Hepatocellular carcinoma: Putative interactive mechanism between aflatoxins and hepatitis viral infections implicating oxidative stress during the onset and progression of cancer

Graphical Abstract

The association of hepatocellular carcinoma (HCC) with hepatitis B virus (HBV), hepatitis C virus (HCV) and mycotoxins, especially aflatoxins, has been established. Mycotoxins are commonly encountered by the consumption of mycotoxin-contaminated food by African and Asian populations. A number of mechanisms contribute to the high risk of HCC in individuals with both aflatoxin B1 (AFB1)-DNA adducts and hepatitis B surface antigen (HBsAg), and a viral-chemical interaction has been confirmed. Among the various suggested mechanisms, oxidative stress exacerbates the co-exposure of aflatoxins and chronic hepatitis infections. This increases the rate of DNA unwinding, supercoiling and/or overstretching. It is hypothesized that these processes are promoted by reactive oxygen/nitrogen species (ROS/RNS) generated during HBV or HCV infections, which allow aflatoxin metabolites to intercalate between DNA strands with their hydroxyl radicals. The aflatoxin metabolites may attack the ribose in the DNA backbone where the bases reside. These complex reactions result in modification of the energetics of DNA transcription and replication as well as a concomitant mutation in the p53 tumor suppressor gene. The hypothesis described here may generate novel ideas, which could lead to further hypothesis-driven experiments aimed at improving strategies for the prevention and treatment of HCC.


Background
Hepatitis B Virus
Hepatitis B virus (HBV), a hepadnavirus (DNA virus), mostly affects populations living in developing countries in Africa and Asia, and causes an infectious inflammatory disease of the liver. The virus is transmitted via blood and body fluids. The cirrhosis induced by HBV may lead to hepatocellular carcinoma (HCC).

Viral particles composed of lipid and protein form the surface of the virion and are referred to as “hepatitis B surface antigen” (HBsAg). Soon after infection, viral DNA is found in the nucleus. Four known genes are encoded by the genome, called C, P, S and X. While the function of genes C (HBcAg), P (HBpAg) and S (HBsAg) are well known, that of gene X (HBxAg) is not fully understood (1). Hepatitis B virus gene X is believed to stimulate genes involved in cell growth and inactivation of growth-regulating molecules (2). It has been reported that HBV gene X promotes more hepatic tumors in aflatoxin B1 (AFB1)-treated mice than in wild-type mice (3). In areas of high aflatoxin exposure, 50% of HCC cases have been reported to bear a specific AGG to AGT point mutation in codon 249 of the p53 tumor suppressor gene (codon 249ser mutation)3. More research is needed to clarify the sensitisation of hepatocytes by viral infection to the carcinogenic effects of AFB1. This sensitisation is considered one of the mechanisms underlying the association of HCC with HBV/hepatitis C virus (HCV) infection and the consumption of AFB1-contaminated food.

Hepatitis C Virus
About 130-170 million people worldwide are infected with HCV, mostly in Central America, Africa and Asia. Like HBV, HCV is transmitted by transfusion and intravenous drug use. Patients infected with HCV can develop cirrhosis and HCC after many years (4).

A member of the Hepacivirus genus in the Flaviviridae family, HCV is a single-stranded, positive-sense RNA virus with seven genotypes. Unlike HBV, there is currently no vaccine against HCV. Vertical transmission during pregnancy and at delivery from an infected mother to child can also occur (5). Thus, screening blood products before transfusion or organ transplantation can significantly reduce the risk of infection.

Co-infection with HBV, HCV and HIV
The prevalence of cases of histologically-proven cirrhosis among patients with chronic HBV/HCV concurrent infection is higher than those with HCV infection alone (6). Due to severe clinical presentations observed during this concurrent infection and HBV superinfection in HCV chronic carriers, there is a need for HBV vaccination in all HBsAg-negative chronic hepatitis patients (7). In addition, a large number of cases of patients infected with HBV and/or HCV who are co-infected with HIV have been reported (8,9). Due to the high prevalence of multiple viral hepatitis infections (HBV/HCV, HBV/hepatitis D virus [HDV], HBV/HCV/HDV) in HIV-positive patients, management of these patients should take into account the mechanism of action of each drug. Liver enzymes should be monitored during antiretroviral treatment.

 

Figure 1

Figure 1 | Sequence of events implicating oxidative stress in the mechanisms of HCC development: induction of oxidative stress by viral infection, stimulation of DNA unwinding, intercalation of aflatoxin into DNA strands with its concomitant epoxide metabolites, inactivation/mutation of the p53 gene and progression of HCC.

Mycotoxins
Mycotoxins are secondary metabolites generated by microfungi, which can be used as antibiotics or growth promoters, but, unfortunately, they can also cause toxicity and disease in humans and animals. The extent of mycotoxin production depends on certain conditions that favour mould growth such as drought stress, storage in relative humidity and temperature of surroundings (10,11). The optimum temperature for the biosynthesis of most mycotoxins is within a more mesophilic range (20 to 30ºC).

The classification of mycotoxins is based on generic group (teratogens, mutagens, carcinogens) by cell biologists, on their chemical structure (lactones, coumarins) by organic chemists, on their biosynthetic origin (polypeptide, amino acid-derived) by biochemists and on the affected organ (hepatotoxins, nephrotoxins, immunotoxins) by clinicians. Low-dose exposure over a long period of time, inducing chronic toxicity, may result in cancer and other irreversible effects (12). Manifestations of acute effects (e.g., turkey syndrome, human ergotism, stachybotryotoxicosis) have been observed during some mycotoxin episodes. The extent of human exposure can be measured and monitored by the presence of residues, adducts and metabolites in tissues, fluids and excreta (13).

Mycotoxin contamination is distributed worldwide, but human exposure is more pronounced in regions where malnutrition is a problem and where poor methods of handling and storage of foodstuffs are common. These regions unfortunately lack adequate regulations to protect their populations.

Aflatoxins
Aflatoxins are difuranocoumarin derivatives produced by many strains of Aspergilus flavus and A. parasiticus contaminating mostly peanuts, maize and cereals but also figs and oilseeds (10,14). Based on their fluorescence under UV light (blue or green) and their chromatographic mobility on thin-layer chromatography, four major aflatoxins have been characterized: AFB1, AFB2, AFG1 and AFG2, with AFB1 being the most potent natural carcinogen (15).

Activation of Aflatoxin
The metabolic transformation of aflatoxins by cytochrome enzymes (CYP3A4 and 1A2) into the reactive 8,9-epoxide form in the liver renders them more toxic and carcinogenic for humans and animals (16). CYP3A4, the predominant cytochrome P450 enzyme, produces two main metabolites from AFB1: AFB1-8,9-exo-epoxide and AFB1-8,9-endo-epoxide. However, binding to DNA occurs only with the first metabolite, which forms the predominant 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-AFB1 (AFB1-N7-Gua) adduct (17). Other metabolites from AFB1, including AFQ1, AFM1 and AFP1, and from aflatoxins G1, B2 and G2 are also produced, but they are less mutagenic and carcinogenic than AFB1-N7-Gua. Aflatoxin intoxication, called aflatoxicosis, can be acute and may result in death, whereas chronic aflatoxicosis may result in cancer or immunosuppression (17). Extra-hepatic (placenta, small intestine) metabolization of AFB1 has also been reported (18).

Epidemiology of Hepatitis Viruses, Aflatoxin and HCC
Cancer due to ingestion of aflatoxin-contaminated food has been associated with the presence of HBV and HCV (19). Vaccination against HBV has been reported to be a more cost-effective strategy for preventing liver cancer than removing aflatoxins from the diet (20). Inactivation of the p53 tumor suppressor gene induced by free radicals constitutes an important factor in the development of HCC (21,22). Cohort studies in Asia and a large case-control study in Africa have shown an interactive association between chronic HBV infection and aflatoxin in relation to HCC risk (23). HBsAg carriers are more likely to develop HCC (24). The presence of both the codon 249ser mutation in the p53 gene and HBV infection was associated with an odds ratio of 399 (95% CI: 48.6-3270) (25). Non-hepatic (lung cancer) effects of aflatoxins have been also reported (26). It would be of interest to study the prevalence of HCC with/without HBV/HCV/HIV co-infections in individuals exposed to aflatoxin contamination. The detection of HBsAg in these subjects would be of value, especially when compared with subjects who have been vaccinated.

Reactive Oxygen (ROS) and Nitrogen Species (RNS)
Free radicals are generated from endogenous oxidants produced by mitochondria, cytochrome P450 metabolism, peroxisomes and inflammatory activation, and from exogenous oxidants produced by food, air pollutants, tobacco, smoke, exercise, ionizing radiation, infrared radiation and sun. While acting as inducers of cellular senescence and apoptosis, and functioning as anti-tumorigenic species, their role as secondary messenger in intracellular signalling cascades has been recognized (27,28).

Atoms comprising free radicals are mostly in the ground state, where every electron in the outermost shell has a complementary electron that spins in the opposite direction. A free radical is, indeed, any atom with at least one unpaired electron in the outermost shell, and is capable of independent existence (29). Any free radical involving oxygen or nitrogen can be referred to as ROS or RNS. Oxygen-centred radicals contain two unpaired electrons in the outer shell. When free radicals abstract an electron from a surrounding compound or molecule, a new free radical is formed in its place. In turn, the newly formed unstable radical then tends to return to its ground state by abstracting electrons with anti-parallel spins from cellular structures or molecules (30,31). The electron transport chain (ETC), found in the inner mitochondrial membrane, utilizes oxygen to generate energy in the form of adenosine triphosphate (ATP); oxygen acts as the terminal electron acceptor within the ETC.

Two to five percent of the total oxygen intake both at rest and during exercise has the ability to form the highly damaging superoxide radical (O2-) through electron escape. Free radicals of importance in living organisms include the hydroxyl radical (OH), superoxide anion (O2-), nitric oxide (NO), thiyl radical (RS) and peroxyl radical (ROO). Although peroxynitrite (ONOO), hypochlorous acid (HOCl), hydrogen peroxide (H2O2), singlet oxygen (¹O2-) and ozone (O3) are not free radicals, they can, however, lead to a free radical reaction. The most common ROS involved in iron overload include the superoxide anion, the hydroxyl radical, singlet oxygen and hydrogen peroxide (32). Hydroxyl radical has been reported to react with DNA components (purine bases, pyrimidine bases and deoxyribose backbone), inducing single- or double-stranded DNA breaks and modifications of DNA components (33). 8-hydroxy-2’-deoxyguanosine (8-OHdG), the main metabolite of DNA damage, can be detected in human urine.

Free radicals can play a dual role as both beneficial and deleterious species (34). They are used by leucocytes against bacterial infections (35). In contrast to this antibacterial effect, ROS/RNS can be harmful by inducing structural alterations in DNA, producing gross chromosomal alterations (36,37), affecting cytoplasmic and nuclear signal transduction pathways, especially the mitogen-activated-phosphokinase (MAPK) pathway, and modulating the activity of proteins and stress genes, all of which are associated with carcinogenesis (38,39).

To prevent or control all these effects, living systems have built a defence mechanism, including enzymatic antioxidants (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx]) and non-enzymatic antioxidants (vitamin C, vitamin E, thiol/sulphydryl [-SH] group, glutathione [GSH]). Maintaining the SH group of proteins in a reduced state protects cells from oxidation (40). Similarly, membrane lipids can be stabilized from free radical attack by using ß-carotene as an antioxidant, which suppresses singlet oxygen and scavenges peroxide radicals (41,42).

The role of free radicals as the primary species causing damage to DNA in the mechanism of carcinogenesis has been confirmed (35).

Oxidative Stress
Oxidative stress may result from a cellular redox imbalance between the production of ROS and RNS and the cellular antioxidant defence mechanisms. Deregulation of cytokines, mitochondrial electron transfer chain (ETC) and xanthine oxidase can, indeed, activate nicotinamide adenine dinucleotide (phosphate) reduced form (NAD(P)H) and nitrite oxide synthase (NOS) to overproduce, respectively, superoxide radical and peroxynitrite.

The redox state of the biological system should be kept within a narrow range under normal conditions. The intracellular “redox buffering” or “redox homeostasis” capacity is achieved primarily by GSH and thioredoxin (TRX) and secondarily by the activities of enzymatic and non-enzymatic antioxidants. Activities of GSH and TRX reductases maintain high ratios of reduced to oxidized GSH and TRX, counteracting the intracellular oxidative stress by reducing both hydrogen and lipid peroxides.

Fluctuations in the cell redox environment mediated by intracellular changes in the GSH concentration can be reflected in the cell cycle. Depletion of GSH, rendering the cellular environment more oxidizing, will trigger apoptosis. Accumulation of ROS and especially the reactive 8,9-epoxide form of AFB1 in cancer cells will then alter redox regulation of signalling cascades and exacerbate the oxidative stress conditions.

ROS may also initiate an increase in toxic biochemical reactions such as peroxidation of membrane lipids and extensive damage to proteins causing intracellular protein aggregation and precipitation (43).

The resulting changes in cellular environment play an important role in signal transduction, enzyme activation, DNA and RNA synthesis, DNA repair and differentiation, cell growth and proliferation, differentiation and apoptosis. The oxidative stress leads to uncontrolled cell divisions triggered by inactivation of the p53 gene, the “guardian of the genome” and “policeman of the oncogenes” (44).

Free radicals are implicated in the pathogenesis of several diseases, including diabetes mellitus (especially the complications of type II diabetes), atherosclerosis, aging, cancer and inflammatory diseases (45,46,47). It would therefore be of interest to understand how overproduction of the reactive 8,9-epoxide of AFB1, intercalating into DNA strand breaks induced by free radical assault generated from HBV/HCV, can exacerbate the oxidative stress, which will finally inactivate the p53 tumor suppressor gene in the onset and progression of HCC.

Introduction
A considerable number of studies have confirmed the association of HCC with hepatitis viral infection (HBV, HCV) and the consumption of mycotoxin-contaminated food, especially aflatoxins (AFB1) (23,48-50). Other contributing risk factors to HCC that have been reported include alcohol intake, cigarette smoking and familial tendency. The HBsAg carrier consecutive to the HBV infection is the most important indicator in many countries (3,24,51,52). The high risk of HCC in individuals with both AFB1-DNA adducts and HBsAg has indeed confirmed the viral-chemical interaction (53). Wild and Turner (54) described the mechanisms of aflatoxin activation and HCC induction due to mutation changes. However, the mechanism underlying the onset and progression of this type of cancer as a result of the association or the interaction between mycotoxin ingestion and HBV or HCV infections as etiological factors is not well understood. Nonetheless, substantial progress has been made to understand the mechanisms of this interaction and, more recently, to explain its multiplicative aspects (55). Five mechanisms have been suggested, including: (i) fixation of AFB1- induced mutations in the presence of liver regeneration and hyperplasia induced by chronic HBV infection (3,56-59); (ii) direct or indirect sensitisation of hepatocytes to the carcinogenic effects of AFB1 by HBV infection (56,60-63); (iii) predisposition of HBV-infected hepatocytes to aflatoxin-induced DNA damage (55,64); (iv) increase in susceptibility to chronic HBV infection in aflatoxin-exposed individuals (65); and (v) exacerbation of oxidative stress by co-exposure to aflatoxins and chronic hepatitis infection (66-72).

The hypothesis described here focuses on the implication of oxidative stress in the mechanisms of HCC development with the following sequence of events: induction of oxidative stress by viral infection; stimulation of DNA unwinding due to the assault of free radicals; intercalation of aflatoxin into DNA strands with its concomitant epoxide metabolites; and inactivation/mutation of the p53 gene and progression of HCC.

Relation of the Sequences of the Hypothesis
Regulation of the Cell Cycle
Regulation of the cell cycle involves two classes of regulatory molecules, cyclin and cyclin-dependent kinases (CDKs) (73). These complexes perform phosphorylation that activates or inactivates target proteins for participation in the cell cycle. Cyclins are synthesized at specific stages of the cell cycle, in response to various molecular signals (74). Upon receiving a pro-mitotic extracellular signal, G1cyclin-CDK complexes become active and ready to prepare the cell for the S phase of the cell cycle and promote the expression of transcription factors that stimulate the expression of S cyclins and enzymes required for DNA replication. At this stage, these complexes can degrade by ubiquitination molecules functioning as inhibitors at the S phase. Once ubiquitinated, these inhibitor molecules are prone to undergo a proteolytic degradation by the proteasomes. The rate of DNA unwinding induced by ROS/RNS generated from HBV/HCV infection and, more especially, by the 8,9-epoxide of AFB1 may modify the activity of these kinases as the replication process requires DNA to be unwound.

Checkpoints
Monitoring and regulation of the cell cycle are performed by cell cycle checkpoints (75). DNA damage is controlled at two main checkpoints (G1-S and G2-M), where p53 plays an important role. Inactivated and mutated p53 induced by DNA strand breaks will affect the function of these specific checkpoints (76). Failure of the checkpoints leads to the continuation of cell division despite the DNA damage; breakage of DNA strands results in genetic damage (77,78). Under these particular circumstances, apoptosis (programmed cell death) and/or the development of cancer may occur as a result of p53 inactivation by destruction of its sulphydryl group and checkpoint-activated cycle cell arrest.

Epigenetic Mechanisms Affecting the Cell Cycle
DNA Methylation, DNA Acetylation and Histone Modifications
Free radical attack of the DNA backbone, exacerbating the unwinding of DNA, induces DNA damage, which will affect epigenetic events such as DNA methylation, DNA acetylation and histone modifications. These modifications induced by free radicals (oxidative stress) may also affect the cell cycle, especially at the G1-S checkpoint, and play a role in the regulation of gene expression and transcriptional activation.

Epigenetic cross-talk, a result of interplay between DNA methylation and histone modifications during gene silencing, involves protection against genetic changes in response to environmental genotoxins and has implications for cancer therapy and prevention (79). This cross-talk may not take place or may be inadequately performed under oxidative stress where p53 protein accumulates in the nucleus and is activated as a transcription factor (80,81). This tumor suppressor protein is normally maintained at low levels in unstressed cells. On the other hand, there is an increased phosphorylation of p53 catalyzed by kinases in response to DNA damage (82,83). Kinases such as serine/threonine kinases and checkpoint kinase (chk) 1 and 2, which act downstream of ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia related), phosphorylate human p53 at Ser20 and possibly at other residues (84).

Epigenetic modifications include DNA hypomethylation, DNA hypermethylation, DNA acetylation and histone modifications. For instance, DNA hypomethylation of the cancer antigen genes (CAGE) and histone modifications (histone H3 lysine 9 tri-methylation, histone H4 lysine 20 methylation, histone H3 lysine 4 tri-methylation) have been associated with HCC (85).

In our hypothesis, HBV, HCV, aflatoxins and, especially, aflatoxin metabolites intercalating between strand breaks and increasing the rate of DNA unwinding can affect epigenetic states such as DNA methylation, histone acetylation and chromatin organization in the progression of HCC (86). Aberrant DNA methylation, increased genomic hypomethylation, inaccurate transmission of chromatin states, gene induction and silencing can occur under oxidative stress conditions as a result of these epigenetic modifications considered heritable marks (84).

While DNA methylation is involved in many steps of cancer promotion, DNA hypomethylation correlates with tumor progression and metastasis. These two events contribute to the development of environmental and occupational carcinogenesis (87).

Taken together, at the onset and during the progression of HCC, alterations in DNA methylation and histone modifications due to oxidative stress will affect the silencing of tumor suppressor genes and/or activation of proto-oncogenes.

In addition to direct inactivation of p53 by free radical assault, hypermethylation-induced silencing of this tumor suppressor gene occurring during cancer progression may aggravate its activation and degradation during methylation-mediated silencing of another gene, p14ARF (88,89). This hypermethylation occurs mainly in the GC-rich DNA regions called “CpG islands” (90).

Telomeres, Cell Cycle Arrest and Apoptosis
Telomeres are stretches of DNA at the ends of chromosomes, and are part of highly specialized nucleoprotein structures that maintain genomic stability by stabilizing and protecting the ends of linear chromosomes. However, due to the limitation of the replication system in synthesizing new DNA to the very end of linear chromosomes, replication results in progressive erosion of telomeric DNA (91,92). Under oxidative stress, their length may be shortened further, thus signalling more rapidly for cell death and/or cancer by activating telomerase, which prevents the shortening of telomeres. Telomeres bind to telomeric-repeat binding factor (TRF), and their stability is maintained by telomerase, which protects telomeres from the loss of sequences during DNA replication (93). Inhibition of these factors by oxidative stress leads to the activation of ATM kinase and may induce cell cycle arrest and apoptosis because they will be considered broken DNA ends under these circumstances. Various DNA damage responses, including histone H2AX and ChK2 phosphorylation, p53 accumulation and apoptosis, can occur (94,95). In particular, apoptosis will occur as a result of inhibition of telomeric-repeat binding factor 2 (TRF2). In some cells, shortening of telomeres, occurring at each cell division, is associated with cancer and may trigger cell death (96). One can then imagine that the increased rate of DNA unwinding consecutive to DNA assault by ROS/RNS may accelerate the shortening of telomeres during replication by telomerase activation. In addition, telomere attrition and hypoxia contribute to checkpoint activation and genomic instability (97).

In conclusion, epigenetic changes, including DNA breakage, checkpoint failure, aberrant DNA methylation and histone modifications, which increase the frequency of permanent genetic mutation will, indeed, lead to the development of cancer.

Damage to DNA, Proteins, Lipids and Other Macromolecules
DNA damage has been associated with oxidative stress, induced by some chemicals acting through free radicals after being metabolically activated. During chronic inflammatory hepatic disease or infection, activated macrophages and leucocytes release ROS/RNS, which damage DNA (98), despite a complex mechanism of histone modifications that have evolved to protect DNA from spontaneous breakages (99,100). It has been demonstrated that HCV expression, replication and infection can induce oxidative stress in vitro and also in vivo (101,102,103). The viral infection seems to be particularly associated with double-stranded DNA (dsDNA) damage pathways because of its core E1 and NS proteins that are reported to be potent inducers of ROS/RNS (104,105). Oxidized proteins and genotoxic products generated by lipid peroxidation of polyunsaturated fatty acids within cell membranes, as a result of free radical attack, may also be important mediators of DNA damage that contribute significantly to the induction of cancer (106,107).

Malondialdehyde (MDA), a by-product of lipid peroxidation, and/or M1G deoxyribose, resulting from the combination of MDA and deoxyribonucleoside together with 8-hydroxy-2’-deoxyguanosine (8-OHdG), are major DNA damage factors, which also provide free radicals that may be involved in cancer development (69,108).

All these reactive products are generated from the breakdown of DNA, proteins and lipids that may have detrimental effects on the cell.

DNA Unwinding
The role of oxidative stress has recently been demonstrated in the progression of liver fibrosis and cirrhosis, especially in the correlation between the downregulation of antioxidant protein and the later stages of liver fibrosis (102). The DNA unwinding process involves helicases, which yield the transient single-stranded DNA (ssDNA) intermediates required for replication, recombination and repair (109,110,111).

The rate of DNA unwinding has been used to study DNA repair after exposure of peripheral blood lymphocytes (PBL) to low doses of radiation, ultraviolet light and various carcinogenic agents known to induce strand breakages by reactive oxygen species such as hydroxyl radicals (112-116). The metabolites of DNA damage (8-OHdG) and lipid peroxidation (MDA, lipid peroxides) may increase the rate of DNA unwinding, supercoiling and/or overstretching of the DNA, leading to the induction of DNA strand breakages. These metabolites have the capacity to convert the chemical energy of nucleotide triphosphate (NTP) hydrolysis (a reaction with a negative ΔG) into mechanical force for motion and strand separation, together with helicases, which are considered motor enzymes. During their translocation along ssDNA, they encounter a range of nucleic acid sequences and, like helicases, promote the unwinding process of dsDNA or RNA strands (117,118). It has been reported that the unwinding of these nucleic acids is initiated by the HCV helicase that uses ATP hydrolysis (99,100). The T7 helicase, for example, works in association with a strong motor protein like DNA polymerase, and may have evolved to rely on the cooperative action of two motor proteins to unwind DNA.

Wang and colleagues (109) were the first to report the direct link between a step in the ATPase cycle and unwinding by RNA helicase, and defined stages for the elucidation of the mechanochemical coupling mechanism employed by RNA helicases.

Hepatitis C virus helicase NS3 (HCVNS3) is a complex protein containing helicase and protease domains. The helicase domain NS3h, which belongs to the superfamily II, can use two mechanisms (passive and active) to unwind DNA strands (110,111). Addition of ATP to a reaction containing activated complexes leads to multiple cycles of unwinding and pausing that can be triggered by successive attack by ROS/RNS generated from carcinogenic metabolites, thus promoting oncogenesis (119,120). The DNA unwinding process constitutes the first reaction of DNA to multiple attacks by ROS/RNS, thus allowing aflatoxin metabolites to intercalate between DNA strands.

Generation and Intercalation of AFB1-adducts into dsDNA
The diet of the majority of populations living in tropical and subtropical regions consists largely of maize and cassava flour. In addition to being deficient in essential amino acids and important vitamins and micronutrients, maize and cassava flour are prone to fungal infestation and thus to contamination by mycotoxins, which increases the risk of cancer development.

Mycotoxins such as AFB1 are activated in humans by cytochrome P450 microsomal enzymes into AFB1-exo-8,9-epoxide, which acts as a DNA intercalating agent to bind to cellular macromolecules (proteins, DNA) to form adducts (121,122,123). The binding to the N7 position of guanine yields a trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxy AFB1 (124). Reaction of this epoxide with duplex DNA has shown a sequence selectivity between two sequences: the d(ATCGAT)2 and the isomeric d(ATGCAT)2 with one or two equivalents of epoxide reacting metabolites (125). The mechanism of pre-covalent intercalation of the epoxide on the 5’ face of guanine puts the epoxide in close proximity and in the proper orientation to the N7 position of guanine, thus facilitating an SN2 reaction, which has been confirmed by recent nuclear magnetic resonance studies (61). The subsequent reactions of DNA can then induce G:C to T:A mutation of the p53 tumor suppressor gene. Their interaction with the HBV carrier has been demonstrated in animal models (62,63,64), as well as in humans, especially in subjects with both AFB1-DNA adducts and HBsAg with higher risk of HCC (19,23,51,61,126). It has been reported that both chronic HBsAg carrier status and liver adducts were significantly higher in patients than in control subjects (9) and the risk of HCC was greatest in subjects with both AFB1-DNA adducts and HBsAg, suggesting a viral interaction (154). The intercalation of AFB1-DNA adducts into DNA strands with subsequent damage to nitrogen bases or breakage in the phosphodiester DNA backbone can explain the high risk of HCC in individuals exposed to aflatoxin and especially in those individuals with HBsAg.

Inactivation/Mutation of the p53 Gene and Induction of G to T Transversion
The high prevalence of the 249ser p53 mutation observed in some African and Asian regions is due to high aflatoxin exposure. In general, free radicals including aflatoxin epoxide-reacting metabolite are known to oxidize the SH group of the p53 gene, leading to its inactivation and/or mutation. As a stress-responsive transcription factor, the p53 gene undergoes post-translational modifications (acetylation of lysines, phosphorylation of serine/threonine residues, oxidation and covalent modification of cysteines, nitration of tyrosine) (127). The extent of aflatoxin B1 exposure acting through its epoxides promotes the induction of G to T transversion in codon 249 of this protective tumor suppressor gene (127-131). It has been reported that this particular induction is associated with HBV infection in developing countries where the contamination of food by mycotoxins, especially aflatoxins, is a problem (21,22,55,132).

The progression to HCC may occur as a result of specific mutations in the p53 gene that cannot trigger cell death via apoptosis in the case of DNA repair failure. It has also been reported that binding of the HBV x protein (HBx) to the p53 gene completely blocks this gene in the cytoplasm, thus inhibiting its translocation into the nucleus 133,134. It remains to be established if this lack of translocation affects the pathway of p53-mediated transcriptional regulation and apoptosis. In addition, the mechanism implicating M1G deoxyribose, the major MDA-DNA adduct (135,136), in the induction of G to T transversions is not yet fully understood despite the increased frequency of mutation triggered by its activity (2,137). These mutations and cellular DNA damage induced by an increase in ROS/RNS accelerate the development of HCC (104). In view of these sequences of events, one link of the chain is missing in the implication of oxidative stress in the interaction between AFB1 and HBV and/or HCV in HCC development.

How can a large carcinogenic metabolite such as the aflatoxin-adduct be incorporated into the DNA and inactivate/mutate the p53 gene leading to the induction of HCC?

Hypothesis
The rate of DNA unwinding, supercoiling and/or overstretching is promoted by ROS/RNS generated during HBV or HCV infections, allowing aflatoxin metabolites to easily intercalate between DNA strands by their hydroxyl radicals; this results in either direct damage to nitrogen bases or breakage in the phosphodiester DNA backbone. The process involving free radicals may play an important role in the energetics of DNA transcription and replication (Fig 1).

In addition, the high rate of p53 gene mutation concomitantly induced by this process may inhibit not only the apoptosis genes and DNA repair, but also the activation of cellular checkpoints required to block cells, especially tumor cells, during one of various phases of the cell cycle, particularly at the G1-S checkpoint in response to DNA damage (133,138). This inhibition leads to replication of further mutated genomes and increased risk of tumor formation as a result of the accumulation of mutations.

Strengths of the Hypothesis
Failure of DNA Damage Repair Mechanism
Oxidative DNA damage occurring in normal tissue is due to some by-products of lipid peroxidation (MDA, M1G deoxyribose) and to ROS/RNS. The highly reactive oxygen species (ROS) and hydroxyl radical (•OH) react with DNA by addition to double bonds of DNA bases and by abstraction of a hydrogen atom from the methyl group of thymine and from each of the C-H bonds of 2’-deoxyribose. These reactive products interact with cellular biomolecules, inducing DNA strand breaks. During replication, an activation of a set of mechanisms takes place in the cell to repair the damaged site. The tumor suppressor gene p53 is activated as part of the normal DNA damage response to keep pre-cancerous lesions under control. AFB1-DNA adducts generated in individuals exposed to AFB1 are then repaired by the nucleoside excision repair (NER) pathway for the reduction of the mutation frequency. However, in NER-deficient bacteria, this mutation is, indeed, more frequently induced (137,139).

This particular deficiency also affects the rate of DNA unwinding, which increases with the incapacity of DNA polymerase to fill the gap created by the cleavage of DNA into bases. The interference of the HBx protein with the NER pathway together with the rapid cell turnover observed in chronic hepatitis contribute to the transcription of the CDK inhibitor p21waf1/cip1. p21waf1/cip1-mediated cell cycle arrest at the G1-S checkpoint can also compromise this important pathway (139-140). In addition, due to the inactivation of the p53 gene by oxidation of its sulphydryl group, the transcription of p21 is repressed by the HBx protein, leading to uncontrolled cell proliferation (141). Expression of this protein correlates with the frequency of DNA mutations in transgenic mice exposed to AFB1 (141,142). Failure of the DNA damage repair mechanism is the main cause of further modifications in the cells, involving an increase in the rate of DNA unwinding, p53 gene mutation and progression to cell proliferation.

Consequences of DNA Damage in the Progression of the Disease
Following DNA damage due to oxidative stress, AFB1-DNA adducts increase directly or indirectly viral DNA integration into the host genome (143). These carcinogenic adducts also affect the susceptibility of subjects to viral infections 70).

In patients with HCC and exposed to AFB1, the level of AFB1-DNA adducts has been reported to be higher compared to that of patients with HCC not exposed to AFB1, patients carrying HBsAg and control subjects (53). Similarly, the level of 8-OHdG has been reported to be high in patients with HCC when AFB1 is suspected and confirmed (144).

DNA damage activates the p53 gene that keeps pre-cancerous lesions under control. This is in contrast with the observation of Christophorou et al. (144) and Efeyan et al. (76), who found that ADP Ribosylation Factor (ARF), a tumor suppressor triggered by oncogenic disruption of the cell cycle, is sufficient to activate the p53 gene (111,120). The oncogene activation can perturb DNA replication specifically by premature termination of DNA replication forks (111,120,145).

Decrease of Glutathione-S-Transferase (GST) Activity

Glutathione protects the body against a number of harmful chemical substances that induce drug toxicities. It has been reported that its metabolism is often correlated with cellular sensitivity to anticancer agents. Indeed, glutathione is protective against drug cytotoxicity. Aflatoxins are metabolized in the liver by CYP450 detoxifying enzymes such as CYP1A2 and CYP3A4 and also by GSTs and epoxide hydrolase. In addition to their function in catalysis and transport, GSTs react with many toxic substances, including free radicals and cancer agents such as aflatoxins. While increasing excretion of AFB1-GSH conjugate as a mercapturic acid, GST activity is significantly reduced in the presence of HBV infection (146).

Glutathione-S-transferase mu 1 (GSTM1) and theta 1 (GSTT1) null genotypes involved in the metabolism of a wide range of carcinogens have been reported to be associated with an increased risk of HCC in a special geographic environment. Deletion of these genes has been reported to be common in the population. The risk of HCC has been reported to be double in subjects having the two null genotypes combined.

Transfection of the HBx protein gene into HepG2 cells decreased the expression of GSTa class enzymes (146). GST activity has been reported to be lower in the presence of HBV DNA, presumably due to the oxidation of its sulphydryl groups (147). There is, indeed, an altered balance of activation and detoxification of carcinogens during a viral infection. This is reflected in the decrease of the ability of hepatocytes to detoxify chemical carcinogens (147).

Induction of Cytochrome P450s (CYPs) Activity

Cytochrome P450s (CYP1A2 and CYP3A4) metabolize AFB1 to reactive AFB1-8,9 epoxide, which then binds to DNA and protein to form, respectively, AFB1-N7-guanine DNA and AFB1-alb adducts. HBV or HCV infections alter the hepatic expression of aflatoxin-metabolizing enzymes and increase the binding of its metabolites to DNA and to albumin (58,61,148). Studies in HBV transgenic mice have shown that there is induction of specific CYP450s associated with liver injury, induced by overexpression of HBsAg (149,150).

Importance of the Hypothesis
The hypothesis stresses the role of oxidative damage induced by HBV or HCV integration into the host DNA at all stages of infection, the interaction of these viruses with AFB1 adducts and their effects on p53 gene inactivation/mutation in pre-cancer and cancer progression stages (151,152). The understanding of the molecular mechanisms underlying this interaction during those stages may help with designing better strategies for the prevention of HCC. Various epidemiologic studies have, indeed, demonstrated the presence of HBsAg or HBV DNA with liver injury induced by overexpression of HBsAg (23,51,53,149-151). According to Kremsdorf and colleagues (153), HBx protein affects the p53 gene and oxidative damage in HBV-related HCC with the above-mentioned antigen and virus DNA complex (152).

Evaluation of the Hypothesis
Prevention
Due to the interaction of AFB1 with HBV in the occurrence and progression of HCC, HBV vaccination seems to be important but it is difficult to demonstrate its efficacy. Reduction of aflatoxin exposure remains the practical way for reducing the incidence of HCC (154). Good hygiene practice (e.g., sterilization, use of sterile needles) is also important (155,156).

Several strategies have been used to minimize pre- and post-harvest aflatoxin contamination of foodstuffs (157,158). The first strategy consists of minimizing fungal infestation and aflatoxin contamination by: (i) cultivating Aspergilus flavus and A. parasiticus resistant varieties, which will limit fungal invasion and toxin production during crop growth; (ii) controlling field infection using phytosanitarian measures (treatment of seeds and application of fungicides); (iii) lowering the moisture content of seeds after harvest and during storage; and (iv) adding preservatives to prevent contamination during storage. The second strategy for eliminating fungal contamination consists of: (i) sorting contaminated pods and kernels, which reduces aflatoxin contamination in the final product; (ii) redrying groundnut pods and kernels, which limits fungal growth and aflatoxin synthesis; and (iii) facilitating the binding of aflatoxin by dietary supplements or using ammonia to induce a chemical inactivation of aflatoxin.

Chemoprevention
Various attempts have been made to reduce the absorption of aflatoxin or to modify its metabolism and to find specific compounds bearing detoxification properties (159,160). A reduction of 55% in urinary AFB1-N7-Gua has been observed in a chemoprevention trial in China using chlorophyllin as an absorption agent (160). By scavenging the hydroxyl radicals, green tea containing polyphenols has been shown to reduce both urinary AFM1 and aflatoxin-albumin adducts in one intervention trial in China (161). Looking at the molecular mechanism, Oltipraz acts by blocking both aflatoxin DNA adduct formation and hepatocellular carcinogenesis in animal models by modifying the level of detoxifying enzymes such as GSTs (162). Broccoli sprouts containing glucosinolate when taken at the individual level were also useful in reducing AFB1-N7-Gua excretion in a randomized clinical trial in a Chinese township (163). In chemoprevention with respect to oxidative stress, attention must be paid to having an adequate balance of some antioxidants, which may prevent the progression of this disease.

Previous Experiments and Further Investigations that would Corroborate the Hypothesis
The hypothesis has been partially verified in some of the preliminary experiments showing that both viral and chemical carcinogens are involved through free radicals in the induction of HCC. Working on the mechanism of direct hepatocarcinogenicity induced by iron overload in rats, it has been reported that the rate of DNA unwinding and the level of 8-OHdG were two-fold higher in the iron supplemented group than in the control group (Fig 2). There was a positive correlation (Tables 1 and 2) between these two parameters positively influenced by the level of serum iron (164).

Table1a Weak positive correlation
b Positive correlation
c Strong positive correlation

Abbreviations: LOOH: lipid hydroxyl; MDA: malondialdeyde; 8-IP: 8-isoprostane; 8-OHdG: 8-hydroxy-2’-deoxyguanosine; FADU: fluorimetric analysis of DNA unwinding; AMES test, ALT: alanine transferase; AST: aspartic transferase.

(From Asare GA et al. Toxicology 2006; 219:41-52)

Table 2a Weak positive correlation
b Positive correlation
c Strong positive correlation

Abbreviations: LOOH: lipid hydroxyl; MDA: malondialdeyde; 8-IP: 8-isoprostane; 8-OHdG: 8-hydroxy-2’-deoxyguanosine; FADU: fluorimetric analysis of DNA unwinding; AMES test, ALT: alanine transferase; AST: aspartic transferase.

(From Asare GA et al. Toxicology 2006; 219:41-52)

Studies have also demonstrated (Fig 2) that vitamins A and E reduce the effects of oxidative stress in an animal model of iron overload inducing HCC for a limited period of time, suggesting the involvement of ROS/RNS (165). In addition, MDA and M1G-deoxyribose concentration increased with the extent of lipid oxidation observed in patients with HCC. Due to the transversion of G to T induced by these two compounds, inactivated p53 will then bind more to the HBx protein in patients with HBV or HCV than in uninfected individuals. As explained above, this process will inhibit the p53 gene for inducing apoptosis in these patients, who may further develop HCC. In this case, as in bacteria deficient in NER, an increased rate of mutation will be observed.

Figure 2

Figure 2 | Interaction plot of time and group: Fluorimetric Analysis of DNA Unwinding (FADU) of iron-overloaded Wistar rat liver homogenate and control rats (p<0.05, 16 months). The increased rate of DNA unwinding (δf/min) observed in iron and copper overloaded groups is reduced upon supplementation with exogenous antioxidants: Vit A and Vit E for a limited period of time.

It would be of interest to compare the extent of this induction of G to T transversion by MDA and M1G-deoxyribose among these two groups of patients (HBV and HCV), as well as among the populations of countries heavily exposed, or not exposed, to AFB1.

Can enhanced DNA-damage signalling be better protective against cancer?
Does DNA damage play a stronger role in the progression of HCC in the case of the association of aflatoxin and HBV or HCV?

The role played by DNA damage response in all its aspects of the onset and the progression of HCC should be investigated.

Conclusion
If the hypothesis is verified, the rate of AFB1 intercalation into DNA breaks, inducing DNA unwinding, may be expected to be less extensive in healthy patients despite their exposure to mycotoxins compared with patients with advanced HCC. In contrast, the extent of AFB1 intercalation in patients with HBV and HCC should correspond to the progression of the disease and should be related to the DNA damage dependent free radical attack and to the response of patients to the oxidative stress factors. In this case, the HBx protein may bind and block the p53 gene in the nucleus, with all the consequences of repression of transcription and the non-induction of cell cycle arrest leading to uncontrolled cell proliferation. The ability to rejoin the DNA strand breakages reflects the capacity of DNA damage repair, which may be directly correlated with the total antioxidant capacity and the GST activity and inversely correlated with the extent of DNA unwinding.

Abbreviations
HBV: hepatitis B virus; HBx: HBV X protein; HBsAg: hepatitis B surface antigen; HCC: hepatocellular carcinoma; HCV: hepatitis C virus; AFB: aflatoxin B; NER: nucleotide excision repair; CYP: Cytochrome P; CYP450: cytochrome P450; AFB1-N7-Gua: 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-AFB1; HIV: human immunodeficiency virus; ETC: electron transport chain; ROS: reactive oxygen species; O2•-: superoxide radical (anion); OH: hydroxyl radical; RNS: reactive nitrogen species; NO: nitric oxide radical; RS: thyl radical; ROO: peroxyl radical; ONOO-: peroxynitrite radical; HOCl: hypochlorous acid; H2O2: hydrogen peroxide; ¹O2•- : singlet oxygen; O3: ozone; MAPK: mitogen-activated protein kinase; SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; SH: thiol/sulphydryl group; GSH: glutathione; GST: glutathione-s-transferase; NAD(P)H: nicotinamide dinucleotide (phosphate) reduced form; NOS: nitrite oxide synthase; TRX: thioredoxin; CDKs: cyclin-dependent kinases; ATM: ataxia telangiectasia mutated; ATR: ataxia telangiectasia related; CAGE: cancer antigen; ARF: ADP ribosylation factor; TRF: telomeric-repeat binding factor: TRF2: telomeric-repeat binding factor 2; CDK: cyclin dependent kinase; ssDNA: single stranded DNA; dsDNA: double stranded DNA; MDA: malondialdehyde; M1G D: malonyl-1-guanosine-deoxyribose; 8-OHdG: 8-hydroxy-2’-deoxyguanosine; ATP: adenosine triphosphate; ATPase: adenosine triphosphatase; NTP: nucleoside triphosphate.

Acknowledgments
The financial support of the UNESCO Global Network for Molecular and Cell Biology, the Oppenheimer Trust, the National Research Foundation-South Africa, and the Seoul National University College of Pharmacy Department of Biochemistry and Molecular Toxicology is gratefully acknowledged.

Author declares no conflict of interest

About the author
Professor KSA Mossanda’s previous appointments include: Postdoctoral Fellow in many universities in Europe, USA, Asia and Africa; Professor, Dean of Faculty of Pharmacy, and Head of Clinical Biology Department at the University of Kinshasa, Congo.

He is presently working as Research Coordinator at the Walter Sisulu University (South Africa), Supervisor of postgraduate students and External examiner of Master’s and PhD dissertations from various South African Institutions and abroad.

He is member of various scientific and professional societies and reviewer of more than 20 African and international journals.

His research expertise includes: bio-chemistry, toxicology, carcinogenesis, mutagenesis, traditional medicine, chemoprevention and anti-inflammatory activities of medicinal plants.

His current focus includes viral infection, complications of diabetes and cancer. He has published 75 papers in peer-reviewed journals and refereed/peer-reviewed conference proceedings, 3 chapters in books and is co-author of a scientific book.

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