The chemistry and biology of aflatoxin B 1 : from mutational spectrometry to carcinogenesis

Maryann E.Smela, Sophie S.Currier, Elisabeth A.Bailey world where both aflatoxin and hepatitis B virus (HBV) are prevalent frequently have a single signature mutation (6). and John M.Essigmann1 The first part of this review will focus on the sequence Department of Chemistry and Division of Bioengineering and specificity of AFB1 adduct formation and the types of mutations Environmental Health Massachusetts Institute of Technology, Cambridge, induced by AFB1 in various experimental contexts. Of central MA 02139, USA importance in this discussion will be technology by which the 1To whom correspondence should be addressed genetic effects of individual DNA adducts has been probed Email: jessig@mit.edu in vivo. The review will then cover the synergistic effects of Dietary exposure to aflatoxin B1 (AFB1) is associated with AFB1 exposure and HBV infection on the formation of liver an 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 B finally, will evaluate the different models proposed to explain virus (HBV) is a common occurrence. Most HCC samples this phenomenon. from people living where HBV is prevalent have one striking mutational hotspot: a GC→TA transversion at the The mutational specificities of specific AFB1-DNA lesions third position of codon 249 of the p53 gene. In this review, correlate with the mutational spectrum of activated AFB1 the chemical reaction of an electrophilic derivative of aflatoxin with specific DNA sequences is examined, along Figure 1 details the pathway of AFB1 metabolism leading to with the types of mutations caused by AFB1 and the covalent adduct formation. The AFB1 exo-8,9-epoxide is the sequence context dependence of those mutations. An aflatoxin metabolite that reacts covalently with DNA to form attempt is made to assign the source of these mutations to the adducts that presumably account for the biological effects specific chemical forms of AFB1-DNA damage. In addition, of the toxin. Of the various adducts formed, the most quantitatepidemiological and experimental data are examined ively abundant both in vitro (7–10) and in vivo (9,11–13) is regarding the synergistic effects of AFB1 and HBV on HCC 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7formation and the predominance of one hotspot GC→TA Gua). The positively charged imidazole ring of the principal transversion 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 opens to form the more chemically and biologically stable AFB

in vivo.The review will then cover the synergistic effects of Dietary exposure to aflatoxin B 1 (AFB 1 ) is associated with AFB 1 exposure and HBV infection on the formation of liver an 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 B finally, will evaluate the different models proposed to explain virus (HBV) is a common occurrence.Most HCC samples this phenomenon.from people living where HBV is prevalent have one striking mutational hotspot: a GC→TA transversion at the The mutational specificities of specific AFB 1 -DNA lesions third position of codon 249 of the p53 gene.In this review, correlate with the mutational spectrum of activated AFB 1 the chemical reaction of an electrophilic derivative of aflatoxin with specific DNA sequences is examined, along Figure 1 details the pathway of AFB 1 metabolism leading to with the types of mutations caused by AFB 1 and the covalent adduct formation.The AFB 1 exo-8,9-epoxide is the sequence context dependence of those mutations.An aflatoxin metabolite that reacts covalently with DNA to form attempt is made to assign the source of these mutations to the adducts that presumably account for the biological effects specific chemical forms of AFB 1 -DNA damage.In addition, of the toxin.Of the various adducts formed, the most quantitatepidemiological and experimental data are examined ively abundant both in vitro (7-10) and in vivo (9,11-13) is regarding the synergistic effects of AFB 1 and HBV on HCC 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B 1 (AFB 1 -N7formation and the predominance of one hotspot GC→TA Gua).The positively charged imidazole ring of the principal transversion in the p53 gene of affected individuals.
adduct promotes depurination, giving rise to an apurinic (AP) site.Alternatively, the imidazole ring of AFB 1 -N7-Gua opens to form the more chemically and biologically stable AFB 1 Introduction formamidopyrimidine (AFB 1 -FAPY) (14).While this reaction occurs most favorably under basic conditions, the AFB 1 -FAPY Aflatoxin B 1 (AFB 1 ) is a fungal metabolite that contaminates adduct is a significant product in vivo (13).It is likely that the the food supply in certain areas of the world (1).It is produced initial AFB 1 -N7-Gua adduct, the AFB 1 -FAPY adduct, and the by Aspergillus flavus and related fungi that grow on improperly AP site, individually or collectively, represent the chemical stored foods, such as corn, rice and peanuts.AFB 1 requires precursors to the genetic effects of AFB 1 .Other DNA lesions metabolic conversion to its exo-8,9-epoxide (1,2) in order to form, but at much lower levels than these three products (13).damage DNA (2).As shown in Figure 1, the AFB 1 epoxide As indicated below, the AFB 1 -N7-Gua adduct has mutagenic reacts with guanine to form a number of adducts (3), and one properties that correlate with those of the biologically relevant reasonable model is that these adducts, or secondary DNA DNA lesion(s) of AFB 1 (15).The other two abundant lesions lesions derived from them, lead to heritable genetic changes that formed by AFB 1 , specifically AFB 1 -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 that ively whether or not they play significant roles in aflatoxin forms in cells treated with the toxin, the mutational spectrum toxicology. of the toxin is dominated by one genetic change: the GC→TA In all biological systems evaluated to date, the most fretransversion.It is presumed that these mutations arise from a quently observed mutation induced by chemically reactive guanine adduct, owing to the fact that nearly all, if not all, forms of AFB 1 (e.g., the AFB 1 epoxide or other electrophilic aflatoxin adducts occur at that base. derivatives) is the GC→TA transversion.This is the principal In hepatocellular carcinomas (HCCs), as in many other mutation found in several experimental systems: in the endocancers (4), the p53 gene is mutated in Ͼ50% of the tumors genous lacI gene in an SOS-induced Escherichia coli strain (4,5).Of likely significance is the observation that the principal that contains the mucAB mutagenesis enhancing operon (16); point genetic change in the p53 genes of HCC is the GC→TA in human cells replicating an AFB 1 -modified pS189 shuttle transversion, the same point mutation principally induced by vector (17); in the ras gene of rainbow trout liver (19); in the electrophilic forms of aflatoxin.As a specialized situation, the human Ha-ras proto-oncogene (20); in the transgenic C57BL/ mutated p53 genes of individuals with HCC from areas of the 6N (BigBlue) mouse treated with phorone (a glutathione depleting agent) (21); in the lacI gene in transgenic C57BL/6 the host.After replication in vivo, either intra-or extrachromosomally, 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).It has been reasonably suggested that the premutagenic lesion responsible for the observed GC→TA transversions in bacterial systems would be the AFB 1 -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 (21)(22)(23)(24)(25).In E.coli the mutational properties of the AFB 1 -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 AFB 1 -N7-Gua adduct are targeted to the site of the lesion, a significant fraction (13%) occur at the base 5Ј 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 AFB 1 -N7-Gua is consistent with structural models, indicating that the aflatoxin moiety of the aflatoxin guanine adduct intercalates on the 5Ј face of the guanine residue (Figure 3) (26).These results suggest a molecular mechanism that could explain an important potentially modified guanines.Since guanine residues flanked by AϩT-rich sequences are poor targets for modification by AFB 1 (27)(28)(29), it is conceivable that the majority of the 5Ј 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 5Ј cytosines or guanines.These mutations would mistakenly have been scored as resulting gene (26); and in AFB 1 -exposed rats that either did or did not undergo partial hepatectomy (27).Both GC→TA and GC→AT from either an additional AFB 1 -modified guanine in the same strand or an AFB 1 -modified guanine in the opposite strand.In mutations are induced with equal efficiency in other systems by metabolically activated AFB 1 (28) and the AFB 1 8,9-order to determine whether some of the mutations observed in experiments carried out with globally-modified DNA are dichloride model for the AFB 1 epoxide (29).GC→AT transitions are predominant in the c-Ki-ras oncogene of rat HCCs, the result of a 3Ј-proximal AFB 1 -N7-Gua residue, future studies should involve the examination of the three AFB 1 an 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 to below, 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-stranded DNA, owing to the favorable environment created by base At least three DNA lesions-AFB 1 -N7-Gua, AFB 1 -FAPY and the AP site-are candidate precursors to the mutations pair stacking (30).Double-stranded DNA is a more favorable target for aflatoxin binding than single stranded DNA, and induced by AFB 1 .A number of years ago, systems were developed to allow assessment of the mutational specificity several chemical approaches provide compelling evidence that aflatoxin indeed intercalates on the 5Ј face of the guanine and quantitative mutagenicity of individual lesions formed by DNA damaging agents (17).In its most commonly applied residue in double-stranded DNA (31,32).This intercalation model fits well with the long-standing idea that there is a form (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 and the 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 account for 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 epoxide binding.adduct encounters the replication and repair apparatuses of 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., AFB 1 -N7-Gua, an AP site or AFB 1 -FAPY) is annealed to the genome.The 5Ј and 3Ј 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.
The reactivity of aflatoxin toward different DNA sequences (42)(43)(44)(45)(46).One contradictory report, however, states that cytosine methylation has no influence over aflatoxin reactivity (47).has been studied by a number of groups (27)(28)(29)(33)(34)(35)(36)(37)(38)(39)(40).In one systematic study Benasutti et al. (28) illustrate that the reactivity of AFB 1 toward a guanine residue is highly dependent Can one explain the mutations observed in HCC on the on the base immediately 5Ј, and the base immediately 3Ј to basis of the mutational specificity of the aflatoxin epoxide? the central guanine, with a run of three guanines being the most favorable sequence overall.In the 5Ј position the reactivity There is one striking feature underlying the sequence specificity of mutational hotspots that arise in liver tumors believed to profile is G (1.0) Ͼ C (0.8) Ͼ A (0.4) Ͼ T (0.3), and in the 3Ј position it is G (1.0) Ͼ T (0.8) Ͼ C (0.3) Ͼ A (0.2). (A have been induced by AFB 1 exposure.As indicated earlier, the predominant mutational hotspot found in human HCC is numerical value for relative reactivity is given in parentheses.These values were calculated such that the 3Ј 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 5Ј value to yield a number that can be used to compare the reactivities of different immediate sequences 3Ј base of target G in normal type).The RR value for this context is 0.3, which falls somewhere between weak and on a central G. Herein, these values will be denoted 'relative reactivity', RR.) Significantly, the sequence context-specific intermediate reactivity toward AFB 1 .This mutation results in an Arg→Ser alteration in the p53 protein.Evidence is provided reactivity for AFB 1 holds true for DNA globally modified by AFB 1 (28,35,38,41).The sequence-specificity for AFB 1 that indicates that AFB 1 reacts with the third position of codon 249 of p53 in vitro (34).Position 2 of codon 249 (RR ϭ 0.4) reactivity has been divided into three classes, and these are referred to in the literature as 'weak (RR ~0.23)','intermediate was not reactive towards AFB 1 , 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 AFB 1 reactivity.

It has also been suggested that not only sequence context, but
This study also demonstrates that AFB 1 reacts with 20% of the bases in exons 5-8 of the p53 gene, ~85% of which are also pre-existing modification of neighboring bases, can affect the reactivity of AFB 1 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 AFB 1 adduct formation, 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 yet modulated by the presence of 5-methyl cytosine at a CpG site another study (48), GC→TA and CG→AT transversions were consistent with other studies, whereby different systems were utilized for the analysis of AFB 1 -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 secondary structure, may play a role in AFB 1 modification and/or muta-demonstrating that in different systems, reactivity toward AFB 1 and the correlation between reactivity and mutation may be genesis.As mentioned above, it is possible that an aflatoxinmodified guanine residue at the third position of the AGG different.
Providing further support for the view that AFB 1 binds to sequence may give rise to mutations at the second position as well.This separation of the mutation site from the site of and 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 5Ј to the modified guanine as a consequence of intercalation of mutational hotspots from human xeroderma pigmentosum (XP) cells, observed in the supF gene of the pS189 shuttle vector, the AFB 1 moiety on the 5Ј face of the guanine (15) (Figure 3).There is substantial evidence that the activation of cellular are at AGG sequences.Three of these hotspots are located at the third G [the 3Ј base was either a G (RR ϭ 1.0), a T (RR ϭ ras (c-ras) genes by specific single-base mutations may be an important step in the transformation of normal cells to malig-0.8) or an A (RR ϭ 0.2), contexts that show different reactivities toward AFB 1 ].One AGG sequence contained nant cells (52-54).This activation event is manifested as mutations in codon 12 of the c-Ki-ras gene in DNA in rat hotspots at both the second (RR ϭ 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 codons 11, 12 and 13 of rat, trout and human and the relative reactivity due 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 12 incur mutations in rat, and the second positions of both codons was 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, mutations have been observed at the first and second positions of codon hotspots for predominantly GC→TA mutations are intermediate or strongly reactive towards AFB 1 , not all of these 12 in the Ha-ras proto-oncogene (58).Interestingly, the c-Kiras sequence context (GCAGGA) for both mutations in sites 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 3Ј to the mutated G are different reactive are actually hotspots for mutation.These data are (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 5Ј to the base involved in the mutation.The base 3Ј to the modified 13).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 may not be as crucial as the 5Ј base.Taking a minimalist approach, AFB 1 mutation regardless of the 3Ј 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 hotspot for AFB 1 is a GG sequence with the modification/mutation than adenine 5Ј 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) have described hotspots in just this way.It is noted, however, that structures, which may be more or less accessible to DNA repair machinery.
the AFB 1 -induced mutations in the aforementioned studies are not 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 AFB 1 reactivity occurs.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 Factors that influence AFB 1 mutagenesis and HCC targeted guanine is in the third position of a CGG context, formation with either a C (RR ϭ 0.4), an A (RR ϭ 0.3) or a T (RR ϭ 0.8) in the 3Ј 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 over aflatoxin reactivity in specific contexts, one out of the six sequences 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 compared would be considered intermediate and three out of the six contexts 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 have weak reactivity and the rest of which have intermediate to deficient cells present an opportunity to examine the contributions of lesion formation and misreplication [points (i) and strong reactivity toward AFB 1 .Of all the types of mutations observed, ~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 the were found at CpG sites.Only 17% of the sequences examined in 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 toward 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 AFB 1 may not incur mutations, since preferential repair of some sequences may be taking place.Surprisingly, most of 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 the contexts that were hotspots in repair proficient cells were predicted to have only weak to intermediate reactivity toward study, rats treated with AFB 1 in the presence or absence of the liver regeneration process were evaluated for p53 mutations AFB 1 , 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 out of five (20%) of the sequences examined does not have a G sequences that are more reactive to AFB 1 may also be more prone to undergo DNA repair, again suggesting a role for or a C immediately adjacent to the mutated base (on two occasions, 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, either 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 by binding to repair proteins or to the damaged DNA itself, making it possible that AFB 1 adducts persist preferentially in both base substitution and frameshift mutations occurs in a run of six Gs.These data once again show that although patients who have been exposed to both HBV and AFB 1 (62-64).XRCC1, an enzyme involved in base excision repair, has certain sequence contexts exhibit weak reactivity toward AFB 1 , they may still incur mutations.We note, however, that they several polymorphisms.Individuals with the 399 Gln allele have increased levels of AFB 1 adducts (65).These differences also support Benasutti's rules for preferential AFB 1 binding to GC-rich regions.
in DNA repair are believed to be independent of p53, so it is reasonable to speculate that p53 mutations and the effects of In summary, in all of the aforementioned studies, some of the 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 promote to Benasutti's empirical rules, are also intermediate or strong spots for AFB 1 reactivity.Most of these hotspots for reactivity apoptosis following DNA damage in a cell line containing wild-type p53, while promoting a growth advantage in a cell unique to AFB 1 -induced liver tumors, as tumors presumably induced by other factors fail to consistently show this genetic line containing AFB 1 -induced mutant p53 (66).
The types and positions of the mutations induced by AFB 1 change (66,71).Currently, clinical trials with oltipraz are underway in Qidong (China) where, so far, a 51% decrease in may differ among species due to a difference in AFB 1 activation (67).Human SV40 immortalized cell lines expressing different AFB 1 metabolites has been observed.Further studies will elucidate whether this drug plays a role in reducing HCC human cytochrome P450 (CYP) enzymes (CYP1A2, CYP2A6, CYP2B6 or CYP3A4) gave rise to different mutational spectra, incidence (71).The correlation between exposure to aflatoxin and the in 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, aflatoxin may be particularly reactive with this nucleotide, owing to its globally-modified p53 gene.Indeed, AFB 1 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 to CYPs, which are involved in activation, and glutathione Stransferase (GST), which is involved in detoxification, of AFB 1 the combined effects of AFB 1 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 where exposure to both agents is very common.In both cases, if the human population itself (70), the balance between the activities of the enzymes of AFB 1 activation and detoxification mutations in codon 249 significantly debilitate the function of p53, the loss of function of this protein should give cells a may explain the differences observed in response to the toxin.
selective growth advantage, since p53 is an important tumorsuppressor gene.p53 is a transcriptional activator that has a drug currently in Phase IIb clinical trials, has been shown to decrease AFB 1 metabolites in humans by 51% by inducing been shown to regulate the cell cycle ( 82), to play a role in the apoptosis pathway (83)(84)(85)(86)(87) and to be involved with DNA GST and inhibiting CYP1A2, the CYP isoform that activates AFB 1 to its reactive metabotite (71).
repair (88)(89)(90)(91)(92)(93).AFB 1 and HBV may act in concert, or they may induce completely different pathways, the combined A general pattern in the types and sequence-dependence of AFB 1 -induced mutations can be inferred from the studies effects of which lead to the selection of the hallmark p53 mutation 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 with increased incidence of HCC.In general, a person with HBV for AFB 1 intercalation (72).The data indicate that AFB 1 induces a predominance of GC→TA transversions within GC-is four times more likely to develop liver cancer, regardless of their level of exposure to AFB 1 (6).Thus, the association rich sequence contexts.However, not all sequence contexts that are strong sites for modification by AFB 1 are necessarily of HBV infection and HCC formation has been the focus of many studies.Several groups have shown that one of the gene hotspots 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 AFB 1 reactivity toward different sequences, experiments suggest that the binding of HBx to the C-terminal region of p53 inhibits p53 sequence-specific DNA binding potential differences in repair of various AFB 1 -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 may structures on AFB 1 reactivity should shed further light on the correlation between hotspots for AFB 1 modification and AFB 1 inhibit p53-induced apoptosis (96).In contrast, another group has shown that p53, in cells exposed to HBV, can still activate mutations.some genes to some extent in vivo (97).Other work, while failing to observe a direct interaction between p53 and HBx Aflatoxin B 1 , hepatitis B virus and hepatocellular carcinoma (63,73,(98)(99)(100), has indeed determined an alteration in either the localization, the phosphorylation status or the transcription Codon 249 of p53 is a hotspot for AFB 1 modification and AFB 1 -induced mutation, specifically AGGC→AGTC.This of wild-type p53 during HBx expression.Yet others have probed 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 no effect has been observed on p53 RNA ( 101).An increase in over 2000 HCC samples from all over the world have been examined for this mutation (6, [73][74][75].In regions where expo-the number of hallmark codon 249 mutations has been observed when a cell line that expresses the HBx protein is exposed to sure to aflatoxin is high-namely Qidong and Tongan (China), India, Southern Africa, The Gambia, Mozambique and increasing levels of AFB 1 (66).In addition oltipraz has been shown to inhibit HBV transcription and to upregulate Senegal-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 is not the direct association of HBx with p53, but some other third position of codon 249 of the p53 gene (74)(75)(76)(77)(78).In contrast, in regions of low exposure to aflatoxin-namely, effect that HBx has on the cell, such as an inhibition of apoptosis 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 positive site (74,(78)(79)(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 study indicates that patients with positive urinary AFB 1 -N7-Gua Singapore, 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, patients with positive HBV surface antigen (HBsAg) are about seven examined 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 AFB 1 and HBsAg are present, patients are 60 times more likely to Would animal models be of value to probe the importance of p53 in the induction of HCC by aflatoxin?develop this disease (103).Thus, there seems to be a synergism between HBV and AFB 1 exposure for the development of p53 is at least 92% homologous across the species listed in HCC.Interestingly, a relationship has been observed between Table II (106).Codons 248 and 249 in the animal models the presence of HBsAg and the mutational hotspot at codon discussed here are analogous to human codons 248 and 249, 249 of p53 (6, 74,75).Twelve percent of the 2019 HCC cases and they are believed to be within the DNA binding regions examined for the mutational hotspot at codon 249 were of the animals' proteins, which is also the case with the human examined for the presence of HBsAg.For the samples taken protein.The GC→TA hotspot mutation at the codon analogous from regions of high aflatoxin exposure, 47% (82 out of 175 to the human codon 249 in the p53 genes of other species has total) of HBV-positive HCC samples examined had a GC→TA not been identified.To date, all attempts to recreate this hotspot transversion 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 of regions of moderate aflatoxin exposure, only 35% (15 out of different species (Table II).A different sequence context may 43 total) of the HBsAg-positive samples had the GC→TA alter the reactivity of AFB 1 toward the analogous codon 249 transversion.None of the 11 HBsAg-negative samples in these sequence.Table II illustrates, from a compilation of data, that regions exhibited the mutation.Other work (73) has shown there is no mutation analogous to the human p53 codon 249 that in areas of low aflatoxin exposure, HCC samples from mutation (Arg→Ser) in primates, rats, mice, ducks, woodpeople who are positive for the HBsAg also have p53 mutations, chucks, tree shrews or ground squirrels when these animals although they have not yet determined what these mutations are exposed to aflatoxin.A possible reason that no analogous are.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 codon mutants to modulate in trans the transcriptional activity of results in a silent mutation.Additionally, some of the species wild-type p53 (p53 acts functionally as a tetramer) on a tested do not have a species-specific form of hepatitis B. The reporter construct in HCC cell lines (HEP-3B) possessing an HBV status of several species studied is also shown in Table integrated HBx gene (105).It has not been confirmed, however, II.If HBV and AFB 1 are both needed for the selection of a that 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 contract R248W mutant enhanced the level of transcription to 132% HBV-mediated disease and to have a similar p53 codon of wild type, while the R249S mutant drastically reduced the sequence in the DNA binding region.Similar to the woodchuck level of transcription to 18% of wild type.Control experiments and ground squirrel models noted above, ducks contract a were carried out in tumor cell lines from lung, prostate and form of hepatitis B, duck hepadnavirus B (DHVB), but the mammary 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 activity type), and a milder effect in mesothelial (52% of wild type), (106).In the case of primates and rats, even though the mammary (75% of wild type) and prostate (80% of wild type) mutation at codon 249 would give rise to an amino acid tissues.When any of the mutants are expressed in a cell change of Arg→Ser, studies were carried out in the absence line that does not contain wild-type p53, no transcriptional of HBV (107,108).Currently, rats transgenic for HBV are activation of the reporter construct is seen.Thus, other mutant being developed (109).Mice naturally have an Arg→Leu sequences 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 codon described above.
246, which is analogous to the human Arg→Ser mutation at or HBV status affect p53 are still not completely clear.The increasingly powerful tools used in biochemistry and genetics codon 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 carcinogen were simultaneously exposed to AFB 1 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 AFB 1 (from 0 to 14% when homozygous for wild-type p53; from 14 to 71% when example, mice have different proportions of metabolizing and  carcinogenesis, but the details of how aflatoxin exposure and/

Fig. 2 .
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., AFB 1 -N7-Gua, an AP site or AFB 1 -FAPY) is annealed to the genome.The 5Ј and 3Ј 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.

Fig. 3 .
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 DNA on the 5Ј 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.The major product (shown) of this reaction is AFB 1 -N7-Gua.(B) The NMR structure of an oligonucleotide containing AFB1-N7-Gua is shown.To the right in this panel is the sequence context in which mutagenesis studies were done (see text).Note the position of the intercalated AFB1 residue.(C) The major adduct 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 5Ј to the site of covalent bonding (which is a cytosine).This panel provides a rationalization for the second site mutation.During replication, it is proposed that the aflatoxin residue drops into the position of the 5Ј cytosine, everting the cytosine from the helix.The restructured complex is shown with the original positions of the bases 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.

Table I .
Species-specific sequence differences among ras codons: correlation of predicted reactivity with reactive forms of AFB 1 RR of the first G in the codon.b RR of the second G in the codon. a

Table II .
Expected amino acid changes arising from GC→TA mutations at p53 codons 248 and 249 in different species a Actual mutations observed only in humans.Mutations shown in other species are proposed AFB 1 -induced GC→TA transversions.b Studies 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 did not involve aflatoxin.c Codons noted are function equivalent of p53 codons 248 and 249 in humans d The first base of codon 250 in each species above is a C. e Sequence context RR: CGG ϭ 0.8, CGC ϭ 0.3, GGA ϭ 0.3, CGA ϭ 0.3, CGT ϭ 0.6, GGC ϭ 0.3. a (111)(112)(113)(114)reen,C.L., Croy,R.G., Fowler,K.W., Buchi,G.H. and protective enzymes, so most of the aflatoxin they ingest is Wogan,G.N. (1983) Interactions of aflatoxin B1 and alkylating agents inactivated and excreted before it can form DNA adducts with DNA: structural and functional studies.Cold Spring Harbor Symp.(111)(112)(113)(114).On the other hand, woodchucks show an increase Quant.Biol., 47, 327-337. in conversion of AFB 1 to the epoxide when they are exposed 4. Hsu,I.C., Metcalf,R.A., Sun,T., Welsh,J.A., Wang,N.J. and Harris,C.C. Nature, 350, 429-431.6. Shen,H.-M.and Ong,C.-N.(1996) Mutations of the p53 tumor suppressor with HBV and exposed to AFB 1 .Of four tumors from animals gene and ras oncogenes in aflatoxin hepatocarcinogenesis. Mutat.Res., that were positive for both AFB 1 and HBV exposure, no codon the same synergism that humans do when they are infected