A Bioinspired Single-Atom Fe Nanozyme with Excellent Laccase-Like Activity for Efficient Aflatoxin B1 Removal.

The applications of natural laccases are greatly restricted because of their drawbacks like poor biostability, high costs, and low recovery efficiency. M/NC single atom nanozymes (M/NC SAzymes) are presenting as great substitutes due to their superior enzyme-like activity, excellent selectivity and high stability. In this work, inspired by the catalytic active center of natural enzyme, a biomimetic Fe/NC SAzyme (Fe-SAzyme) with O2-Fe-N4 coordination is successfully developed, exhibiting excellent laccase-like activity. Compared with their natural counterpart, Fe-SAzyme has shown superior catalytic efficiency and excellent stability under a wide range of pH (3.0-9.0), temperature (4-80 °C) and NaCl strength (0-300 mm). Interestingly, density functional theory (DFT) calculations reveal that the high catalytic performance is attributed to the activation of O2 by O2-Fe-N4 sites, which weakened the O─O bonds in the oxygen-to-water oxidation pathway. Furthermore, Fe-SAzyme is successfully applied for efficient aflatoxin B1 removal based on its robust laccase-like catalytic activity. This work provides a strategy for the rational design of laccase-like SAzymes, and the proposed catalytic mechanism will help to understand the coordination environment effect of SAzymes on laccase-like catalytic processes.

Evaluation of antifungal activity of natural compounds on growth and aflatoxin B1 production of Aspergillus parasiticus and Aspergillus flavus.

BACKGROUND: Aspergillus species cause broad spectrum infections especially invasive lethal infections in immunocompromised patients. This study aimed to assess the antifungal activity of plants and compounds including Aloe vera, Thyme, carvacrol, and nano-encapsulation of carvacrol on the growth and production of aflatoxin B1 production by Aspergillus parasiticus and Aspergillus flavus. METHODS AND RESULTS: Minimum inhibitory concentrations of extracts Aloe vera, Thyme, carvacrol, and nanocarvacrol, and fluconazole as a control were determined according to Clinical and Laboratory Standards Institute by serial microdilution protocol. Then, the effect of inhibitory concentrations of these compounds on the aflatoxin B1 production level was evaluated by real-time PCR and high-performance liquid chromatography. Our results indicate that the Aspergillus parasiticus and Aspergillus flavusare sensitive to selected plants and compounds. CONCLUSION: Our findings showed that the compounds are appropriate alternative candidates against growth and production of aflatoxin of Aspergillus spp.

Glass bead system to study mycotoxin production of Aspergillus spp. on corn and rice starches.

Mycotoxin production by aflatoxin B1 (AFB1) -producing Aspergillus flavus Zt41 and sterigmatocystin (ST) -hyperproducer Aspergillus creber 2663 mold strains on corn and rice starch, both of high purity and nearly identical amylose-amylopectin composition, as the only source of carbon, was studied. Scanning electron microscopy revealed average starch particle sizes of 4.54 ± 0.635 µm and 10.9 ± 2.78 µm, corresponding to surface area to volume ratios of 127 1/µm for rice starch and 0.49 1/µm for corn starch. Thus, a 2.5-fold difference in particle size correlated to a larger, 259-fold difference in surface area. To allow starch, a water-absorbing powder, to be used as a sole food source for Aspergillus strains, a special glass bead system was applied. AFB1 production of A. flavus Zt41 was determined to be 437.6 ± 128.4 ng/g and 90.0 ± 44.8 ng/g on rice and corn starch, respectively, while corresponding ST production levels by A. creber 2663 were 72.8 ± 10.0 µg/g and 26.8 ± 11.6 µg/g, indicating 3-fivefold higher mycotoxin levels on rice starch than on corn starch as sole carbon and energy sources. KEY POINTS: • A glass bead system ensuring the flow of air when studying powders was developed. • AFB1 and ST production of A. flavus and A. creber on rice and corn starches were studied. • 3-fivefold higher mycotoxin levels on rice starch than on corn starch were detected.

Camel milk or silymarin could improve the negative effects that experimentally produced by aflatoxin B1 on rat's male reproductive system.

BACKGROUND: Camel milk and silymarin have many different beneficial effects on several animal species. Meanwhile, Aflatoxins are mycotoxins with extraordinary potency that pose major health risks to several animal species. Additionally, it has been documented that aflatoxins harm the reproductive systems of a variety of domestic animals. The present design aimed to investigate the impact of aflatoxin B1 (AFB1) on rat body weight and reproductive organs and the ameliorative effects of camel milk and silymarin through measured serum testosterone, testes pathology, and gene expression of tumor necrosis factor (TNF-α), luteinizing hormone receptor (LHR), and steroidogenic acute regulatory protein (StAR) in the testes. A total of sixty mature male Wister white rats, each weighing an average of 83.67 ± 0.21 g, were used. There were six groups created from the rats. Each division had ten rats. The groups were the control (without any treatment), CM (1 ml of camel milk/kg body weight orally), S (20 mg silymarin/kg b. wt. suspension, orally), A (1.4 mg aflatoxin/kg diet), ACM (aflatoxin plus camel milk), and AS (aflatoxin plus silymarin). RESULTS: The results indicated the positive effects of camel milk and silymarin on growth, reproductive organs, and gene expression of TNF-α, LHR, and StAR with normal testicular architecture. Also, the negative effect of AFB1 on the rat's body weight and reproductive organs, as indicated by low body weight and testosterone concentration, was confirmed by the results of histopathology and gene expression. However, these negative effects were ameliorated by the ingestion of camel milk and silymarin. CONCLUSION: In conclusion, camel milk and silymarin could mitigate the negative effect of AFB1 on rat body weight and reproductive organs.

New insights into aflatoxin B1 mechanistic toxicology in cattle liver: an integrated approach using molecular docking and biological evaluation in CYP1A1 and CYP3A74 knockout BFH12 cell lines.

Aflatoxin B1 (AFB1) is a pro-carcinogenic compound bioactivated in the liver by cytochromes P450 (CYPs). In mammals, CYP1A and CYP3A are responsible for AFB1 metabolism, with the formation of the genotoxic carcinogens AFB1-8,9-epoxide and AFM1, and the detoxified metabolite AFQ1. Due to climate change, AFB1 cereals contamination arose in Europe. Thus, cattle, as other farm animals fed with grains (pig, sheep and broiler), are more likely exposed to AFB1 via feed with consequent release of AFM1 in milk, posing a great concern to human health. However, knowledge about bovine CYPs involved in AFB1 metabolism is still scanty. Therefore, CYP1A1- and CYP3A74-mediated molecular mechanisms of AFB1 hepatotoxicity were here dissected. Molecular docking of AFB1 into CYP1A1 model suggested AFB1 8,9-endo- and 8,9-exo-epoxide, and AFM1 formation, while docking of AFB1 into CYP3A74 pointed to AFB1 8,9-exo-epoxide and AFQ1 synthesis. To biologically confirm these predictions, CYP1A1 and CYP3A74 knockout (KO) BFH12 cell lines were exposed to AFB1. LC-MS/MS investigations showed the abolished production of AFM1 in CYP1A1 KO cells and the strong increase of parent AFB1 in CYP3A74 KO cells; the latter result, coupled to a decreased cytotoxicity, suggested the major role of CYP3A74 in AFB1 8,9-exo-epoxide formation. Finally, RNA-sequencing analysis indirectly proved lower AFB1-induced cytotoxic effects in engineered cells versus naïve ones. Overall, this study broadens the knowledge on AFB1 metabolism and hepatotoxicity in cattle, and it provides the weight of evidence that CYP1A1 and CYP3A74 inhibition might be exploited to reduce AFM1 and AFBO synthesis, AFB1 toxicity, and AFM1 milk excretion.

Aflatoxin B1 inhibited the development of primary myoblasts of grass carp (Ctenopharyngodon idella) by degrading extracellular matrix.

According to the International Agency for Research on Cancer (IARC), aflatoxin B1 (AFB1) has been recognized as a major contaminant in food and animal feed and which is a common mycotoxin with high toxicity. Previous research has found that AFB1 inhibited zebrafish muscle development. However, the potential mechanism of AFB1 on fish muscle development is unknown, so it is necessary to conduct further investigation. In the present research, the primary myoblast of grass carp was used as a model, we treated myoblasts with AFB1 for 24 h. Our results found that 5 µM AFB1 significantly inhibited cell proliferation and migration (P < 0.05), and 10 µM AFB1 promoted lactate dehydrogenase (LDH) release (P < 0.05). Reactive oxygen species (ROS), protein carbonyl (PC) and malondialdehyde (MDA) levels were increased in 15, 5 and 10 µM AFB1 (P < 0.05), respectively. Catalase (CAT), glutathione peroxidase (GPx) and total superoxide dismutase (T-SOD) activities were decreased in 10, 10 and 15 µM AFB1 (P < 0.05), respectively. Furthermore, 15 µM AFB1 induced oxidative damage by Nrf2 pathway, also induced apoptosis in primary myoblast of grass carp. Meanwhile, 15 µM AFB1 decreased MyoD gene and protein expression (P < 0.05). Importantly, 15 µM AFB1 decreased the protein expression of collagen Ⅰ and fibronectin (P < 0.05), and increased the protein levels of urokinase plasminogen activator (uPA), matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 2 (MMP-2), and p38 mitogen-activated protein kinase (p38MAPK) (P < 0.05). As a result, our findings suggested that AFB1 damaged the cell morphology, induced oxidative damage and apoptosis, degraded ECM components, in turn inhibiting myoblast development by activating the p38MAPK/urokinase-type plasminogen activator (uPA)/matrix metalloproteinase (MMPs)/extracellular matrix (ECM) signaling pathway.

The role of larvae of black soldier fly and house fly and of feed substrate microbes in biotransformation of aflatoxin B1.

Over the past few years, there has been growing interest in the ability of insect larvae to convert various organic side-streams containing mycotoxins into insect biomass that can be used as animal feed. Various studies have examined the effects of exposure to aflatoxin B1 (AFB1) on a variety of insect species, including the larvae of the black soldier fly (BSFL; Hermetia illucens L.; Diptera: Stratiomyidae) and the housefly (HFL; Musca domestica L.; Diptera: Muscidae). Most of these studies demonstrated that AFB1 degradation takes place, either enzymatic and/or non-enzymatic. The possible role of feed substrate microorganisms (MOs) in this process has thus far not been investigated. The main objective of this study was therefore to investigate whether biotransformation of AFB1 occurred and whether it is caused by insect-enzymes and/or by microbial enzymes of MOs in the feed substrate. In order to investigate this, sterile and non-sterile feed substrates were spiked with AFB1 and incubated either with or without insect larvae (BSFL or HFL). The AFB1 concentration was determined via LC-MS/MS analyses and recorded over time. Approximately 50% of the initially present AFB1 was recovered in the treatment involving BSFL, which was comparable to the treatment without BSFL (60%). Similar patterns were observed for HFL. The molar mass balance of AFB1 for the sterile feed substrates with BSFL and HFL was 73% and 78%, respectively. We could not establish whether non-enzymatic degradation of AFB1 in the feed substrates occurred. The results showed that both BSFL and substrate-specific MOs play a role in the biotransformation of AFB1 as well as in conversion of AFB1 into aflatoxin P1 and aflatoxicol, respectively. In contrast, HFL did not seem to contribute to AFB1 degradation. The obtained results contribute to our understanding of aflatoxin metabolism by different insect species. This information is crucial for assessing the safety of feeding fly larvae with feed substrates contaminated with AFB1 with the purpose of subsequent use as animal feed.

Baicalin protects against hepatocyte injury caused by aflatoxin B1 via the TP53-related ferroptosis Pathway.

OBJECTIVE: Baicalin has antioxidative, antiviral, and anti-inflammatory properties. However, its ability to alleviate oxidative stress (OS) and DNA damage in liver cells exposed to aflatoxin B1 (AFB1), a highly hepatotoxic compound, remains uncertain. In this study, the protective effects of baicalin on AFB1-induced hepatocyte injury and the mechanisms underlying those effects were investigated. METHODS: Stable cell lines expressing CYP3A4 were established using lentiviral vectors to assess oxidative stress levels by conducting assays to determine the content of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD). Additionally, DNA damage was evaluated by 8-hydroxy-2-deoxyguanosine (8-OHdG) and comet assays. Transcriptome sequencing, molecular docking, and in vitro experiments were conducted to determine the mechanisms underlying the effects of baicalin on AFB1-induced hepatocyte injury. In vivo, a rat model of hepatocyte injury induced by AFB1 was used to evaluate the effects of baicalin. RESULTS: In vitro, baicalin significantly attenuated AFB1-induced injury caused due to OS, as determined by a decrease in ROS, MDA, and SOD levels. Baicalin also considerably decreased AFB1-induced DNA damage in hepatocytes. This protective effect of baicalin was found to be closely associated with the TP53-mediated ferroptosis pathway. To elaborate, baicalin physically interacts with P53, leading to the suppression of the expression of GPX4 and SLC7A11, which in turn inhibits ferroptosis. In vivo findings showed that baicalin decreased DNA damage and ferroptosis in AFB1-treated rat liver tissues, as determined by a decrease in the expression of γ-H2AX and an increase in GPX4 and SLC7A11 levels. Overexpression of TP53 weakened the protective effects of baicalin. CONCLUSIONS: Baicalin can alleviate AFB1-induced OS and DNA damage in liver cells via the TP53-mediated ferroptosis pathway. In this study, a theoretical foundation was established for the use of baicalin in protecting the liver from the toxic effects of AFB1.

Hesperetin protects hippocampal neurons from the neurotoxicity of Aflatoxin B1 in mice.

Aflatoxin B1 (AFB1) is a major food and feed pollutant that endangers public health. Previous studies have shown that exposure to AFB1 causes neurotoxicity in the body. However, the mechanism of neurotoxicity caused by AFB1 is not well understood, and finding a workable and practical method to safeguard animals from AFB1 toxicity is essential. This study confirmed that AFB1 caused endoplasmic reticulum stress (ER stress) and apoptosis in hippocampal neurons using C57BL/6 J mice and HT22 cells as models. In vitro experiments showed that the aryl hydrocarbon receptor (AHR) plays a significant role in the cytotoxicity of AFB1. Finally, we assessed how hesperetin protecting against the neurotoxicity caused by AFB1. Our findings demonstrated that AFB1 increased the levels of BAX and Cleaved-Caspase3 proteins, while decreasing the levels of BCL2 protein in the CA1 and CA3 regions of the hippocampus. The AFB1 increased the expression of AHR and activated nuclear translocation. It also elevated the expression levels of Chop, GRP78, p-IRE1/ Xbp1s, and p-PERK/p-EIF2a. Importantly, we also discovered for the first time that blocking AHR in HT22 cells dramatically reduced the level of ER stress and apoptosis caused by AFB1. In vivo and in vitro studies, supplementation of hesperetin effectively reversed AFB1-induced cytotoxicity. We have demonstrated that hesperetin effectively restored the imbalance in the GSH/GST system in HT22 cells treated with AFB1. Furthermore, we observed that elevated GSH levels facilitated the formation of AFB1-GSH complexes, which enhanced the excretion of AFB1. Therefore, hesperetin improves ER stress-induced apoptosis by reducing AFB1 activation of AHR.

Integrated network toxicology, molecular docking, and in vivo experiments to elucidate molecular mechanism of aflatoxin B1 hepatotoxicity.

Due to the rise in temperature and sea level caused by climate change, the detection rate of aflatoxin B1 (AFB1) in food crops has increased dramatically, and the frequency and severity of aflatoxicosis in humans and animals are also increasing. AFB1 has strong hepatotoxicity, causing severe liver damage and even cancer. However, the mechanism of AFB1 hepatotoxicity remains unclear. By integrating network toxicology, molecular docking and in vivo experiments, this research was designed to explore the potential hepatotoxicity mechanisms of AFB1. Thirty-three intersection targets for AFB1-induced liver damage were identified using online databases. PI3K/AKT1, MAPK, FOXO1 signaling pathways, and apoptosis were significantly enriched. In addition, the proteins of ALB, AKT1, PIK3CG, MAPK8, HSP90AA1, PPARA, MAPK1, EGFR, FOXO1, and IGF1 exhibited good affinity with AFB1. In vivo experiments, significant pathological changes occurred in the liver of mice. AFB1 induction increased the expression levels of EGFR, ERK, and FOXO1, and decreased the expression levsls of PI3K and AKT1. Moreover, AFB1 treatment caused an increase in Caspase3 expression, and a decrease in Bcl2/Bax ratio. By combining network toxicology with in vivo experiments, this study confirms for the first time that AFB1 promotes the FOXO1 signaling pathway by inactivating PI3K/AKT1 and activating EGFR/ERK signaling pathways, hence aggravating hepatocyte apoptosis. This research provides new strategies for studying the toxicity of environmental pollutants and new possible targets for the development of hepatoprotective drugs.

Salvia miltiorrhiza polysaccharide mitigates AFB1-induced liver injury in rabbits.

Aflatoxin B1 (AFB1) is one of the common dietary contaminants worldwide, which can harm the liver of humans and animals. Salvia miltiorrhiza polysaccharide (SMP) is a natural plant-derived polysaccharide with numerous pharmacological activities, including hepatoprotective properties. The purpose of this study is to explore the intervention effect of SMP on AFB1-induced liver injury and its underlying mechanisms in rabbits. The rabbits were administered AFB1 (25 µg/kg/feed) and or treatment with SMP (300, 600, 900 mg/kg/feed) for 42 days. The results showed that SMP effectively alleviated the negative impact of AFB1 on rabbits' productivity by increasing average daily weight gain (ADG) and feed conversion rate (FCR). SMP reduced aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels in serum, ameliorating AFB1-induced hepatic pathological changes. Additionally, SMP enhanced superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) activity, and inhibited reactive oxygen species (ROS), malondialdehyde (MDA), 4-Hydroxynonenal (4-HNE), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression, thus mitigating AFB1-induced oxidative stress and inflammatory responses. Moreover, SMP upregulated the expression of nuclear factor E2 related factor 2 (Nrf2), heme oxygenase 1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1) and B-cell lymphoma 2 (Bcl2) while downregulating kelch like ECH associated protein 1 (Keap1), cytochrome c (cyt.c), caspase9, caspase3, and Bcl-2-associated X protein (Bax) expression, thereby inhibiting AFB1-induced hepatocyte apoptosis. Consequently, our findings conclude that SMP can mitigate AFB1-induced liver damage by activating the Nrf2/HO-1 pathway and inhibiting mitochondria-dependent apoptotic pathway in rabbits.

Omics analysis revealed the intestinal toxicity induced by aflatoxin B1 and aflatoxin M1.

Aflatoxin B1 (AFB1), a common mycotoxin, can occur in agricultural products. As a metabolite of AFB1, aflatoxin M1 (AFM1) mainly exist in dairy products. These two mycotoxins threaten human health, although it is unclear how they affect the function of the intestinal barrier. In this study, mice were exposed to AFB1 (0.3 mg/kg body b.w.) and AFM1(3.0 mg/kg b.w.) either individually or in combination for 28 days to explore the main differentially expressed proteins (DEPs) and the associated enriched pathways. These findings were preliminarily verified by the transcriptomic and proteomic analyses in differentiated Caco-2 cells. The results revealed that AFB1 and AFM1 exposure in mice disrupted the function of the intestinal barrier, and the combined toxicity was greater than that of each toxin alone. Further proteomic analysis in mice demonstrated that the mechanisms underlying these differences could be explained as follows: (i) lipid metabolism was enriched by AFB1-induced DEPs. (ii) protein export pathway was stimulated by AFM1-induced DEPs. (iii) cell metabolic ability was inhibited (as evidenced by changes in UDP-GT1, UDP-GT2, and Gatm6), apoptosis was induced (MAP4K3), and epithelial cell integrity was disrupted (Claudin7 and IQGAP2), resulting in more extensive intestinal damage after combined treatment. In conclusion, the hazardous impact of co-exposure to AFB1 and AFM1 from proteomic perspectives was demonstrated in the present study.

Allicin attenuates the oxidative damage induced by Aflatoxin B1 in dairy cow hepatocytes via the Nrf2 signalling pathway.

Aflatoxin B1 (AFB1) is known to inhibit growth, and inflict hepatic damage by interfering with protein synthesis. Allicin, has been acknowledged as an efficacious antioxidant capable of shielding the liver from oxidative harm. This study aimed to examine the damage caused by AFB1 on bovine hepatic cells and the protective role of allicin against AFB1-induced cytotoxicity. In this study, cells were pretreated with allicin before the addition of AFB1 for co-cultivation. Our findings indicate that AFB1 compromises cellular integrity, suppresses the expression of nuclear factor erythroid 2-related factor 2 (Nrf2). In addition, allicin attenuates oxidative damage to bovine hepatic cells caused by AFB1 by promoting the expression of the Nrf2 pathway and reducing cell apoptosis. In conclusion, the results of this study will help advance clinical research and applications, providing new options and directions for the prevention and treatment of liver diseases.

Lactobacillus salivarius Ameliorates AFB1-induced hepatotoxicity via PINK1/Parkin-mediated mitophagy in Geese.

Aflatoxin B1 (AFB1) is commonly found in feed ingredients and foods all over the world, posing a significant threat to food safety and public health in animals and humans. Lactobacillus salivarius (L. salivarius) was recorded to improve the intestinal health and performance of chickens. However, whether L. salivarius can alleviate AFB1-induced hepatotoxicity in geese was unknown. A total of 300 Lande geese were randomly assigned to five groups: control group, AFB1 low-dose group (L), L. salivarius+AFB1 low-dose group (LL), AFB1 high dosage groups (H), L. salivarius+AFB1 high dosage groups (LH), respectively. The results showed that the concentrations of ALT, AST, and GGT significantly increased after exposure to AFB1. Similarly, severe damage of hepatic morphology was observed including the hepatic structure injury and inflammatory cell infiltration. The oxidative stress was evidenced by the elevated concentrations of MDA, and decreased activities of GSH-Px, GSH and SOD. The observation of immunofluorescence, real-time PCR, and western blotting showed that the expression of PINK1 and the value of LC3II/LC3I were increased, but that of p62 significantly decreased after AFB1 exposure. Moreover, the supplementation of L. salivarius effectively improved the geese performance, ameliorated AFB1-induced oxidative stress, inhibited mitochondrial mitophagy and enhanced the liver restoration to normal level. The present study demonstrated that L. salivarius ameliorated AFB1-induced the hepatotoxicity by decreasing the oxidative stress, and regulating the expression of PINK1/Parkin-mediated mitophagy in the mitochondria of the geese liver. Furthermore, this investigation suggested that L. salivarius might serve as a novel and safe additive for preventing AFB1 contamination in poultry feed.

Compound probiotics regulate the NRF2 antioxidant pathway to inhibit aflatoxin B1-induced autophagy in mouse Sertoli TM4 cells.

This study investigated the effects of compound probiotics (CP) on AFB1-induced cytotoxicity in Sertoli TM4 cells. The L9 (3 × 3) orthogonal test was conducted to determine the optimal CP required for high AFB1 degradation in the artificial gastrointestinal fluid in vitro. The maximal AFB1 degradation rate was 40.55 % (P < 0.05) when the final viable count was 1.0 × 105 CFU/mL for Bacillus subtilis, Lactobacillus casein, and Saccharomyces cerevisiae. The effects of CP and the CP supernatant (CPS) on TM4 cell viability were evaluated to achieve the optimal protective conditions. When CPS4 (corresponding to CP viable counts of 1.0 × 104 CFU/mL) was added to the TM4 cells for 24 h, the cell viability reached 108.86 % (P < 0.05). AFB1 reduced TM4 cell viability in a concentration- and time-dependent manner at an AFB1 concentration ranging from 0 to 1.5 µM after 48-h AFB1 exposure. The optimal AFB1 concentration/times for low- and high damage models were 0.5 and 1.25 µM both for 24 h, which decreased viability to 76.04 % and 65.35 %, respectively. however, CPS4 added to low- and high-damage models increased the cell viability to 97.43 % and 75.12 %, respectively (P < 0.05). Transcriptome sequencing was performed based on the following designed groups: the control, 0.5 µM AFB1, 1.25 µM AFB1, CPS4, and CPS4+0.5 µM AFB1. The Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis was further performed to identify significantly enriched signaling pathways, which were subsequently verified. It was shown that AFB1 induced apoptosis by blocking the PI3K-AKT-mTOR pathway and upregulating autophagy proteins such as LC3B, Beclin1, and ATG5 while inhibiting autophagic flux. CPS4 promoted AFB1 degradation, activated the p62-NRF2 antioxidant, and inhibited ROS/TRPML1 pathways, thereby reducing ROS production and inflammation and ultimately alleviating AFB1-induced autophagy and apoptosis. These findings supports the potential of probiotics to protect the male reproductive system from toxin damage.

Theoretical insights into the mechanism underlying aflatoxin B1 transformation by the BsCotA-methyl syringate system.

Global concern exists regarding the contamination of food and animal feed with aflatoxin B1 (AFB1), which poses a threat to the health of both humans and animals. Previously, we found that a laccase from Bacillus subtilis (BsCotA) effectively detoxified AFB1 in a reaction mediated by methyl syringate (MS), although the underlying mechanism has not been determined. Therefore, our primary objective of this study was to explore the detoxification mechanism employed by BsCotA. First, the enzyme and mediator dependence of AFB1 transformation were studied using the BsCotA-MS system, which revealed the importance of MS radical formation during the oxidation process. Aflatoxin Q1 (AFQ1) resulting from the direct oxidation of AFB1 by BsCotA, was identified using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results of UPLC-MS/MS and density functional theory calculations indicated that the products included AFQ1, AFB1-, and AFD1-MS-coupled products in the BsCotA-MS system. The toxicity evaluations revealed that the substances derived from the transformation of AFB1 through the BsCotA-MS mechanism exhibited markedly reduced toxicity compared to AFB1. Finally, we proposed a set of different AFB1-transformation pathways generated by the BsCotA-MS system based on the identified products. These findings greatly enhance the understanding of the AFB1-transformation mechanism of the laccase-mediator system.

The relationship between aflatoxin B1 with the induction of extrinsic/intrinsic pathways of apoptosis and the protective role of taraxasterol in TM3 leydig cell line.

Aflatoxins B1 (AFB1) a dangerous type of aflatoxin, poses a serious threat to human health. Meanwhile, Taraxasterol, a bioactive compound in dandelion, exhibits strong anti-inflammatory and antioxidant activity. Therefore, the aim of this study was to investigate the impact of AFB1 on the intrinsic and extrinsic pathways of apoptosis, as well as evaluate the protective role of taraxasterol in the TM3 Leydig cell line. Cell viability was evaluated using an MTT assay, measuring the effects of 3.6 µM AFB1 and varying concentrations of taraxasterol. Expression levels of Caspase 3,8, and 9 were analyzed with RT-qPCR, and flow cytometry was used to assess cell cycle progression and apoptotic alterations. The findings of this study demonstrated that exposure to 3.6 µM of AFB1 resulted in an upregulation of Caspase 3 and Caspase 9 expression, indicating an activation of apoptotic pathways in TM3 cells. Additionally, the analysis of apoptosis revealed a significant increase in cellular apoptosis at this AFB1 concentration. However, when TM3 cells were exposed to 5 µM of taraxasterol, a downregulation of Caspase 3 and Caspase 9 expression was observed, suggesting a protective effect against apoptosis. Moreover, the apoptotic rate in TM3 cells was reduced in the presence of 5 µM of taraxasterol. Consequently, this study highlights the potential of taraxasterol as a protective agent against AFB1-induced apoptosis and suggest its potential application in regulating cell survival and apoptosis-related processes. Further investigations are necessary to elucidate the underlying mechanisms and evaluate the clinical implications of taraxasterol in the context of fertility disorders and other conditions associated with AFB1 exposure.

Aflatoxin B1-exposed hepatocyte-derived extracellular vesicles: Initiating hepatic stellate cell-mediated liver fibrosis through a p53-Parkin-dependent mitophagy pathway.

Environmental aflatoxin B1 (AFB1) exposure has been proposed to contribute to hepatocellular carcinoma by promoting liver fibrosis, but the potential mechanisms remain to be further elucidated. Extracellular vesicles (EVs) were recognized as crucial traffickers for hepatic intercellular communication and play a vital role in the pathological process of liver fibrosis. The AFB1-exposed hepatocyte-derived EVs (AFB1-EVs) were extracted, and the functional effects of AFB1-EVs on the activation of hepatic stellate cells (HSCs) were explored to investigate the molecular mechanism of AFB1 exposure-induced liver fibrogenesis. Our results revealed that an environment-level AFB1 exposure induced liver fibrosis via HSCs activation in mice, while the AFB1-EVs mediated hepatotoxicity and liver fibrogenesis in vitro and in vivo. AFB1 exposure in vitro increased PINK1/Parkin-dependent mitophagy in hepatocytes, where upregulated transcription of the PARK2 gene via p53 nuclear translocation and mitochondrial recruitment of Parkin, and promoted AFB1-EVs-mediated mitochondria-trafficking communication between hepatocytes and HSCs. The knockdown of Parkin in HepaRG cells reversed HSCs activation by blocking the mitophagy-related AFB1-EVs trafficking. This study further revealed that the hepatic fibrogenesis of AFB1 exposure was rescued by genetic intervention with siPARK2 or p53's Pifithrin-α (PFTα) inhibitors. Furthermore, AFB1-EVs-induced HSCs activation was relieved by GW4869 pharmaceutic inhibition of EVs secretion. These results revealed a novel mechanism that AFB1 exposure-induced p53-Parkin signal axis regulated mitophagy-dependent hepatocyte-derived EVs to mediate the mitochondria-trafficking intercellular communication between hepatocytes and HSCs in the local hepatotoxic microenvironment to promote the activated HSCs-associated liver fibrogenesis. Our study provided insight into p53-Parkin-dependent pathway regulation and promised an advanced strategy targeting intervention to EVs-mediated mitochondria trafficking for preventing xenobiotics-induced liver fibrosis.

Efficacy of atoxigenic Aspergillus flavus from southern China as biocontrol agents against aflatoxin contamination in corn and peanuts.

Aspergillus flavus is a ubiquitous facultative pathogen that routinely infects important crops leading to formation of aflatoxins during crop development and after harvest. Corn and peanuts in warm and/or drought-prone regions are highly susceptible to aflatoxin contamination. Controlling aflatoxin using atoxigenic A. flavus is a widely adopted strategy. However, no A. flavus genotypes are currently approved for use in China. The current study aimed to select atoxigenic A. flavus endemic to Guangxi Zhuang Autonomous Region with potential as active ingredients of aflatoxin biocontrol products. A total of 204 A. flavus isolates from corn, peanuts, and field soil were evaluated for ability to produce the targeted mycotoxins. Overall, 57.3% could not produce aflatoxins while 17.15% were incapable of producing both aflatoxins and CPA. Atoxigenic germplasm endemic to Guangxi was highly diverse, yielding 8 different gene deletion patterns in the aflatoxin and CPA biosynthesis gene clusters ranging from no deletion to deletion of both clusters. Inoculation of corn and peanuts with both an aflatoxin producer and selected atoxigenic genotypes showed significant reduction (74 to 99%) in aflatoxin B1 (AFB1) formation compared with inoculation with the aflatoxin producer alone. Atoxigenic genotypes also efficiently degraded AFB1 (61%). Furthermore, atoxigenic isolates were also highly efficient at reducing aflatoxin concentrations even when present at lower concentrations than aflatoxin producers. The use of multiple atoxigenics was not always as effective as the use of a single atoxigenic. Effective atoxigenic genotypes of A. flavus with known mechanisms of atoxigenicity are demonstrated to be endemic to Southern China. These A. flavus may be utilized as active ingredients of biocontrol products without concern for detrimental impacts that may result from introduction of exotic fungi. Field efficacy trials in the agroecosystems of Southern China are needed to determine the extent to which such products may allow the production of safer food and feed.

Effects of aflatoxin B1 on metabolism- and immunity-related gene expression in Hermetia illucens L. (Diptera: Stratiomyidae).

Contamination of food products with mycotoxins such as aflatoxin B1 (AFB1) poses a severe risk to human health. Larvae of the black soldier fly (BSFL), Hermetia illucens (Diptera: Stratiomyidae), can successfully metabolize AFB1 without any negative consequences on their survival or growth. Organic waste streams contaminated with mycotoxins can be upcycled into protein-rich BSFL as an alternative feed for livestock and the left-over feed residue into nutrient-rich crop fertilizers. However, the underlying mechanisms that allow BSFL to metabolize AFB1 are unknown. In this study, five-day-old BSFL were fed with either a control or an AFB1-spiked (20 µg/kg) diet to elucidate the underlying mechanisms. Larval samples were collected at three timepoints (6 h, 24 h and 72 h) and subjected to RNA-Seq analysis to determine gene expression patterns. Provision of an AFB1-spiked diet resulted in an up-regulation of 357 and a down-regulation of 929 unique genes. Upregulated genes include multiple genes involved in AFB1 metabolism in other (insect) species. Downregulated genes were generally involved in the insects' growth, development, and immunity. BSFL possesses a diverse genetic arsenal that encodes for enzymes capable of metabolizing AFB1 without trade-offs on larval survival. In conclusion, the adverse impact of AFB1 exposure on immunity-related processes is observed in the transcriptomic response, and is indicative of a trade-off between detoxification and immune responses.