The chemistry and biology of aflatoxin B1: from mutational spectrometry to carcinogenesis
Maryann E. Smela,
Sophie S. Currier,
Elisabeth A. Bailey and
John M. Essigmann,
Department of Chemistry and Division of Bioengineering and Environmental Health Massachusetts Institute of Technology, Cambridge, MA 02139, USA
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Abstract
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Dietary exposure to aflatoxin B1 (AFB1) is associated with an increased incidence of hepatocellular carcinoma (HCC), especially in populations in which exposure to hepatitis B virus (HBV) is a common occurrence. Most HCC samples from people living where HBV is prevalent have one striking mutational hotspot: a GC
TA transversion at the third position of codon 249 of the p53 gene. In this review, the chemical reaction of an electrophilic derivative of aflatoxin with specific DNA sequences is examined, along with the types of mutations caused by AFB1 and the sequence context dependence of those mutations. An attempt is made to assign the source of these mutations to specific chemical forms of AFB1-DNA damage. In addition, epidemiological and experimental data are examined regarding the synergistic effects of AFB1 and HBV on HCC formation and the predominance of one hotspot GC
TA transversion in the p53 gene of affected individuals.
Abbreviations: AFB1, aflatoxin B1; AP, apurinic; CYP, human cytochrome P450; FAPY, formamidopyrimidine; HBsAg, HBV surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma.
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Introduction
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Aflatoxin B1 (AFB1) is a fungal metabolite that contaminates the food supply in certain areas of the world (1). It is produced by Aspergillus flavus and related fungi that grow on improperly stored foods, such as corn, rice and peanuts. AFB1 requires metabolic conversion to its exo-8,9-epoxide (1,2) in order to damage DNA (2). As shown in Figure 1
, the AFB1 epoxide reacts with guanine to form a number of adducts (3), and one reasonable model is that these adducts, or secondary DNA lesions derived from them, lead to heritable genetic changes that help a cell along the pathway toward malignant transformation. Despite the structurally varied population of DNA adducts that forms in cells treated with the toxin, the mutational spectrum of the toxin is dominated by one genetic change: the GC
TA transversion. It is presumed that these mutations arise from a guanine adduct, owing to the fact that nearly all, if not all, aflatoxin adducts occur at that base.

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Fig. 1. Pathway of metabolic activation leading to DNA adduct formation by AFB1. CYP isoenzymes metabolize AFB1 to the 8,9-epoxide, which reacts with DNA. While a number of products are formed, the initial major adduct is AFB1-N7-Gua. This adduct has a destabilized glycosidic bond and depurinates to the AP site shown. Alternatively, the primary adduct can suffer opening of its imidazole ring, giving rise to the chemically and biologically stable formamidopyrimidine adduct, AFB1-FAPY.
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In hepatocellular carcinomas (HCCs), as in many other cancers (4), the p53 gene is mutated in >50% of the tumors (4,5). Of likely significance is the observation that the principal point genetic change in the p53 genes of HCC is the GC
TA transversion, the same point mutation principally induced by electrophilic forms of aflatoxin. As a specialized situation, the mutated p53 genes of individuals with HCC from areas of the world where both aflatoxin and hepatitis B virus (HBV) are prevalent frequently have a single signature mutation (6).
The first part of this review will focus on the sequence specificity of AFB1 adduct formation and the types of mutations induced by AFB1 in various experimental contexts. Of central importance in this discussion will be technology by which the genetic effects of individual DNA adducts has been probed in vivo. The review will then cover the synergistic effects of AFB1 exposure and HBV infection on the formation of liver tumors that harbor the hallmark mutation in the p53 gene and, finally, will evaluate the different models proposed to explain this phenomenon.
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The mutational specificities of specific AFB1-DNA lesions correlate with the mutational spectrum of activated AFB1
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Figure 1
details the pathway of AFB1 metabolism leading to covalent adduct formation. The AFB1 exo-8,9-epoxide is the aflatoxin metabolite that reacts covalently with DNA to form the adducts that presumably account for the biological effects of the toxin. Of the various adducts formed, the most quantitatively abundant both in vitro (710) and in vivo (9,1113) is 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua). The positively charged imidazole ring of the principal adduct promotes depurination, giving rise to an apurinic (AP) site. Alternatively, the imidazole ring of AFB1-N7-Gua opens to form the more chemically and biologically stable AFB1 formamidopyrimidine (AFB1-FAPY) (14). While this reaction occurs most favorably under basic conditions, the AFB1-FAPY adduct is a significant product in vivo (13). It is likely that the initial AFB1-N7-Gua adduct, the AFB1-FAPY adduct, and the AP site, individually or collectively, represent the chemical precursors to the genetic effects of AFB1. Other DNA lesions form, but at much lower levels than these three products (13). As indicated below, the AFB1-N7-Gua adduct has mutagenic properties that correlate with those of the biologically relevant DNA lesion(s) of AFB1 (15). The other two abundant lesions formed by AFB1, specifically AFB1-FAPY and the AP site, have not been studied in sufficient detail to conclude definitively whether or not they play significant roles in aflatoxin toxicology.
In all biological systems evaluated to date, the most frequently observed mutation induced by chemically reactive forms of AFB1 (e.g., the AFB1 epoxide or other electrophilic derivatives) is the GC
TA transversion. This is the principal mutation found in several experimental systems: in the endogenous lacI gene in an SOS-induced Escherichia coli strain that contains the mucAB mutagenesis enhancing operon (16); in human cells replicating an AFB1-modified pS189 shuttle vector (17); in the ras gene of rainbow trout liver (19); in the human Ha-ras proto-oncogene (20); in the transgenic C57BL/6N (BigBlue) mouse treated with phorone (a glutathione depleting agent) (21); in the lacI gene in transgenic C57BL/6 mice and F344 rats given a single dose of AFB1 (2.5 mg/kg in mice; 0.125 mg/kg in rats) (22); in an intra-sanguinous host-mediated assay (23); in human hepatocytes grown in culture (24); in human liver tumors (46); in the human HPRT gene (26); and in AFB1-exposed rats that either did or did not undergo partial hepatectomy (27). Both GC
TA and GC
AT mutations are induced with equal efficiency in other systems by metabolically activated AFB1 (28) and the AFB1 8,9-dichloride model for the AFB1 epoxide (29). GC
AT transitions are predominant in the c-Ki-ras oncogene of rat HCCs, an observation that is consistent with the types of mutations observed opposite an AP site in NIH 3T3 cells. As indicated below, these data would support a role for an aflatoxin-induced AP site in the generation of mutations.
At least three DNA lesionsAFB1-N7-Gua, AFB1-FAPY and the AP siteare candidate precursors to the mutations induced by AFB1. A number of years ago, systems were developed to allow assessment of the mutational specificity and quantitative mutagenicity of individual lesions formed by DNA damaging agents (17). In its most commonly applied form (Figure 2
) this technology involves synthesis of a short oligonucleotide (e.g. 520 nt) with a known single DNA lesion at a specific site. The oligonucleotide is inserted into a gap placed by using recombinant DNA techniques into the genome of a virus or a plasmid. The adduct-containing vector is introduced into a bacterial or mammalian cell, where the adduct encounters the replication and repair apparatuses of the host. After replication in vivo, either intra- or extra-chromosomally, the progeny of the vector containing the adduct are recovered and analyzed for the type, the amount and, in some cases, the genetic requirements for mutagenesis. Recently, the approach described above has been applied in an attempt to dissect the mutagenic properties of two of the three principal candidates for the mutagenic lesion of aflatoxin (18).

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Fig. 2. Construction and uses of viral genomes containing DNA lesions at specific sites. A single-stranded viral genome is cleaved at a unique site and the product is annealed to an oligonucleotide in a manner that results in the formation of a gap of known size. An oligonucleotide containing a DNA lesion (e.g., AFB1-N7-Gua, an AP site or AFB1-FAPY) is annealed to the genome. The 5' and 3' ends of the oligonucleotide are in perfect alignment with ends of the genome and are ligated to form a covalently continuous and biologically viable viral genome with a site-specific adduct. The lesion containing genome is transfected into cells and mutant progeny are isolated, characterized and enumerated.
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It has been reasonably suggested that the premutagenic lesion responsible for the observed GC
TA transversions in bacterial systems would be the AFB1-induced AP site, since dAMP is the most common base inserted opposite AP sites in E.coli induced for the SOS response (19,20). We note, however, that the mutational specificity of the AP site in mammalian cells is different; in fact, studies conducted in different mammalian systems have shown different preferences for the base inserted opposite an AP site (2125). In E.coli the mutational properties of the AFB1-N7-Gua lesion and the AP site (15) were compared when each lesion was situated at a specific site within the bacteriophage M13 genome. The predominant mutation for both lesions is a GC [or (AP)C, in the case of the AP site] to TA transversion. The data on the AP site are in accord with previous studies on the mutational specificity of this lesion in E.coli (19). Interestingly, while most of the mutations caused by the AFB1-N7-Gua adduct are targeted to the site of the lesion, a significant fraction (13%) occur at the base 5' to the modified guanine. In contrast, the AP site-containing genome gives rise only to mutations targeted at the site of damage. The mutational asymmetry observed for AFB1-N7-Gua is consistent with structural models, indicating that the aflatoxin moiety of the aflatoxin guanine adduct intercalates on the 5' face of the guanine residue (Figure 3
) (26). These results suggest a molecular mechanism that could explain an important early step in the carcinogenicity of AFB1. The observation that mutations occur at the 5' base suggests that mutational spectra, obtained in studies carried out with DNA globally modified with AFB1, may harbor mutations at bases 5' to potentially modified guanines. Since guanine residues flanked by A+T-rich sequences are poor targets for modification by AFB1 (2729), it is conceivable that the majority of the 5' mutations would reside in 5' cytosines or guanines. These mutations would mistakenly have been scored as resulting from either an additional AFB1-modified guanine in the same strand or an AFB1-modified guanine in the opposite strand. In order to determine whether some of the mutations observed in experiments carried out with globally-modified DNA are the result of a 3'-proximal AFB1-N7-Gua residue, future studies should involve the examination of the three AFB1 lesions in a sequence context-specific system.
It is well known that most molecules with the ability to intercalate into DNA prefer to interact with double-stranded DNA, owing to the favorable environment created by base pair stacking (30). Double-stranded DNA is a more favorable target for aflatoxin binding than single stranded DNA, and several chemical approaches provide compelling evidence that aflatoxin indeed intercalates on the 5' face of the guanine residue in double-stranded DNA (31,32). This intercalation model fits well with the long-standing idea that there is a precovalent association between the aflatoxin molecule and the base to which it eventually becomes covalently bonded. This notion may allow the principle of mass action to account for the different reactivities of different guanine residues and, therefore, for the sequence specificity of aflatoxin epoxide binding.
The reactivity of aflatoxin toward different DNA sequences has been studied by a number of groups (2729,3340). In one systematic study Benasutti et al. (28) illustrate that the reactivity of AFB1 toward a guanine residue is highly dependent on the base immediately 5', and the base immediately 3' to the central guanine, with a run of three guanines being the most favorable sequence overall. In the 5' position the reactivity profile is G (1.0) > C (0.8) > A (0.4) > T (0.3), and in the 3' position it is G (1.0) > T (0.8) > C (0.3) > A (0.2). (A numerical value for relative reactivity is given in parentheses. These values were calculated such that the 3' value can be multiplied by the 5' value to yield a number that can be used to compare the reactivities of different immediate sequences on a central G. Herein, these values will be denoted `relative reactivity', RR.) Significantly, the sequence context-specific reactivity for AFB1 holds true for DNA globally modified by AFB1 (28,35,38,41). The sequence-specificity for AFB1 reactivity has been divided into three classes, and these are referred to in the literature as `weak (RR ~0.23)',`intermediate (RR ~0.44)' or `strong (RR ~0.5)' sites for AFB1 reactivity. It has also been suggested that not only sequence context, but also pre-existing modification of neighboring bases, can affect the reactivity of AFB1 toward a particular sequence. Several reports in the literature demonstrate that AFB1 adduct formation, as well as several other types of DNA damage, is modulated by the presence of 5-methyl cytosine at a CpG site (4246). One contradictory report, however, states that cytosine methylation has no influence over aflatoxin reactivity (47).
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Can one explain the mutations observed in HCC on the basis of the mutational specificity of the aflatoxin epoxide?
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There is one striking feature underlying the sequence specificity of mutational hotspots that arise in liver tumors believed to have been induced by AFB1 exposure. As indicated earlier, the predominant mutational hotspot found in human HCC is a GC
TA transversion in the third position of codon 249 of the p53 gene (AGGC: codon 249 in bold, targeted G underlined, 3' base of target G in normal type). The RR value for this context is 0.3, which falls somewhere between weak and intermediate reactivity toward AFB1. This mutation results in an Arg
Ser alteration in the p53 protein. Evidence is provided that indicates that AFB1 reacts with the third position of codon 249 of p53 in vitro (34). Position 2 of codon 249 (RR = 0.4) was not reactive towards AFB1, even though Benasutti's rules predict it to be 1.3 times more reactive than the third base. This study also demonstrates that AFB1 reacts with 20% of the bases in exons 58 of the p53 gene, ~85% of which are guanines that are in several different sequence contexts. Another group has also observed that significant adduct formation occurred in several codons in exons 7 and 8 of the p53 gene, as well as at the third position of codon 249 (29). In yet another study (48), GC
TA and CG
AT transversions were observed at positions adjacent to the hallmark mutation site at codon 249 of p53 (AGG
ATG and AGGC
AGGA), demonstrating that in different systems, reactivity toward AFB1 and the correlation between reactivity and mutation may be different.
Providing further support for the view that AFB1 binds to and causes mutations in certain sequence contexts, data from Levy et al. (33) illustrate that four out of seven of the mutational hotspots from human xeroderma pigmentosum (XP) cells, observed in the supF gene of the pS189 shuttle vector, are at AGG sequences. Three of these hotspots are located at the third G [the 3' base was either a G (RR = 1.0), a T (RR = 0.8) or an A (RR = 0.2), contexts that show different reactivities toward AFB1]. One AGG sequence contained hotspots at both the second (RR = 0.4) and third G. It is not clear whether mutations at both of the guanine positions are due to aflatoxin binding at one site or at two different sites. Using a polymerase arrest assay, this study showed that there was more blockage at the third G than at the second G of the AGG sequence. This indicates that although the sites that are hotspots for predominantly GC
TA mutations are intermediate or strongly reactive towards AFB1, not all of these sites have incurred the level of mutations consistent with their reactivity, and some sequences that should only be weakly reactive are actually hotspots for mutation. These data are consistent with other studies, whereby different systems were utilized for the analysis of AFB1-induced mutations (29,4951), implying that another factor, such as DNA secondary structure, may play a role in AFB1 modification and/or mutagenesis. As mentioned above, it is possible that an aflatoxin-modified guanine residue at the third position of the AGG sequence may give rise to mutations at the second position as well. This separation of the mutation site from the site of covalent modification may be due to distortion of the base 5' to the modified guanine as a consequence of intercalation of the AFB1 moiety on the 5' face of the guanine (15) (Figure 3
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There is substantial evidence that the activation of cellular ras (c-ras) genes by specific single-base mutations may be an important step in the transformation of normal cells to malignant cells (5254). This activation event is manifested as mutations in codon 12 of the c-Ki-ras gene in DNA in rat liver tumors (55,56). Table I
illustrates the sequences of codons 11, 12 and 13 of rat, trout and human and the relative reactivity of each guanine. The first and second positions of codon 12 incur mutations in rat, and the second positions of both codons 12 and 13 incur mutations in trout (57). In humans, mutations have been observed at the first and second positions of codon 12 in the Ha-ras proto-oncogene (58). Interestingly, the c-Ki-ras sequence context (GCAGGA) for both mutations in rainbow trout is analogous to that of codon 249 of p53 (AGGC), except that the bases 3' to the mutated G are different (an A in the case of codon 12 and a T in the case of codon 13). It is possible, as suggested by these data and others (33), that the third position of the sequence AGG is a hotspot for AFB1 mutation regardless of the 3' base. Rats and humans do not incur mutations in codon 13, possibly due to a base other than adenine 5' to the GG sequences. It is also a possibility that the different sequences form different DNA secondary structures, which may be more or less accessible to DNA repair machinery.
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Table I. Species-specific sequence differences among ras codons: correlation of predicted reactivity with reactive forms of AFB1
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In vivo studies conducted with the lacI gene in transgenic rats and mice examined sequences surrounding mutated guanines to determine if the mutations observed were sequence context dependent (59). Mutations were observed in six different sequence contexts in mice. In half of these contexts, the targeted guanine is in the third position of a CGG context, with either a C (RR = 0.4), an A (RR = 0.3) or a T (RR = 0.8) in the 3' position. With regard to the empirical rules for aflatoxin reactivity in specific contexts, one out of the six sequences would be considered strong, two out of the six would be considered intermediate and three out of the six contexts would be considered weak. Mutations were also observed in 25 different sequence contexts in rats, eight of which have weak reactivity and the rest of which have intermediate to strong reactivity toward AFB1. Of all the types of mutations observed, ~48% of those in mice and 66% of those in rats were found at CpG sites. Only 17% of the sequences examined in mice and 4% of the sequences examined in rats did not have at least one G or C immediately adjacent to the mutated G. Sequences that have at least one G or C adjacent to a central G have an average RR of 0.44, while sequences without an adjacent C or G have an average RR of 0.18. In a separate study, rats treated with AFB1 in the presence or absence of the liver regeneration process were evaluated for p53 mutations (60). Only one mutation (20%) occurs at a CpG site. One out of five (20%) of the sequences examined does not have a G or a C immediately adjacent to the mutated base (on two occasions, the mutated base was a C; these mutations could have been due to the Gs in the opposite strand). In a third study, carried out in the human HPRT gene (61), a hotspot for both base substitution and frameshift mutations occurs in a run of six Gs. These data once again show that although certain sequence contexts exhibit weak reactivity toward AFB1, they may still incur mutations. We note, however, that they also support Benasutti's rules for preferential AFB1 binding to GC-rich regions.
In summary, in all of the aforementioned studies, some of the mutational hotspots occurred in sequences that, according to Benasutti's empirical rules, are also intermediate or strong spots for AFB1 reactivity. Most of these hotspots for reactivity and mutation are within sequences where a G is located 5' to the base involved in the mutation. The base 3' to the modified G appears less consistently associated with mutation and may not be as crucial as the 5' base. Taking a minimalist approach, one could say that all that is required for a mutational hotspot for AFB1 is a GG sequence with the modification/mutation occurring at the second G. In fact Levy et al. (33) have described hotspots in just this way. It is noted, however, that the AFB1-induced mutations in the aforementioned studies are not observed at every site, within the particular gene analyzed, where a strong or intermediate sequence for AFB1 reactivity occurs.
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Factors that influence AFB1 mutagenesis and HCC formation
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The shape of the mutational spectrum of aflatoxin is influenced by at least three factors: (i) the preference of one G over another for reaction, (ii) the preference for a base to be erroneously replicated more frequently in one context compared with another and (iii) the preference for repair of an adduct in one context over others. One would expect that DNA repair-deficient cells present an opportunity to examine the contributions of lesion formation and misreplication [points (i) and (ii)] separated from the complications of preferential repair [point (iii)]. Mutational spectra were studied in cells with the DNA nucleotide excision repair deficiency disorder, XP (33). All sequence contexts that should be strongly reactive toward AFB1 may not incur mutations, since preferential repair of some sequences may be taking place. Surprisingly, most of the contexts that were hotspots in repair proficient cells were predicted to have only weak to intermediate reactivity toward AFB1, whereas in XP cells, most of the hotspots occurred in intermediate to strong contexts. This result implies that sequences that are more reactive to AFB1 may also be more prone to undergo DNA repair, again suggesting a role for secondary structure. It has also been observed that the hepatitis B x protein (HBx) can inhibit nucleotide excision repair, either by binding to repair proteins or to the damaged DNA itself, making it possible that AFB1 adducts persist preferentially in patients who have been exposed to both HBV and AFB1 (6264). XRCC1, an enzyme involved in base excision repair, has several polymorphisms. Individuals with the 399 Gln allele have increased levels of AFB1 adducts (65). These differences in DNA repair are believed to be independent of p53, so it is reasonable to speculate that p53 mutations and the effects of HBV play a role in HCC induction via separate pathways. In support of this hypothesis, HBx has been shown to promote apoptosis following DNA damage in a cell line containing wild-type p53, while promoting a growth advantage in a cell line containing AFB1-induced mutant p53 (66).
The types and positions of the mutations induced by AFB1 may differ among species due to a difference in AFB1 activation (67). Human SV40 immortalized cell lines expressing different human cytochrome P450 (CYP) enzymes (CYP1A2, CYP2A6, CYP2B6 or CYP3A4) gave rise to different mutational spectra, in terms of the proportions of transitions and transversions at each position that was mutated in codons 249 and 250 of a globally-modified p53 gene. Indeed, AFB1 exposure itself alters expression of some genes, including those for certain CYPs, which are involved in activation, and glutathione S-transferase (GST), which is involved in detoxification, of AFB1 (68). HBV infection is also thought to influence the regulation of such genes (69). From species to species, and even within the human population itself (70), the balance between the activities of the enzymes of AFB1 activation and detoxification may explain the differences observed in response to the toxin. Oltipraz [4-methyl-5-(2-pyrazinyl)-3-H-1,2-dithiole-3-thione], a drug currently in Phase IIb clinical trials, has been shown to decrease AFB1 metabolites in humans by 51% by inducing GST and inhibiting CYP1A2, the CYP isoform that activates AFB1 to its reactive metabotite (71).
A general pattern in the types and sequence-dependence of AFB1-induced mutations can be inferred from the studies carried out above. We note that the atomic environment of some DNA sequences may render certain sites more favorable for AFB1 intercalation (72). The data indicate that AFB1 induces a predominance of GC
TA transversions within GC-rich sequence contexts. However, not all sequence contexts that are strong sites for modification by AFB1 are necessarily hotspots for mutation. In addition, some sequence contexts that are weak sites for modification are actually hotspots for mutation. Further investigation of potential differences in the mechanism of AFB1 reactivity toward different sequences, potential differences in repair of various AFB1-modified sequences and the influence of different DNA secondary structures on AFB1 reactivity should shed further light on the correlation between hotspots for AFB1 modification and AFB1 mutations.
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Aflatoxin B1, hepatitis B virus and hepatocellular carcinoma
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Codon 249 of p53 is a hotspot for AFB1 modification and AFB1-induced mutation, specifically AGGC
AGTC. This mutation has been found in up to 50% of HCC samples in areas of the world where aflatoxin exposure is high. To date, over 2000 HCC samples from all over the world have been examined for this mutation (6,7375). In regions where exposure to aflatoxin is highnamely Qidong and Tongan (China), India, Southern Africa, The Gambia, Mozambique and Senegal116 out of 262 (44%) of the total HCC cases examined show a predominance of GC
TA mutations at the third position of codon 249 of the p53 gene (7478). In contrast, in regions of low exposure to aflatoxinnamely, Australia, Europe, Japan and the USAonly 17 out of 1273 (1%) of the HCC samples examined had mutations at this site (74,7880). In regions where exposure to aflatoxin is moderatenamely, Beijing, Shanghai, Xi'an, Hong Kong, Singapore, South Korea, Taiwan, Thailand, Vietnam, Southern Asia, South Africa and Egypt40 out of 568 (7%) HCC cases examined had mutations at the third position of codon 249 of the p53 gene (6,65,77,81). This p53 mutation appears to be unique to AFB1-induced liver tumors, as tumors presumably induced by other factors fail to consistently show this genetic change (66,71). Currently, clinical trials with oltipraz are underway in Qidong (China) where, so far, a 51% decrease in AFB1 metabolites has been observed. Further studies will elucidate whether this drug plays a role in reducing HCC incidence (71).
The correlation between exposure to aflatoxin and the hotspot at codon 249 suggests two possibilities. First, aflatoxin may be particularly reactive with this nucleotide, owing to its surrounding sequence, as discussed in the preceding section. Second, it is possible that this mutation is due somehow to the combined effects of AFB1 exposure and HBV infection, either directly or through separate pathways, since this mutation is observed much more often in areas of the world where exposure to both agents is very common. In both cases, if mutations in codon 249 significantly debilitate the function of p53, the loss of function of this protein should give cells a selective growth advantage, since p53 is an important tumor-suppressor gene. p53 is a transcriptional activator that has been shown to regulate the cell cycle (82), to play a role in the apoptosis pathway (8387) and to be involved with DNA repair (8893). AFB1 and HBV may act in concert, or they may induce completely different pathways, the combined effects of which lead to the selection of the hallmark p53 mutation and the ultimate endpoint of HCC.
HBV infection has been shown to be associated with increased incidence of HCC. In general, a person with HBV is four times more likely to develop liver cancer, regardless of their level of exposure to AFB1 (6). Thus, the association of HBV infection and HCC formation has been the focus of many studies. Several groups have shown that one of the gene products of HBV, HBx, binds to and inactivates the p53 protein (94,95). Although there is some controversy over this issue, experiments suggest that the binding of HBx to the C-terminal region of p53 inhibits p53 sequence-specific DNA binding and, thus, inhibits its role as a transcriptional activator (95). The same group has also shown that expression of HBx may inhibit p53-induced apoptosis (96). In contrast, another group has shown that p53, in cells exposed to HBV, can still activate some genes to some extent in vivo (97). Other work, while failing to observe a direct interaction between p53 and HBx (63,73,98100), has indeed determined an alteration in either the localization, the phosphorylation status or the transcription of wild-type p53 during HBx expression. Yet others have probed total cellular RNA in liver cell lines expressing HBx, searching for genes that are up- or down-regulated, and no effect has been observed on p53 RNA (101). An increase in the number of hallmark codon 249 mutations has been observed when a cell line that expresses the HBx protein is exposed to increasing levels of AFB1 (66). In addition oltipraz has been shown to inhibit HBV transcription and to upregulate expression of wild-type p53 in cell culture (70). Perhaps it is not the direct association of HBx with p53, but some other effect that HBx has on the cell, such as an inhibition of apoptosis in p53 mutant cells, that promotes mutant selection.
Although exposure to HBV alone increases the risk of HCC, the risk is even higher in an individual who is both positive for HBV and is exposed to aflatoxin (6,102104). One study indicates that patients with positive urinary AFB1-N7-Gua antigen are three times more likely to develop HCC, patients with positive HBV surface antigen (HBsAg) are about seven times more likely to develop HCC and when both AFB1 and HBsAg are present, patients are 60 times more likely to develop this disease (103). Thus, there seems to be a synergism between HBV and AFB1 exposure for the development of HCC. Interestingly, a relationship has been observed between the presence of HBsAg and the mutational hotspot at codon 249 of p53 (6,74,75). Twelve percent of the 2019 HCC cases examined for the mutational hotspot at codon 249 were examined for the presence of HBsAg. For the samples taken from regions of high aflatoxin exposure, 47% (82 out of 175 total) of HBV-positive HCC samples examined had a GC
TA transversion at position three of codon 249 of p53. About 42% (18 out of 43) of the HBV-negative HCC samples from these regions had the same mutation. For the samples taken from regions of moderate aflatoxin exposure, only 35% (15 out of 43 total) of the HBsAg-positive samples had the GC
TA transversion. None of the 11 HBsAg-negative samples in these regions exhibited the mutation. Other work (73) has shown that in areas of low aflatoxin exposure, HCC samples from people who are positive for the HBsAg also have p53 mutations, although they have not yet determined what these mutations are. These studies help establish the relationship between this hotspot mutation in tumors and HBV infection.
Experiments have examined the ability of different p53 mutants to modulate in trans the transcriptional activity of wild-type p53 (p53 acts functionally as a tetramer) on a reporter construct in HCC cell lines (HEP-3B) possessing an integrated HBx gene (105). It has not been confirmed, however, that these cells actually do express the HBx protein. The p53 mutants tested included R248W and R249S mutations. The R248W mutant enhanced the level of transcription to 132% of wild type, while the R249S mutant drastically reduced the level of transcription to 18% of wild type. Control experiments were carried out in tumor cell lines from lung, prostate and mammary epithelial tissues, none of which express HBx. The R249S mutant exhibited a similar effect in lung (20% of wild type), and a milder effect in mesothelial (52% of wild type), mammary (75% of wild type) and prostate (80% of wild type) tissues. When any of the mutants are expressed in a cell line that does not contain wild-type p53, no transcriptional activation of the reporter construct is seen. Thus, other mutant sequences should be examined in the experimental system described above.
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Would animal models be of value to probe the importance of p53 in the induction of HCC by aflatoxin?
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p53 is at least 92% homologous across the species listed in Table II
(106). Codons 248 and 249 in the animal models discussed here are analogous to human codons 248 and 249, and they are believed to be within the DNA binding regions of the animals' proteins, which is also the case with the human protein. The GC
TA hotspot mutation at the codon analogous to the human codon 249 in the p53 genes of other species has not been identified. To date, all attempts to recreate this hotspot by exposing animals to aflatoxin have not been successful. However, it is possible that this failure to generate the hotspot is due solely to the differences among the p53 sequences of different species (Table II
). A different sequence context may alter the reactivity of AFB1 toward the analogous codon 249 sequence. Table II
illustrates, from a compilation of data, that there is no mutation analogous to the human p53 codon 249 mutation (Arg
Ser) in primates, rats, mice, ducks, woodchucks, tree shrews or ground squirrels when these animals are exposed to aflatoxin. A possible reason that no analogous mutation has been found may be thatat least in woodchucks, tree shrews (opposite strand mutation) and ground squirrelsa GC
TA transversion in the third position of the codon results in a silent mutation. Additionally, some of the species tested do not have a species-specific form of hepatitis B. The HBV status of several species studied is also shown in Table II
. If HBV and AFB1 are both needed for the selection of a p53-inactivating mutation at the third position of codon 249, it is important for an animal model to be able both to contract HBV-mediated disease and to have a similar p53 codon sequence in the DNA binding region. Similar to the woodchuck and ground squirrel models noted above, ducks contract a form of hepatitis B, duck hepadnavirus B (DHVB), but the predominant mutation in ducks at codon 249 is Arg
Leu, which may not be as detrimental as Arg
Ser for p53 activity (106). In the case of primates and rats, even though the mutation at codon 249 would give rise to an amino acid change of Arg
Ser, studies were carried out in the absence of HBV (107,108). Currently, rats transgenic for HBV are being developed (109). Mice naturally have an Arg
Leu amino acid change in codon 249; we note, however, that mice have been engineered to have an Arg
Ser mutation at codon 246, which is analogous to the human Arg
Ser mutation at codon 249 (AGGC
AGTC) (110). One study has been done where mice either with or without the codon 246 mutation were simultaneously exposed to AFB1 and were positive for the HBsAg. This mutation causes an increase in high-grade liver tumor formation in the presence of AFB1 (from 0 to 14% when homozygous for wild-type p53; from 14 to 71% when heterozygous for wild-type p53), and even more so in the presence of both AFB1 and HBsAg (from 62 to 100% when homozygous for wild-type p53). Again, in these studies, it has not been proven that HBx is expressed in these animals.
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Table II. Expected amino acid changes arising from GC TA mutations at p53 codons 248 and 249 in different speciesa
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In animal models it is important for the species-specific form of hepadnavirus to have all the characteristics of human HBV, including the HBx protein, and this protein should be similar to that found in human HBV. It is known that DHVB does not have the HBx protein. Different species may differ in their ability to metabolize and repair AFB1 (111122). For example, mice have different proportions of metabolizing and protective enzymes, so most of the aflatoxin they ingest is inactivated and excreted before it can form DNA adducts (111114). On the other hand, woodchucks show an increase in conversion of AFB1 to the epoxide when they are exposed to woodchuck hepadnavirus B (WHVB) (120). Recently, the tree shrew has been proposed as an animal model that can be infected with human HBV (123,124). This animal model shows the same synergism that humans do when they are infected with HBV and exposed to AFB1. Of four tumors from animals that were positive for both AFB1 and HBV exposure, no codon 249 mutations have been observed. Three of the tumors have mutations elsewhere in p53, and only one of these is in the DNA binding domain of the protein (124). This result implies there may be some other factor, such as a co-carcinogen or phenotypic trait, that may play a role specifically in the development of human HCC and may somehow lead to the selection of the codon 249 mutation. Further studies should help elucidate the mechanisms of known contributors to HCC and suggest other possible factors in the etiology of this disease.
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Concluding comments
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In the past 25 years much has been learned about the relationship between DNA damage by aflatoxin and the biological endpoints of mutagenesis and carcinogenesis. The pathway of metabolic activation of AFB1 to the primary toxic metabolite, the 8,9-oxide, as well as the pathways that suppress toxicity, were discovered. Moreover, the DNA products that arise from covalent modification have been identified and monitored in various sequence contexts in vitro, in culture, within the tissues of animals and, under certain circumstances, in humans. Toxicity modulators such as oltipraz have been discovered that exploit the aforementioned knowledge of aflatoxin metabolism in a manner that may reduce the carcinogenic risk of aflatoxin to humans. Genetic studies have revealed specific genetic changes in tumors that are thought to have arisen in response to aflatoxin. Those changes, finally, have been associated with the mutational specificities of the DNA lesions formed by the toxin in vivo. Taken together, we now have a good framework within which to pose a set of deeper biological questions concerning the mechanism by which this powerful toxin and carcinogen, either alone or in conjunction with other factors such as HBV, exerts its effects. We know that p53 status probably determines the biology of hepatocellular carcinogenesis, but the details of how aflatoxin exposure and/or HBV status affect p53 are still not completely clear. The increasingly powerful tools used in biochemistry and genetics should foster the efforts that, over the next quarter of a century, will address the question of how this important carcinogen affects the human population.
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Notes
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1 To whom correspondence should be addressed Email: jessig{at}mit.edu 
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Acknowledgments
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We are especially indebted to Professor Michael P.Stone of Vanderbilt University for molecular modeling. This work was supported by grants CA80024/ES09546 and ES07020 from the National Institutes of Health.
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Received September 12, 2000;
accepted November 3, 2000.