**8. Mechanism of carcinogenesis of AFB1**

AFB1 is a known genotoxic hepatocarcinogen that causes genetic damage such as formation of DNA adducts, albumin adduct, gene mutations, micronucleus formation, sister chromatid exchange and mitotic recombination which result in genetic changes in the target cells, which then cause DNA damage and ultimately cancer [26].

When AFB1 is ingested by humans and other susceptible animals, it is transported to the liver where the CYP 450 enzymes convert AFB1 into ROS endo-AFB1–8, 9-epoxide and exo-AB1–8, 9-epoxide the latter being more toxic than former, thus this confers genotoxic characteristics on AFB1 [62]. The exo-AFB1–8, 9 epoxide has affinity for the N<sup>7</sup> atom of guanine and so bind with it leading to the formation of primary DNA adduct (AFB1-N7 -Gua adduct) [63]. The AFB1-N7 -Gua adduct is transformed into two minor compounds namely an apurinic (AP) site and a stable ring-opened AFB1- Formamidopyrimidine (AFB1-FAPY) adduct the latter being more mutagenic than the former [21]. The AP and AFB1-FAPY adduct are mended by nucleotide excision repair (NER) or base excision repair (BER) [64, 65].

Conversely, when the mending process is improperly done, it results in AGG to AGT transversion mutations with these mutations taking place at codon 249 in the tumor suppressor gene *TP53*. When these mutations occur, the amino acid arginine in the tumor suppressor protein p53 become replaced with serine (R249S) [66, 67]. When the mutated R249S p53 is expressed, apoptosis is inhibited, p53 mediated transcription is also inhibited and liver cells are stimulated to grow uncontrollably resulting in HCC [68]. Studies have reported that the R249S mutation is mostly found in more than 50% of HCC cases especially in China and Africa where the incidence of HCC is high [66, 69]. On the other hand, the R249S mutation is rare in the regions of the world where aflatoxins exist at extremely low levels in the diet and in cancers other than HCC [70]. The TP53 directs the synthesis of p53 protein. When the conditions within the cells are normal, the p53 is kept at low levels through it binding to ubiquitin-ligases such as Mdm2 (also referred to as Hdm2 in humans) and then degraded by proteasome enzymes [71]. On the other hand, in the presence of some

stress factors, the p53 goes through certain processes such as phosphorylation on serine 15 (Pser15-p53) and become activated after it has been produced. The activated p53 binds specific DNA response elements resulting in trans-activation of genes that play key roles in programmed cell death, the arrest of cell cycle, repair of DNA repair or aging [72]. These responses lead to repair of damage that have been caused to DNA which help to maintain the genetic integrity of the cells. The response may also stimulate apoptosis of damaged cells resulting in their elimination from the system.

Some studies reported that AFB1 impair miRNA biogenesis; the authors also reported that AFB1 suppress Wnt/β-catenin signaling pathway by inducing overexpression of miR-34a and thus causing liver cancer [73]. Other studies showed that AFB1 promote HCC cell multiplication through an IGF-2-dependent signal axis [74]. AFB-mediated DNA damage results in the deregulation of the cell cycle and cause HCC through the up-regulation of pro-apoptotic pathways including p53, NF-kB, BCl2, c-Myc, CDK, Ras, protein kinase C, Cyclins and CKI's [75, 76]. All these mechanisms of actions take place in the liver.
