The Mechanism of Mycotoxin (Aflatoxin B1)

The Mechanism of Mycotoxin (Aflatoxin B1)

Mycotoxin

Abstract

            Mycotoxins which are secondary metabolites of moulds causing diseases referred to mycotoxicoses. The occurrence of mycotoxin is majorly in hot and humid climates. This is because this kind of climate favors the growth of the mould. Ingestion is the major exposure of mycotoxin. This paper focuses on aflatoxin B1 which is caused by mycotoxins and discusses in detail the molecular mechanism of the same.

Introduction

Aflatoxin B1 (AFB1) is a hepatoxic disease that is prevalent in many parts of the world. It is a toxic metabolite produced by strains of Aspergillus flavus, A. parasiticus, and A. nomius. Aflatoxins B1, B2, G1, G2, and M1 can be produces by strains of A. flavus and A. parasiticus (Wilson and Payne, 1994). AFB1 is capable of contaminating a wide variety of food supply such peanuts, rice, corn, and a number of cereals. This contamination is due to colonization of the fungus to susceptible crops as mentioned. It is also possible for the contamination to happen during harvesting, storage or drying and this happens when the moisture levels in food supply is high thus enabling the fungal growth. Number of studies have linked AFB1 with human carcinogens more so with human hepatocellular carcinoma which is a much known cancer leading to a number of deaths worldwide. One of the most prevalent and also regarded as carcinogenic of the aflatoxin compounds is Aflatoxin B1 (AFB1).

Target Organ

According to Williams et al., (2004) the International Agency for Research on Cancer have classified AFB1 as a Group carcinogen alternatively known as an agent which is considered to be carcinogenic to human beings. It has been established that the liver is the chief target organ for Aflatoxin B1, but some research also show that lungs can also be targeted due dietary and inhalation exposure and this has been supported by epidemiological and laboratory evidence (Massey et al., 2000). Not all hepatocellular carcinoma is because of the action of AFB1. All the possible risk factors should be evaluated before making a conclusion on the AFB1 as the contributing risk factor in human hepatocellular carcinoma despite the fact that they have found to be carcinogenic in a number of experimental models as stated by Kumar et al., (2000). The analysis of the toxic effects of the mycotoxin effect more so in the case of Aflatoxin B1 has shown the following in liver histopathology, Centrilobular necrosis, fatty changes in midzonal region, polymorphonuclear, infiltration and fibrin in sinusoids, bile duct proliferation with periductal fibrosis, bile stasis gastrointestinal bleeding in bile ducts, multinucleated giant cells dilated biliary canaliculi, foamy cytoplasm, extensive fibrosis, giant, cell transformation of

hepatocytes, moderate to severe cholestasis , Cholangiolar proliferation.

Molecular mechanism

1. DNA adduction formation

            Toxicity, carcinogenicity and disposition of AFB1 are always as result of biotransformation as stated by Eton et al., (1994). The metabolite actions that are able to react with cellular micromolecules are the driving force behind the toxicity and carcinogenic effect of Aflatoxin B1 (Eton et al., 1994). Of greatest importance in bioactivation is the AFB1 bioactivation by prostaglandin H synthase and lipoxygenase which is more important than the bioactivation by P450 (Donnelly, 1996). The bioactivation of AFB1 by epoxidation involving terminal furan ring double bond leads to the production of electrophilic intermediate AFB1-8,9-epoxide. This is a stereoisomer existing in both the exo and endo conformations as stated in Baertschi et al., (1988). Despite the fact that AFB1-exo epoxide is a weakly mutagenic, it has the ability to alkylate nucleic acids and proteins and its existence is from is from the structures of stable products since it is not an isolated in intact from from biological systems (Degen et al., 1978). In some time back, the chemical synthesis of AFB1-exo-epoxide was not possible, but currently, this has been made possible by utilizing dimethyldioxirane and two-phase buffered m-chloroperbenzoic acid system (Kolb et al., 2001). It is noted that it is possible to crystallize AFB1 readily in high yields and that it is quiet stable in aprotic and even nucleophilic solvents. Despite the fact that it has a half life of 1s in aqueous buffer, it can undergo reaction with DNA which has high concentration resulting to AFB1-DNA adducts and a yield of 98 percent as stated by Johnson and Guengerich (1997). The existing difference in reactivity between AFB1- exo-epoxide and endo isomer is because the intercalation of the furanocoumarin entity of the epoxide between the bases in DNA orients the epoxide for SN2 attack by N7 of guanine, this results in the formation of trans-8, 9- dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1- N7-Gua) as the primary AFB1–DNA adduct (Croy et al., 1978). Because of the blocking of the nucleophilic attack by guanine N7 by Oxirane ring of the endo-epoxide when it intercalates into DNA, the reaction of AFB1-endo-epoxide with DNA only leads to the formation of low levels of adducts according to Iyer et al., (1994). This kind of adduct is labile and this is attributed to the positive charge on the imidazole ring of AFB1-N7-Gua. Some of the key reactions that it undergoes in vitro involve the release of AFB1-8,9-dihydrodiol and the restoration of guanylic sites in DNA, second is the depurination which result to the formation of purinic site in DNA and the third involves base catalyzed hydrolysis which result into the opening of imidazole ring and consequently, the formation of a stable AFB1–formamidopyrimidine adduct (AFB1–FAPY) (Groopman et al., 1981). In the in vivo adducts, AFB1-FAPY is vital part as a result of exposure to AFB1. Two rotameric forms which are the stereoisomers resulting from the restricted rotation about a single bond make AFB1-FAPY an equilibrium mixture. These are always separable by chromatography assigned as AFB1-FAPY minor and AFB1-FAPY major. While the structure of AFB1-FAPY major is known, the structure of AFB1-FAPY minor has not been well established (Mao et al., 1998).  In vivo treatment has shown that AFB1 has the capability to trigger 8 hydroxy-20-deoxyguanosine (8-OHdG) formations in rat and duck liver (Yarborough et al., 1996). In a number of mutations triggered by AFB1, DAN alkylation by AFB1-exo-epoxide and the formation of AFB1-N7-Gua have been to lead to G to T transformations, nonetheless, 8-OHdG also produces chiefly G to T transversion mutations (Cheng et al., 1992). A number of reactive oxygen species which include superoxide radical anion, hydrogen peroxide and lipid hydroperoxides do not undergo any kind of interaction with the DNA and are only known to be precursors to hydroxyl radicals while the AFB1 triggered oxidative DNA damage results to AFB1 carcinogenesis (Halliwell and Gutteridge, 1999). When hydroxyl radicals react with DNA, anumber of products can be generated which include pyrimidines and purines and even guanine residues to make 8-OHdG. In vivo rat liver study and rat hepatocytes reveal that AFB1-triggered reactive oxygen species formation needs metabolism by cytochrome P450 for the formation of AFB1 exo-epoxide and hhydroxylated metabolite AFM_1.   More light need to be shed on the mechanism that results to AFB triggered oxidative stress and 8-OHdG contribution to the AFB1 tumourigenesis (Bedard and Massey, 2006).

• Point mutation in TP₅₃

According to Ozturk (1991), the mutation in T₅₃ codon 249 exon 7 is closely associated with the exposure to aflatoxin B1 (AGG to AGT, arg to ser). This is due to the mutation of p53 tumor suppressor gene. This kind of mutation is associated with a number of incidences of hepatocellular carcinoma. The generation of adducts at guanine in TP₅₃ causes T to G transversions. Of interest is R249S hotspot in structural mutants.  The mutations at TP53 gene are majorly due to missense base changes compared to others in tumor suppressor genes are due to deletion according to Bennett et al (1999). The DNA damage (point mutation) caused on the TP53 gene due to exposure to aflatoxin B1 can result to the triggering of a number of cellular responses and these would determine whether the cell will be able to eliminate the damage or to active apoptosis processes. It is the formation of adducts as discussed above that result to HCC (due to point mutation at gene TP53).

It should be noted that hepatocellular carcinoma is mostly associated with mutation R249S which major results due to exposure to aflatoxin and most importantly AFB1. It is the embryonic stem cells that have so far shown a targeted mutation results to the expression of murine p53R246S which is the as the same as the human p53R249S according to Lee and Sabapathy, (2008). A transgenic p53R246S mouse is an indication that the mutant may be a promoting agent for cases of aflatoxin-induced hepatocarcinogenesis according to Ghebranious and Sell, (1998).  The enhanced tendency to aggregate defined the property of mutant proteins in this group and the mutants such as R175H, R249S and R282W had the ability to aggregate in the cytoplasm (Xu et al., 2011).

Hepatocellular Carcinoma

            As discussed above on the molecular mechanism, the resultant effect of the mechanism is hepatocellular carcinoma (HCC). This is always the manifestation of a malignant liver cancer with aflatoxin B1 is the chief factor for HCC. As discussed the mutation activity at p53 tumor suppressor gene-TP53; MIM# 191170, has a major role in heptocarcinogenesis. The initiation of the tumor is characterized by DNA mutations and in most cases; the p53 pathway becomes defective resulting to HCC and most human cancers. 249^ser mutation of the p53 tumor suppressor gene is the main cause of hepatocellular carcinoma due to exposure to aflatoxin B1 (Kress et al., 1992). The promutagenic formation N7dG is due to metabolic activation of aflatoxin B1 to 8.9-epoxide binding to the DNA.

Oxygen and nitrogen oxide species can result to the damage of DNA. AFB1 triggers reactive oxygen species due to the metabolism by cytocrome P450 leading to the formation of AFB1 exo-epoxide and hydroxylated metabolites. The involvement of AFB1 in oxidative stress needs to be investigated. This kind of reaction during inflammation is characterized by the breaking of DSB’s double strand, SSB’s, Single strand and 8-oxo-dG, 8-hydroxy-deoxy-guanosine. The oxyradical species are the major cause of p53 mutational load. Signal transduction pathways which are associated with transcriptional triggering of oncogenes can be activated by oxygen species.

Conclusion

Aflatoxin B1 as a mycotoxin caused disease has been documented to be the factor in HCC with liver as the target organ. The two molecular mechanisms (DNA adduct formation and point mutation at TP53) are very vital in understanding carcinogenicity of aflatoxin B1. G:C to T:A transversions at the third base of p53 at codon 249 is what leads to liver cancer in exposure to AFB1. Ways of reversing the mutation caused by aflatoxin B1 should be explored as this would help in solving HCC due to exposure to aflatoxin B1.

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