Aflatoxins, Aflatoxin B1, and Their Impact on Poultry

Aflatoxins were first identified in early 1960s and since then have been the most studied mycotoxins. Being mainly produced by certain strains of Aspergillus parasiticus, Aspergillus flavus, and Aspergillus nomius, these mycotoxins are majorly found in agricultural products in tropical and subtropical regions [1–3]. Aflatoxins are often present in feedstuffs and cause some adverse effects, which can range from vomiting, weight loss, and acute necrosis of parenchyma cells to various types of carcinoma and immunosupression in large animals, pets, and poultry birds [4, 5]. Multiple reports are available about aflatoxins, their types, biotransformation, and toxicity in various animal species [6–10], but in avian species, data is scattered and needs to be summarized.

Aflatoxin B1 (AFB1), among the four major types of aflatoxins, is the most toxic and potent carcinogen in humans and animals [11]. AFB1 causes series of pathophysiological changes in an organism such as lower growth rate, malnutrition, silenced immune response, and disturbed gastrointestinal tract. Also, AFB1 can induce various histopathological manifestations of hepatocytes such as proliferation of the bile duct, centrilobular necrosis and fatty degeneration of the hepatocytes, and even hepatoma [12–15]. AFB1 is shown to induce hepatocellular carcinoma in many species of animals including fishes (rainbow trout, sock eye salmon, and guppy), poultry (turkeys, ducks, and geese), non-human primates (rhesus, cynomolgus, African green, and squirrel monkeys), and rodents (rats, mice, and tree shrews) [4, 16]. In poultry, AFB1 mainly affects the liver, kidney, immune organs (spleen, bursa of fabricius, and thymus), and gastrointestinal system. Details of these effects are discussed in the later sections of this review.

Poultry industry is one of the largest, most organized, fastest-growing, and vibrant segments of agro-industries, generating direct and indirect employment and income for millions of people, in developing and underdeveloping countries [17–19]. However, the poultry industry is persistently facing many challenges [20], majorly being the diseases caused by virus, bacteria, protozoa, parasites, and fungus, among which mycotoxin contamination of feed is one of the most important aspects. According to an estimate by the Food and Agriculture Organization (FAO), 25% of the world’s food crops are affected by mycotoxins, and the rate of mycotoxin contamination is likely to increase in line with the trend seen in preceding years [21–25]. A worldwide mycotoxin survey in 2013 revealed that 81% of around 3000 grain and feed samples analyzed had at least one mycotoxin, which was higher than the 10-year average (from 2004 to 2013) of 76% in a total of 25,944 samples. The most notorious mycotoxins are aflatoxins, which often result in low performance in poultry and decreased quality of egg and meat production and then cause significant economic losses [26–28]. In broilers, aflatoxins drastically affect almost all valuable production factors including weight gain, feed intake, and feed conversion ratio (FCR) and induce immunosupression, which is directly related to reduced effectiveness of vaccination programs, increased risk of infectious diseases, and high mortality. In layers, aflatoxins cause the decrease in egg production, egg size, and egg quality.

Biotransformation and Mechanism of AFB1 Toxicity in Poultry

Many controversies exist about the metabolism of AFB1 in avian species [29] and still need extensive research to decipher the exact mechanism of AFB1 biotransformation in poultry species. In general, AFB1 passes through phase I and phase II metabolism. Phase I involves the addition of a small polar group, containing both positive and negative charges by any of the following reactions: epoxidation, hydration, hydroxylation, O-demethylation and reduction. Phase II involves the addition of another substance such as endogenous glutathione to the product of phase I (conjugation) to generate a polar or water-soluble product that can easily be excreted out by the kidneys [5, 6, 30]. Studies have shown that in phase I, AFB1 undergoes microsomal and cytosolic biotransformation in the liver of poultry species [31] where it gets converted to toxic metabolites. Microsomal biotransformation results in AFB1 exo-8,9-epoxide (AFBO), aflatoxin Q1 (AFQ1), aflatoxin B2a (AFB2a), aflatoxin M1 (AFM1), and aflatoxin P1 (AFP1) metabolites, and cytosolic biotransformation leads to formation of aflatoxicol (AFL) metabolite [29]. Studies showed that AFQ1, AFB2a, AFM1, and AFP1 are less toxic in comparison to AFBO [5]. There is a conflict on biological effects of AFL, and it largely remains unknown in humans and animals. Some authors believe it to be less toxic compared to AFBO and to be easily excreted out through conjugation reactions by kidneys [32], while others considered it not the detoxification product but toxic metabolite [29]. Among all metabolites, exo-AFB1-8,9-epoxide (AFBO) is considered as the most toxic metabolite of AFB1, but again, the controversy exists on the specific cytochrome P450 (CYP450) enzyme subfamilies responsible for biotransformation of AFB1 into AFBO. More recently, a series of studies revealed that the avian CYP2A6 ortholog is the main CYP450 enzyme responsible for the bioactivation of AFB1 into AFBO in all poultry species [29, 33, 34]. AFBO can bind with particular cellular compounds (proteins, DNA, and RNA) to influence normal cellular activities and is thought to be responsible for the carcinogenicity and mutagenicity of aflatoxins if not excreted from the body by phase II metabolic reactions [3, 5, 7, 30, 33]. Phase II metabolism of AFB1 involves conjugation of AFB1 with endogenous glutathione by a chemical reaction catalyzed by glutathione S-transferases; the most important family of enzymes, involved in protecting humans and most animals from potentially toxic chemicals such as drugs and carcinogens [35, 36]. Glutathione reduces the toxic effects of AFBO and helps to excrete it out from the body. Some evidences support the fact that it is the conjugation of epoxide by GSH, which is the major rate-limiting factor in species susceptibility to AFB1, rather than the level of P-450-mediated bioactivation [37]. A schematic diagram of cytosolic and microsomal biotransformation of AFB1 in the liver is presented in Fig. 1.

Fig. 1
figure 1

Cytosolic and microsomal biotransformation of AFB1 in liver coupled with mechanism of toxicity and carcinogenicity induction

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After biotransformation of AFB1 by CYP450 enzymes in the liver, DNA binding is the most common mechanism of AFB1 action [38, 39]. As a highly electrophilic intermediate, AFBO has an affinity for DNA, RNA, and proteins, resulting in toxigenicity, carcinogenicity, and mutagenicity [35] (Fig. 1). Another widely reported action mechanism of AFB1 is the induction of ROS formation, which leads to oxidation of DNA bases, causing DNA damage [40]. The toxicity and carcinogenicity of AFB1 are closely associated with the rate at which it is activated and the rate at which AFB1 is detoxified and metabolized at primary and secondary levels.

Systemic Effects of AFB1 in Poultry

Liver

As a major xenobiotic-metabolizing and xenobiotic-detoxifying organ in the body, the liver is mainly affected after the ingestion of AFB1 in poultry species [32, 41]. AFB1 is delivered to the liver through blood circulation after its absorption across the cell membrane. In almost all animal species including birds, AFB1 is hepatotoxic, causing pallor discoloration, enlargement, congestion, and necrosis of the liver, along with proliferation of bile duct and infiltration of mononuclear and heterophilic cells [42, 43]. Decreased activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR), and increased malondialdehyde (MDA) contents have also been reported in the liver of poultry birds while being fed with AFB1 [44]. One study demonstrated that AFB1 (0.1 mg/kg body weight) disturbed mitochondria and caused hepatocyte mitochondrial antioxidant dysfunction in duckling [44]. Another study documented that AFB1 can provoke liver impairment by promoting hepatocyte apoptosis and disturbing cellular enzymatic activities in ducklings administrated with 0.1 mg/kg body weight of AFB1 [45]. From previously discussed reports, it is clear that exposure to AFB1 can alter liver functions on various cellular and molecular levels and provoke undesirable pathological effects in the liver of poultry birds.

Kidney

Second to the liver, the kidney is the most vulnerable organ due to its ability to filter large amount of blood and contribution to the body’s homeostasis by eliminating metabolic waste products [46]. Different studies have shown that AFB1 can disturb the renal functions via increasing the relative weight of kidneys and inducing congestion in renal sinusoids [47, 48]. In Africa, birds exposed to AFB1 developed fatty and hemorrhagic kidney syndrome, characterized by thickening of the glomerular basement membrane, abnormal development of glomerular epithelial cells, degenerative changes in renal tubular cells, congestion, and parenchymal hemorrhage [47, 49]. AFB1 induces the degenerative and necrotic changes in renal tubular epithelium and reduces the glomerular filtration rate, thereby altering the avian renal functions [50–52]. Also, AFB1 causes impairment of blood biochemical parameters, including reduced concentrations of calcium, inorganic phosphate, sodium and potassium, and an increase in urea, creatinine, and uric acid [49, 53, 54]. Chickens fed on AFB1 displayed excessive apoptosis, cell cycle blockage, and cell proliferation in renal cells and increased levels of Bax and caspase-3 messenger RNA (mRNA) [46].

Gastrointestinal Tract

The gastrointestinal tract (GIT) is the main route of entry after the ingestion of AFB1. This mycotoxin affects the GIT in multiple aspects such as changes in gut morphology, digestive ability, activities of digestive enzymes, the intestinal innate immunity, and gut microbiota. There are very less reports available about the effects of AFB1, and the results have been controversial [55, 56]. Some found that AFB1 induced GIT pathological lesions, while others suggested that AFB1 did not induce any lesions of the GIT. This discrepancy is probably the result of different sections of GIT used for histopathological examinations, different exposure times, and the different strains of chickens used in various studies. AFB1 could decrease digestive ability by affecting the active transport of nutrients across the intestinal membranes, and the effect is dose and time dependent. Three-week feeding of 1.25 to 5 mg AFB1/kg diet had no effect on in vitro absorption of glucose and methionine in the intestine of broilers, whereas a higher dose of 10 mg AFB1/kg diet, for more than 1 week, increased both the mediated and diffusion components of glucose and methionine absorption [57]. Few studies support the hypothesis that the decreased nutrient absorption in AFB1-affected broilers is because of the effect of toxin on systemic metabolism and not an effect on digestive functionality [58, 59], which needs further studies. AFB1 also exerts its effects on the GIT by interacting with digestive enzymes; however, controversy exists on this aspect too. AFB1 has not been considered seriously for its effects on the intestinal innate immunity. Limited data related with intestinal health suggests that AFB1 can only moderately affect transepithelial electrical resistance (TEER) during acute exposure to the toxin [60]. A recent study showed that 0.3 mg/kg concentration of AFB1 in chicken feed can induce a decrease in T cell subsets and the mRNA contents of IL-2, IL-6, and TNF-α and also impairs ileum mucosa [61].

Immune System

The immune system is crucial for defense against invading organisms, and AFB1 has shown to evoke and agitate immune responses in a number of studies conducted in past decade [62–64]. Exposure to AFB1 provokes damaging effects on primary and secondary lymphoid tissues of birds including thymus, bursa of fabricius, spleen, and bone marrow. Enough evidences support that consumption of feed containing AFB1 lowers the disease resistance and antibody-mediated function in avian species [31, 65–67]. In poultry, aflatoxin-contaminated feed generates a series of cell-mediated immune responses involving suppressed phagocytic efficiency of the phagocytes, delayed hypersensitivity reactions along with bursal involution, and depletion of cell populations of the thymus [66]. Studies have shown that AFB1 negatively affected bursa and thymus by increasing the expression of caspase-3 and enhancing the apoptotic cell percentage in both immune organs. Furthermore, 0.3 mg/kg AFB1 in the diet can induce histopathological changes, decrease mature lymphocytes, and increase apoptotic percentage of lymphocytes in broiler [68]. Poultry birds have shown decreased relative weight of spleen, histopathological changes, increased splenic apoptotic cell percentage, and decreased activities of GSH-Px, total SOD, GR, and CAT when exposed to AFB1 [69]. Studies also showed that daily dietary exposure of AFB1 could bring down antibody titers to vaccines for different diseases including Newcastle disease, infectious bronchitis, and infectious bursal disease [70, 71].

Effect of AFB1 on Growth, Performance, and Production

A number of studies have focused on negative effects of AFB1 on growth and performance in poultry. Susceptibility variation has been found among different age groups of birds in same species. Young birds are more susceptible and showed more damages as compared to older birds [72]. Lower growth rate, poor nutrient usage, decreased weight gain and egg production, and increased bruising after exposure to feed contaminated with AFB1 have also been shown in many studies [73, 74]. A study [75] indicated that AFB1 in feed can result in decreased growth performance and reduced body weight in duck and proposed that these changes are related to the decreased digestibility of nutrients. Despite of a scattered percentage of decreased body weights in different age groups and at different concentrations of AFB1/kg diet in various reports [74, 76–80], there is a general agreement that dietary AFB1 reduces weight gain and feed intake and decreases the FCR.

Along with producing hepatotoxic, nephrotoxic, gastrointestinal, and immunosuppression effects on liver, kidney, gastrointestinal, and immune systems, respectively, AFB1 could induce adverse effects in other systems such as cardiovascular, nervous, respiratory, and endocrine systems as well. However, most of the studies about negative effect of AFB1 on these systems were carried out on domestic animals and rodents, and deep understanding of AFB1- produced effects on these systems in different poultry species calls for further investigation.

Detoxification of Aflatoxins with Nutritional Supplements

A successful detoxification process must be economical and capable of eliminating maximum traces of toxin without leaving harmful residues and without impairing the nutritional quality of the commodity. As a result, more efforts are focused on finding effective means of biological degradation of aflatoxins. Many detoxification methods of aflatoxin-contaminated feedstuffs are recommended over last two decades and include various physical (mechanical separation of contaminated seeds and heat treatment), chemical (extraction using solvents, detoxification using chemical agents, and added sorbents), and biological treatments [81, 82]. In this context, a number of feed supplements have provided protection against the damage caused by AFB1. A study [83] showed that vitamins A, E, K, and D could be used in protection against aflatoxins. Supplementation by vitamins A, E, and C has resulted in enhanced antioxidative effect in poultry birds and protects the immune cells from oxidative damage induced by AFB1 [84]. Plenty of studies worldwide are compiled together in a comprehensive meta-analysis in poultry, where the nutrient supplements are used against AFB1 in broiler, and some of them could be considered as cost-effective and useful to ameliorate the undesirable effects of AFB1 [85].

Selenium (Se) and zinc (Zn) are two under studied trace elements for their protective roles against oxidative stresses and other adverse effects induced by AFB1. A number of studies have documented the importance of Se and Zn in human and animal biology when used optimally.

Selenium, Its Detoxifying Effects, and Protection Mechanism

Selenium is an essential nutrient of fundamental importance in human and animal biology. It is crucial for the ideal functioning of the immune, cardiovascular, and reproductive systems. Also, it ensures defense against infections, tumors, and prevents inflammatory and allergic conditions in both humans and animals [86–90].

Se is a significant feed-derived natural antioxidant in poultry, and adequate level of Se is crucial for chicken health, productive and reproductive characteristics (embryonic development and sperm quality), and optimal functioning of immune system [91]. Two major Se sources, which are inorganic (selenite or selenate) and organic selenium (selenomethionine), are used in poultry [92]. The protective effect of Se against various toxic agents in various animal species along with poultry is well known [93–95]. Liao et al. investigated that AFB1 exposure induced liver dysfunction by disturbing the tissue enzyme activity and enhanced apoptosis, but the Se coadministration protected liver tissues against AFB1-induced toxicity [45]. A number of studies conducted on various organs in poultry birds demonstrated the protective effects of Se against AFB1 [61, 69, 96]. The dietary sodium selenite in the feed of broiler has excellent effects on oxidative stress and apoptosis and can amend the immunosuppression effects induced by AFB1 in spleen of broiler [69]. Se supplementation has improved AFB1-induced apoptosis at a concentration of 0.4 mg/kg [96]. Also, Se supplementation in broiler diet provided protection against AFB1-induced changes in the ileum and sodium selenite improved the cellular immune functioning of the AFB1-affected ileum mucosa [61]. In their review, Galvano et al. mentioned several reports on in vitro and in vivo studies documenting that Se inhibits AFB1-DNA binding and adducts formation, and sodium selenite and Se-enriched yeast extract protect cells from AFB1 cytotoxicity [35].

Se exerts protective functions through its direct ability to enhance immunity, by affecting the metabolism of carcinogens, by playing a crucial role in protein synthesis, cell division, and the formation of anticancer metabolites, and last but not the least, its antioxidative abilities [97]. Out of all the functions, antioxidant and antitumor abilities are the most important roles played by Se. The anticarcinogenic action of Se is mediated by selenoproteins and smaller non-proteins, which could reduce metabolites through GSH-Px, modify carcinogen metabolism, modulate immune functions, inhibit enzymes that catalyze cell proliferation, and induce apoptosis [98]. Also, Se may prevent the binding of DNA with carcinogens as well as reactive Se metabolites can render the carcinogens into non-carcinogenic. Dietary Se has been shown to protect chicks from AFB1-induced liver injury by inhibiting CYP450 enzyme, which is responsible for the activation of AFB1 to toxic AFBO [99]. Alongside, the mitochondrial redox equilibrium is modulated by catalytic redox Se metabolites, which ultimately induce apoptosis in non-regulatory cancer cells [100].

Zinc, Its Detoxifying Effects, and Protection Mechanism

Essentiality of Zn for humans and animals has been known for many decades due to its principal role in individual’s growth, development, and optimal functioning of various physiological processes. Indeed, the past two decades have seen a rapid growth in knowledge of the underlying mechanisms, whereby Zn exerts its ubiquitous effects on immune function, disease resistance, and general health [101–103]. Its involvement in such fundamental activities probably accounts for the essentiality of Zn for all forms of life.

Zn in avian species is necessary for normal growth and maintenance including bone development, feathering, enzyme structure and function, and appetite regulation in chicks [104]. Zn is important for proper functioning of the components of the cellular immune system including heterophils, mononuclear phagocytes, and T lymphocytes, which are important for disease resistance in poultry [105–108]. The deficiency of Zn in poultry results in suppressed immune system, poor shell quality and poor feathering, dermatitis, and infertility [109–111]. In poultry, Zn serves not only as a nutrient but can also be used as a dietary supplement to manipulate the reproductive system of the birds [112, 113]. Although a bunch of studies had already been carried out, demonstrating protective effects of zinc against a range of noxious agents in human and different laboratory animal [114–118], only few studies focused on protective effects of Zn against AFB1. Zinc amended growth retardation, thymic involution, and impaired peripheral immune efficiency in piglets exposed to AFB1, and these protective effects were likely to play a key role in immune responses [119]. Another study showed significant enhancement in growth performance represented by improved relative body weight gain and feed efficiency in AFB1-intoxicated birds when fed with Zn supplementation [119]. It is clear from all the aforementioned studies that Zn is a potential trace element which plays a crucial role against adverse effects induced by various agents. Also, its function in enhancing various systems of the body could be used as amending tool against AFB1 intoxication.

Action mechanism of Zn involves its functions in the body in three different areas: catalytic, structural, and regulatory. In its catalytic role, Zn is a critical component of the catalytic site of more than 300 different metalloenzymes. These metalloenzymes play crucial roles in different biological processes [82], making it highly important cofactor for different protective mechanisms including protection against toxicity. In its structural role, Zn facilitates protein folding and produces “Zn fingers.” When bound to a protein, Zn can either directly take part in chemical catalysis or support protein structure and stability [120]. Zn is involved in the regulation of nucleoproteins and the activity of various inflammatory cells, in its regulatory role [121]. For instance, Zn regulates the expression of metallothionein, which has multiple functions including intracellular Zn compartmentalization and antioxidant function [122].

Conclusion

Poultry is highly sensitive and susceptible to aflatoxins particularly AFB1, which produces acute, chronic, mutagenic, and teratogenic toxicity along with causing millions of dollars per year damage to the poultry industry worldwide. Nutritional supplementation could be a cost-effective and efficient method to bring down the toxic effects of AFB1, which has already been proved by various reports. Se and Zn are potential antioxidants that take part in enhancing various biological systems in humans and animals by being an important component of several essential enzymes. These two less studied yet important trace elements have been highly effective against the adverse effects produced by various noxious agents including AFB1. A number of studies are being carried out on a global scale to optimize the concentration of these trace elements in feed, in order to gain the benefits against AFB1. This comprehensive review will provide information about the harmful effects of AFB1 in different organ systems of poultry birds, as well as the counter effects of various nutritional supplementations with a special focus on Se and Zn along with their protective mechanisms.