Liver diseases associated with chronic hepatitis B virus (HBV) infection, including hepatocellular carcinoma, account for more than 1 million deaths annually worldwide. In addition to HBV infection, other risk factors are involved in the etiology of hepatocellular carcinoma and, among these, dietary exposure to the carcinogenic aflatoxins is of particular importance in certain regions of southeast Asia and sub- Saharan Africa. The relative contributions of these two risk factors and the mechanism of the interaction between them in the pathogenesis of hepatocellular carcinoma are still poorly understood. The recently developed individual biochemical and molecular markers of aflatoxin exposure, i.e., aflatoxin-albumin adducts in blood and a specific GC to TA transversion mutation in codon 249 of the p53 gene (249 ser p53 mutation) in hepatocellular carcinomas, permit a better quantitative estimation of aflatoxin exposure in different populations of the world. A comprehensive summary of the data from our laboratory and the literature, based on a large number (>1000) of individual cases of hepatocellular carcinoma, is presented here and shows the following: 1) A high level and high prevalence of exposure to aflatoxins occur in West Africa, Mozambique, and some regions of China; 2) a high prevalence of the 249 ser p53 mutation is detected in these countries; and 3) hepatocellular carcinomas from countries with low or no exposure to aflatoxins show a very low prevalence of the 249 ser p53 mutation and distinctly different p53 mutation spectra, probably indicating different etiologies. Experimental and epidemiologic studies demonstrate an interaction between HBV infection and aflatoxins in hepatocarcinogenesis. The relevance of the biochemical/ molecular markers of aflatoxin exposure, HBV vaccination, and the reduction of aflatoxin exposure, in addition to the interaction between HBV infection and other risk factors in liver carcinogenesis, are discussed with regard to the implementation of measures for primary prevention.
Vital registration data indicate that adult mortality is higher in developing countries than in industrialized countries and is an important target for the implementation of public health intervention measures (1). It has been estimated that, of a total of 50 million deaths in the world in 1990, 10 million occurred in individuals in the adult age range. Liver diseases associated with chronic hepatitis B virus (HBV) infection, including hepatocelVital registration data indicate that adult mortality is higher in developing countries than in industrialized countries and is an important target for the implementation of public health intervention measures ( 1 ) . It has been estimated that, of a total of 50 million deaths in the world in 1990, 10 million occurred in individuals in the adult age range. Liver diseases associated with chronic hepatitis B virus (HBV) infection, including hepatocellular carcinoma, account for more than 1 million deaths annually ( 2 ) . In addition to HBV infection, other risk factors are involved in the etiology of hepatocellular carcinoma ( 3 ) , and, among these, dietary exposure to the carcinogenic aflatoxins is of particular importance in certain regions of southeast Asia and sub- Saharan Africa ( 4 ) .
Aflatoxins are produced by Aspergillus parasiticus (aflatoxins B 1 , B 2 , G 1 , and G 2 ) and Aspergillus flavus (aflatoxins B 1 and B 2 ) and occur, therefore, as mixtures in foods; aflatoxin B 1 is the major component of these mixtures. Aflatoxin M 1 , a hydroxylated metabolite of aflatoxin B 1 , is also found in milk. Aflatoxin B 1 and, to a lesser degree, aflatoxins G 1 and M1 are the most biologically active aflatoxins by virtue of their activation to a reactive epoxide form, a reaction that does not occur with aflatoxins B 2 and G 2 ( 4 ) . Because aflatoxins occur naturally as mixtures in the diet, we generally refer to them collectively in this review unless studies have used a specific compound, in which case the compound is identified.
The relative contributions and the mechanism of interaction between aflatoxins and HBV infection in the etiopathogenesis of hepatocellular carcinoma are still poorly understood. Recently, individual biochemical and molecular markers of aflatoxin exposure, namely, DNA adducts and protein adducts and aflatoxin metabolites in body fluids ( 5 , 6 ) , have been developed, and an AGG to AGT mutation affecting the third nucleotide of codon 249 in the p53 gene (249 ser p53 mutation) in hepatocellular carcinoma ( 7 , 8 ) has been specifically associated with exposure to aflatoxins. These findings, together with the previously available serologic markers of HBV infection, have provided valuable tools in molecular epidemiologic studies designed to address this issue. Our review describes the existing data on these markers and discusses their contribution both to understanding the nature of the interaction between HBV infection and aflatoxin exposure in the molecular pathogenesis of hepatocellular carcinoma and to implementing primary prevention measures to reduce mortality from this cancer.
Aflatoxin Exposure Worldwide
Measurement of aflatoxin exposure on the basis of food analysis is problematic, both because a variety of dietary items can be the sources of aflatoxins and because of difficulties in determining how representative the sampling of the dietary components is at the population level. Furthermore, estimation of human exposure also requires a knowledge of the patterns of consumption of contaminated foods within a given region or population and of seasonal and annual fluctuations in aflatoxin levels. Because of these difficulties, the majority of countries do not have comprehensive data on population exposures to aflatoxins; this is particularly true of those populations with the highest exposures. Nevertheless, epidemiologic studies of specific populations have combined measurements of aflatoxins in foods with food intake estimates to provide calculated population exposures ( 4 ) . These calculated exposures generally range from 10 to 200 ng/kg body weight per day in sub-Saharan Africa and southeast Asia to less than 3 ng/kg body weight per day in the United States.
An alternative basis for estimating dietary exposures is to use individual biomarkers of exposure. These biomarkers provide a more objective and representative measure of aflatoxin exposure, circumventing as they do the above-named difficulties in developing reliable estimates from dietary assessment ( 9 , 10 ) . The biomarker field is particularly advanced for the assessment of aflatoxin exposure, with measurement of covalent binding of aflatoxin to albumin and measurement of urinary aflatoxin metabolites having proved valuable in numerous field studies ( 5 ) and having been shown to be predictive of hepatocellular carcinoma risk in prospective studies ( 11 , 12 ) . The aflatoxin-albumin measurements provide information on individual exposure over the previous 2-3 months, based on the half-life of albumin. Experimental data have also shown that this biomarker reflects the formation of the reactive aflatoxin B 1 metabolite and the level of DNA damage occurring in the livers of rats treated with aflatoxin B 1 ( 13 ) .
Data from a number of different populations, summarized in Fig. 1 , demonstrate that, in some populations in southern China and sub-Saharan Africa, all individuals are exposed to aflatoxins at high levels beginning in the perinatal period and continuing throughout the lifespan of the individual. These biomarker data in individuals have been effective in drawing attention to the sheer magnitude of the exposure to these carcinogens in these parts of the world. Seasonal variations in exposure have also been observed, with higher levels found several months after harvest because of the consumption of stored crops ( 14 ) . The data on individual exposures are broadly in line with the estimated levels of exposure based on food analyses in different parts of the world.
249 ser p53 Mutations in Hepatocellular Carcinoma
Mutations in the p53 tumor suppressor gene are present in many human cancers ( 15 ) , and it has been proposed that some of these mutations are fingerprints of past exposure to a given carcinogen ( 16 – 18 ) . Hsu et al. ( 8 ) and Bressac et al. ( 7 ) have shown that mutation of the third nucleotide in codon 249 in hepatocellular carcinoma is specifically associated with exposure to aflatoxin, and, since then, a considerable amount of data has accumulated in the literature on the occurrence of such p53 mutations in hepatocellular carcinomas in many regions of the world. Experimental studies in bacteria, in cultured cells, and in animals ( see below ) are also supportive of this association.
A total of 316 p53 mutations in various codons have been detected in over 1000 hepatocellular carcinomas, with the prevalence ranging from 25%-32% in Europe, Japan, and Taiwan to 45% in sub-Saharan Africa and certain regions of China. A high frequency of GC to TA transversions has been detected ( seeTable 1 and Fig. 2 ). It is extraordinary that more than 50% of the GC to TA transversions occur at one base pair, the third nucleotide of codon 249, resulting in the amino acid substitution of serine for arginine. The prevalence of the aflatoxin-specific p53 mutation, 249 ser , varies greatly in hepatocellular carcinomas from different populations of the world, and Table 1 shows the relationship of this mutation to the incidences of hepatocellular carcinoma and the levels of exposure to aflatoxin. This particular mutation occurred in 48 cases of a total of 92 hepatocellular carcinomas examined from Qidong (China), Mozambique, and Senegal, whereas 28 of 291 hepatocellular carcinomas with this mutation have been observed in Thailand, Mexico, Shanghai, and Taiwan. In contrast, only one 249 ser p53 mutation was detected in 168 cases of hepatocellular carcinoma from Europe, the United States, or Australia. It is informative to correlate aflatoxin exposure, as determined by biomarker measurements ( Fig. 1 ), with the prevalence of this specific mutation in the p53 gene. Populations in southern China (Guanxi, Qidong, and Haimen), West Africa (The Gambia, Senegal, and Guinea-Conakry), and East Africa (Kenya) have the highest recorded levels of exposure, as determined by aflatoxin-albumin adduct measurements, and, as noted above, these countries are among those with the highest prevalence of 249 ser p53 mutations. The only country with a high prevalence of 249 ser p53 mutations and for which individual biomarker data are unavailable is Mozambique. However, it is known that food levels of aflatoxins are particularly high in this country (38-184 ng aflatoxin B 1 /kg body weight per day) ( 4 ) . In what can be classified as regions of intermediate exposure on the basis of biomarker data (Thailand, Taiwan, and Shanghai), mutation data reveal an intermediate prevalence of 249 ser p53 mutations. Dietary analysis for aflatoxins in Mexico suggests a relatively high level of exposure because of the frequent consumption of corn (10-35 g corn/day), and this assessment is supported by a limited analysis of sera from 16 patients with hepatocellular carcinoma. All of the sera were positive for aflatoxin-albumin adducts at high levels ( 19 ) . In Taiwan, qualitative data on aflatoxin-albumin adducts, obtained by means of an enzyme-linked immunosorbent assay of similar sensitivity to the one that was used to generate the data shown in Fig. 1 , revealed a prevalence of 34% of sera positive for this marker ( 20 ) . Exposure levels to aflatoxin B 1 are low in Europe, the United States, and Egypt and correlate with the low prevalence of 249 ser p53 mutations in hepatocellular carcinomas from these countries.
When these data are all considered, a clear relationship is observed between the prevalence of 249 ser p53 mutations, exposure to aflatoxins, and the incidence of hepatocellular carcinoma ( Table 1 ). It should be noted that this analysis is essentially an ecological correlation, and there is a possibility of confounding caused by concomitant exposures to other environmental factors. A correlation at the individual level between aflatoxin exposure and the presence of the 249 ser p53 mutation in hepatocellular carcinoma would provide a better opportunity to control for such confounding and would provide more direct evidence of a causal association. Attempts have been made to establish such a correlation by measuring aflatoxin B 1 -DNA adducts in the liver tissue of patients with hepatocellular carcinoma ( 21 , 22 ) . However, the limitations of such an approach include the possible influence of the disease state on adduct levels (by alteration of diet or metabolism) and the fact that recent exposure, as measured by the biomarker, may not reflect past exposure. A prospective study design would be more appropriate to examine this question.
The great majority of the hepatocellular carcinoma case patients in the above-mentioned studies were positive for hepatitis B virus surface antigen (HBsAg), a marker of chronic HBV infection. While relatively few patients with hepatocellular carcinoma were HBsAg negative, a higher frequency of 249 ser p53 mutations did seem to occur in HBsAg-positive patients ( 23 ) . However, as Eaton and Gallagher ( 23 ) have noted, this analysis should be interpreted with caution because of the difficulty in clearly defining the HBV status of patients with hepatocellular carcinoma on the basis of published data. Nevertheless the tendency to higher levels of 249 ser p53 mutations in HBsAgpositive individuals is suggestive of a synergistic interaction between HBV infection and aflatoxin exposure in the induction of hepatocellular carcinoma. This synergy is in turn consistent with epidemiologic data ( 11 , 12 ) and experimental studies in the woodchuck ( 24 ) and in HBV-transgenic mice ( 25 ) that show an increased incidence or an earlier onset of tumors in cases of combined exposure. However, it is also possible that the association between the 249 ser p53 mutation and HBsAg positivity is a fortuitous one, in that areas of high aflatoxin exposure are those with a high prevalence of HBV infection.
In Alaska and Japan, areas with a low exposure to aflatoxins and a low prevalence of 249 ser p53 mutations (in two of a total of 496 HCC cases of hepatocellular carcinoma; seeTable 1 ), the incidence of hepatocellular carcinoma is, nevertheless, relatively high. This high incidence rate is likely to be associated with other risk factors, namely, a high prevalence of hepatitis C virus (HCV) infection and/or interaction between HBV/HCV infection and alcohol consumption ( 26 , 27 ) .
p53 Mutation Spectra in Hepatocellular Carcinoma
As already mentioned, in regions of high exposure to aflatoxins, such as Qidong-Guanxi (China) and Mozambique, a high proportion of the p53 mutations detected in heptocellular carcinomas consisted of GC to TA transversions (43 of 46), and 40 of these were specifically 249 ser mutations. It is, however, of interest to note (Fig. 2 ) that, in addition to these 249 ser mutations, significant differences in the overall p53 mutation spectra are also observed among hepatocellular carcinomas from various regions of the world. In hepatocellular carcinomas from Europe and the United States, a low prevalence (<10%) of GC to TA tranversions and a 20%-30% prevalence of GC to AT transitions are observed together with an increased level of mutations involving AT base pairs. In Japan, a higher prevalence of AT base pair mutations and an increase in overall GC to TA tranversions are seen. It is also interesting that, in hepatocellular carcinomas from Taiwan, a particularly high prevalence of AT to TA tranversions is found. This p53 mutation profile indicates that risk factors other than aflatoxin exposure, such as DNA damage attributable to oxidative free radicals, lipid peroxidation, and alcohol intake ( 28 , 29 ) , are involved in generating the mutations detected in different regions of the world.
The presence of the 249 ser p53 mutation has been examined in hepatocellular carcinomas induced by aflatoxin B 1 in various experimental systems. Nonhuman primates have a DNA sequence at codons 247-250 that is identical to that found in humans, but the data with respect to 249 ser p53 mutations following aflatoxin B 1 exposure are uninformative because of the small number of cases examined. In rhesus and cynomolgus monkeys, no 249 ser p53 mutations were detected in a total of four cases of hepatocellular carcinoma, two of which originated in a single animal; other tumors reported as induced by aflatoxin B 1 in this study (namely, two cholangiocarcinomas, a spindle cell carcinoma of the bile duct, a hemangioendothelial sarcoma of the liver, and an osteogenic sarcoma of the tibia) were also analyzed with negative results ( 30 ) . The prevalence of 249 ser p53 mutations in human hepatocellular carcinomas reaches a maximum of 50%-60% ( seeTable 1 ); thus, the number of nonhuman primate hepatocellular carcinomas analyzed is too small to ensure the reliability of these negative findings. The ground squirrel, the woodchuck, the duck, and the rat have a different p53 DNA sequence at codons 247-250 than humans, and no 249 ser p53 mutations were detected in hepatocellular carcinomas (or liver nodules, in the case of the rat) from these animals following exposure to aflatoxin B 1 ( 31 – 35 ) . In the rat and the woodchuck, a G:C to T:A transversion at the equivalent of the third nucleotide in codon 249 would, in any case, result in a silent mutation. In summary, because of the small number of hepatocellular carcinomas examined in monkeys and because of the limitations of the other experimental systems used, the experimental data on 249 ser p53 mutations in hepatocellular carcinomas induced by aflatoxin B 1 do not contradict the observations in human hepatocellular carcinomas.
An additional issue that requires consideration in crossspecies comparisons is the role of p53 in carcinogenesis and, particularly, the functional importance of the 249 ser p53 mutation in hepatocytes. In rats treated with carcinogens, including aflatoxin B 1 , there has generally been an absence of p53 mutations in liver tumors, or contradictory findings have been reported. In murine hepatocellular carcinomas, no p53 mutations have been detected ( 36 , 37 ) . A high prevalence of p53 mutations was, however, observed in hepatocellular carcinomas induced in rats treated with tamoxifen and 2-acetylaminofluorene ( 38 , 39 ) . The 249 ser p53 mutation may also have a specific functional effect on, for example, mitotic activity in human hepatocytes that is not found in hepatocytes from other species ( 40 ) . Although these findings indicate that the molecular pathogenesis of liver cancer in rodents and humans may proceed through different pathways, further genetic analysis with appropriate techniques is required to substantiate the conclusion of such a difference.
The nature of the mutations induced by aflatoxin B 1 has been examined in several systems, including human cells. These data show that the predominant mutations are G:C to T:A transversions ( 41 – 45 ) , that the aflatoxin B 1 -N7-G adduct is the primary DNA adduct responsible for this mutation ( 46 ) , and that the nucleotide sequence influences the target for aflatoxin B 1 binding ( 44 , 47 , 48 ) . There is also evidence that GC to TA transversions are targeted to codon 249 of p53, in comparison with surrounding codons, both in plasmid DNA treated with aflatoxin B 1 in vitro ( 49 ) and in human hepatocytes treated with aflatoxin B 1 in culture ( 50 ) . In addition, the complex balance of cytochrome P450-, epoxide hydrolase-, and glutathione Stransferase- mediated metabolism of aflatoxin B 1 and DNA repair processes is an important determinant in the formation of DNA damage and mutations caused by this carcinogen ( 51 ) . The expression of the enzymes in these metabolic and repair pathways is critical in determining cross-species differences in the formation of aflatoxin-DNA and aflatoxin-protein adducts as well as in the susceptibility to aflatoxin-induced carcinogenesis ( 52 ) . The frequency of different polymorphisms in some of the aflatoxin-metabolizing enzymes also varies within the human population (e.g., between ethnic groups), and the impact of these variations on the susceptibility to aflatoxins at the individual and population level is still poorly documented ( 53 ) .
Mutations in codon 249 of p53 are not exclusive to hepatocellular carcinoma, and such mutations account for about 5% of all p53 mutations in human cancers, as reported in the International Agency for Research on Cancer p53 mutation database ( 15 ) . It is of interest to note that the two carcinogens, benzo[ a ]- pyrene and aflatoxin B 1 , produce within this codon different substitutions for arginine. Benzo[ a ]pyrene produces a substitution of methionine for arginine (AGG to ATG) ( 54 ) , whereas aflatoxin B 1 is associated with an arginine-to-serine substitution. This difference probably reflects differential binding of these carcinogens to a given DNA sequence and differential repair of these DNA adducts.
Arginine 249 plays an essential structural role in the folding of the DNA-binding domain of p53 ( 55 ) . In contrast with Arg 248 and Arg 273, the most commonly mutated arginines in human cancers, Arg 249 does not make direct contact with specific DNA bases, but it belongs to the highly critical L3 loop and is adjacent to Arg 248, which contacts DNA in the minor groove. Arg 249 makes contacts with four other amino acid residues distributed in different secondary structural motifs, i.e., His 162 and Glu 171 (both in L2 loop) and Met 246 and Gly 245 (both in L3 loop) ( 55 ) . Analysis of the tertiary structure of p53 indicates that these contacts contribute to the stabilization of the L3 loop and to its correct orientation with respect to the DNAbinding surface. Mutation of this residue is expected to have major structural consequences and to destabilize the domain of p53 that makes contacts with the minor groove of DNA [Hainaut P: unpublished results; ( 56 ) ]. Experimentally, the mutant Ser 249 polypeptide has been shown to have a significant dominantnegative effect on wild-type p53 in several cell types, including one hepatocellular carcinoma cell line ( 40 ) . These structural and functional implications of mutations at codon 249 may contribute to the explanation of why these mutations are specifically selected in hepatocellular carcinoma and why they are retained during cancer development.
The data summarized here clearly indicate the strong association between aflatoxin exposure and the occurrence of the 249 ser p53 mutation in hepatocellular carcinoma originating in certain areas of China and sub-Saharan Africa. This association is supported by the use of biomarker data on aflatoxin exposure, which provide a more reliable and a more precise picture than the dietary data used previously. It is, however, apparent that genetic alterations in genes other than the p53 tumor suppressor gene, as well as altered expression of various cellular proteins, also occur in the natural history of hepatocellular carcinoma ( 36 , 57 – 60 ) .
The types of oncogenes or tumor suppressor genes involved and the temporal occurrence of alterations (early versus late) in these genes vary greatly among different types of tumors ( 61 ) . Inactivation of the p53 gene seems to occur late in the development of colon cancers ( 62 ) and early in glioblastomas ( 63 ) and esophageal cancers ( 64 ) . It is reasonable to assume that the temporal occurrence of these genetic, as well as epigenetic, events may differ in hepatocellular carcinomas with different etiologies. In the case of hepatocellular carcinomas in regions of high exposure to aflatoxins, there is sound evidence that the 249 ser p53 mutation is an early event, as shown by the presence of this mutation in normal hepatic tissue from patients with hepatocellular carcinoma in regions with high exposure to aflatoxin B 1 ( 65 ) . This observation is consistent with data from The Gambia ( 66 ) showing that exposure to aflatoxin B 1 occurs even during the perinatal period and continues for the entire lifespan in the great majority of the population. The exposure to this carcinogen and the concomitant high proportion of HBV chronic carriers are probably associated with the early occurrence (already at the age of 12-15 years) and the peak incidence in young adults (aged 35-45 years) of hepatocellular carcinoma reported in The Gambia, Guinea-Conakry, Zimbabwe, and Qidong and Guanxi (China) ( 11 , 67 – 70 ) . Experimental studies in HBV transgenic mice have shown that transplacental aflatoxin B 1 exposure is associated with the occurrence of alterations in minisatellite DNA sequences detected in hepatocellular carcinomas developing in adult mice ( 71 ) . In addition, the possibility that HBV infection alters aflatoxin metabolism has been suggested from studies in HBV transgenic mice ( 72 , 73 ) . The mechanism by which HBV infection is responsible for the induction of hepatocellular carcinoma is still unclear. The development of hepatocellular carcinoma as a consequence of chronic HBV infection has been associated in only rare cases with insertional mutagenesis ( 74 ) . Some evidence indicates that the HBV X protein may bind to and inactivate the p53 protein ( 75 – 79 ) . Another mechanism, and at present the most plausible, is that recurrent liver cell proliferation occurring among HBV chronic carriers is the critical rate-limiting factor favoring the selective clonal development of cells mutated by aflatoxin B 1 in certain geographic regions or mutated by other exogenous or endogenous sources of cellular DNA damage in other regions ( 18 , 80 ) . Further evidence supporting the role of cell proliferation is provided by the fact that HCV infection results in chronic hepatitis and cirrhosis with an increased rate of cell death and cell division and the fact that HCV infection is associated, as is the case with HBV, with a high risk of hepatocellular carcinoma ( 26 ) . While HBV is a DNA virus, HCV is an RNA virus, and there is no evidence of integration of HCV sequences into cellular DNA or for an HCVencoded transactivating protein.
The availability of biologic markers of exposure to aflatoxin and to HBV infection and an understanding of their role in the pathogenesis of hepatocellular carcinoma have a direct bearing on the assessment of the relative importance of the different causative agents and, consequently, on the implementation of measures for primary prevention. A program of vaccination against HBV in The Gambia was progressively introduced from 1986 as part of the World Health Organization's Expanded Programme of Immunisation, and the results to date show a high degree of protection against primary HBV infection and the chronic carrier state ( 68 , 81 , 82 ) . The next decade will provide evidence of the degree of protection against the development of hepatocellular carcinoma. The study in The Gambia has also shown the high cost-effectiveness of HBV vaccination in preventing deaths from chronic liver diseases or liver cancer ( 83 ) . Thus, economic considerations should not be an impediment to integrating HBV vaccination into national programs of immunization in regions of high prevalence of HBV carriers, as recommended by the World Health Organization ( 2 ) .
It remains to be clarified what proportion of hepatocellular carcinomas is attributable solely to aflatoxin exposure and how much can be attributed to an interaction with HBV infection. If the latter is the major contributor, HBV vaccination may be sufficient to reduce the incidence of hepatocellular carcinoma associated with aflatoxin exposure. At the same time, there are some 300–400 million HBV chronic carriers currently present in the world who will continue to be at risk of hepatocellular carcinoma, and high proportions of these are exposed to aflatoxins. In addition, aflatoxins may be relevant to diseases other than cancer ( 9 , 84 ) . Thus, in regions such as West Africa and in some regions of China with high exposures to aflatoxins, implementing measures to reduce exposure to this class of potent carcinogens must be a priority.
Intervention measures against aflatoxins can be targeted at the individual or the community level. At the individual level, in societies where the dietary staple is contaminated with aflatoxins, avoidance of the exposure by dietary change is unrealistic. An alternative strategy would be chemoprevention, using compounds that limit the deleterious consequences of aflatoxins once ingested. This approach has been evaluated in experimental models with the anti-schistosomal drug oltipraz, which modulates aflatoxin detoxification ( 85 ) , and a clinical trial has been conducted in the People's Republic of China using this compound ( 86 ) . The realistic possibility of applying this chemopreventive approach at the population level is still doubtful. More promising as a long-term strategy for intervention at the community level in developing countries are attempts to limit the formation of aflatoxins before or after harvest. These attempts include sorting methods, biocontrol methods, and chemical and physical methods for the destruction and degradation of aflatoxaflatoxins ( 87 ) . The various attempts in this direction have not been successful for foods for human consumption because of the chemical stability of the aflatoxin molecule under normal cooking conditions and the consequence that decontamination or detoxification procedures render the food unsuitable for human consumption. Nevertheless, there are data that indicate that simple, low technology pre- and post-harvest improvements in agricultural and food storage practices might result in lower levels of aflatoxin contamination in human foods ( 88 ) . A further promising alternative in the long term is the development of transgenic plants that are resistant to infestation by Aspergillus or are resistant to aflatoxin biosynthesis ( 89 ) . Validated markers of human exposure to aflatoxins, such as aflatoxin-albumin adducts and the 249 ser p53 mutation, would be valuable surrogate end points in trials conducted to test the efficiency of the above intervention procedures.