**6. Immunomodulatory effects of AFB1 in humans**

Data or information on how AFBs and the related compounds regulate the immune system is scanty. Reports indicate that AFBs and other mycotoxins can suppress the immune system through the inhibition of DNA replication, transcription and translation of genes that are required to switch on the innate and acquired immune system response by using myriad of mechanisms [39]. Studies have indicated that the AFB1–8, 9-exo-epoxide that is formed when AFB1 is metabolized reacts with DNA found in the mitochondria rather than with DNA found in the nucleus of the cell and this hinders the synthesis of ATP [40, 41]. The binding of the AFB1–8, 9-exoepoxide to DNA of the mitochondria results in the formation AFB1-mitochondrial DNA adduct which leads to mutations in the membranes causing ballooning of the cell as well as disrupting the production of ATP [40, 42].

Studies have reported that AFB1 and its intermediate products inhibit translation when they bind to important enzymes that are needed in the translation of mRNAs into proteins. AFB1 and its intermediate products also suppress translation by blocking the activities of translocase in ribosomes and this suppress translation [43]. Additionally, it has been reported that aflatoxins negatively affect protein synthesis by interfering with substrates and enzymes that are needed for initiation, transcription and translation [44].

Furthermore, several research works have looked at how AFB1 inhibit the immune system in humans. In 2015, Jiang et al. conducted a study to evaluate how the regulation of some pro-inflammatory cytokines such as IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ and TNF-α in the bowels of broiler birds could be affected by AFB1. They reported that the transcript levels of the studied pro-inflammatory cytokines in broiler birds treated with AFB1 were much lower than the levels in broiler birds that were not treated with AFB1. Furthermore, Jiang et al. indicated that broiler birds that were treated with AFB1 exhibited reduced amount of T-cells in the intestines in comparison with broiler birds that were not treated. Several studies have also reported the suppression or inhibition of transcription and translation of IL-4, IL-6 and IL-10 genes respectively by AFB1in the peritoneal macrophages, splenic lymphocytes and macrophage cell lines [45–47].

On the contrarily, a study conducted by Li et al. [48] indicated that broiler birds that were fed with livestock feed containing 0.074 mg/kg showed an increase in the levels of IL-6, IFN-γ and TNF-α mRNA and protein expression in the spleen and serum. In 2014, Qian et al. [49] undertook a study to determine the impact of AFB1 on the splenic lymphocyte phenotypes and the inflammatory cytokine production in male F344 rats. They indicated that rats that were exposed to AFB1 showed a dosedependent reduction in the level of IL-4 produced by CD4<sup>+</sup> T cells. Also Bruneau et al. [50] reported that AFB1 induced the suppression or inhibition of IFN-γ and TNF-α expression by CD4<sup>+</sup> T cells and CD3− CD8a NK cells respectively. Bruneau et al. [45] in addition indicated that murine macrophages that were exposed to AFB1 *in vitro* showed a reduction in the level of anti-inflammatory cytokine IL-10 but rather increased the level of pro-inflammatory cytokine IL-6. Taken together these studies suggest aflatoxins are immunosuppressive agents.

#### *Aflatoxin B1: An Immunomodulator and Cancer Agent DOI: http://dx.doi.org/10.5772/intechopen.106833*

A study was conducted by Forouharmehr, Harkinezhad [50] to determine the impact of AFB1 on how the expression of STAT5A can be affected by treating bovine mammary epithelial cells with AFB1 and quantifying the mRNA levels of STAT5A using RT-qPCR. They indicated that cells that were treated with AFB1 showed a great decline in the mRNA levels of STAT5A in a dose-dependent manner. They further reported that the suppression of the mRNA levels of STAT5A minimized the proliferation and differentiation of mammary epithelial cells, thus affecting the amount and the quality of milk protein that is produced.

In 1999, Rossano et al. [51] treated human monocytes that have been activated with lipopolysaccharide of bacteria with 0.01–1.0 pg/mL of AFB1 in order to determine how AFB1 could affect the expression and release of IL-1α, IL-6α, TNF-α. They reported that at 0.05 pg/mL of AFB1, the levels of IL-1α, IL-6α, and TNF-α were greatly reduced and that AFB1 totally shut off the transcription of their mRNAs. Rossano et al. further reported that transcript levels of β-actin remained unchanged by AFB1. These findings made the researchers to make a conclusion that AFB1 exerts it effects on the expression of cytokines likely by suppressing the transcription of certain mRNAs without affecting translation.

The type I interferon signaling response pathway of the innate immune system plays a significant part in eliminating disease causing microorganisms and cancer cells in the human system. The type I interferons exhibit antiviral as well as anti-cancer properties. The anti-cancer activities of type I interferon led to their use to treat cancers such as Kaposi sarcoma in AIDS patients, cancer of the bone marrow (hairy cell leukemia) and other forms of cancers [14]. In 1979, Hahon et al. [52] conducted a study to determine how AFB1 could affect the induction of interferon production in monkey kidney cells (LLC-MK) that had been infected with influenza virus. They reported that AFB1 inhibited influenza viral induction of interferon dose-dependently.

In other to understand the mechanism of AFB1 inhibition of the interferon signaling response pathway, Narkwa et al., demonstrated in a study that AFB1 suppressed IFN-α induced ISRE (interferon stimulated response element) signaling in a dose dependent manner using luciferase reporter gene assay (**Figure 3**), [53]. Further using RT-qPCR Narkwa et al. [53] showed that AFB1 inhibits transcript expression levels of key signaling elements such as STAT1, JAK1 and OAS3 genes of the JAK– STAT-ISRE arm of the type 1 IFN response pathway. Some studies have reported that post-transcriptional processes may be involved in the translation of mRNA into protein [54]. This suggests that low mRNA expression may not directly results in lower expression of protein and oppositely. Consequently, after demonstrating that AFB1 suppresses the transcript expression levels of STAT1, JAK1 and OAS3, Western blot assay was used to determine whether the suppression of transcript expression level of STAT1 by AFB1 would ultimately affect its translation into protein. The authors observed that AFB1 suppressed the translation of STAT1 mRNA into protein. The type I IFN signaling has been reported to exert its anti-cancer and antiviral response through the activation of the JAK–STAT-ISRE arm of the pathway (**Figure 4**) [53]. One component of the JAK–STAT-ISRE signaling pathway considered to have tumor suppressor function is STAT1 [55]. When activated, STAT1 suppresses tumor development by inducing programmed cell death [56] and also inhibit tumor maturation or growth [57]. Therefore the suppression/inhibition of STAT1 by AFB1 as demonstrated by Narkwa et al., would definitely weaken the capacity of STAT1 to coordinate the expression of multitude of genes necessary to stimulate programmed cell death, prohibit multiplication and maturation of cells in response to AFB1. Therefore, the above stated studies provide overwhelming evidence that aflatoxins in general and

#### **Figure 3.**

*AFB1 suppresses the antiviral and anticancer type I interferon response signaling in a dose dependent manner. Source: [34].*

#### **Figure 4.**

*Key antiviral and anticancer elements of the innate immune type I interferon signaling response pathway suppressed by AFB1. Source: [34].*

AFB1 in particular suppress the immune system and predispose individuals to diseases including cancers.
