Aflatoxins: Toxicity, Occurrences and Chronic Exposure

Abstract

Aflatoxins, a carcinogenic group of mycotoxins, are naturally occurring toxic fungi that cause illness in both animals and humans. Predominantly found in hot and humid areas, aflatoxins are generated by Aspergillus fungi and are found in a large percentage of the world’s food supply. Aflatoxin B1 (AFB1), being the most potent of the over 18 aflatoxins discovered, is most noted for its role in the development of hepatocellular carcinoma (HCC) in humans and animals, unfortunately, many features of the illnesses it causes and the mechanisms that produce them, remain unexplained. This review examined AFB1 metabolism; its epoxidation and DNA adduction, its correlations to cancer initiation and the mechanisms that underpin it, the synergistic interactions with stunted growth associated with AFB1 intake and kwashiorkor, involvement of oxidative stress and reactive oxygen species. Its harmful effects, including growth retardation, starvation, and immunotoxicity, were also discussed, delving into new findings of AFB1 contamination in worldwide food sources. This review indicated that AFB1 is commonly found in high concentrations in food supplies, notably in maize. To lessen the global burden of AFB1 toxicity, data gathered through this review emphasized the necessity to apply novel and existing techniques to prevent these toxins on other diseases.

1. Introduction

Mycotoxins are unrelated structural secondary metabolites produced by various molds, most of which belong to one of three genera; Aspergillus, Penicillium, or Fusarium [1]. There are two kinds of mycotoxin-producing fungi; field fungi, such as Fusarium species, produce mycotoxins on crops that are still growing, and storage fungi, such as Aspergillus and Penicillium species which produce mycotoxins after the crops have been harvested [2].

Aflatoxins, and being widespread in many human foods, are the biggest threat to food safety. Aflatoxins, since their discovery, have been found to be highly toxic to humans; being linked to liver cancer in humans for instance. Being common in agricultural products, such as maize, groundnut, millets, and their derivatives serving as the primary carriers, aflatoxins are prevalent in tropical and subtropical countries, posing high risks as food and feed pollutants, resulting in an excessive frequency of several deadly chronic diseases and aflatoxicosis epidemics [3].

2. Occurrence in food

The FAO’s forecast for world cereal production in 2021 has been reduced by 2.1 million tonnes since November, to 2 791 million tonnes, still 0.7 percent (19.2 million tonnes) higher than the previous year’s output and a new high. However, the forecast for world coarse grains production remains 1.4 percent higher on an annual basis. In the case of wheat, recent reports from Brazil and the United Kingdom of Great Britain and Northern Ireland indicating smaller harvests than expected have resulted in a slightly lower global production forecast, now pegged at 769.6 million tonnes, reinforcing an expected 1 percent year-over-year decline. In the case of rice, government estimates in Pakistan show that a record crop was harvested this season, defying FAO predictions of a minor decline in output due to water restrictions in several areas. This change, when combined with a yield-based increase for the United States of America, compensates for somewhat lower output projections in Thailand [4]. These figures demonstrate the significance of cereals and other grains in the world food supply. AFB1, on the other hand, is known to infect cereals and grains products, as well as other commonly consumed foods like groundnuts, dried fruits, and spices. The studies that found AFB1 contamination in these commodities are listed below.

2.1 Maize

Maize has the highest observed concentrations of AFB1. In Croatia, Pakistan, and the Democratic Republic of Congo, maximum AFB1 levels >1000 g/kg were discovered (2072, 1405.3, and 1401.45 μg/kg, respectively). For the investigation in the Democratic Republic of Congo, samples were evaluated after harvest, during transportation, and lastly at the market. The incidence of AFB1 infection increased considerably between freshly harvested maize (32%), and market samples (100%) [5]. Both examinations that used Pakistani samples discovered high levels of contamination. Maximum values of 1405.3 μg/kg were discovered in Lahore, Pakistan, while maximum levels of 409.3 μg/kg were discovered in Punjab, Pakistan [6]. The maize samples were collected during an unusually hot and dry season, according to the Croatian study, which could explain why the levels were so high (2072 μg /kg) [7]. In investigations into maize contamination, 46.1% of samples tested positive for AFB1, with an average maximum concentration of 553.9 μg/kg [8].

2.2 Rice

AFB1 contamination in rice has recently been identified in various Asian nations. Aflatoxin-producing fungus and aflatoxins were discovered in 187 rice samples in a Brazilian analysis. In these samples, 383 Aspergillus fungus strains were found, with 17% of those strains capable of producing type B aflatoxins. AFB1 contamination was discovered in 14% of rice samples, with AFB1 levels ranging from 0 to 63.32 μg/kg [9]. 230 rice samples were collected from various locations in Brazil during an outbreak in 2007–2009, according to another study. Many samples were contaminated with mycotoxins like ochratoxin A, deoxynivalenol, and zearalenone, and up to 180.74 g/kg of AFB1 was found [10]. AFB1 was detected in 56 of the 199 rice samples examined in Canada, with concentrations ranging from 7.1 to 7.1 μg/kg. AFB1 levels in Chinese rice ranged from 0.1 to 136.8 g/kg, with fumonisin B1 infection found in several of the samples. Ecuadorian rice samples had amounts as high as 47.4 grams per kilogram, while Iran and India reported levels as high as 6.3 and 308 grams per kilogram, respectively [11]. Three distinct investigations on rice contamination with AFB1 were conducted in Pakistan. AFB1 contamination was discovered in 35 percent, 52 percent, and 95.4 percent of the samples, with maximum AFB1 levels of 21.3, 32.9, and 24.54 μg/kg, respectively, in these three studies. Rice samples from Sweden and Malaysia were also found to be contaminated with AFB1 [11].

2.3 Wheat/Sorghum/Cereals

In a few recent studies, AFB1 has been discovered in wheat, sorghum, and cereals. Even though it was only evaluated in a few trials, sorghum had the highest average frequency of AFB1 contamination (67.3%) and the second-highest average maximum concentration (83.6 μg/kg) of all food products [12]. Wheat had the lowest average maximum concentration of 6.0 μg/kg, although having the highest AFB1 contamination rate (44.8%) [13].

2.4 Groundnuts

The contamination of groundnuts with AFB1 has been studied. Peanuts are the most commonly contaminated groundnuts; however, pistachios and hazelnuts have also been discovered to be contaminated. AFB1 was found in ten of twenty-one peanut butter samples in a Japanese study, albeit the amounts were not higher than 2.59 μg/kg. Aflatoxin contamination was not found in unprocessed peanut samples, which were also analyzed in the study [11]. The occurrence of AFB1 was investigated in three different areas of China in one study. AFB1 contamination was discovered in 100 percent of the peanut samples tested in this study, however at modest levels (up to 0.7 g/kg). Malaysia has also identified peanut contamination, with levels as high as 15.33 μg/kg. Groundnut contamination was investigated in Burkina Faso and Mozambique in a study. Burkina Faso had moderate AFB1 levels of up to 15.5 μg/kg, whilst Mozambique had high AFB1 levels of up to 123 μg/kg [11]. Turkey’s groundnuts were the topic of two separate studies in 2014 and 2016. Contamination was discovered in 16.9% of the 302 samples tested in the previous study, with AFB1 levels ranging from 0.16 to 368 μg/kg. In the latter study, only 6.5 percent of the 170 samples tested positive for AFB1 contamination, with values ranging from 0.09 to 10.6 μg/kg [14]. Thailand samples revealed a low percentage of groundnut contamination (9%), whereas Zimbabwe samples had high AFB1 levels of up to 175.9 g/kg, although having a low degree of contamination (12.5%) [15].

2.5 Fruits/spices

Although fruits and spices have been studied, they are not a source of AFB1 exposure. Spices (cumin, black pepper, and chili pods/powder) had the second-highest average frequency of contamination (64.4%), as well as the highest average maximum AFB1 concentration (25.4 μg/kg) [11]. Dried fruits (such as figs, raisins, currants, sultanas, plums, dates, and apricots) had the second-lowest frequency (36.0%) and average maximum value of 16.3 μg/kg [16].

2.6 Reports of AFB1 occurrence in food commodities

Table for rice

Country	Total number of samples	Total number of positive AFB1	Mininum AFB1 levels (μg/kg)	Maximum AFB1 levels (μg/kg)	Positive percentage for AFB1	References
Brazil	187	21	Null	63.32	11.2	[10]
Pakistan	208	73	0.04	21.4	35.1	[17]
Pakistan	1027	189	1.1	32.9	18.4	[18]
Ecuador	43	3	4.9	47.4	7.0	[19]

Table for maize

Country	Total number of samples	Total number of positive AFB1	Minimum AFB1 levels	Maximum AFB1 levels	Positive percentage for AFB1	References
Democratic Republic of Congo	50 (harvest)	16	1.5	51.23	32.0	[5]
South Africa	114	15	1	133	13.2	[20]
Tanzania	Null	Null	0.53	364	29.0	[21]
Pakistan	75	73	0.5	409.3	97.3	[6]
Croatia	633	241	1.1	2072	38.1	[7]
Pakistan	100	52	2	1405.3	52.0	[18]
Mozambique	13	6	16.3	363	46.2	[22]
Burkina faso	26	13	3.4	636	50	[22]

Table for wheat

Country	Total number of samples	Total number of positive AFB1	Minimum AFB1 levels	Maximum AFB1 levels	Positive percentage for AFB1	References
China	178	11	0.03	0.12	6.2	[12]

Table for cereal

Country	Total number of samples	Total number of positive AFB1	Minimum AFB1 levels	Maximum AFB1 levels	Positive percentage for AFB1	References
Pakistan	237	98	0.04	6.9	41.1	[17]
Spain, Italy, Morocco, Tunisia	173	14	6.4	66.7	8.1	[11]

Table for Sorghum

Country	Total number of samples	Total number of positive AFB1	Minimum AFB1 levels	Maximum AFB1 levels	Positive percentage for AFB1	References
India	1606	1173	0.01	263.98	73.0	[23]
Sudan	60	17	0.06	12.29	28.3	[11]
Ethiopia	90	85	null	33.1	94.4	[13]

Table for nuts

Country	Total number of samples	Total number of positive AFB1	Minimum AFB1 levels	Maximum AFB1 levels	Positive percentage for AFB1	References
Zimbabwe	208	26	0.7	175.9	12.5	[15]
Mozambique	23	3	3.4	123	13.0	[22]
Turkey	170	11	0.09	10.6	6.5	[14]
Pakistan	180	83	0.04	14.5	46.1	[24]
Turkey	302	51	0.16	368	16.9	[25]
Thailand	25	9	0.04	4.74	36.0	[26]
Burkina Faso	9	2	5.6	15.5	22.2

Table for spice

Country	Total number of samples	Total number of positive AFB1	Minimum AFB1 levels	Maximum AFB1 levels	Positive percentage for AFB1	References
Thailand	60	40	0.1	53.62	66.7	[26]
Italy	130	11	0.08	>15	8.5	[27]
Malaysia	58	49	0.01	28.43	84.5	[28]

Table for dried fruits

Country	Total number of samples	Total number of positive AFB1	Minimum AFB1 levels	Maximum AFB1 levels	Positive percentage for AFB1	References
Iran	88	50	0.3	8.4	56.8	[16]
Turkey	130	16	0.1	12.5	12.3	[14]
Pakistan	77	33	0.04	9.8	42.9	[24]
Greece	26	6	null	<2	23.1	[29]

3. Toxicology

Aflatoxins are a set of compounds that are very similar with tiny molecular differences. Aflatoxin B1 is the most prevalent and powerful of these poisons (AFB1). In mammals, the toxin is processed by various distinct pathways [30] . The fraction of the dose routed into the several possible pathways defines aflatoxin sensitivity across human and animal species, with harmful “biological” exposure the result of epoxide activation and epoxide interaction with proteins and DNA. There’s also evidence that dosage affects the fractions that follow different possible pathways, maybe due to the saturation of the most chemically competitive processes [31]. The youth are more susceptible to Aflatoxin, and there are considerable differences across species, individuals within the same species (based on their ability to detoxify aflatoxin via biochemical mechanisms), and the sexes (according to the concentrations of testosterone). The variation in aflatoxin toxicity depends on the difference in nutritional parameters because aflatoxin exposure slows recovery from protein deficiency [32]. Aflatoxism is the term for the toxicity induced by aflatoxins. Two forms of aflatoxicosis have been identified: acute severe intoxication, which causes immediate liver damage and eventually illness or death, and chronic sub-symptomatic exposure. The dose and duration of aflatoxin exposure have a major impact on toxicity and can result in a variety of outcomes, according to a review of the literature across all species: Large doses cause acute sickness and death, primarily owing to liver cirrhosis; chronic sub-lethal doses create nutritional and immunologic issues; and all doses raise cancer risk [3] (Figure 1).

3.1 Mechanisms of toxicity

The majority of aflatoxins’ toxicological implications and mechanisms remain unknown. An extensive study of the causes of aflatoxins’ toxicity was done to provide a scientific foundation for the development of preventive and control strategies. Authorities in charge of food safety might use a deep grasp of the subject as a scientific tool to attain regulatory objectives. The majority of study on AFB1 has focused on its mutagenesis capabilities, which have been related to the AFB1-exo-8,9 epoxide since its discovery as an intermediate metabolite (AFBO) [3]. AFBO mixes with biological macromolecules as nucleic acids, proteins, and phospholipids to affect genetic, metabolic, signaling, and cell structure [33]. However, new evidence is developing that AFB1 causing oxidative stress (OS) has an equal or higher influence on cell function and integrity [34]. Figure 2 summarizes the AFB1 toxicity pathways that operate on genomic DNA, other functional macromolecules, and immunocompetent cells to generate genotoxicity, immunotoxicity, and acute intoxication.

Figure 1.

Aflatoxins have a metabolic route that involves protein binding (toxicity) or DNA reactions (cancers). Hydroxylated metabolites, such as aflatoxin M1, GSH, glutathione, and the epoxide, are some of the hazardous secondary products of this system.

Figure 2.

Main aflatoxin B1 toxicity mechanisms are mediated by oxidative stress and AFB1-exo-8,9 epoxide. NB: ROS also affects proteins, RNA molecules, and immunity as does AFBO. Abbreviations: AFBO: Aflatoxin B1-exo-8,9-epoxide; NA: Nucleic acids; ROS: Reactive oxygen species; LPO: Lipid peroxidation; ODD: Oxidative DNA damage; Acr: Acrolein; Cro: Crotonaldehyde; Acet: Acetaldehyde; HNE: 4-Hydroxy-2-Nonenal; uFA: Unsaturated fatty acids; IL1β: Interleukin 1β, IL6: Interleukin 6; TNFα: Tumor necrotizing factor α; P-dG: Cyclic Propano-Deoxyguanosine; Igs: Immunoglobulins [3].

3.2 Aflatoxins lead to other chronic infections

Chronic ailments result from a lifetime of low-dose aflatoxins exposure, the most prevalent and deadly of which is cancer. While aflatoxins have long been linked to primary liver malignancies including HCC and bile duct hyperplasia, they have also been linked to cancers of the kidney, pancreas, bladder, bone, and viscera [3]. Again, aflatoxins have been linked to lung and skin malignancies in workers who breathe them or come into close contact with them. Immunosuppression, teratogenicity, mutagenicity, cytotoxicity, and estrogenic effects are induced in mammals due to long-term exposure to aflatoxins. Aflatoxins have also been thought to contribute to childhood diseases such as kwashiorkor and growth failure by interfering with micronutrient absorption, protein synthesis, and metabolic enzyme performance [3].

3.3 Acute toxicity

Although the cause of acute aflatoxicosis is unknown, when aflatoxins interrelate with large biological molecules such as proteins, phospholipids, and nucleic acids, they form various adducts that interfere with the physiological and structural functions of these biological molecules. Aflatoxin-protein adducts have been related to acute intoxication because they inhibit protein synthesis, particularly enzymes implicated in essential functions such as metabolic pathways, protein synthesis, DNA replication and repair, and immunological response. There is a growing body of evidence that cell, mitochondrial, and endoplasmic reticulum membrane disruption is due to aflatoxin-phospholipid adducts and ROS-induced LPO [33]. As reported by a scientific study on AFB1’s acute toxicity in chicken birds, aflatoxin–dihydrodiol (AF–dhd) is the main metabolite responsible for acute aflatoxicosis since it is the important metabolite that leads to the formation of aflatoxin–albumin adducts [35]. AFB2a has shown a covalent association with cellular proteins and phospholipids, resulting in the linkage of long-chain fatty acids and protein adducts, which may lead to acute aflatoxicosis [33]. Long-term exposure to low levels of aflatoxins, on the other hand, can cause symptoms similar to acute aflatoxicosis; however, as previously mentioned, these symptoms can be mitigated by the removal of harmful substances by phase II enzymes and cellular absorption of free radicals, as well as DNA repair to prevent mutations. Alternatively, these effects may build over time with repeated low-dose exposure, eventually leading to liver cancer as a common side effect [3]. When the dose is excessively high, a rapid rise in a short time might cause acute aflatoxicosis. Excessive amounts of aflatoxins can overwhelm the cell’s detoxification capacity, driving the toxins’ metabolism toward the production of toxic metabolites, resulting in severe DNA damage, cell growth disruption, asexual cloning by the DNA, metabolic disorders, cytotoxicity, and tissue necrosis, eventually leading to organ failure in a short time. This is especially important because the harmful effects of aflatoxin accumulate over time (Colakoglu and Donmez, 2012), which could lead to more devastating situations than cancers that have been more established.

3.4 Cancers caused by prolonged aflatoxin exposure

Aflatoxin has been speculated to cause liver cancer in humans, but it can also cause lung cancer in people who work with infected crops. Mutations in the tumor-suppressing gene P53, as well as the activation of dominant oncogenes, induce hepatomas [37]. The cancer risk from aflatoxin exposure has been well documented and is based on a lifetime dose [38]. The International Cancer Research Institute has categorized aflatoxin as a Class 1 carcinogen, resulting in its regulation to very low levels in traded commodities (20 ppb in grains and 0.5 ppb in milk in the United States; 4 ppb in foods in several European nations) [37]. Hepatitis B and C virus (HBV/HCV) outbreaks, on the other hand, affect roughly 20% of the population in several poor countries, appearing to have a good synergy with these biological agents for liver cancer. Aflatoxin is 30 times more potent in people with hepatitis B surface antigen than in people without the virus, and when HBV infection and aflatoxin exposure are coupled, the relative risk of cancer in HBV patients climbs from 5 to 60 [18]. In some areas where aflatoxin contamination and HBV coexist, hepatomas are the most common malignancy (64 percent of malignancies; 25) and may be the primary cause of mortality.

Aflatoxin B1 is expected to cause between 25,200 and 155,000 cases of liver cancer per year, with 40% of cases occurring in Sub-Saharan Africa, where aflatoxin-induced liver cancer accounts for one-third of all liver cancer occurrences [39]. Aflatoxin B1 is expected to cause between 25,200 and 155,000 cases of liver cancer per year, with 40% of cases occurring in Sub-Saharan Africa, where aflatoxin-induced liver cancer accounts for one-third of all liver cancer occurrences [40].

3.5 Teratogenicity

Aflatoxin exposure in pregnant women or birds can affect unfertilized eggs or embryos in utero, resulting in a variety of poor health effects and abnormal gestation/incubation outcomes [41]. Aflatoxin or its metabolites are transmitted to the infant during pregnancy and processed using the same mechanisms as adults [42]. In pregnant women, it has been demonstrated by scientific kinds of literature that, aflatoxins can be transferred from mothers to offspring through blood circulations. In fetal cord blood and maternal blood samples, aflatoxin metabolites, aflatoxin-DNA, and aflatoxin–albumin adducts, as well as biomarkers derived from them, were found [42]. As a result, fetal growth restriction, fetal loss, or premature birth may occur in significantly exposed mothers’ pregnancies. An adverse association between birth weight and the levels of suitable biomarkers in the cord blood has been extensively documented in people and animals when growth restriction is present [43]. However, little research has shown excess aflatoxin accumulations by pregnant women to stillbirth, and research on the link between excess aflatoxin consumption by pregnant women and premature birth and fetal loss is confusing or contradictory [44]. Furthermore, an enriched aflatoxin diet harms pregnant women’s state of complete physical, mental and social well-being and exposes their fetuses to congenital defects as a result of indirect impacts. Increased systemic inflammation, for example, is caused by overexpression of maternal pro-inflammatory cytokines and/or downregulation of anti-inflammatory cytokines, which affects and causes placental insufficiency, resulting in poor fetal growth, miscarriage, stillbirth, or premature birth [41]. Anemia and high aflatoxin intake were found to be linked in a cross-sectional study of Ghanaian women, as evaluated by the AFB-albumin adduct in the mothers’ serum [45]. However, there is no evidence of a relationship between aflatoxins exposure and inflammation-induced anemia in pregnant women [3].

3.6 Genotoxicity caused by oxidative stress

Although the creation of aflatoxin-N7-gua DNA adducts has been attributed to the majority of aflatoxins’ mutagenicity, it is becoming clear that oxidative stress (OS) created by AFB1 metabolism is also a role [46]. The OS can cause oxidative DNA damage (ODD) either directly on DNA or indirectly through membrane phospholipid lipid peroxidation by-products (LPO). OS is caused by the release of large amounts of reactive oxygen species (ROS) from the breakdown of AFB1 by CYP450 enzymes in the liver, which can damage DNA’s nitrogen bases and deoxyribose moieties, resulting in in in over 100 distinct DNA adducts [3]. The most well-known and examined of these adducts is 7,8-dihydro-8-oxo-20-deoxyguanosine (8-hydroxydeoxyguanosine, 8-oxo-dG, 8-OH-dG), which is commonly employed as a biomarker for oxidative DNA damage [3]. Intraperitoneal injection of AFB1 into rats elevated 8-oxo-dG levels in the liver in a dose- and time-dependent manner, which was avoided by pre-treatment of animals with the antioxidants selenium and deferoxamine, establishing the relationship between the adduct and Aflatoxin-induced oxidative stress [3]. latest scientific work found no notable increase in seven ROS-modified bases in the liver tissues of rats treated with 7.5 mg/kg AFB1, including 8-oxo-dG, when compared to control rats (untreated); however, levels of 8,50 -cyclo-20 -deoxyadenosine, another DNA adduct from the oxidative attack of the adenine base, increased significantly [47]. By organisms, organs, tissue, sub-cellular component, and cell cycle, the quantity of oxidative DNA damage, the kind of adduct produced, and the effectiveness and speed of DNA repair have all been found to differ [3]. AFG1 increased the expression of tumor necrosis factor (TNF)- and CYP2A13 in mouse alveolar type II (AT-II) cells of lung tissues, as well as in vitro in human AT-II-like cells (A549), which mediate inflammation by increasing the number of -H2AX- and 8-OHdG-positive cells in inflamed tissues, according to a recent scientific study [48]. The inflammatory response generated by TNF increases the expression of CYP2A13, which keeps AFG1 active and causes ODD, as seen by increased expression of the DNA damage marker -H2AX. GT transversion mutations are caused by 8-oxo-dG lesions, which are similar to AFBO-derived DNA adducts but do not pick out the p53 gene and necessitate the use of additional processes and DNA polymerases [35].

3.7 Aflatoxins and the immune system’s relationship

Reduced vaccination efficacy, evidence of aflatoxins affecting both innate and acquired/adaptive immunity was found to have an increased incidence and severity of infectious infections, as well as prolonged healing times [3]. According to a study, AFB1 immunotoxicity is mediated by AFBO, as well as interchanging with immunocompetent cells throughout the body, altering their fast growth and/or the manufacture of immune reaction mediators, disrupting innate and adaptive immunity. Although these mechanisms were established using animal studies, AFB1’s immunotoxicity has also been confirmed in vitro on human cell lines and in case–control studies in heavily exposed areas such as Ghana [3, 49]. In rats, a ten-fold higher dose of 1 mg AFB1/kg bw increased the number of CD8+ (cytotoxic T cells) while not affecting other immunological markers [3]. Other scientific studies, on the other hand, have demonstrated that the immune response can be altered even at low levels of aflatoxins and shorter exposure times. For example, rats were fed a portion of food that contain about 5 to 75 g AFB1/kg bw for five weeks [50], Other research, on the other hand, has demonstrated that the immune response can be altered even at low levels of aflatoxins and shorter exposure intervals. For five weeks, rats were fed a diet with 5 to 75 g AFB1/kg bw [51]. Although the preponderance of evidence suggests that aflatoxins mostly impair immune function, in vitro and in vivo investigations have revealed that they can also dysregulate immune responses through immunostimulatory effects [52].

3.8 The link between aflatoxins and innate immunity

In vivo and in vitro, structurally barriers example: skin and intestinal epithelial cells are damaged, causing the weakened structure–function against microbial and toxin intrusions. The production of intra-epidermal vesicles and squamous cell carcinoma have been connected to contact with the skin of a variety of animals [53]. Pigs fed an aflatoxins-contaminated diet for 28 days exhibited crusting and skin ulceration on their snouts, lips, and buccal commissures (AFB1, AFB2, AFG1, and AFG2) [3]. Aflatoxins have been shown to impair the intestine’s mechanical barrier by interrupting cell cycle development or damaging intestinal epithelial cells and the tight junctions that hold them together in scientific review studies. Broilers fed 0.6 mg AFB1/kg food for exactly 3 weeks had their cell growth interrupted at the G2/M phase, leading to a decrease in jejunum height and a decrease in the villus height/crypt ratio, jeopardizing their function as a selective barrier [54]. The mechanical, chemical, and immunological barriers that protect the gut mucosa from external assaults at the molecular level are affected by aflatoxins. The CacO-2 human cell line was treated in vitro with 1–100 M AFB1 for 48 hours, which decreased trans-epithelial electrical resistance (TEER). As a result, paracellular permeability increased and survival decreased [55]. After 48 hours of exposure to varying doses of AFM1, CacO-2 cells’ selective permeability was likewise impaired (0.2 to 20 M) [56]. Cell viability, function, or gene expression of cytokines and chemokines in immune cells like monocytes, macrophages, dendritic cells (DC), and natural killer (NK) cells, all of which play important roles in innate immunity has been shown by a certain secondary metabolite (Flavonoids). TLR-2, TLR-4, and TLR-7 transcription are suppressed in broilers exposed to AFB1, showing a suppressive effect on innate immunity. These receptor proteins have a role in sentinel cells like macrophages and dendritic cells recognizing external invaders, which is a crucial step in initiating an immune response [57]. Human monocytes were pre-treated for 24 hours with as little as 0.1 pg. AFB1/mL before being cultured with Candida albicans for 30 minutes at 37°C [3]. In addition, the aflatoxins AFB1, AFB2, and/or AFM1 have been shown in other studies to reduce macrophage viability, proliferation, cytotoxicity, and phagocytic activity, as well as the expression of cytokines like TNF-, IL-1, and IL-6, and the inducible nitric oxide synthase (iNOS), which mediate intracellular pathogen killing during phagocytosis [3]. When dairy cow neutrophils were given low doses of AFB1 for 18 hours (0.01, 0.05, and 0.5 ng/mL), their phagocytic and cytotoxic capacities against Staphylococcus aureus and Escherichia coli were drastically diminished. This was attributed to the reduction of reactive oxygen species (ROS) in neutrophil cytoplasm, which is important for pathogen killing during phagocytosis [58]. In numerous mammals, aflatoxins blocked the complement system, which is a critical component of innate defense that causes the phagocytosis of dangerous microorganisms [3]. Complement activity was observed to be decreased in cattle and poultry fed at varying threshold levels [59]. After ducklings were fed AFB1 at doses of 0.5 or 0.8 mg/kg feed for 40 days, the APCA was activated for the first 15 days, then suppressed for the remaining days of the study. In contrast, the effect of aflatoxins on the complement system appears to be very reliant on the host, as rabbits fed a 24 mg/kg diet for 28 days showed no significant change in serum hemolytic activity (CH50) [3].

3.9 The link between aflatoxins and adaptive immunity

The decrease of adaptive/acquired immunity that occurs as a result of aflatoxins exposure is well documented, implying that exposed hosts are more susceptible to infectious pathogens and that vaccine protection is reduced or nonexistent [60]. In contrast to a control group fed an aflatoxin-free diet, vaccination failed to protect pigs against Erysipelothrix rhusiopathiae when fed AFB1-contaminated feed [3]. Humans and animals have shown reduced lymphocyte fast growth, activation, and/or function. In adaptive immunity, lymphocytes are the most significant immune cells. Apoptosis was seen in human peripheral blood cells treated at diverse times with different dosages of AFG1 (3.12–2000 g/L) [61]. In vitro treatment of human lymphocytes with AFB1 at concentrations ranging from 5 to 165 uM increased the frequency of apoptotic and necrotic lymphocytes in a dose-dependent manner, with a considerable increase in cell necrosis beginning at 50 uM (15.6 mg/L) after 24 hours [62]. T-cell proliferation was decreased in a dose-dependent manner starting at 15 M in vitro culture of the human lymphoblastoid Jurkat T-cell line with AFB1 or AFM1 at 3-50 M concentrations range for 72 hours, but no apoptosis or necrosis was seen [63]. When compared to negative control cells cultivated in the absence of aflatoxins, AFB1 and AFM1 dramatically enhanced the expression of IL-8, a cytokine implicated in innate immunity, while adaptive immunity was unaffected, as seen by unchanged levels of interferon (INF)- and IL-2 cytokine [3]. AFB1 and AFM1 significantly increased the expression of IL-8, a cytokine implicated in innate immunity, when compared to negative control cells cultivated in the absence of aflatoxins, while adaptive immunity was unaffected, as seen by unchanged levels of interferon (INF)- and IL-2 cytokine [64]. The suppression of adaptive CMI has been researched in lab animals such as chickens and rats, with results showing a decrease in the amount of distinct T-cell lymphocyte subsets as well as the cytokines they release, both of which are important components of this form of the immune response. Reduced delayed-type hypersensitivity (DTH) in a variety of species, including chicken and rats, at doses ranging from 0.3 to 1.0 mg/kg feed, supported adaptive CMI suppression by aflatoxins, meaning a reduction in the frequency of adaptive CMI cases [65]. Rats given AFB1 dosages ranging from 5 to 75 g/kg bw for five weeks had decreased proliferation and cytokine production in splenic helper T cells (CD4+) engaging in acquired cellular immunity. In laboratory animals, adaptive CMI has been studied [50]. AFM1 decreased DTH and related T lymphocyte subsets (CD3+, CD4+, CD8+, CD19+, and CD49 b), as well as the interleukins they release, such as INF-, IL-10, and IL-4, in mice administered 25 or 50 g/kg bw intraperitoneally [49]. A decrease in CD3+ and CD19+ lymphocyte subsets bearing the D69 activation marker (i.e., CD3 + CD69+ and CD19 + CD69+), as well as CD8+ T-cells, which play a key role in vaccination and immune response against pathogens, was highly correlated with high levels of AFB1, as measured by the concentrations of AFB1-albumin adduct in the serum [66].

3.10 Aflatoxins, malnutrition, and neurodegenerative diseases are linked

Aflatoxins have been linked to a variety of diseases, each with its own set of processes and risk factors. Malnutrition problems include malnutrition (faltering and stunting), physical and mental maturation issues, reproductive and sexuality troubles, and nervous system abnormalities, among others (neurodegenerative diseases and neuroblastoma) [67, 68]. Chronic aflatoxins exposure has been related to neurological illnesses, according to a growing body of scientific evidence. In neuronal brain cells, oxidative stress caused by aflatoxins, as well as AFBO and ROS produced by CYP450 enzymes, react with functional macromolecules, restricting lipid and protein synthesis and causing degeneration [69]. Aflatoxins have also been shown to disrupt the structure and function of mitochondria in brain cells, causing oxidative phosphorylation to be inhibited and cell [70]. As with vitamins A, C, and E, aflatoxin interferes with vitamin and mineral absorption, worsening low nutritional status, and selenium deficiency inhibits children’s growth [71]. As a result, children exposed during pregnancy may develop growth abnormalities that remain throughout adulthood, including stunted and delayed physical and mental maturation [72].

3.11 Aflatoxin and kwashiorkor investigations in the past

A possible link between aflatoxin exposure and childhood kwashiorkor, a disorder characterized by the protein-energy shortage, was debated decades ago. Kwashiorkor and marasmus (another malnutrition-related childhood disease prevalent in impoverished countries) are both severe malnutrition diseases. Although protein deficiency is a fundamental cause of both kwashiorkor and marasmus, one key difference between the two conditions is that kwashiorkor can occur even when the children’s calorie intake is adequate, whereas marasmus can only be caused by low caloric intake [73]. Fatty liver and edema, both frequent kwashiorkor signs, are less likely in children with marasmus. Kwashiorkor’s symptoms include anorexia and light-colored hair and skin [74]. Marasmic kwashiorkor is defined as edema from kwashiorkor combined with wasting from marasmus [75]. According to a scientific study, children with kwashiorkor had higher amounts of aflatoxins or their metabolites in their blood or urine than children with other protein malnutrition-related illnesses such as marasmus. Furthermore, aflatoxins were identified in autopsies of children who died from kwashiorkor in their lungs and livers, but not in their kidneys, but not at statistically significant levels, compared to those who died from other diseases or other forms of malnutrition [76]. Kwashiorkor patients were paired with children who did not show any indications or symptoms of protein-energy deficit. All of the children’s serum and/or urine contained aflatoxins. Although the controls had a higher proportion of urine aflatoxins than the kwashiorkor group, the kwashiorkor group had a much higher serum/urine ratio. Rather than aflatoxin playing a direct role in the production of kwashiorkor, these data could imply that kwashiorkor has decreased liver function, which could lead to abnormalities in aflatoxin metabolism. Indeed, it has been proposed that children with kwashiorkor are more susceptible to the hazards of Aflatoxin in the diet [74].

4. Conclusions

Aflatoxins being very common and highly toxic, pose a great threat to food safety, more research would aid in a better understanding of their toxicity incidence, patterns, and resultant correlations with foods and other illnesses to appropriately address their negative effects on public health and the economy. With the growing prevalence of aflatoxin in developing countries where agroclimatic zones encourage aflatoxin growth in cash crops such as peanut, maize, sorghum, and sunflower; contamination of farm produce in endemic regions continues to be a major impediment to international trade and food security, as it not only affects local populations but also has the potential to spread to other parts of the world by either exporting highly contaminated goods or restricting their marketability, both of which contribute to rising prices and limiting access. Interventions can be made to target the inhibitions of these fungi on the field and in their storage produce if the mechanism of actions is well understood. The data presented through this research aims to delve more into the growing body of evidence associating teratogenicity, immunotoxicity, malnutrition (‘kwashiorkor’), neurological disease, and aflatoxin exposure with respect to cancers. More research is needed to determine the mechanism that connects aflatoxins to the many diseases they cause. The link between Aflatoxin exposure and the immune system reveals that this fugal’s effect is lethal and should be handled with prudence. Furthermore, studies show that aflatoxins impair immune function in humans who are exposed to these natural fungal toxins.