Epigallocatechin Gallate and Glutathione Attenuate Aflatoxin B1-Induced Acute Liver Injury in Ducklings via Mitochondria-Mediated Apoptosis and the Nrf2 Signalling Pathway

Aflatoxin B₁ (AFB₁) exists widely in feed and food with severe hazards, posing a serious threat to human and animal health. Epigallocatechin gallate (EGCG) and glutathione (GSH) have been reported as having anti-oxidative and other functions. The present study aimed to investigate the detoxification effect of EGCG and GSH alone or in combination on AFB₁ exposure in ducklings. Fifty one-day-old male ducklings were randomly assigned into five experimental groups (n = 10): 1. Control (CTR); 2. 0.3 mg/kg BW AFB₁ (AFB₁); 3. 0.3 mg/kg BW AFB₁ + 100 mg/kg BW EGCG (AFB₁ + EGCG); 4. 0.3 mg/kg BW AFB₁ + 30 mg/kg BW GSH (AFB₁ + GSH); 5. 0.3 mg/kg BW AFB₁ + 100 mg/kg BW EGCG + 30 mg/kg BW GSH (AFB₁ + EGCG + GSH). The experiment lasted for seven days. Compared with the CTR group, AFB₁ reduced growth performance, total serum protein and albumin content, increased serum enzyme activity (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyl transpeptidase), and caused pathological damage to the ducklings’ livers. AFB₁ exposure increased malondialdehyde content and decreased superoxide dismutase, total antioxidant capacity, catalase, glutathione peroxidase activities, and glutathione content in the liver. EGCG and GSH alone or in combination mitigated these adverse effects. Meanwhile, EGCG and GSH attenuate apoptosis of hepatocytes, and regulated AFB₁-induced changes in the abundance of genes contained in the Keap1/Nrf2 signalling and apoptotic pathways. Collectively, these results suggest that EGCG and GSH alleviate the hepatocyte injury induced by AFB₁ by inhibiting oxidative stress and attenuating excessive mitochondria-mediated apoptosis.     

Simplified Synthesis and Stability Assessment of Aflatoxin B1-Lysine and Aflatoxin G1-Lysine

Aflatoxins B₁ (AFB₁) and G₁ (AFG₁) are carcinogenic mycotoxins that contaminate crops such as maize and groundnuts worldwide. The broadly accepted method to assess chronic human aflatoxin exposure is by quantifying the amount of aflatoxin adducted to human serum albumin. This has been reported using ELISA, HPLC, or LC-MS/MS to measure the amount of AFB₁-lysine released after proteolysis of serum albumin. LC-MS/MS is the most accurate method but requires both isotopically labelled and unlabelled AFB₁-lysine standards, which are not commercially available. In this work, we report a simplified synthetic route to produce unlabelled, deuterated and ¹³C₆ ¹⁵N₂ labelled aflatoxin B₁-lysine and for the first-time aflatoxin G₁-lysine. Additionally, we report on the stability of these compounds during storage. This simplified synthetic approach will make the production of these important standards more feasible for laboratories performing aflatoxin exposure studies.     

Aflatoxin B1 Exposure in Sheep: Insights into Hepatotoxicity Based on Oxidative Stress,Inflammatory Injury,Apoptosis, and Gut Microbiota Analysis

The widespread fungal toxin Aflatoxin B₁ (AFB₁) is an inevitable pollutant affecting the health of humans, poultry, and livestock. Although studies indicate that AFB₁ is hepatotoxic, there are few studies on AFB₁-induced hepatotoxicity in sheep. Thus, this study examined how AFB₁ affected sheep liver function 24 h after the animals received 1 mg/kg bw of AFB₁ orally (dissolved in 20 mL, 4% v/v ethanol). The acute AFB₁ poisoning caused histopathological injuries to the liver and increased total bilirubin (TBIL) and alkaline phosphatase (AKP) levels. AFB₁ also markedly elevated the levels of the pro-inflammatory cytokines TNF-α and IL-6 while considerably reducing the expression of antioxidation-related genes (SOD-1 and SOD-2) and the anti-inflammatory gene IL-10 in the liver. Additionally, it caused apoptosis by dramatically altering the expression of genes associated with apoptosis including Bax, Caspase-3, and Bcl-2/Bax. Notably, AFB₁ exposure altered the gut microbiota composition, mainly manifested by BF311 spp. and Alistipes spp. abundance, which are associated with liver injury. In conclusion, AFB₁ can cause liver injury and liver dysfunction in sheep via oxidative stress, inflammation, apoptosis, and gut-microbiota disturbance.     

Screening a Strain of Aspergillus niger and Optimization of Fermentation Conditions for Degradation of Aflatoxin B1

Aflatoxin B₁, a type of highly toxic mycotoxin produced by some species belonging to the Aspergillus genus, such as Aspergillus flavus and Aspergillus parasiticus, is widely distributed in feed matrices. Here, coumarin was used as the sole carbon source to screen microorganism strains that were isolated from types of feed ingredients. Only one isolate (ND-1) was able to degrade aflatoxin B₁ after screening. ND-1 isolate, identified as a strain of Aspergillus niger using phylogenetic analysis on the basis of 18S rDNA, could remove 26.3% of aflatoxin B₁ after 48 h of fermentation in nutrient broth (NB). Optimization of fermentation conditions for aflatoxin B₁ degradation by selected Aspergillus niger was also performed. These results showed that 58.2% of aflatoxin B₁ was degraded after 24 h of culture under the optimal fermentation conditions. The aflatoxin B₁ degradation activity of Aspergillus niger supernatant was significantly stronger than cells and cell extracts. Furthermore, effects of temperature, heat treatment, pH, and metal ions on aflatoxin B₁ degradation by the supernatant were examined. Results indicated that aflatoxin B₁ degradation of Aspergillus niger is enzymatic and this process occurs in the extracellular environment.     

Fisetin Suppresses Macrophage-Mediated Inflammatory Responses by Blockade of Src and Syk

Flavonoids, such as fisetin (3,7,3′,4′-tetrahydroxyflavone), are plant secondary metabolites. It has been reported that fisetin is able to perform numerous pharmacological roles including anti-inflammatory, anti-microbial, and anti-cancer activities; however, the exact anti-inflammatory mechanism of fisetin is not understood. In this study, the pharmacological action modes of fisetin in lipopolysaccharide (LPS)-stimulated macrophage-like cells were elucidated by using immunoblotting analysis, kinase assays, and an overexpression strategy. Fisetin diminished the release of nitric oxide (NO) and reduced the mRNA levels of inducible NO synthase (iNOS), tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in LPS-stimulated RAW264.7 cells without displaying cytotoxicity. This compound also blocked the nuclear translocation of p65/nuclear factor (NF)-κB. In agreement, the upstream phosphorylation events for NF-κB activation, composed of Src, Syk, and IκBα, were also reduced by fisetin. The phospho-Src level, triggered by overexpression of wild-type Src, was also inhibited by fisetin. Therefore, these results strongly suggest that fisetin can be considered a bioactive immunomodulatory compound with anti-inflammatory properties through suppression of Src and Syk activities.     

Aflatoxin B1 Toxicity in Zebrafish Larva (Danio rerio): Protective Role of Hericium erinaceus

Aflatoxin B1 (AFB1), a secondary metabolite produced by fungi of the genus Aspergillus, has been found among various foods as well as in fish feed. However, the effects of AFB1 on fish development and its associated toxic mechanism are still unclear. In the present study, we confirmed the morphological alterations in zebrafish embryos and larvae after exposure to different AFB1 doses as well as the oxidative stress pathway that is involved. Furthermore, we evaluated the potentially protective effect of Hericium erinaceus extract, one of the most characterized fungal extracts, with a focus on the nervous system. Treating the embryos 6 h post fertilization (hpf) with AFB1 at 50 and 100 ng/mL significantly increased oxidative stress and induced malformations in six-day post-fertilization (dpf) zebrafish larvae. The evaluation of lethal and developmental endpoints such as hatching, edema, malformations, abnormal heart rate, and survival rate were evaluated after 96 h of exposure. Hericium inhibited the morphological alterations of the larvae as well as the increase in oxidative stress and lipid peroxidation. In conclusion: our study suggests that a natural extract such as Hericium may play a partial role in promoting antioxidant defense systems and may contrast lipid peroxidation in fish development by counteracting the AFB1 toxicity mechanism.     

Toxic Effects of Aflatoxin B1 in Chinese Sea Bass (Lateolabrax maculatus)

This study was performed to assess the effects of dietary aflatoxin B₁ (AFB₁) on the growth, antioxidant and immune response, digestive enzyme activities, and intestinal morphology of Lateolabrax maculatus during a 56-day feeding trial. Four diets were formulated including 0, 0.1, 0.5, and 1.0 mg/kg of AFB₁. Each diet was randomly assigned to 3 fish tanks with 40 fish per tank. Results indicated that the fish’s final body weight, weight gain rate, specific growth rate, feed intake, condition factor, viscerosomatic index, hepatosomatic index, and intestinesomatic index decreased (p < 0.01) as dietary AFB₁ increased. AFB₁ levels in diets increased (p < 0.05) serum total antioxidant capacity (TAOC), superoxide (SOD), catalase, malondialdehyde (MDA), alkaline phosphatase (AKP), and lysozyme (LZM), and increased (p < 0.05) the TAOC, SOD, MDA, AKP, LZM, and immunoglobulin M in the livers of the fish. Dietary AFB₁ decreased (p < 0.05) intestinal trypsin activity and induced intestinal injury. In summary, dietary AFB₁ up to 1.0 mg/kg was toxic to L. maculatus as judged by reduced growth, enhanced antioxidant and immune response, decreased intestinal trypsin activity, and impaired intestinal morphology.     

Mechanisms of butylated hydroxytoluene chemoprevention of aflatoxicosis--inhibition of aflatoxin B1 metabolism

Chemoprevention of toxicoses and/or cancer through the use of nutrients or pharmacologic compounds is the subject of intense study. Among the many compounds examined, food additives such as antioxidants are being considered due to their ability to reduce disease formation by either induction or inhibition of key enzyme systems. One such compound, butylated hydroxytoluene (BHT), has been found to protect against cancer formation caused by exposure to aflatoxin B₁ (AFB₁) in rodents. We have shown that dietary BHT protects against clinical signs of aflatoxicosis in turkeys, a species that is very susceptible to this mycotoxin. In this study, the effect of BHT on AFB₁ metabolism and other cytochrome P450 (CYP)-related enzyme activities in turkey liver microsomes was examined to discern possible mechanisms of BHT-mediated protection against aflatoxicosis. Ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), prototype activities for CYP1A1 and 1A2, respectively, were decreased in the BHT fed (4000 ppm) animals, while oxidation of nifedipine, a prototype activity for CYP3A4, was increased. However, BHT added to microsomal incubations inhibited these CYP activities in a concentration-related manner. Importantly, BHT inhibited conversion of AFB₁ to the reactive intermediate AFB₁-8,-9-epoxide (AFBO), exhibiting Michaelis-Menton competitive inhibition kinetics (Ki = 0.81 μM). Likewise, microsomes prepared from turkeys fed BHT were significantly less active in AFBO formation compared to those from control birds. When turkeys were fed BHT for up to 40 days, residual BHT was present in liver, breast meat, thigh meat and abdominal fat in concentrations substantially below U.S. FDA guidelines for this antioxidant, but in concentrations greater than the Ki, likely sufficient to inhibit bioactivation of AFB₁in vivo. BHT-induced hydropic degeneration in the livers of BHT fed animals was significantly greater in birds that remained on BHT treatment for up to 30 days, but this lesion diminished in animals fed for 40 days or when returned to a control diet. The data indicate that the observed chemopreventive properties of BHT in turkeys may be due, at least in part, to its ability to inhibit hepatic AFB₁ epoxidation and also that the BHT-induced hydropic degeneration is reversible and does not appear to cause long-term effects.     

Temporal Variation and Association of Aflatoxin B1 Albumin-Adduct Levels with Socio-Economic and Food Consumption Factors in HIV Positive Adults

The association between aflatoxin exposure and alteration in immune responses observed in humans suggest that aflatoxin could suppress the immune system and work synergistically with HIV to increase disease severity and progression to AIDS. No longitudinal study has been conducted to assess exposure to aflatoxin (AF) among HIV positive individuals. We examined temporal variation in AFB₁ albumin adducts (AF-ALB) in HIV positive Ghanaians, and assessed the association with socioeconomic and food consumption factors. We collected socioeconomic and food consumption data for 307 HIV positive antiretroviral naive adults and examined AF-ALB levels at recruitment (baseline) and at six (follow-up 1) and 12 (follow-up 2) months post-recruitment, by age, gender, socioeconomic status (SES) and food consumption patterns. Generalized linear models were used to examine the influence of socioeconomic and food consumption factors on changes in AF-ALB levels over the study period, adjusting for other covariates. AF-ALB levels (pg/mg albumin) were lower at baseline (mean AF-ALB: 14.9, SD: 15.9), higher at six months (mean AF-ALB: 23.3, SD: 26.6), and lower at 12 months (mean AF-ALB: 15.3, SD: 15.4). Participants with the lowest SES had the highest AF-ALB levels at baseline and follow up-2 compared with those with higher SES. Participants who bought less than 20% of their food and who stored maize for less than two months had lower AF-ALB levels. In the adjusted models, there was a statistically significant association between follow up time and season (dry or rainy season) on AF-ALB levels over time (p = 0.04). Asymptomatic HIV-positive Ghanaians had high plasma AF-ALB levels that varied according to season, socioeconomic status, and food consumption patterns. Steps need to be taken to ensure the safety and security of the food supply for the population, but in particular for the most vulnerable groups such as HIV positive people.     

Kalopanaxsaponin B Ameliorates TNBS-Induced Colitis in Mice

The stem-bark of Kalopanax pictus (KP, family Araliaceae), of which main constituent is kalopanaxsaponin B, has been used for asthma, rhinitis, and arthritis in Chinese traditional medicine. To clarify anticolitic effect of KP, we examined anti-inflammatory effect of KP extract and kalopanaxsaponin B in lipopolysaccharide (LPS)-stimulated peritoneal macrophage and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice. Of KP extracts, KP BuOH-soluble fraction most potently inhibited LPS-induced IL-1β, IL-6 and TNF-α expression, as well as NF-κB activation. However, KP BuOH fraction increased IL-10, an anti-inflammatory cytokine. KP BuOH fraction also inhibited colon shortening and myeloperoxidase activity in TNBS-induced colitic mice. KP BuOH fraction also potently inhibited the expression of the pro-inflammatory cytokines, IL-1β, IL-6 and TNF-α as well as the activation of NF-κB. Kalopanaxsaponin B, a main constituent of KP, inhibited TNBS-induced colonic inflammation, including colon shortening, and TNBS-increased myeloperoxidase activity pro-inflammatory cytokine expression and NF-κB activation in mice. Based on these findings, KP, particularly its main constituent, kalopanaxsaponin B, may ameliorate colitis by inhibiting NF-κB pathway.     

Removal of Aflatoxin B1 by Edible Mushroom-Forming Fungi and Its Mechanism

Aflatoxins (AFs) are biologically active toxic metabolites, which are produced by certain toxigenic Aspergillus sp. on agricultural crops. In this study, five edible mushroom-forming fungi were analyzed using high-performance liquid chromatography fluorescence detector (HPLC-FLD) for their ability to remove aflatoxin B₁ (AFB₁), one of the most potent naturally occurring carcinogens known. Bjerkandera adusta and Auricularia auricular-judae showed the most significant AFB₁ removal activities (96.3% and 100%, respectively) among five strains after 14-day incubation. The cell lysate from B. adusta exhibited higher AFB₁ removal activity (35%) than the cell-free supernatant (13%) after 1-day incubation and the highest removal activity (80%) after 5-day incubation at 40 °C. In addition, AFB₁ analyses using whole cells, cell lysates, and cell debris from B. adusta showed that cell debris had the highest AFB₁ removal activity at 5th day (95%). Moreover, exopolysaccharides from B. adusta showed an increasing trend (24–48%) similar to whole cells and cell lysates after 5- day incubation. Our results strongly suggest that AFB₁ removal activity by whole cells was mainly due to AFB₁ binding onto cell debris during early incubation and partly due to binding onto cell lysates along with exopolysaccharides after saturation of AFB₁ binding process onto cell wall components.     

Risk Assessment on Dietary Exposure to Aflatoxin B1 in Post-Harvest Peanuts in the Yangtze River Ecological Region

Based on the 2983 peanut samples from 122 counties in six provinces of China’s Yangtze River ecological region collected between 2009–2014, along with the dietary consumption data in Chinese resident nutrition and health survey reports from 2002 and 2004, dietary aflatoxin exposure and percentiles in the corresponding statistics were calculated by non-parametric probability assessment, Monte Carlo simulation and bootstrap sampling methods. Average climatic conditions in the Yangtze River ecological region were calculated based on the data from 118 weather stations via the Thiessen polygon method. The survey results found that the aflatoxin contamination of peanuts was significantly high in 2013. The determination coefficient (R²) of multiple regression reflected by the aflatoxin B₁ content with average precipitation and mean temperature in different periods showed that climatic conditions one month before harvest had the strongest impact on aflatoxin B₁ contamination, and that Hunan and Jiangxi provinces were greatly influenced. The simulated mean aflatoxin B₁ intake from peanuts at the mean peanut consumption level was 0.777–0.790 and 0.343–0.349 ng/(kg·d) for children aged 2–6 and standard adults respectively. Moreover, the evaluated cancer risks were 0.024 and 0.011/(100,000 persons·year) respectively, generally less than China’s current liver cancer incidence of 24.6 cases/(100,000 persons·year). In general, the dietary risk caused by peanut production and harvest was low. Further studies would focus on the impacts of peanut circulation and storage on aflatoxin B₁ contamination risk assessment in order to protect peanut consumers’ safety and boost international trade.     

Betulinic Acid Ameliorates the T-2 Toxin-Triggered Intestinal Impairment in Mice by Inhibiting Inflammation and Mucosal Barrier Dysfunction through the NF-κB Signaling Pathway

T-2 toxin, a trichothecene mycotoxin produced by Fusarium, is widely distributed in crops and animal feed and frequently induces intestinal damage. Betulinic acid (BA), a plant-derived pentacyclic lupane-type triterpene, possesses potential immunomodulatory, antioxidant and anti-inflammatory biological properties. The current study aimed to explore the protective effect and molecular mechanisms of BA on intestinal mucosal impairment provoked by acute exposure to T-2 toxin. Mice were intragastrically administered BA (0.25, 0.5, or 1 mg/kg) daily for 2 weeks and then injected intraperitoneally with T-2 toxin (4 mg/kg) once to induce an intestinal impairment. BA pretreatment inhibited the loss of antioxidant capacity in the intestine of T-2 toxin-treated mice by elevating the levels of CAT, GSH-PX and GSH and reducing the accumulation of MDA. In addition, BA pretreatment alleviated the T-2 toxin-triggered intestinal immune barrier dysregulation by increasing the SIgA level in the intestine at dosages of 0.5 and 1 mg/kg, increasing IgG and IgM levels in serum at dosages of 0.5 and 1 mg/kg and restoring the intestinal C3 and C4 levels at a dosage of 1 mg/kg. BA administration at a dosage of 1 mg/kg also improved the intestinal chemical barrier by decreasing the serum level of DAO. Moreover, BA pretreatment improved the intestinal physical barrier via boosting the expression of ZO-1 and Occludin mRNAs and restoring the morphology of intestinal villi that was altered by T-2 toxin. Furthermore, treatment with 1 mg/kg BA downregulated the expression of p-NF-κB and p-IκB-α proteins in the intestine, while all doses of BA suppressed the pro-inflammatory cytokines expression of IL-1β, IL-6 and TNF-α mRNAs and increased the anti-inflammatory cytokine expression of IL-10 mRNA in the intestine of T-2 toxin-exposed mice. BA was proposed to exert a protective effect on intestinal mucosal disruption in T-2 toxin-stimulated mice by enhancing the intestinal antioxidant capacity, inhibiting the secretion of inflammatory cytokines and repairing intestinal mucosal barrier functions, which may be associated with BA-mediated inhibition of the NF-κB signaling pathway activation.     

Adsorbents Reduce Aflatoxin M1 Residue in Milk of Healthy Dairy Cow Exposed to Moderate Level Aflatoxin B1 in Diet and Its Exposure Risk for Humans

This study investigated the effect of moderate risk level (8 µg/kg) AFB₁ in diet supplemented with or without adsorbents on lactation performance, serum parameters, milk AFM₁ content of healthy lactating cows and the AFM₁ residue exposure risk in different human age groups. Forty late healthy lactating Holstein cows (270 ± 22 d in milk; daily milk yield 21 ± 3.1 kg/d) were randomly assigned to four treatments: control diet without AFB₁ and adsorbents (CON), CON with 8 μg/kg AFB₁ (dry matter basis, AF), AF + 15 g/d adsorbent 1 (AD1), AF + 15 g/d adsorbent 2 (AD2). The experiment lasted for 19 days, including an AFB₁-challenge phase (day 1 to 14) and an AFB₁-withdraw phase (day 15 to 19). Results showed that both AFB₁ and adsorbents treatments had no significant effects on the DMI, milk yield, 3.5% FCM yield, milk components and serum parameters. Compared with the AF, AD1 and AD2 had significantly lower milk AFM₁ concentrations (93 ng/L vs. 46 ng/L vs. 51 ng/L) and transfer rates of dietary AFB₁ into milk AFM₁ (1.16% vs. 0.57% vs. 0.63%) (p < 0.05). Children aged 2–4 years old had the highest exposure risk to AFM₁ in milk in AF, with an EDI of 1.02 ng/kg bw/day and a HI of 5.11 (HI > 1 indicates a potential risk for liver cancer). Both AD1 and AD2 had obviously reductions in EDI and HI for all population groups, whereas, the EDI (≥0.25 ng/kg bw/day) and HI (≥1.23) of children aged 2–11 years old were still higher than the suggested tolerable daily intake (TDI) of 0.20 ng/kg bw/day and 1.00 (HI). In conclusion, moderate risk level AFB₁ in the diet of healthy lactating cows could cause a public health hazard and adding adsorbents in the dairy diet is an effective measure to remit AFM₁ residue in milk and its exposure risk for humans.     

Degradation of Aflatoxin B1 during the Fermentation of Alcoholic Beverages

Aflatoxin B₁ (AFB₁) is a contaminant of grain and fruit and has one of the highest levels of carcinogenicity of any natural toxin. AFB₁ and the fungi that produce it can also contaminate the raw materials used for beer and wine manufacture, such as corn and grapes. Therefore, brewers must ensure strict monitoring to reduce the risk of contamination. In this study, the fate of AFB₁ during the fermentation process was investigated using laboratory-scale bottom and top beer fermentation and wine fermentation. During fermentation, cool wort beer samples and wine must samples were artificially spiked with AFB₁ and the levels of AFB₁ remaining after fermentation were analyzed. AFB₁ levels were unchanged during both types of fermentation used for beer but were reduced to 30% of their initial concentration in wine. Differential analysis of the spiked and unspiked wine samples showed that the degradation compound was AFB_2a, a hydrated derivative of AFB₁. Thus, the results showed that the risk of AFB₁ carryover was still present for both types of beer fermentation but was reduced in the case of wine fermentation because of hydration.     

Decrease in Aflatoxin M1 Concentration in Milk during Cholesterol Removal by Application of β-Cyclodextrin

Approximately one-third of humankind is chronically exposed to the carcinogenic aflatoxin M₁ contained in milk. As β-cyclodextrin is frequently used in the food industry, its effect on aflatoxin M₁ concentration was investigated during cholesterol removal from milk due to the similarity among the physicochemical properties of aflatoxin M₁ and cholesterol. Moreover, the elimination of cholesterol using β-cyclodextrin has been successfully applied in many studies without any substantial effect on the quality of the treated milk. Therefore, milk samples were spiked with aflatoxin M₁ within the range from 0.20 to 2.00 µg/kg, and cholesterol removal was carried out by 2.0% (w/w) β-cyclodextrin addition, as this concentration is enough for the sufficient removal of cholesterol. It was found that the mean cholesterol concentration decreased by 92.3%, while the aflatoxin M₁ concentration decreased to 0.53 ± 0.04 µg/kg, i.e., by 39.1% after treatment (n = 2). This mitigation procedure itself is easy and inexpensive and thus is fully applicable with a high potential for complete decontamination of aflatoxin M₁ milk. This method will therefore considerably improve the food safety issues associated with aflatoxin M₁ presence in milk and dairy products.