Chemistry and Biology of Afb1

Carcinogenesis vol.22 no.4 pp.535–545, 2001

The chemistry and biology of aflatoxin B1: from mutationalspectrometry to carcinogenesis

Maryann E.Smela, Sophie S.Currier, Elisabeth A.Bailey world where both aflatoxin and hepatitis B virus (HBV) areprevalent frequently have a single signature mutation (6).and John M.Essigmann1

The first part of this review will focus on the sequenceDepartment of Chemistry and Division of Bioengineering and

specificity of AFB1 adduct formation and the types of mutationsEnvironmental Health Massachusetts Institute of Technology, Cambridge,induced by AFB1 in various experimental contexts. Of centralMA 02139, USAimportance in this discussion will be technology by which the1To whom correspondence should be addressedgenetic effects of individual DNA adducts has been probedEmail: [email protected] vivo. The review will then cover the synergistic effects ofDietary exposure to aflatoxin B1 (AFB1) is associated withAFB1 exposure and HBV infection on the formation of liveran increased incidence of hepatocellular carcinoma (HCC),tumors that harbor the hallmark mutation in the p53 gene and,especially in populations in which exposure to hepatitis Bfinally, will evaluate the different models proposed to explainvirus (HBV) is a common occurrence. Most HCC samplesthis phenomenon.from people living where HBV is prevalent have one

striking mutational hotspot: a GC→TA transversion at theThe mutational specificities of specific AFB1-DNA lesionsthird position of codon 249 of the p53 gene. In this review,correlate with the mutational spectrum of activated AFB1the chemical reaction of an electrophilic derivative of

aflatoxin with specific DNA sequences is examined, along Figure 1 details the pathway of AFB1 metabolism leading towith the types of mutations caused by AFB1 and the covalent adduct formation. The AFB1 exo-8,9-epoxide is thesequence context dependence of those mutations. An aflatoxin metabolite that reacts covalently with DNA to formattempt is made to assign the source of these mutations to the adducts that presumably account for the biological effectsspecific chemical forms of AFB1-DNA damage. In addition, of the toxin. Of the various adducts formed, the most quantitat-epidemiological and experimental data are examined ively abundant both in vitro (7–10) and in vivo (9,11–13) isregarding the synergistic effects of AFB1 and HBV on HCC 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-formation and the predominance of one hotspot GC→TA Gua). The positively charged imidazole ring of the principaltransversion in the p53 gene of affected individuals. adduct promotes depurination, giving rise to an apurinic (AP)

site. Alternatively, the imidazole ring of AFB1-N7-Gua opensto form the more chemically and biologically stable AFB1

Introduction formamidopyrimidine (AFB1-FAPY) (14). While this reactionoccurs most favorably under basic conditions, the AFB1-FAPYAflatoxin B1 (AFB1) is a fungal metabolite that contaminatesadduct is a significant product in vivo (13). It is likely that thethe food supply in certain areas of the world (1). It is producedinitial AFB1-N7-Gua adduct, the AFB1-FAPY adduct, and theby Aspergillus flavus and related fungi that grow on improperlyAP site, individually or collectively, represent the chemicalstored foods, such as corn, rice and peanuts. AFB1 requiresprecursors to the genetic effects of AFB1. Other DNA lesionsmetabolic conversion to its exo-8,9-epoxide (1,2) in order toform, but at much lower levels than these three products (13).damage DNA (2). As shown in Figure 1, the AFB1 epoxideAs indicated below, the AFB1-N7-Gua adduct has mutagenicreacts with guanine to form a number of adducts (3), and oneproperties that correlate with those of the biologically relevantreasonable model is that these adducts, or secondary DNADNA lesion(s) of AFB1 (15). The other two abundant lesionslesions derived from them, lead to heritable genetic changes thatformed by AFB1, specifically AFB1-FAPY and the AP site,help a cell along the pathway toward malignant transformation.have not been studied in sufficient detail to conclude definit-Despite the structurally varied population of DNA adducts thatively whether or not they play significant roles in aflatoxinforms in cells treated with the toxin, the mutational spectrumtoxicology.of the toxin is dominated by one genetic change: the GC→TA

In all biological systems evaluated to date, the most fre-transversion. It is presumed that these mutations arise from aquently observed mutation induced by chemically reactiveguanine adduct, owing to the fact that nearly all, if not all,forms of AFB1 (e.g., the AFB1 epoxide or other electrophilicaflatoxin adducts occur at that base.derivatives) is the GC→TA transversion. This is the principalIn hepatocellular carcinomas (HCCs), as in many othermutation found in several experimental systems: in the endo-cancers (4), the p53 gene is mutated in �50% of the tumorsgenous lacI gene in an SOS-induced Escherichia coli strain(4,5). Of likely significance is the observation that the principalthat contains the mucAB mutagenesis enhancing operon (16);point genetic change in the p53 genes of HCC is the GC→TAin human cells replicating an AFB1-modified pS189 shuttletransversion, the same point mutation principally induced byvector (17); in the ras gene of rainbow trout liver (19); in theelectrophilic forms of aflatoxin. As a specialized situation, thehuman Ha-ras proto-oncogene (20); in the transgenic C57BL/mutated p53 genes of individuals with HCC from areas of the6N (BigBlue) mouse treated with phorone (a glutathionedepleting agent) (21); in the lacI gene in transgenic C57BL/6Abbreviations: AFB1, aflatoxin B1; AP, apurinic; CYP, human cytochromemice and F344 rats given a single dose of AFB1 (2.5 mg/kgP450; FAPY, formamidopyrimidine; HBsAg, HBV surface antigen; HBV,

hepatitis B virus; HCC, hepatocellular carcinoma. in mice; 0.125 mg/kg in rats) (22); in an intra-sanguinous

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the host. After replication in vivo, either intra- or extra-chromosomally, the progeny of the vector containing the adductare recovered and analyzed for the type, the amount and, insome cases, the genetic requirements for mutagenesis. Recently,the approach described above has been applied in an attemptto dissect the mutagenic properties of two of the three principalcandidates for the mutagenic lesion of aflatoxin (18).

It has been reasonably suggested that the premutageniclesion responsible for the observed GC→TA transversions inbacterial systems would be the AFB1-induced AP site, sincedAMP is the most common base inserted opposite AP sites inE.coli induced for the SOS response (19,20). We note, however,that the mutational specificity of the AP site in mammalian cellsis different; in fact, studies conducted in different mammaliansystems have shown different preferences for the base insertedopposite an AP site (21–25). In E.coli the mutational propertiesof the AFB1-N7-Gua lesion and the AP site (15) were comparedwhen each lesion was situated at a specific site within thebacteriophage M13 genome. The predominant mutation forboth lesions is a GC [or (AP)C, in the case of the AP site] toTA transversion. The data on the AP site are in accord withprevious studies on the mutational specificity of this lesion inE.coli (19). Interestingly, while most of the mutations causedby the AFB1-N7-Gua adduct are targeted to the site of thelesion, a significant fraction (13%) occur at the base 5� to themodified guanine. In contrast, the AP site-containing genomegives rise only to mutations targeted at the site of damage.The mutational asymmetry observed for AFB1-N7-Gua isconsistent with structural models, indicating that the aflatoxinmoiety of the aflatoxin guanine adduct intercalates on the 5�face of the guanine residue (Figure 3) (26). These resultssuggest a molecular mechanism that could explain an importantFig. 1. Pathway of metabolic activation leading to DNA adduct formation

by AFB1. CYP isoenzymes metabolize AFB1 to the 8,9-epoxide, which early step in the carcinogenicity of AFB1. The observationreacts with DNA. While a number of products are formed, the initial major that mutations occur at the 5� base suggests that mutationaladduct is AFB1-N7-Gua. This adduct has a destabilized glycosidic bond and

spectra, obtained in studies carried out with DNA globallydepurinates to the AP site shown. Alternatively, the primary adduct canmodified with AFB1, may harbor mutations at bases 5� tosuffer opening of its imidazole ring, giving rise to the chemically and

biologically stable formamidopyrimidine adduct, AFB1-FAPY. potentially modified guanines. Since guanine residues flankedby A�T-rich sequences are poor targets for modification byAFB1 (27–29), it is conceivable that the majority of the 5�host-mediated assay (23); in human hepatocytes grown in

culture (24); in human liver tumors (4–6); in the human HPRT mutations would reside in 5� cytosines or guanines. Thesemutations would mistakenly have been scored as resultinggene (26); and in AFB1-exposed rats that either did or did not

undergo partial hepatectomy (27). Both GC→TA and GC→AT from either an additional AFB1-modified guanine in the samestrand or an AFB1-modified guanine in the opposite strand. Inmutations are induced with equal efficiency in other systems

by metabolically activated AFB1 (28) and the AFB1 8,9- order to determine whether some of the mutations observedin experiments carried out with globally-modified DNA aredichloride model for the AFB1 epoxide (29). GC→AT trans-

itions are predominant in the c-Ki-ras oncogene of rat HCCs, the result of a 3�-proximal AFB1-N7-Gua residue, futurestudies should involve the examination of the three AFB1an observation that is consistent with the types of mutations

observed opposite an AP site in NIH 3T3 cells. As indicated lesions in a sequence context-specific system.It is well known that most molecules with the ability tobelow, these data would support a role for an aflatoxin-induced

AP site in the generation of mutations. intercalate into DNA prefer to interact with double-strandedDNA, owing to the favorable environment created by baseAt least three DNA lesions—AFB1-N7-Gua, AFB1-FAPY

and the AP site—are candidate precursors to the mutations pair stacking (30). Double-stranded DNA is a more favorabletarget for aflatoxin binding than single stranded DNA, andinduced by AFB1. A number of years ago, systems were

developed to allow assessment of the mutational specificity several chemical approaches provide compelling evidence thataflatoxin indeed intercalates on the 5� face of the guanineand quantitative mutagenicity of individual lesions formed by

DNA damaging agents (17). In its most commonly applied residue in double-stranded DNA (31,32). This intercalationmodel fits well with the long-standing idea that there is aform (Figure 2) this technology involves synthesis of a short

oligonucleotide (e.g. 5–20 nt) with a known single DNA lesion precovalent association between the aflatoxin molecule andthe base to which it eventually becomes covalently bonded.at a specific site. The oligonucleotide is inserted into a gap

placed by using recombinant DNA techniques into the genome This notion may allow the principle of mass action to accountfor the different reactivities of different guanine residues and,of a virus or a plasmid. The adduct-containing vector is

introduced into a bacterial or mammalian cell, where the therefore, for the sequence specificity of aflatoxin epoxidebinding.adduct encounters the replication and repair apparatuses of

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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 theproduct 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 thegenome and are ligated to form a covalently continuous and biologically viable viral genome with a site-specific adduct. The lesion containing genome istransfected into cells and mutant progeny are isolated, characterized and enumerated.

The reactivity of aflatoxin toward different DNA sequences (42–46). One contradictory report, however, states that cytosinemethylation has no influence over aflatoxin reactivity (47).has been studied by a number of groups (27–29,33–40). In

one systematic study Benasutti et al. (28) illustrate that thereactivity of AFB1 toward a guanine residue is highly dependent Can one explain the mutations observed in HCC on theon the base immediately 5�, and the base immediately 3� to basis of the mutational specificity of the aflatoxin epoxide?the central guanine, with a run of three guanines being themost favorable sequence overall. In the 5� position the reactivity There is one striking feature underlying the sequence specificity

of mutational hotspots that arise in liver tumors believed toprofile is G (1.0) � C (0.8) � A (0.4) � T (0.3), and in the3� position it is G (1.0) � T (0.8) � C (0.3) � A (0.2). (A have been induced by AFB1 exposure. As indicated earlier,

the predominant mutational hotspot found in human HCC isnumerical value for relative reactivity is given in parentheses.These values were calculated such that the 3� value can be a GC→TA transversion in the third position of codon 249 of

the p53 gene (AGGC: codon 249 in bold, targeted G underlined,multiplied by the 5� value to yield a number that can be usedto compare the reactivities of different immediate sequences 3� base of target G in normal type). The RR value for this

context is 0.3, which falls somewhere between weak andon a central G. Herein, these values will be denoted ‘relativereactivity’, RR.) Significantly, the sequence context-specific intermediate reactivity toward AFB1. This mutation results in

an Arg→Ser alteration in the p53 protein. Evidence is providedreactivity for AFB1 holds true for DNA globally modifiedby AFB1 (28,35,38,41). The sequence-specificity for AFB1 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)reactivity has been divided into three classes, and these arereferred to in the literature as ‘weak (RR ~0.23)’,‘intermediate was not reactive towards AFB1, even though Benasutti’s rules

predict it to be 1.3 times more reactive than the third base.(RR ~0.44)’ or ‘strong (RR ~0.5)’ sites for AFB1 reactivity.It has also been suggested that not only sequence context, but This study also demonstrates that AFB1 reacts with 20% of

the bases in exons 5–8 of the p53 gene, ~85% of which arealso pre-existing modification of neighboring bases, can affectthe reactivity of AFB1 toward a particular sequence. Several guanines that are in several different sequence contexts.

Another group has also observed that significant adduct forma-reports in the literature demonstrate that AFB1 adduct forma-tion, as well as several other types of DNA damage, is tion 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 yetmodulated by the presence of 5-methyl cytosine at a CpG site

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Fig. 3. Interaction of the exo-epoxide of AFB1 with DNA. (A) The epoxide (gray) approaches a reactive guanine (black). As the toxin intercalates into DNAon the 5� face of the guanine residue, the free electron pair of N7 of the attacking guanine is aligned to conduct an SN2 displacement on C8 of aflatoxin. Themajor product (shown) of this reaction is AFB1-N7-Gua. (B) The NMR structure of an oligonucleotide containing AFB1-N7-Gua is shown. To the right inthis panel is the sequence context in which mutagenesis studies were done (see text). Note the position of the intercalated AFB1 residue. (C) The majoradduct produces either the G→T transversion at the site of reaction (the black guanine) and a second site mutation at the position of the base 5� to the site ofcovalent bonding (which is a cytosine). This panel provides a rationalization for the second site mutation. During replication, it is proposed that the aflatoxinresidue drops into the position of the 5� cytosine, everting the cytosine from the helix. The restructured complex is shown with the original positions of thebases shown in broken lines. The polymerase inserts adenine (box entering from the lower left) opposite the aflatoxin residue resulting in a C→T transition,which is the mutation observed in vivo.

another study (48), GC→TA and CG→AT transversions were consistent with other studies, whereby different systems wereutilized for the analysis of AFB1-induced mutations (29,49–observed at positions adjacent to the hallmark mutation site

at codon 249 of p53 (AGG→ATG and AGGC→AGGA), 51), implying that another factor, such as DNA secondarystructure, may play a role in AFB1 modification and/or muta-demonstrating that in different systems, reactivity toward AFB1

and the correlation between reactivity and mutation may be genesis. As mentioned above, it is possible that an aflatoxin-modified guanine residue at the third position of the AGGdifferent.

Providing further support for the view that AFB1 binds to sequence may give rise to mutations at the second position aswell. This separation of the mutation site from the site ofand causes mutations in certain sequence contexts, data from

Levy et al. (33) illustrate that four out of seven of the covalent modification may be due to distortion of the base 5�to the modified guanine as a consequence of intercalation ofmutational hotspots from human xeroderma pigmentosum (XP)

cells, observed in the supF gene of the pS189 shuttle vector, the AFB1 moiety on the 5� face of the guanine (15) (Figure 3).There is substantial evidence that the activation of cellularare 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 � ras (c-ras) genes by specific single-base mutations may be animportant step in the transformation of normal cells to malig-0.8) or an A (RR � 0.2), contexts that show different

reactivities toward AFB1]. One AGG sequence contained nant cells (52–54). This activation event is manifested asmutations in codon 12 of the c-Ki-ras gene in DNA in rathotspots at both the second (RR � 0.4) and third G. It is not

clear whether mutations at both of the guanine positions are liver tumors (55,56). Table I illustrates the sequences of codons11, 12 and 13 of rat, trout and human and the relative reactivitydue to aflatoxin binding at one site or at two different sites.

Using a polymerase arrest assay, this study showed that there of each guanine. The first and second positions of codon 12incur mutations in rat, and the second positions of both codonswas more blockage at the third G than at the second G of the

AGG sequence. This indicates that although the sites that are 12 and 13 incur mutations in trout (57). In humans, mutationshave been observed at the first and second positions of codonhotspots for predominantly GC→TA mutations are inter-

mediate or strongly reactive towards AFB1, not all of these 12 in the Ha-ras proto-oncogene (58). Interestingly, the c-Ki-ras sequence context (GCAGGA) for both mutations insites have incurred the level of mutations consistent with their

reactivity, and some sequences that should only be weakly rainbow trout is analogous to that of codon 249 of p53(AGGC), except that the bases 3� to the mutated G are differentreactive are actually hotspots for mutation. These data are

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Table I. Species-specific sequence differences among ras codons: correlation of predicted reactivity with reactive forms of AFB1

Species Rat Trout Humans

Codon 11 12 13 11 12 13 11 12 13Sequence GCT GGT (Gly) GGC GCA GGA (Gly) GGT (Gly) GCC GGC (Gly) GGTRR G1

a 0.2 0.2 0.3 0.3 0.8 0.8RR G2

a 0.8 0.4 0.3 0.8 0.4 0.8Mutation GAT (Asp) GTA (Val) GTT (Val) TGC (Trp)

TGT (Cys) GTC (Val)GTT (Val)

aRR of the first G in the codon.bRR of the second G in the codon.

(an A in the case of codon 12 and a T in the case of codon and mutation are within sequences where a G is located 5� tothe base involved in the mutation. The base 3� to the modified13). It is possible, as suggested by these data and others (33),

that the third position of the sequence AGG is a hotspot for G appears less consistently associated with mutation and maynot be as crucial as the 5� base. Taking a minimalist approach,AFB1 mutation regardless of the 3� base. Rats and humans do

not incur mutations in codon 13, possibly due to a base other one could say that all that is required for a mutational hotspotfor AFB1 is a GG sequence with the modification/mutationthan adenine 5� to the GG sequences. It is also a possibility

that the different sequences form different DNA secondary occurring at the second G. In fact Levy et al. (33) havedescribed hotspots in just this way. It is noted, however, thatstructures, which may be more or less accessible to DNA

repair machinery. the AFB1-induced mutations in the aforementioned studies arenot observed at every site, within the particular gene analyzed,In vivo studies conducted with the lacI gene in transgenic rats

and mice examined sequences surrounding mutated guanines to where a strong or intermediate sequence for AFB1 reactivityoccurs.determine if the mutations observed were sequence context

dependent (59). Mutations were observed in six differentsequence contexts in mice. In half of these contexts, the Factors that influence AFB1 mutagenesis and HCCtargeted guanine is in the third position of a CGG context, formationwith 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 The shape of the mutational spectrum of aflatoxin is influenced

by at least three factors: (i) the preference of one G overaflatoxin reactivity in specific contexts, one out of the sixsequences would be considered strong, two out of the six another for reaction, (ii) the preference for a base to be

erroneously replicated more frequently in one context comparedwould be considered intermediate and three out of the six con-texts would be considered weak. Mutations were also observed with another and (iii) the preference for repair of an adduct in

one context over others. One would expect that DNA repair-in 25 different sequence contexts in rats, eight of which haveweak reactivity and the rest of which have intermediate to deficient cells present an opportunity to examine the contribu-

tions of lesion formation and misreplication [points (i) andstrong reactivity toward AFB1. Of all the types of mutationsobserved, ~48% of those in mice and 66% of those in rats (ii)] separated from the complications of preferential repair

[point (iii)]. Mutational spectra were studied in cells with thewere found at CpG sites. Only 17% of the sequences examinedin mice and 4% of the sequences examined in rats did not DNA nucleotide excision repair deficiency disorder, XP (33).

All sequence contexts that should be strongly reactive towardhave at least one G or C immediately adjacent to the mutatedG. Sequences that have at least one G or C adjacent to a AFB1 may not incur mutations, since preferential repair of

some sequences may be taking place. Surprisingly, most ofcentral G have an average RR of 0.44, while sequences withoutan adjacent C or G have an average RR of 0.18. In a separate the contexts that were hotspots in repair proficient cells were

predicted to have only weak to intermediate reactivity towardstudy, rats treated with AFB1 in the presence or absence ofthe liver regeneration process were evaluated for p53 mutations AFB1, whereas in XP cells, most of the hotspots occurred in

intermediate to strong contexts. This result implies that(60). Only one mutation (20%) occurs at a CpG site. One outof five (20%) of the sequences examined does not have a G sequences that are more reactive to AFB1 may also be more

prone to undergo DNA repair, again suggesting a role foror a C immediately adjacent to the mutated base (on twooccasions, the mutated base was a C; these mutations could secondary structure. It has also been observed that the hepatitis

B x protein (HBx) can inhibit nucleotide excision repair, eitherhave been due to the Gs in the opposite strand). In a thirdstudy, carried out in the human HPRT gene (61), a hotspot for by binding to repair proteins or to the damaged DNA itself,

making it possible that AFB1 adducts persist preferentially inboth base substitution and frameshift mutations occurs in arun of six Gs. These data once again show that although patients who have been exposed to both HBV and AFB1 (62–

64). XRCC1, an enzyme involved in base excision repair, hascertain sequence contexts exhibit weak reactivity toward AFB1,they may still incur mutations. We note, however, that they several polymorphisms. Individuals with the 399 Gln allele

have increased levels of AFB1 adducts (65). These differencesalso support Benasutti’s rules for preferential AFB1 binding toGC-rich regions. in DNA repair are believed to be independent of p53, so it is

reasonable to speculate that p53 mutations and the effects ofIn summary, in all of the aforementioned studies, some ofthe mutational hotspots occurred in sequences that, according HBV play a role in HCC induction via separate pathways. In

support of this hypothesis, HBx has been shown to promoteto Benasutti’s empirical rules, are also intermediate or strongspots for AFB1 reactivity. Most of these hotspots for reactivity apoptosis following DNA damage in a cell line containing

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wild-type p53, while promoting a growth advantage in a cell unique to AFB1-induced liver tumors, as tumors presumablyinduced by other factors fail to consistently show this geneticline containing AFB1-induced mutant p53 (66).

The types and positions of the mutations induced by AFB1 change (66,71). Currently, clinical trials with oltipraz areunderway in Qidong (China) where, so far, a 51% decrease inmay differ among species due to a difference in AFB1 activation

(67). Human SV40 immortalized cell lines expressing different AFB1 metabolites has been observed. Further studies willelucidate whether this drug plays a role in reducing HCChuman cytochrome P450 (CYP) enzymes (CYP1A2, CYP2A6,

CYP2B6 or CYP3A4) gave rise to different mutational spectra, incidence (71).The correlation between exposure to aflatoxin and thein terms of the proportions of transitions and transversions at

each position that was mutated in codons 249 and 250 of a hotspot at codon 249 suggests two possibilities. First, aflatoxinmay be particularly reactive with this nucleotide, owing to itsglobally-modified p53 gene. Indeed, AFB1 exposure itself

alters expression of some genes, including those for certain surrounding sequence, as discussed in the preceding section.Second, it is possible that this mutation is due somehow toCYPs, which are involved in activation, and glutathione S-

transferase (GST), which is involved in detoxification, of AFB1 the combined effects of AFB1 exposure and HBV infection,either directly or through separate pathways, since this mutation(68). HBV infection is also thought to influence the regulation

of such genes (69). From species to species, and even within is observed much more often in areas of the world whereexposure to both agents is very common. In both cases, ifthe human population itself (70), the balance between the

activities of the enzymes of AFB1 activation and detoxification mutations in codon 249 significantly debilitate the function ofp53, the loss of function of this protein should give cells amay explain the differences observed in response to the toxin.

Oltipraz [4-methyl-5-(2-pyrazinyl)-3-H-1,2-dithiole-3-thione], selective growth advantage, since p53 is an important tumor-suppressor gene. p53 is a transcriptional activator that hasa drug currently in Phase IIb clinical trials, has been shown

to decrease AFB1 metabolites in humans by 51% by inducing been shown to regulate the cell cycle (82), to play a role inthe apoptosis pathway (83–87) and to be involved with DNAGST and inhibiting CYP1A2, the CYP isoform that activates

AFB1 to its reactive metabotite (71). repair (88–93). AFB1 and HBV may act in concert, or theymay induce completely different pathways, the combinedA general pattern in the types and sequence-dependence of

AFB1-induced mutations can be inferred from the studies effects of which lead to the selection of the hallmark p53mutation and the ultimate endpoint of HCC.carried out above. We note that the atomic environment of

some DNA sequences may render certain sites more favorable HBV infection has been shown to be associated withincreased incidence of HCC. In general, a person with HBVfor AFB1 intercalation (72). The data indicate that AFB1

induces a predominance of GC→TA transversions within GC- is four times more likely to develop liver cancer, regardlessof their level of exposure to AFB1 (6). Thus, the associationrich sequence contexts. However, not all sequence contexts

that are strong sites for modification by AFB1 are necessarily of HBV infection and HCC formation has been the focus ofmany studies. Several groups have shown that one of the genehotspots for mutation. In addition, some sequence contexts

that are weak sites for modification are actually hotspots for products of HBV, HBx, binds to and inactivates the p53 protein(94,95). Although there is some controversy over this issue,mutation. Further investigation of potential differences in the

mechanism of AFB1 reactivity toward different sequences, experiments suggest that the binding of HBx to the C-terminalregion of p53 inhibits p53 sequence-specific DNA bindingpotential differences in repair of various AFB1-modified

sequences and the influence of different DNA secondary and, thus, inhibits its role as a transcriptional activator (95).The same group has also shown that expression of HBx maystructures on AFB1 reactivity should shed further light on the

correlation between hotspots for AFB1 modification and AFB1 inhibit p53-induced apoptosis (96). In contrast, another grouphas shown that p53, in cells exposed to HBV, can still activatemutations.some genes to some extent in vivo (97). Other work, whilefailing to observe a direct interaction between p53 and HBxAflatoxin B1, hepatitis B virus and hepatocellular carcinoma(63,73,98–100), has indeed determined an alteration in eitherthe localization, the phosphorylation status or the transcriptionCodon 249 of p53 is a hotspot for AFB1 modification and

AFB1-induced mutation, specifically AGGC→AGTC. This of wild-type p53 during HBx expression. Yet others haveprobed total cellular RNA in liver cell lines expressing HBx,mutation has been found in up to 50% of HCC samples in

areas of the world where aflatoxin exposure is high. To date, searching for genes that are up- or down-regulated, and noeffect has been observed on p53 RNA (101). An increase inover 2000 HCC samples from all over the world have been

examined for this mutation (6,73–75). In regions where expo- the number of hallmark codon 249 mutations has been observedwhen a cell line that expresses the HBx protein is exposed tosure to aflatoxin is high—namely Qidong and Tongan (China),

India, Southern Africa, The Gambia, Mozambique and increasing levels of AFB1 (66). In addition oltipraz hasbeen shown to inhibit HBV transcription and to upregulateSenegal—116 out of 262 (44%) of the total HCC cases

examined show a predominance of GC→TA mutations at the expression of wild-type p53 in cell culture (70). Perhaps it isnot the direct association of HBx with p53, but some otherthird position of codon 249 of the p53 gene (74–78). In

contrast, in regions of low exposure to aflatoxin—namely, effect that HBx has on the cell, such as an inhibition ofapoptosis in p53 mutant cells, that promotes mutant selection.Australia, Europe, Japan and the USA—only 17 out of 1273

(1%) of the HCC samples examined had mutations at this Although exposure to HBV alone increases the risk of HCC,the risk is even higher in an individual who is both positivesite (74,78–80). In regions where exposure to aflatoxin is

moderate—namely, Beijing, Shanghai, Xi’an, Hong Kong, for HBV and is exposed to aflatoxin (6,102–104). One studyindicates that patients with positive urinary AFB1-N7-GuaSingapore, South Korea, Taiwan, Thailand, Vietnam, Southern

Asia, South Africa and Egypt—40 out of 568 (7%) HCC cases antigen are three times more likely to develop HCC, patientswith positive HBV surface antigen (HBsAg) are about sevenexamined had mutations at the third position of codon 249 of

the p53 gene (6,65,77,81). This p53 mutation appears to be times more likely to develop HCC and when both AFB1 and

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Table II. Expected amino acid changes arising from GC→TA mutations at p53 codons 248 and 249 in different speciesa

Species Human Primate Rat Mouseb Wood chuck Duck Tree shrew Ground squirrel

Codonc,d 248 249 248 249 248 249 248 249 248 249 248 249 248 249 248 249Sequencee CGG AGG CGG AGG CGC CGG CGC CGA CGA CGG CGT CGC CGG CGC CGC CGGAmino acid Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg ArgBase change CGT AGT CGT AGT CTC CGT CTC CTA CTA CGT CTT CTC CGT CTC CTC CGTAmino acid Arg Ser Arg Ser Leu Ser Leu Leu Leu Arg Leu Leu Arg Leu Leu ArgBase change CTG ATG CTG ATG CTG CTG CTG CTGAmino acid Leu Met Leu Met Leu Leu Leu LeuViral status HBV Non Transgenic Transgenic WHVB DHVB HBV GSHV

for HbsAg for HBVenvelope

aActual mutations observed only in humans. Mutations shown in other species are proposed AFB1-induced GC→TA transversions.bStudies in mice transgenic for the HBV envelope protein did not evaluate the third position of codon 249 of p53, and those that did evaluate codon 249 didnot involve aflatoxin.cCodons noted are function equivalent of p53 codons 248 and 249 in humansdThe first base of codon 250 in each species above is a C.eSequence context RR: CGG � 0.8, CGC � 0.3, GGA � 0.3, CGA � 0.3, CGT � 0.6, GGC � 0.3.

HBsAg are present, patients are 60 times more likely to Would animal models be of value to probe the importanceof p53 in the induction of HCC by aflatoxin?develop this disease (103). Thus, there seems to be a synergism

between HBV and AFB1 exposure for the development ofp53 is at least 92% homologous across the species listed inHCC. Interestingly, a relationship has been observed betweenTable II (106). Codons 248 and 249 in the animal modelsthe presence of HBsAg and the mutational hotspot at codondiscussed here are analogous to human codons 248 and 249,249 of p53 (6,74,75). Twelve percent of the 2019 HCC casesand they are believed to be within the DNA binding regionsexamined for the mutational hotspot at codon 249 wereof the animals’ proteins, which is also the case with the humanexamined for the presence of HBsAg. For the samples takenprotein. The GC→TA hotspot mutation at the codon analogousfrom regions of high aflatoxin exposure, 47% (82 out of 175to the human codon 249 in the p53 genes of other species hastotal) of HBV-positive HCC samples examined had a GC→TAnot been identified. To date, all attempts to recreate this hotspottransversion at position three of codon 249 of p53. About 42%by exposing animals to aflatoxin have not been successful.(18 out of 43) of the HBV-negative HCC samples from these However, it is possible that this failure to generate the hotspot

regions had the same mutation. For the samples taken from is due solely to the differences among the p53 sequences ofregions of moderate aflatoxin exposure, only 35% (15 out of different species (Table II). A different sequence context may43 total) of the HBsAg-positive samples had the GC→TA alter the reactivity of AFB1 toward the analogous codon 249transversion. None of the 11 HBsAg-negative samples in these sequence. Table II illustrates, from a compilation of data, thatregions exhibited the mutation. Other work (73) has shown there is no mutation analogous to the human p53 codon 249that in areas of low aflatoxin exposure, HCC samples from mutation (Arg→Ser) in primates, rats, mice, ducks, wood-people who are positive for the HBsAg also have p53 mutations, chucks, tree shrews or ground squirrels when these animalsalthough they have not yet determined what these mutations are exposed to aflatoxin. A possible reason that no analogousare. These studies help establish the relationship between this mutation has been found may be that—at least in woodchucks,hotspot mutation in tumors and HBV infection. tree shrews (opposite strand mutation) and ground squirrels—

Experiments have examined the ability of different p53 a GC→TA transversion in the third position of the codonmutants to modulate in trans the transcriptional activity of results in a silent mutation. Additionally, some of the specieswild-type p53 (p53 acts functionally as a tetramer) on a tested do not have a species-specific form of hepatitis B. Thereporter construct in HCC cell lines (HEP-3B) possessing an HBV status of several species studied is also shown in Tableintegrated HBx gene (105). It has not been confirmed, however, II. If HBV and AFB1 are both needed for the selection of athat these cells actually do express the HBx protein. The p53 p53-inactivating mutation at the third position of codon 249,mutants tested included R248W and R249S mutations. The it is important for an animal model to be able both to contractR248W mutant enhanced the level of transcription to 132% HBV-mediated disease and to have a similar p53 codonof wild type, while the R249S mutant drastically reduced the sequence in the DNA binding region. Similar to the woodchucklevel of transcription to 18% of wild type. Control experiments and ground squirrel models noted above, ducks contract awere carried out in tumor cell lines from lung, prostate and form of hepatitis B, duck hepadnavirus B (DHVB), but themammary epithelial tissues, none of which express HBx. The predominant mutation in ducks at codon 249 is Arg→Leu,R249S mutant exhibited a similar effect in lung (20% of wild which may not be as detrimental as Arg→Ser for p53 activitytype), and a milder effect in mesothelial (52% of wild type), (106). In the case of primates and rats, even though themammary (75% of wild type) and prostate (80% of wild type) mutation at codon 249 would give rise to an amino acidtissues. When any of the mutants are expressed in a cell change of Arg→Ser, studies were carried out in the absenceline that does not contain wild-type p53, no transcriptional of HBV (107,108). Currently, rats transgenic for HBV areactivation of the reporter construct is seen. Thus, other mutant being developed (109). Mice naturally have an Arg→Leusequences should be examined in the experimental system amino acid change in codon 249; we note, however, that mice

have been engineered to have an Arg→Ser mutation at codondescribed above.

541

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246, which is analogous to the human Arg→Ser mutation at or HBV status affect p53 are still not completely clear. Theincreasingly powerful tools used in biochemistry and geneticscodon 249 (AGGC→AGTC) (110). One study has been done

where mice either with or without the codon 246 mutation should foster the efforts that, over the next quarter of a century,will address the question of how this important carcinogenwere simultaneously exposed to AFB1 and were positive for

the HBsAg. This mutation causes an increase in high-grade affects the human population.liver tumor formation in the presence of AFB1 (from 0 to 14%when homozygous for wild-type p53; from 14 to 71% when Acknowledgementsheterozygous for wild-type p53), and even more so in the

We are especially indebted to Professor Michael P.Stone of Vanderbiltpresence of both AFB1 and HBsAg (from 62 to 100% whenUniversity for molecular modeling. This work was supported by grantshomozygous for wild-type p53). Again, in these studies, it has CA80024/ES09546 and ES07020 from the National Institutes of Health.

not been proven that HBx is expressed in these animals.In animal models it is important for the species-specific

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Received September 12, 2000; accepted November 3, 2000Clin. Oncol., 116, 581–590.

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