1.1, 3.7) [73]. A recent meta-analysis has shown that the population attributable risk of develop‐ ing HCC in individuals exposed to dietary aflatoxins is 17%, with the risk being 21% in 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 tumour tissue was shown by histochemical staining to be positive for AFB1-N7 -gua adducts were on average 10 years younger than those with adduct-negative tumours [10].

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

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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

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

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].

alone is insufficient to result in the development of the 249ser mutation [82].

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

hepatitis virus infection did not show enhanced activation of AFB1 [75, 44].

stress induced by chronic viral hepatitis.

extremely uncommon in tumors other than HCC [58].

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 carcinogens did not [67, 47].

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 young children [81].

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