Research progress in toxicological effects and mechanism of aflatoxin B₁ toxin

Hepatotoxicity

Liver is the main target organ for AFB₁. Dietary supplementation of AFB₁ in rats lead to irreversible liver damage in a dose-dependent manner (Qian et al., 2016; Lu et al., 2013) by inducing fat deposition, fatty acid oxidation (Zhang et al., 2011) and telomere shortening (Ali et al., 2021). Recent studies have also shown that AFB₁ can trigger massive production of reactive oxygen species (ROS) in the liver cells, leading to oxidative stress, inflammation, and liver damage (Singh, Maurya & Trigun, 2015). Furthermore, AFB₁ exposure enhanced apoptosis of liver cells, activated the resident Kupffer cells, and promoted an inflammatory response in the liver through dephosphorylated-cyclooxygenase-2 (COX2) (Zhang et al., 2019a). Prolonged exposure to AFB₁ disrupted lipid and lipoprotein metabolism (Rotimi et al., 2017), and resulted in extensive damage to mitochondrial lipids and reduced antioxidant capacity in the rat liver (Rotimi et al., 2019). Likewise, mice exposed to AFB₁ showed mitochondrial dysfunction and increased rates of mitochondria-dependent apoptosis in the liver (Xu et al., 2021). In one study, primary broiler hepatocytes (PBHs) treated with different concentrations of AFB₁ showed mitochondrial dysfunction, oxidative stress, and ROS-dependent mitochondrial apoptosis through the nuclear factor-erythroid 2-related factor-2 (Nrf2) signaling pathway (Liu & Wang, 2016). Epidemiological studies have shown that AFB₁ is one of the important risk factors of primary liver cancer (Kucukcakan & Hayrulai-Musliu, 2015). Furthermore, there is evidence that AFB₁ and chronic hepatitis B virus can synergistically induce mutations in the p53 gene and initiate liver cancer (Liu et al., 2012). To summarize, AFB₁ can induce liver damage and even liver cancer by inducing oxidative stress, inflammation, and mitochondrial dysfunction by targeting the p53, ROS, COX2, Nrf2 and other signaling pathways.

Enterotoxicity

The gut barrier function maintains the homeostasis between the resident immune cells and commensal microorganisms via the intestinal epithelial cells (IECs) (Peterson & Artis, 2014). Long-term exposure to AFB₁ has been associated with chronic intestinal diseases. AFB₁ caused intestinal mucosal injury and inhibited IECs proliferation in mice (Gaikwad & Pillai, 2004). In addition, AFB₁ and AFM₁ can damage the intestinal barrier in mice through clathrin-mediated endocytosis through synergistic and additive interactions (Gao et al., 2021). AFB₁ also altered the composition of the intestinal microbiota of male F344 rats in a dose-dependent manner, and significantly decreased the abundance of the probiotic lactic acid bacteria (Wang et al., 2016a). The microbial-related metabolic changes in the gut microbiota of these AFB₁-treated rats were analyzed by ion fragmentation spectroscopy. AFB₁ significantly increased the number of inflammatory fecal liposomes, and altered intestinal microbiota-dependent biliary cholesterol metabolism, degradation of bilirubin and fatty acids, and glycolysis. The structural changes in the fecal microflora induced by AFB₁ are similar to that observed in IBD (inflammatory bowel disease) patients. The combination of metabolic dysfunction, loss of IECs and glandular atrophy caused by AFB1 can lead to chronic intestinal diseases (Zhou, Tang & Wang, 2021).

The gastrointestinal system in poultry is especially sensitive to AFB₁. Broilers fed with different doses of AFB₁ exhibit severe damage to the intestinal villi characterized by lower density and absorption area (Yunus et al., 2011; Kana, Teguia & Tchoumboue, 2010), increased atrophy and shedding, and a significant reduction in height (Yin et al., 2016). Moreover, the jejunum of chickens exposed to AFB₁ showed histopathological changes, increased apoptosis rates, and altered expression levels of death receptors, endoplasmic reticulum (ER) molecules and apoptotic factors (Zheng et al., 2017). Ingestion of AFB₁-contaminated feed can also affect the absorption capacity of the small intestine and impair its innate immune function (Wang et al., 2018c). Furthermore, infiltration of inflammatory cells into the small intestine leads to muco-enteritis (Kumar & Balachandran, 2009). Ducks fed with AFB₁-contaminated corns showed longer and wider jejunum villi, which was accompanied by lower average daily gain (ADG) and average daily feed intake (ADFI), resulting in reduced growth and development. Furthermore, the relative weight of the digestive organs, the activity of digestive enzymes and the biochemical indices of intestinal development were also altered (Feng et al., 2017). Thus, AFB₁induced intestinal damage can restrict development, disturb the intestinal microflora, and lead to metabolic disorders or chronic intestinal diseases.

Nephrotoxicity

AFB₁ is absorbed by the kidneys, and its accumulation in the renal tissues leads to the up-regulation of p21 by MYC, PLK1 and PLD1, resulting in S-phase cell cycle arrest and renal injury (Huang et al., 2019). HEK293 cells treated with AFB₁ showed increased apoptosis and DNA fragmentation, which corresponded to the up-regulation of p53, Bax and caspases (Dlamini et al., 2021). AFB₁ and AFM₁ synergistically increased oxidative stress and the apoptosis pathway in renal cells by regulating the expression level of L-proline (Li et al., 2018). Furthermore, the combination of AFB₁ exposure and low protein diet additively reduced weight gain and promoted renal dysfunction in rats, and exacerbated oxidative stress (Rotimi et al., 2018). Exposure to AFB₁ and DON synergistically increased oxidative stress in the liver, kidney, and spleen of carp by upregulating Nrf2. Interestingly, ROS generation occurred earlier in the kidneys compared to the liver and spleen (Kövesi et al., 2020). In conclusion, the nephrotoxicity of AFB₁ is mainly manifested as oxidative stress induced by p21, L-proline, Nrf2 and other genes. Moreover, AFB₁ can have synergistic nephrotoxic effects along with nutrient level and other mycotoxins.

Neurotoxicity

Intragastric administration of AFB₁ once weekly for 8 weeks significantly impaired brain function in rats by inducing pathological changes in the cerebral cortex and hippocampus (Bahey, Abd Elaziz & Gadalla, 2015). Long-term exposure to AFB₁ may allow it to penetrate the blood–brain barrier, resulting in neurotoxic effects and even chronic neurodegeneration such as that observed in Alzheimer’s disease (Alsayyah et al., 2019). AFB₁ inhibits proliferation of human astrocytes by inducing cell cycle arrest and mitochondria-dependent apoptosis (Park et al., 2020). Environmental AFB₁ exposure may trigger neuroinflammatory responses by activating the microglia, and increase the susceptibility to neurodegenerative diseases (Mehrzad, Hosseinkhani & Malvandi, 2018a). The neurotoxic effects of AFB₁ exposure have also been observed in zebrafish embryos by nuclear magnetic resonance (NMR) (Zuberi et al., 2019). AFB₁ exposure decreased the survival rate of embryos by inhibiting oligodendrocyte development (Park et al., 2020). In addition, neuroblastoma cells (IMR-32 cell line) treated with AFB₁ also presented intracellular ROS accumulation, DNA damage, S phase arrest and apoptosis (Huang et al., 2020). To summarize, AFB₁ can inhibit neural cell development, promote apoptosis, disrupt the homeostasis of the nervous system, and increase the susceptibility to neurodegenerative diseases.

Immunotoxicity

Oxidative stress and apoptosis play key roles in AFB₁-induced immunotoxicity (Chen et al., 2013). Oral administration of AFB₁ downregulated IFN and TNF in the spleen of mice, increased IL4 levels, and damaged the thymus and spleen, eventually resulting in an impaired immune function (Jebali et al., 2015). AFB₁ exposure in rats increased ROS generation and secretion of pro-inflammatory cytokines (TNFα) in the liver cells (Mohammad et al., 2017), which are conducive HCC development (Qin et al., 2016). AFB₁ exposure affected transcription of key functional genes in human microglia cell line (CHME5) and human monocyte-derived dendritic cells (MDDCs), and increased apoptosis (Mehrzad, Hosseinkhani & Malvandi, 2018a; Mehrzad et al., 2018b). Similarly, AFB₁ treatment decreased the viability of the mouse macrophage RAW264.7 cells in a dose- and time-dependent manner by increasing production of ROS and malondialdehyde (MDA) and decreasing GSH levels. These changes correlated with upregulation of NOS2, TNFα and CXCL2 mRNAs, and downregulation of CD86. AFB₁-induced oxidative stress in macrophages also impaired the mitochondrial respiratory chain, leading to activation of the inflammatory response pathways (Ma et al., 2021). AFB₁ also decreased the phagocytic capacity of 3D4/21 cells, and induced apoptosis, pro-inflammatory cytokine secretion, DNA damage and oxidative stress. In addition, 3D4/21 cells treated with AFB₁ expressed high levels of DNA methyltransferase DNMT1 and DNMT3a, which led to the activation of the JAK2/STAT3 signaling pathway. Inhibition of p-JAK2 and p-STAT3 by blocking DNMT1 and DNMT3a alleviated AFB₁-induced immunotoxicity (Zhou et al., 2019). The combination of AFB₁ and ochratoxin A (OTA) increased production of TNFα and IL6 in these cells, and decreased lactate dehydrogenase secretion and the phagocytotic index in a concentration-dependent manner. In addition, the combination treatment significantly decreased the production of GSH, increased ROS levels, and promoted IκBa degradation, NF-κB phosphorylation and nuclear translocation of NF-κB. Thus, AFB₁ and OTA can synergistically aggravate immunotoxicity by activating of the NF-κB signaling pathway (Hou et al., 2018). AFB₁ also impaired the physiological functions of freshly isolated swine alveolar macrophages (SAM), and consumption of AFB₁-contaminated feed increased the risk of secondary infections in pigs (Pang, Chiang & Chang, 2020). In addition, AFB₁ activated the release of heterophil extracellular traps (HETs) in chicks, and induced the expression of TNFα, IL6 and IL1β, iNOS, COX2, NLRP3, caspase1, caspase3 and caspase11, which resulted in liver and kidney damage (Gao et al., 2022).

Studies show that intake of low doses of AFB₁ in animals adversely affect immune organs, decrease antibody titers and complement activity, and cause lymphoid tissue damage. Monogastric animals, especially poultry and pigs, are more sensitive to AFB₁-induced immunotoxicity. Broilers exposed to AFB₁ showed increased apoptosis of thymocytes due to mitochondrial and death receptor-mediated signaling pathways, as well as DNA damage (Peng et al., 2016). AFB₁ also induced tissue damage, cell cycle arrest (Hu et al., 2018) and apoptosis in the Bursa of Fabricius of broilers, which damaged their immune system (Yuan et al., 2016). Ingestion of AFB₁-contaminated feed significantly decreased the body weight and lymphocyte activity of pigs, increased the levels of pro-inflammatory cytokines such as TNFα, IL6, IL1β and IFN γ in the spleen, and decreased that of the regulatory factor IL10 (Meissonnier et al., 2008). The immunotoxic effects of AFB₁ in livestock may decrease the efficacy of vaccines and increase disease prevalence. These findings indicate that AFB₁ affects immune cells and organs, and its immunotoxicity depends on the mitochondrial signaling, death receptor, endoplasmic reticulum, apoptosis, cell cycle and inflammatory pathways.

Reproductive toxicity

AFB₁ can impair spermatogenesis through oxidative stress and mitochondria-dependent apoptosis. Mice exposed to AFB₁ show impaired blood-testis barrier due to lower levels of the BTB-related junction protein, increased apoptosis in the testes, and the oxidative stress-mediated p38 MAPK signaling pathway, which ultimately affected spermatogenesis (Huang et al., 2021). In addition, AFB₁ exposure decreased spermatogenesis in mice by inducing oxidative stress, decreasing mitochondrial content, and upregulating the pro-apoptotic Bax, p53 and Caspase 3 (Yasin, Mazdak & Mino, 2018). AFB₁ also induced testicular damage and testicular dysfunction in Dorper rams (Lin et al., 2022), and impaired spermatogenesis in white leghorn cockerels (Ashraf et al., 2022). Exposure to low concentrations of AFB₁ for several hours can decrease spermatozoa motility, hyperpolarize mitochondrial membranes, and increase DNA fragmentation (Komsky-Elbaz, Saktsier & Roth, 2018). In addition, AFB₁ interferes with porcine oocyte maturation by inducing epigenetic modifications, oxidative stress, excessive autophagy, and apoptosis (Liu et al., 2015a), and affects early embryo development through oxidative stress, DNA damage, apoptosis, and autophagy (Shin et al., 2018). AFB₁ can cross the placental barrier in humans (Wild et al., 1991), and AFB₁ exposure during pregnancy or lactation can adversely affect the health of the mother as well as the infants. In addition, pregnancy in mice can modulate both phase I and II metabolism, and alter the biological potency of AFB₁, thereby increasing liver damage (Sriwattanapong et al., 2017). Prenatal exposure to AFB₁ reduces the body weight of neonatal rats, disrupts lipid and hormone levels, and affect the methylation levels of p53 and growth-regulator H19 in the liver and serum. The pathological changes may increase the risk of HCC in the offspring (Rotimi et al., 2021). These studies indicate that exposure of sexually mature animals to AFB₁ affects gamete production, gamete quality, and gamete maturation, and AFB₁ also disrupts embryonic development, posing a long-term health threat to both pregnant animal mothers and offspring. The reproductive toxicity of AFB₁ depends on oxidative stress, DNA damage and repair, apoptosis, autophagy, and epigenetic modification.

AFB₁ and multi-omics analysis

Transcriptomic analyses have helped elucidate the mechanisms underlying the pathological effects of AFB₁. A total of 1,452 differentially expressed genes (DEGs) have been identified in the liver tissues of AFB₁-treated versus healthy mice. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the DEGs were enriched in functions such as cell adhesion, cell proliferation and cell cycle regulation (Xu et al., 2001). In addition, several lncRNAs involved in the regulation of genes related to apoptosis and DNA repair were upregulated following AFB₁ exposure (Shi et al., 2016). Transcriptomic analysis of AFB₁-exposed rat tissues indicated that the genes affected by AFB₁ were mainly enriched in the p53 signaling pathway, bladder cancer-related signaling pathways, inflammatory response, antioxidant response, cell proliferation, and DNA repair. Metabolomic analysis showed that AFB₁ dysregulated gluconeogenesis and lipid metabolism (Lu et al., 2013). AFB₁ also induced transcriptomic changes in the genes related to cancer development, apoptosis, inflammation, biological activation, and detoxification in the bovine fetal hepatocyte-derived cell line (BFH12) (Pauletto et al., 2020). Finally, the transcriptomes of AFB₁-exposed laying hens showed an upregulation of genes involved in hepatic fat deposition and hepatocyte apoptosis, including those related to the mTOR, FoxO, PPAR, fatty acid degradation and fatty acid metabolism pathways (Liu et al., 2020).

AFB₁ and the encoding genes

AFB₁ can alter the expression levels of oncogenes (such as ras and c-fos) and tumor suppressor genes (such as p53 and survivin) (Su et al., 2004; Duan et al., 2005), and induce genomic instability and mutations by forming DNA adducts, inhibiting DNA repair enzymes and increasing ROS production. It is bio-transformed to AFBO via cytochrome p450 enzymes, which then forms the carcinogenic adducts. In fact, the p53 gene is mutated in the majority of AFB₁ induced HCC cases (Engin & Engin, 2019). In a recent study, transcriptomics and functional genomics identified p53 as the critical transcription factor driving the DNA damage response after exposure to benzo (A) pyrene and AFB₁ (Smit et al., 2017).

The transcription factor Nrf2 regulates antioxidant/stress response genes and detoxification genes, and Nrf2 knockout rats are highly sensitive to AFB₁ (Taguchi et al., 2016). Primary broiler hepatocytes (PBHs) and broiler cardiomyocytes (BCMs) showed a significant decrease in viability, increased mitochondrial dysfunction, ROS generation and high apoptosis rates following AFB₁ treatment, all of which are mediated by the Nrf2 pathway (Liu & Wang, 2016; Wang et al., 2017).

Caveolin-1 (CAV1) is a key mediator of AFB₁-induced hepatotoxicity. The human hepatocyte L02 cell line showed a marked decline in viability due to increased apoptosis and oxidative stress after AFB₁ exposure, which was accompanied by increased expression of CAV1. The latter mediates AFB₁-induced oxidative stress through its interaction with Nrf2, leading to the downregulation of cellular antioxidant enzymes and activation of apoptotic pathways. In addition, CAV1 regulates AFB₁-induced autophagy via the EGFR/PI3K-AKT/mTOR signaling pathway. Taken together, CAV1 plays a crucial role in AFB₁-induced hepatocyte apoptosis by regulating oxidative stress and autophagy, and is therefore a potential therapeutic target against AFB₁-related hepatotoxicity (Xu et al., 2020).

AFB₁ induces COX2 expression, promotes mitochondrial autophagy and impairs mitochondrial lipid metabolism in hepatocytes, leading to hepatic steatosis (Ren, Han & Meng, 2020). In addition, AFB₁ can induce apoptosis and trigger an “eicosanoid and cytokine storm” in the liver, which can initiate tumor growth. Furthermore, AFB₁-generated cellular debris can upregulate COX2, soluble epoxide hydrolase (sEH) and ER stress-response genes (BiP, CHOP and PDI) in macrophages (Fishbein et al., 2020). Meanwhile, the expression of COX2 during pregnancy is crucial, and exposure to AFB₁ may induce physiological changes controlled by COX2 (Zhu, Tan & Leung, 2016).

AFB₁ and non-coding RNAs

Non-coding RNAs play regulatory roles in mediating the toxic effects of AFB₁. For instance, miR-429 and miR-24 are up-regulated in liver cancer tissues exposed to AFB₁, and correlate to tumor size. Overexpression of both increased AFB₁-DNA adducts in the cancer cells, inhibited apoptosis and promoted their proliferation. Thus, miR-429 and miR-24 are reliable biomarkers of AFB₁-induced liver cancer (Huang et al., 2013; Liu et al., 2014). Other miRNAs significantly associated with HCC include hsa-miR-96-5p, hsa-miR-30a-3p, hsa-miR-34a-5p, hsa-miR-34b-5p, hsa-miR-222a-3p, hsa-miR-199a-3p and hsa-miR-4286 (Zhang et al., 2019b). Ectopic expression of miR-34a-5p in liver tumor-bearing rats led to cell cycle arrest by inhibiting MET, CCNE2 and CCND1, and promoted repair of AFB₁-induced DNA damage in the liver (Liu et al., 2015b). High expression of miR-33a and miR-34a down-regulated the Wnt/β-catenin signaling pathway in AFB₁-stimulated HCC cells (Fang et al., 2013; Zhu et al., 2015). In addition, overexpression of lncRNA H19 promoted HCC cell proliferation and infiltration (Lv et al., 2014). MiR-138-1* inhibits the proliferation, colony formation, migration, and invasion of P50 B-2A13 cells (immortalized human bronchial epithelial cells stably expressing CYP2A13), and mediates AFB₁-induced malignant transformation by targeting 3-phosphoinositide-dependent protein kinase-1 (PDK1) (Wang et al., 2016b). MiRNA-429, miRNA-24, miRNA-122, miRNA-33a, miRNA-34a-5p, miRNA-300b-3p, miRNA-138-1 and miRNA-34a regulate AFB₁-induced tumorigenesis via the GSK-3b–C/EBPa–miR-122–IGF-1R regulatory circuitry, p53 DNA repair axis, and the Wnt/β-catenin signaling pathway (Dai et al., 2017). AFB₁-induced fat accumulation and apoptosis in the liver is regulated by the network of lncRNAs, miRNAs and protein-coding genes. Co-expression of lncRNA (TU45776), gga-miR-190a-3p and Bcl-6 gene induce apoptosis in liver cells, whereas co-expression of lncRNA (TU10057), gga-miR-301a-3p, gga-miR-301b-3p and PPARG gene cause fatty liver (Liu et al., 2020). Taken together, AFB₁ regulates the expression of coding genes and non-coding RNAs involved in cell proliferation, apoptosis, and carcinogenesis (Fig. 3).