Aflatoxin B1 and Aflatoxin M1 Induce Compromised Intestinal Integrity through Clathrin-Mediated Endocytosis

With the growing diversity and complexity of diet, humans are at risk of simultaneous exposure to aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1), which are well-known contaminants in dairy and other agricultural products worldwide. The intestine represents the first barrier against external contaminants; however, evidence about the combined effect of AFB1 and AFM1 on intestinal integrity is lacking. In vivo, the serum biochemical parameters related to intestinal barrier function, ratio of villus height/crypt depth, and distribution pattern of claudin-1 and zonula occluden-1 were significantly affected in mice exposed to 0.3 mg/kg b.w. AFB1 and 3.0 mg/kg b.w. AFM1. In vitro results on differentiated Caco-2 cells showed that individual and combined AFB1 (0.5 and 4 μg/mL) and AFM1 (0.5 and 4 μg/mL) decreased cell viability and trans-epithelial electrical resistance values as well as increased paracellular permeability of fluorescein isothiocyanate-dextran in a dose-dependent manner. Furthermore, AFM1 aggravated AFB1-induced compromised intestinal barrier, as demonstrated by the down-regulation of tight junction proteins and their redistribution, particularly internalization. Adding the inhibitor chlorpromazine illustrated that clathrin-mediated endocytosis partially contributed to the compromised intestinal integrity. Synergistic and additive effects were the predominant interactions, suggesting that these toxins are likely to have negative effects on human health.     

Aflatoxin B1 and Sterigmatocystin Binding Potential of Lactobacilli

Due to global climate change, mould strains causing problems with their mycotoxin production in the tropical–subtropical climate zone have also appeared in countries belonging to the temperate zone. Biodetoxification of crops and raw materials for food and feed industries including the aflatoxin B1 (AFB1) binding abilities of lactobacilli is of growing interest. Despite the massive quantities of papers dealing with AFB1-binding of lactobacilli, there are no data for microbial binding of the structurally similar mycotoxin sterigmatocystin (ST). In addition, previous works focused on the detection of AFB1 in extracts, while in this case, analytical determination was necessary for the microbial biomass as well. To test binding capacities, a rapid instrumental analytical method using high-performance liquid chromatography was developed and applied for measurement of AFB1 and ST in the biomass of the cultured bacteria and its supernatant, containing the mycotoxin fraction bound by the bacteria and the fraction that remained unbound, respectively. For our AFB1 and ST adsorption studies, 80 strains of the genus Lactobacillus were selected. Broths containing 0.2 µg/mL AFB1and ST were inoculated with the Lactobacillus test strains. Before screening the strains for binding capacities, optimisation of the experiment parameters was carried out. Mycotoxin binding was detectable from a germ count of 10⁷ cells/mL. By studying the incubation time of the cells with the mycotoxins needed for mycotoxin-binding, co-incubation for 10 min was found sufficient. The presence of mycotoxins did not affect the growth of bacterial strains. Three strains of L. plantarum had the best AFB1 adsorption capacities, binding nearly 10% of the mycotoxin present, and in the case of ST, the degree of binding was over 20%.     

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.     

Assessing the Impacts of Preanalytical Field Sampling Challenges on the Reliability of Serum Aflatoxin B1-Lysine Measurements by Use of LC-MS/MS

Aflatoxin exposure is endemic in developing countries with warm, humid climates that promote toxigenic mold growth on crops and foodstuffs. Estimating human aflatoxin exposure is key to identifying and abating contamination sources. Serum aflatoxin B1 bound to albumin lysine (AFB1-lys) is a preferred exposure biomarker, but field sample collection, processing, transportation, and storage logistics are challenging. We validated an improved LC-MS/MS method for serum AFB1-lys and applied it to three field sampling challenges: transportation/storage (elevated temperature); collection/processing (hemolysis); and sample type substitution (heparinized plasma). Our new LC-MS/MS method had a LOD of 0.03 ng/mL, accuracy (mean spike recovery) of 112%, total imprecision (replicate pool measurements) ≤5% at ≥0.2 ng/mL, and results that were 95.1% similar (mean percentage similarity) to an established method. AFB1-lys in human serum spiked with serum from aflatoxin-dosed rats was stable for 14 days at both ambient (22.5 °C) and elevated (38 °C) temperatures. Simulated hemolysis (adding 0.25–3 mg hemoglobin) did not affect AFB1-lys accuracy at ≥0.5 ng/mL but caused 10–25% signal suppression. Heparinized plasma AFB1-lys was 99.0% similar to serum but interfered with albumin measurements (bromocresol green) causing spurious low bias. Further investigation is warranted, but our findings suggest that AFB1-lys is pre-analytically robust.     

Degradation and Detoxification of Aflatoxin B1 by Tea-Derived Aspergillus niger RAF106

Microbial degradation is an effective and attractive method for eliminating aflatoxin B1 (AFB1), which is severely toxic to humans and animals. In this study, Aspergillus niger RAF106 could effectively degrade AFB1 when cultivated in Sabouraud dextrose broth (SDB) with contents of AFB1 ranging from 0.1 to 4 μg/mL. Treatment with yeast extract as a nitrogen source stimulated the degradation, but treatment with NaNO₃ and NaNO₂ as nitrogen sources and lactose and sucrose as carbon sources suppressed the degradation. Moreover, A. niger RAF106 still degraded AFB1 at initial pH values that ranged from 4 to 10 and at cultivation temperatures that ranged from 25 to 45 °C. In addition, intracellular enzymes or proteins with excellent thermotolerance were verified as being able to degrade AFB1 into metabolites with low or no mutagenicity. Furthermore, genomic sequence analysis indicated that the fungus was considered to be safe owing to the absence of virulence genes and the gene clusters for the synthesis of mycotoxins. These results indicate that A. niger RAF106 and its intracellular enzymes or proteins have a promising potential to be applied commercially in the processing and industry of food and feed to detoxify AFB1.     

Effects of Turmeric Powder on Aflatoxin M1 and Aflatoxicol Excretion in Milk from Dairy Cows Exposed to Aflatoxin B1 at the EU Maximum Tolerable Levels

Due to the climatic change, an increase in aflatoxin B1 (AFB1) maize contamination has been reported in Europe. As an alternative to mineral binders, natural phytogenic compounds are increasingly used to counteract the negative effects of AFB1 in farm animals. In cows, even low dietary AFB1 concentrations may result in the milk excretion of the genotoxic carcinogen metabolite aflatoxin M1 (AFM1). In this study, we tested the ability of dietary turmeric powder (TP), an extract from Curcuma longa (CL) rich in curcumin and curcuminoids, in reducing AFM1 mammary excretion in Holstein–Friesian cows. Both active principles are reported to inhibit AFM1 hepatic synthesis and interact with drug transporters involved in AFB1 absorption and excretion. A crossover design was applied to two groups of cows (n = 4 each) with a 4-day washout. Animals received a diet contaminated with low AFB1 levels (5 ± 1 µg/kg) for 10 days ± TP supplementation (20 g/head/day). TP treatment had no impact on milk yield, milk composition or somatic cell count. Despite a tendency toward a lower average AFM1 milk content in the last four days of the treatment (below EU limits), no statistically significant differences with the AFB1 group occurred. Since the bioavailability of TP active principles may be a major issue, further investigations with different CL preparations are warranted.     

Aflatoxin B1 and Sterigmatocystin Binding Potential of Non-Lactobacillus LAB Strains

Research on the ability of lactic acid bacteria (LAB) to bind aflatoxin B1 (AFB1) has mostly been focusing on lactobacilli and bifidobacteria. In this study, the AFB1 binding capacities of 20 Enterococcus strains belonging to E. casseliflavus, E. faecalis, E. faecium, E. hirae, E. lactis, and E. mundtii, 24 Pediococcus strains belonging to species P. acidilactici, P. lolii, P. pentosaceus, and P. stilesii, one strain of Lactococcus formosensis and L. garviae, and 3 strains of Weissella soli were investigated in MRS broth at 37 °C at 0.2 µg/mL mycotoxin concentration. According to our results, among non-lactobacilli LAB, the genera with the best AFB1 binding abilities were genus Pediococcus, with a maximum binding percentage of 7.6% by P. acidilactici OR83, followed by genus Lactococcus. For AFB1 bio-detoxification purposes, beside lactobacilli, pediococci can also be chosen, but it is important to select a strain with better binding properties than the average value of its genus. Five Pediococcus strains have been selected to compare their sterigmatocystin (ST) binding abilities to AFB1 binding, and a 2–3-fold difference was obtained similar to previous findings for lactobacilli. The best strain was P. acidilactici OR83 with 18% ST binding capacity. This is the first report on ST binding capabilities of non-Lactobacillus LAB strains.     

黄曲霉毒素B1致癌机制及植物性化学物防治的研究进展

黄曲霉毒素B1(AFB1)的长期暴露是肝癌发生的主要危险因素之一,研究发现它可能通过影响ras、c-fos、P53、Survivin等基因的表达而引起肝癌的发生,此外,它还与其它肝癌的高危因素,如乙肝病毒(HBV)的慢性感染等协同作用而促进肝癌的发生和发展。HBx基因的整合及AFB1的攻击可能通过影响DNA修复系统、药物代谢酶系统及细胞色素P450(CYP)代谢酶基因的异常表达导致肝癌的发生。植物化学物,如叶绿酸、茶多酚、茶色素、姜黄素、黄酮类化合物等或/和其含量较高的食物在预防肝癌发生和发展中起重要的作用。本文对AFB1致肝癌的机制及植物化学物防治作用的研究进展作一综述。     

Sequential dietary exposure to aflatoxin B1 and fumonisin B1 in F344 rats increases liver preneoplastic changes indicative of a synergistic interaction

Dietary co-exposure to aflatoxin B1 (AFB1) and fumonisin B1 (FB1) and their interaction on hepatocellular carcinogenesis is of particular concern in toxicology and public health. In this study we evaluated the liver preneoplastic effects of single and sequential dietary exposure to AFB1 and FB1 in the F344 rat carcinogenesis model. Serum biochemical alterations, liver histopathological changes, and the formation of liver glutathione S transferase positive (GST-P+) foci were the major outcome parameters examined. Compared to the AFB1-only treatment, the FB1-only treatment induced less dysplasia, and more apoptosis and mitoses. Sequential AFB1 and FB1 treatment lead to increased numbers of dysplasia, apoptosis and foci of altered hepatocytes, as compared to either mycotoxin treatment alone. More importantly, sequential exposure to AFB1 and FB1 synergistically increased the numbers of liver GTP-P+ foci by approximately 7.3-and 12.9-fold and increased the mean sizes of GST-P+ foci by 6- and 7.5-fold, respectively, as compared to AFB1- or FB1-only treatment groups. In addition, liver ALT and AST levels were significantly increased after sequential treatment as compared to single treatment groups. The results demonstrate the interactive effect of dietary AFB1 and FB1 in inducing liver GST-P+ foci formation and provide information to model future intervention studies.     

Reduction of Aflatoxin B1 and Zearalenone Contents in Corn Using Power Ultrasound and Its Effects on Corn Quality

The degradation of aflatoxin B1 (AFB1) and zearalenone (ZEA) is investigated using power ultrasound to identify suitable methods to reduce the mycotoxin content of corn. AFB1 and ZEA in corn are simultaneously degraded via power ultrasound; thus, this method has a significant effect on corn quality. The power intensity, solid-liquid ratio, and ultrasonic treatment modes significantly affect the degradation rates of AFB1 and ZEA. The dissolution of AFB1 and ZEA in water also facilitates their degradation. At the initial stage of ultrasonic treatment, power ultrasound promotes the dissolution of mycotoxins in water, whereupon they are partially oxidized by free radicals. With a treatment time of 10 min, the reduction rates decreased owing to the dissolution of combined-state mycotoxins. After ultrasonic treatment, the contents of the essential amino acids, the total number of amino acids, and the fatty acids in corn decreased; however, ΔH values decreased during starch gelatinization. In contrast, the amylose content and viscosity of corn significantly increased during gelatinization. Therefore, this method is potentially suitable for the reduction of AFB1 and ZEA contents in corn.     

Evaluation of Growth Performance,Nitrogen Balance and Blood Metabolites of Mutton Sheep Fed an Ammonia-Treated Aflatoxin B1-Contaminated Diet

Experiments were conducted to evaluate the effects of an aflatoxin B1 (AFB1)-contaminated diet treated with ammonia on the diet detoxification and growth performance, nutrient digestibility, nitrogen utilization, and blood metabolites in sheep. Twenty-four female mutton sheep with an initial body weight of 50 ± 2.5 kg were randomly assigned to one of three groups: (1) control diet (C); (2) aflatoxin diet (T; control diet supplemented with 75 μg of AFB1/kg of dry matter); and (3) ammoniated diet (AT; ammoniated aflatoxin diet). The results showed decreases (p < 0.05) in average daily feed intake, nutrient digestibility of dry matter, crude protein and ether extract, and retained nitrogen, and an increase (p < 0.05) in urine nitrogen excretion in sheep fed diet T compared with those fed the other diets. In comparison to C and AT, feeding T decreased (p < 0.05) the concentrations of total protein, immunoglobulin A, immunoglobulin G, immunoglobulin M, superoxide dismutase, and total antioxidants and increased (p < 0.05) the concentrations of alanine amino transferase, malondialdehyde, and interleukin-6. In summary, ammonia treatment has the potential to decrease the concentration of AFB1 and alleviate the adverse effects of AFB1.     

Modulation of aflatoxin toxicity and biomarkers by lycopene in F344 rats

Modulation by lycopene of aflatoxin B(1) (AFB(1))-induced toxic effects, metabolism, and metabolic activations was studied in young F344 rats. Animals were pretreated orally with either corn oil (control group) or lycopene [100 mg/kg body weight (b.w.), intervention group] 5 days/week for 2 weeks. Control animals were then treated daily with AFB(1) (250 microg/kg b.w) alone. Intervention animals were administered lycopene (100 mg/kg b.w.) at 1 h following a daily treatment with AFB(1) (250 mug/kg b.w.). Pretreatment and intervention with lycopene significantly reduced the toxic effect caused by AFB(1) and greatly modulated AFB(1) metabolism and metabolic activation. Urinary excretion of AFB(1) phase 1 metabolites, AFM(1), AFQ(1), and AFP(1), was significantly decreased in lycopene-treated animals. Formation of serum AFB(1)-albumin adducts was also significantly reduced. The rate of reduction was from approximately 30% on day 1 (p<0.05) to 67.7% on day 15 (p<0.001). Lycopene intervention also significantly reduced formation of AFB(1)-DNA adducts in liver compared to control animals, with the highest reduction (52.7%) occurring on day 3 (p<0.05). Levels of AFB(1)-N(7)-guanine excreted in urine were also significantly decreased. Urinary excretion of the phase 2 detoxification metabolite, AFB(1)-mecapturic acid, was significantly increased in lycopene-intervened animals. AFB(1)-induced urinary excretion of 8-hydroxydeoxyguanosine was also reduced to 50% on day 7 after lycopene intervention. Collectively, these results suggest that inhibition of phase 1 metabolism and metabolic activation, as well as induction of phase 2 detoxification enzyme activity are the potential mechanisms for the chemopreventive effects of lycopene.     

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.     

Study of aflatoxicosis reduction: effect of Alchornea cordifolia on biomarkers in an aflatoxin B1 exposed rats

The toxicity of aflatoxins results in cancer and liver disease. Several natural substances such as plants exhibited their ability to inhibit the initiation of aflatoxin carcinogenesis. The aim of this study was to evaluate the effect of Alchornea cordifolia on biomarkers in an aflatoxin B1 (AFB1) exposed rats. The contents of polyphenols, flavonoids and the antioxidant activity of A. cordifolia ethanolic leaf extract (EELac) were assessed. Groups of rats were treated orally with a daily dose of a mixture of AFB1 at a dose of 150?μg/kg body weight and EELac (50, 100 and 300?mg/kg body weight) for 21?days. Biomarkers of AFB1, such as the AFB1-lysine adduct and aflatoxin M1 were assayed in blood and urine, respectively, using an HPLC system with a fluorescence detector. The contents of polyphenols and flavonoids were 6783.23?±?272.76?μg EAG/g and 10.54?±?3.15% of dry matter, respectively. EELac showed a good antioxidant activity (IC50?=?12.65?±?0.13?μg/mL). The administration of the mixture (AFB1?+?EELac) at different doses significantly reduced the level of AFB1-lysine adduct from 14.04?±?2.1 to 4.13?±?0.9?ng/mg albumin and that of Aflatoxin M1 (AFM1) from 456?±?16 to 220?±?24?ng/mL (p?<0.05). The rate of reduction was 70.58% for AFB1-lysine adduct and 51.75% for AFM1. A. cordifolia could be used in the prevention of toxicity induced by AFB1 on account of its high content in phenolic compounds.     

Ouabain pre-treatment modulates B and T lymphocytes and improves survival of melanoma-bearing animals

Ouabain (OUA) is a glycoside shown to modulate B and T lymphocytes. Nevertheless, ouabain effects on B16F10 melanoma immune response, a mouse lineage that mimics human melanoma, are still unknown. Our aim was to study how OUA in vivo treatment modulates lymphocytes and if it improves the response against B16F10 cells. C57BL/6 mice were pre-treated with intraperitoneal (i.p) injection of OUA (0.56 mg/Kg) for three consecutive days. On the 4^th day, 10⁶ B16F10 cells or vehicle were i.p. injected. Animals were euthanized on days 4^th and 21^st for organs removal and subsequent lymphocyte analyses by flow cytometry. In vivo ouabain-treatment reduced regulatory T cells in the spleen in both melanoma and non-melanoma groups. Ouabain preserved the number and percentage of B lymphocytes in peripheral organs of melanoma-injected mice. Melanoma-injected mice pre-treated with OUA also survive longer. Our findings contribute to a better understanding of OUA immunological effects in a melanoma model.     

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.