Volatiles of antagonistic soil yeasts inhibit growth and aflatoxin production of Aspergillus flavus.

Minimizing Aspergillus flavus growth is an effective strategy to mitigate aflatoxin contamination in food and agricultural products. In the present investigation, we attempted to utilize soil-associated yeasts from the Western and Eastern Ghats of India against A. flavus to reduce aflatoxin contamination. Forty-five yeast isolates were screened against A. flavus using overlay and dual plate assays. Among them, 12 isolates effectively inhibited the growth of A. flavus. The 18S rDNA gene sequence analysis identified the twelve antagonistic isolates as belonging to Saccharomyces cerevisiae, Suhomyces xylopsoci, Pichia kudriavzevii, and Candida tropicalis. From the isolated yeasts, S. cerevisiae strains were selected for further evaluation based on the potential antagonistic activity. Volatiles of S. cerevisiae effectively suppressed the mycelial growth of A. flavus (P < 0.05) up to 92.1 % at 7 DAI. Scanning electron microscopic images of the fungus exposed to volatiles showed hyphal deformity and mycelial damage. Aflatoxin B1 (AFB1) production was drastically reduced up to 99.0 % in the volatile-exposed fungus compared to the control. The yeast strain YKK1 showed consistent Aspergillus flavus growth inhibition (80.7 %) and AFB1 production (98.1 %) for 14 days. Gas chromatography-mass spectrophotometry analysis of the yeast volatiles revealed the presence of antimicrobial compounds, including 1-pentanol, 1-propanol, ethyl hexanol, ethanol, 2-methyl-1-butanol, ethyl acetate, dimethyl trisulfide, p-xylene, styrene, and 1,4-pentadiene. The evaluated compounds of yeast volatiles, including ethyl acetate, hexanal, 1-propanol, 1-heptanol, 1-butanol, and benzothiazole, inhibited the fungal growth and AFB1 production of Aspergillus flavus when applied as pure chemicals. Benzothiazole at 5 mM was responsible for a high level of growth inhibition (23.6 %) and reduction of AFB1 synthesis (93.5 %). Hence, volatile compounds produced by soil yeast strains could be a potential biocontrol mechanism against aflatoxin contamination.

PINK1/Parkin-mediated mitophagy is activated to protect against AFB1-induced immunosuppression in mice spleen.

Aflatoxin B1 (AFB1) can cause mitochondrial malfunction and immunosuppression in spleen. Mitochondrial damage can lead to oxidative stress and aggravate immune cell dysfunction. Phosphatase and tensin homolog (PTEN)-induced putative kinase1 (PINK1)/ E3 ubiquitin ligase PARK2 (Parkin)-mediated mitophagy can scavenge damaged mitochondria and alleviate oxidative stress to maintain cellular homeostasis. However, the role of PINK1/Parkin-mediated mitophagy in AFB1-induced immunosuppression in spleen is unclear. In this study, sixty male mice were sensibilized orally with AFB1 at different concentrations [0, 0.5, 0.75, and 1 mg/kg body weight (BW)] for 28 days, and AFB1 caused splenic structure injury and immunosuppression, also led to upregulation of PINK1/Parkin-mediated mitophagy in a dose-dependent manner. Subsequently, thirty male WT C57BL/6 N mice and thirty male Parkin knockout (Parkin-/-) C57BL/6 N mice were sensibilized orally with AFB1 at 0 or 1 mg/kg BW for 28 days, and Parkin-/- inhibited mitophagy and further aggravated AFB1-induced splenic structure injury, immunosuppression, mitochondrial damage and oxidative stress. Collectively, these results indicate that AFB1 exposure activates PINK1/Parkin-mediated mitophagy, which protects against immunosuppression in spleen.

Fabrication, physico-chemical characterization, and bioactivity evaluation of chitosan-linalool composite nano-matrix as innovative controlled release delivery system for food preservation.

The aim of the present study was to encapsulate linalool into chitosan nanocomposite (Nm-linalool) for developing novel controlled release delivery system in order to protect stored rice against fungal infestation, aflatoxin B1 (AFB1) contamination, and lipid peroxidation. The chitosan-linalool nanocomposite showed spherical shapes, smooth surface with monomodal distribution as revealed by SEM and AFM investigation. FTIR and XRD represented peak shifting and changes in degree of crystallinity after incorporation of linalool into chitosan nanocomposite. Nanoencapsulation of linalool showed higher zeta potential and lowered polydispersity index. TGA analysis reflected the stability of Nm-linalool with reduced weight loss at varying temperatures. Biphasic pattern, with initial rapid release followed by sustained release illustrated controlled delivery of linalool from chitosan nanocomposite, a prerequisite for shelf-life enhancement of stored food products. Chitosan nanocomposite incorporating linalool displayed prominent antifungal and antiaflatoxigenic activity during in vitro as well as in situ investigation in rice with improved antioxidant potentiality. Further, Nm-linalool displayed considerable reduction of lipid peroxidation in rice without exerting any adverse impact on organoleptic attributes. In conclusion, the investigation strengthens the application of chitosan-linalool nanocomposite as an innovative controlled nano-delivery system for its practical application as novel environmentally friendly eco-smart preservative in food and agricultural industries.

AHR mediates the aflatoxin B1 toxicity associated with hepatocellular carcinoma.

Aflatoxin exposure is a crucial factor in promoting the development of primary hepatocellular carcinoma (HCC) in individuals infected with the hepatitis virus. However, the molecular pathways leading to its bioactivation and subsequent toxicity in hepatocytes have not been well-defined. Here, we carried out a genome-wide CRISPR-Cas9 genetic screen to identify aflatoxin B1 (AFB1) targets. Among the most significant hits was the aryl hydrocarbon receptor (AHR), a ligand-binding transcription factor regulating cell metabolism, differentiation, and immunity. AHR-deficient cells tolerated high concentrations of AFB1, in which AFB1 adduct formation was significantly decreased. AFB1 triggered AHR nuclear translocation by directly binding to its N-terminus. Furthermore, AHR mediated the expression of P450 induced by AFB1. AHR expression was also elevated in primary tumor sections obtained from AFB1-HCC patients, which paralleled the upregulation of PD-L1, a clinically relevant immune regulator. Finally, anti-PD-L1 therapy exhibited greater efficacy in HCC xenografts derived from cells with ectopic expression of AHR. These results demonstrated that AHR was required for the AFB1 toxicity associated with HCC, and implicate the immunosuppressive regimen of anti-PD-L1 as a therapeutic option for the treatment of AFB1-associated HCCs.

Evaluation of the Multimycotoxin-Degrading Efficiency of Rhodococcus erythropolis NI1 Strain with the Three-Step Zebrafish Microinjection Method.

The multimycotoxin-degrading efficiency of the Rhodococcus erythropolis NI1 strain was investigated with a previously developed three-step method. NI1 bacterial metabolites, single and combined mycotoxins and their NI1 degradation products, were injected into one cell stage zebrafish embryos in the same doses. Toxic and interaction effects were supplemented with UHPLC-MS/MS measurement of toxin concentrations. Results showed that the NI1 strain was able to degrade mycotoxins and their mixtures in different proportions, where a higher ratio of mycotoxins were reduced in combination than single ones. The NI1 strain reduced the toxic effects of mycotoxins and mixtures, except for the AFB1+T-2 mixture. Degradation products of the AFB1+T-2 mixture by the NI1 strain were more toxic than the initial AFB1+T-2 mixture, while the analytical results showed very high degradation, which means that the NI1 strain degraded this mixture to toxic degradation products. The NI1 strain was able to detoxify the AFB1, ZEN, T-2 toxins and mixtures (except for AFB1+T-2 mixture) during the degradation experiments, which means that the NI1 strain degraded these to non-toxic degradation products. The results demonstrate that single exposures of mycotoxins were very toxic. The combined exposure of mycotoxins had synergistic effects, except for ZEN+T-2 and AFB1+ZEN +T-2, whose mixtures had very strong antagonistic effects.

Effect of aflatoxin B1 on bovine spermatozoa's proteome and embryo's transcriptome.

This study aims to evaluate the deleterious effect of the mycotoxin aflatoxin B1 (AFB1) on bull spermatozoa and the carryver effect on the developing embryo. Proteomic analysis of AFB1-treated spermatozoa revealed differential expression of proteins associated with biological processes and cellular pathways that involved in spermatozoon function, fertilization competence and embryonic development. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. To confirm this hypothesis, we have used the annexin V (AV) kit to separate the spermatozoa into apoptotic (AV+) and non-apoptotic (AV-) subpopulations which were found to correlate with high- and low DNA fragmentation, respectively. Fertilization with AV+ AFB1-treated spermatozoa, resulted in no blastocyst formation, whereas fertilization with AV- spermatozoa resulted in reduced cleavage rate and formation of genetically altered blastocysts (POU5F1 and SOX2). Microarray analysis of blastocysts derived from 10 µM AFB1-treated spermatozoa revealed differential expression of 345 genes that involved in cellular pathways such as embryo and placenta development, cell cycle, DNA repair and histone modification, and in signaling pathways, especially calcium signaling pathway. This is the first report on deleterious carrying over effects of AFB1 from the bovine spermatozoa to the formed embryo. Our findings suggest that aside from the damage caused by AFB1 to spermatozoa's DNA integrity, additional damage mechanisms are involved.

Aflatoxin B1 Induces Neurotoxicity through Reactive Oxygen Species Generation, DNA Damage, Apoptosis, and S-Phase Cell Cycle Arrest.

Aflatoxin B1 (AFB1) is a mycotoxin widely distributed in a variety of food commodities and exhibits strong toxicity toward multiple tissues and organs. However, little is known about its neurotoxicity and the associated mechanism. In this study, we observed that brain integrity was markedly damaged in mice after intragastric administration of AFB1 (300 µg/kg/day for 30 days). The toxicity of AFB1 on neuronal cells and the underlying mechanisms were then investigated in the neuroblastoma cell line IMR-32. A cell viability assay showed that the IC50 values of AFB1 on IMR-32 cells were 6.18 µg/mL and 5.87 µg/mL after treatment for 24 h and 48 h, respectively. ROS levels in IMR-32 cells increased significantly in a time- and AFB1 concentration-dependent manner, which was associated with the upregulation of NOX2, and downregulation of OXR1, SOD1, and SOD2. Substantial DNA damage associated with the downregulation of PARP1, BRCA2, and RAD51 was also observed. Furthermore, AFB1 significantly induced S-phase arrest, which is associated with the upregulation of CDKN1A, CDKN2C, and CDKN2D. Finally, AFB1 induced apoptosis involving CASP3 and BAX. Taken together, AFB1 manifests a wide range of cytotoxicity on neuronal cells including ROS accumulation, DNA damage, S-phase arrest, and apoptosis-all of which are key factors for understanding the neurotoxicology of AFB1.

Human multi-organ chip co-culture of bronchial lung culture and liver spheroids for substance exposure studies.

Extrapolation of cell culture-based test results to in vivo effects is limited, as cell cultures fail to emulate organ complexity and multi-tissue crosstalk. Biology-inspired microphysiological systems provide preclinical insights into absorption, distribution, metabolism, excretion, and toxicity of substances in vitro by using human three-dimensional organotypic cultures. We co-cultured a human lung equivalent from the commercially available bronchial MucilAir culture and human liver spheroids from HepaRG cells to assess the potential toxicity of inhaled substances under conditions that permit organ crosstalk. We designed a new HUMIMIC Chip with optimized medium supply and oxygenation of the organ cultures and cultivated them on-chip for 14 days in separate culture compartments of a closed circulatory perfusion system, demonstrating the viability and homeostasis of the tissue cultures. A single-dose treatment of the hepatotoxic and carcinogenic aflatoxin B1 impaired functionality in bronchial MucilAir tissues in monoculture but showed a protective effect when the tissues were co-cultured with liver spheroids, indicating that crosstalk can be achieved in this new human lung-liver co-culture. The setup described here may be used to determine the effects of exposure to inhaled substances on a systemic level.

Effects of Melatonin and Flavonoid-Rich Fractions of Chromolaena odorata on the Alteration of Proinflammatory Cytokines and Nitric Oxide Induced by Aflatoxin B1 in the Gastric Mucosa of Wistar Rats.

In this study, we investigated serum interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) after ingestion of aflatoxin B1 (AFB1) in rats. We also studied the effects of nitric oxide (NO) on the stomach after consumption of AFB1. Therefore, we hypothesized that a standard anti-inflammatory agent-melatonin (MEL), and the flavonoid-rich fractions from Chromolaena odorata (FRFC) could counteract the deleterious effects of IL-1ß, TNF-α, and NO after consumption of AFB1. Thirty-five Wistar rats (211.86 ± 27.23 g) were randomly selected into 5 groups, with 7 rats in each group. Group A (control); all rats in groups B, C, D, and E received 2.5 mg/kg AFB1 each orally on day 5, whereas those of groups C, D, and E received oral administration of 10 mg/kg MEL, 50 mg/kg FRFC1, and 100 mg/kg FRFC2, respectively, for 7 days. All of them were killed on the 8th day, 24 h after last treatment. Serum samples were analyzed for IL-1ß and TNF-α, whereas stomach tissue was evaluated for NO level. Significant (P < 0.5) increase in serum IL-1ß and TNF-α in rats given AFB1 only was recorded when compared with those in the control group. Conversely, we observed significant reduction in serum IL-1ß and TNF-α in all the groups that received MEL, FRFC1, and FRFC2 after pretreatment with AFB1 when compared with those that were given AFB1 only. In addition, there was a significant increase in NO in rats given AFB1 only when compared with control, whereas reduction in NO was significant in the groups C, D, and E that were given MEL, FRFC1, and FRFC2, respectively, when compared with AFB1 group. MEL and FRFC may be responsible for the prevention of increased gastric mucosal NO and inflammatory effects of proinflammatory cytokines induced by AFB1.

Analysis of miRNA-mRNA regulatory network revealed key genes induced by aflatoxin B1 exposure in primary human hepatocytes.

BACKGROUND: Aflatoxin B1 (AFB1) exposure is a crucial factor to initiate hepatocellular carcinoma (HCC). However, comprehensive microRNA (miRNA)-message RNA (mRNA) regulatory network regarding AFB1-associated HCC is still lacking. This work was aimed to identify miRNA-mRNA network in primary human hepatocytes after AFB1 exposure. METHODS: A miRNA expression dataset GSE71540 obtained from the gene expression omnibus (GEO) was used to identify differentially expressed miRNAs (DEMs) after AFB1 exposure using GEO2R. Target genes of these DEMs were identified using TargetScan V_7.2, miRDB, PITA, miRanda, and miRTarBase. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed at Database for Annotation, Visualization and Integrated Discovery (DAVID). miRNA-mRNA regulatory network was established by analyzing three enriched KEGG pathways significantly correlated with HCC onset and then visualized at CytoScape. RESULTS: In this work, nine upregulated and nine downregulated DEMs were identified. Functional enrichment analyses showed that these predicted target genes were significantly associated with cancer development. Analysis of three enriched pathways related to the onset of HCC identified 13 and nine target genes for upregulated DEMs and downregulated DEMs, respectively. Subsequently, the miRNA-mRNA regulatory networks were constructed. CONCLUSIONS: In conclusion, miRNA-mRNA regulatory network was established, which will help to understand the mechanism underlying the AFB1-induced onset of HCC.

Aflatoxin B1 induces S phase arrest by upregulating the expression of p21 via MYC, PLK1 and PLD1.

Aflatoxin B1 (AFB1), a member of the aflatoxin family, is a common contaminant in foods and feeds, and AFB1 exposure is associated with various clinical conditions. Thus far, research on the toxicity of AFB1 has mainly focused on its induction of liver cancer, but little research has been reported on renal toxicity, especially with regards to the underlying molecular mechanisms. In this study, we found that AFB1 treatment significantly induced kidney damage and reduced kidney weight. The human kidney cell line HEK293T was used to further study the molecular mechanism of the toxicity of AFB1 to kidney cells. We found that AFB1 significantly and dose-dependently induced S phase arrest and upregulated p21 mRNA and protein expression. Upstream of p21, three negative regulators, PLK1, MYC, and PLD1, were significantly downregulated under AFB1 treatment. Consistently, p21 was upregulated, and PLK1, MYC and PLD1 were downregulated in mouse kidney after AFB1 treatment. Interestingly, AFB1 also decreased the physical interaction between PLK1 and MYC and weakened the stability of the MYC protein. Importantly, overexpression of PLK1, MYC and PLD1 significantly blocked the upregulation of p21 and attenuated the S phase arrest caused by AFB1. In summary, AFB1 markedly induces kidney damage and strongly induces S phase arrest by upregulating the expression of p21 via PLK1, PLD1 and MYC, which represents a noval mechanism of the renal toxicity of AFB1.

Natural-Product-Inspired Compounds as Countermeasures against the Liver Carcinogen Aflatoxin B1.

Aflatoxin B1 (AfB1) ranks among the most potent liver carcinogens known, and the accidental or intentional exposure of humans and livestock to this toxin remains a serious global threat. One protective measure that had been proposed is employing small-molecule therapeutics capable of mitigating the toxicity of AfB1; however, to date, these efforts have had little clinical success. To identify molecular scaffolds that reduce the toxicity of AfB1, we developed a cell-based high-throughput high-content imaging assay that enabled our team to test natural products (pure compounds, fractions, and extracts) for protection of monolayers and spheroids composed of HepG2 liver cells against AfB1. The spheroid assay showed notable potential for further development, as it afforded greater sensitivity of HepG2 cells to AfB1, which is believed to better mimic the in vivo response of hepatocytes to the toxin. One of the most bioactive compounds to arise from this investigation was alternariol-9-methyl ether (1, purified from an Alternaria sp. isolate), which inspired the synthesis and testing of several structurally related molecules. Based on these findings, it is proposed that several types of natural and synthetic polyarene molecules that have undergone oxidative functionalization (e.g., compounds containing 3-methoxyphenol moieties) are promising starting points for the development of new agents that protect against AfB1 toxicity.

Surface-Enhanced Raman Scattering-Fluorescence Dual-Mode Nanosensors for Quantitative Detection of Cytochrome c in Living Cells.

During apoptosis process, the release of cytochrome c (Cyt c) is considered to be a key factor in the intrinsic pathway and is often defined as no regression point. Quantitative detection of intracellular Cyt c remains a challenge. Herein, we have developed surface-enhanced Raman scattering (SERS)-fluorescence dual-mode nanosensors for the quantitative assay of Cyt c in living cells. Dual signal detection was achieved by constructing gold nanotriangles (AuNTs) nanosensors capable of specifically recognizing Cyt c. The nanosensors were prepared by modifying the aptamer of Cyt c on AuNTs and connecting the complementary strands modified with Cy5. The AuNTs provided both enhanced SERS signals and fluorescence quenching effects. Once cells were induced by external stimulus (such as toxins) to release Cyt c, Cyt c would specifically bind to its aptamer, and the complementary strands modified with Cy5 would detach which would result in weakened SERS signal and recovery of fluorescence signal. The experimental results showed that the nanosensors not only had excellent selectivity and sensitivity but also realized real-time monitoring of Cyt c translocation event from mitochondria to cytoplasm. The SERS and fluorescence intensity showed good linear relationship with Cyt c concentration ranging from 0.044 to 9.95 µM and achieved a minimum limit of detection (LOD) of 0.02 µM in living cells. The accuracy of intracellular Cyt c quantitative results was more than 90% compared with the ELISA results.

Selenium Ameliorates AFB1-Induced Excess Apoptosis in Chicken Splenocytes Through Death Receptor and Endoplasmic Reticulum Pathways.

Aflatoxin B1 (AFB1) can cause hepatotoxicity, genotoxicity, and immunosuppressive effects for a variety of organisms. Selenium (Se), as an essential nutrient element, plays important protective effects against cell apoptosis induced by AFB1. This research aimed to reveal the ameliorative effects of selenium on AFB1-induced excess apoptosis in chicken splenocytes through death receptor and endoplasmic reticulum pathways in vivo. Two hundred sixteen neonatal chickens, randomized into four treatments, were fed with basal diet (control treatment), 0.4 mg/kg Se supplement (+Se treatment), 0.6 mg/kg AFB1 (AFB1 treatment), and 0.6 mg/kg AFB1 + 0.4 mg/kg Se (AFB1 + Se treatment) during 21 days of experiment, respectively. Compared with the AFB1 treatment, the levels of splenocyte apoptosis in the AFB1 + Se treatment were obviously dropped by flow cytometry and TUNEL assays although they were still significantly higher than those in the control or + Se treatments. Furthermore, the mRNA expressions of CASP-3, CASP-8 and CASP-10, GRP78, GRP94, TNF-α, TNF-R1, FAS, and FASL of splenocytes in the AFB1 + Se treatment by qRT-PCR assay were significantly decreased compared with the AFB1 treatment. These results indicate that Se could partially ameliorate the AFB1-caused excessive apoptosis of chicken splenocytes through downregulation of endoplasmic reticulum and death receptor pathway molecules. This research may rich the knowledge of the detoxification mechanism of Se on AFB1-induced apoptosis.

Melatonin protects in vitro matured porcine oocytes from toxicity of Aflatoxin B1.

Aflatoxin B1 (AFB1) is a major food and feed contaminant that threaten public health. Previous studies indicate that AFB1 exposure disrupted oocyte maturation. However, an effective and feasible method is unavailable for protecting oocytes against toxicity of AFB1. In the present study, using in vitro matured porcine oocytes and parthenogenetic embryos as model, we confirmed that AFB1 exposure during in vitro oocyte maturation (IVM) significantly impaired both nuclear and cytoplasmic maturation in a dose- and time-dependent manner. The different concentrations of melatonin were also tested for their protective effects on oocytes against the AFB1-induced toxicity. Our results showed that supplementation of a relative high concentration of melatonin (10-3 mol/L) during IVM efficiently reversed the impaired development rate and blastocyst quality, to the levels comparable to those of the control group. Further analysis indicated that melatonin application efficiently alleviated reactive oxygen species accumulation and initiation of apoptosis induced by AFB1 exposure. In addition, disrupted GSH/GPX system, as well as inhibited mitochondrial DNA (mtDNA) replication and mitochondrial biogenesis in AFB1-treated oocytes, can be notably reversed by melatonin application. Furthermore, cumulus cells may be important in mediating the toxicity of AFB1 to oocytes, and the metabolism of AFB1 in cumulus cells can be depressed by melatonin. To the best of our knowledge, this is the first report to confirm that melatonin application can efficiently protect oocytes from AFB1-induced toxicity. Our study provides a promising and practical strategy for alleviating or reversing AFB1-induced female reproductive toxicity in both clinical treatment and domestic reproductive management.

Biomarker of Aflatoxin Ingestion: ¹H NMR-Based Plasma Metabolomics of Dairy Cows Fed Aflatoxin B1 with or without Sequestering Agents.

The study applied ¹H NMR-based plasma metabolomics to identify candidate biomarkers of aflatoxin B1 (AFB1) ingestion in dairy cows fed no sequestering agents and evaluate the effect of supplementing clay and/or a Saccharomyces cerevisiae fermentation product (SCFP) on such biomarkers. Eight lactating cows were randomly assigned to 1 of 4 treatments in a balanced 4 × 4 Latin square design with 2 squares. Treatments were: control, toxin (T; 1725 µg AFB1/head/day), T with clay (CL; 200 g/head/day), and CL with SCFP (CL + SCFP; 35 g of SCFP/head/day). Cows in T, CL, and CL + SCFP were dosed with AFB1 from d 26 to 30. The sequestering agents were top-dressed from d 1 to 33. On d 30 of each period, 15 mL of blood was taken from the coccygeal vessels and plasma samples were prepared by centrifugation. Compared to the control, T decreased plasma concentrations of alanine, acetic acid, leucine, arginine and valine. In contrast, T increased plasma ethanol concentration 3.56-fold compared to control. Treatment with CL tended to reduce sarcosine concentration, whereas treatment with CL + SCFP increased concentrations of mannose and 12 amino acids. Based on size of the area under the curve (AUC) of receiver operating characteristic and fold change (FC) analyses, ethanol was the most significantly altered metabolite in T (AUC = 0.88; FC = 3.56); hence, it was chosen as the candidate biomarker of aflatoxin ingestion in dairy cows fed no sequestering agent.

Aflatoxin B1 Promotes Influenza Replication and Increases Virus Related Lung Damage via Activation of TLR4 Signaling.

Aflatoxin B1 (AFB1), which alters immune responses to mammals, is one of the most common mycotoxins in feeds and food. Swine influenza virus (SIV) is a major pathogen of both animals and humans. However, there have been few studies about the relationship between AFB1 exposure and SIV replication. Here, for the first time, we investigated the involvement of AFB1 in SIV replication in vitro and in vivo and explored the underlying mechanism using multiple cell lines and mouse models. In vitro studies demonstrated that low concentrations of AFB1 (0.01-0.25 µg/ml) markedly promoted SIV replication as revealed by increased viral titers and matrix protein (M) mRNA and nucleoprotein (NP) levels in MDCK cells, A549 cells and PAMs. In vivo studies showed that 10-40 µg/kg of AFB1 exacerbated SIV infection in mice as illustrated by significantly higher lung virus titers, viral M mRNA levels, NP levels, lung indexes and more severe lung damage. Further study showed that AFB1 upregulated TLR4, but not other TLRs, in SIV-infected PAMs. Moreover, AFB1 activated TLR4 signaling as demonstrated by the increases of phosphorylated NFκB p65 and TNF-α release in PAMs and mice. In contrast, TLR4 knockdown or the use of BAY 11-7082, a specific inhibitor of NFκB, blocked the AFB1-promoted SIV replication and inflammatory responses in PAMs. Furthermore, a TLR4-specific antagonist, TAK242, and TLR4 knockout both attenuated the AFB1-promoted SIV replication, inflammation and lung damage in mice. We therefore conclude that AFB1 exposure aggravates SIV replication, inflammation and lung damage by activating TLR4-NFκB signaling.

Impact of DNA lesion repair, replication and formation on the mutational spectra of environmental carcinogens: Aflatoxin B1 as a case study.

In a multicellular organism, somatic mutations represent a permanent record of the past chemical and biochemical perturbations experienced by a cell in its local microenvironment. Akin to a perpetual recording device, with every replication, genomic DNA accumulates mutations in patterns that reflect: i) the sequence context-dependent formation of DNA damage, due to environmental or endogenous reactive species, including spontaneous processes; ii) the activity of DNA repair pathways, which, depending on the type of lesion, can erase, ignore or exacerbate the mutagenic consequences of that DNA damage; and iii) the choice of replication machinery that synthesizes the nascent genomic copy. These three factors result in a richly contoured sequence context-dependent mutational spectrum that, from appearances, is distinct for most individual forms of DNA damage. Such a mutagenic legacy, if appropriately decoded, can reveal the local history of genome-altering events such as chemical or pathogen exposures, metabolic stress, and inflammation, which in turn can provide an indication of the underlying causes and mechanisms of genetic disease. Modern tools have positioned us to develop a deep mechanistic understanding of the cellular factors and pathways that modulate a mutational process and, in turn, provide opportunities for better diagnostic and prognostic biomarkers, better exposure risk assessment and even actionable therapeutic targets. The goal of this Perspective is to present a bottom-up, lesion-centric framework of mutagenesis that integrates the contributions of lesion replication, lesion repair and lesion formation to explain the complex mutational spectra that emerge in the genome following exposure to mutagens. The mutational spectra of the well-studied hepatocarcinogen aflatoxin B1 are showcased here as specific examples, but the implications are meant to be generalizable.

Characterization of cord blood interleukin 10 on aflatoxinB1-exposed patients with gestational diabetes.

BACKGROUND: Interleukin 10 (IL10) refers to a pleiotropic cytokine exerted immunoregulation. Aflatoxin B1 (AFB1) is a strong carcinogen, marked by causing immunosuppression. We determined the possible association between cord blood IL10 and AFB1-exposed patients with gestational diabetes (GD). METHODS: Cord blood samples from non-GD adults (n = 3) and GD patients (n = 3) were harvested for determining representative serological parameters by use of biochemical assays and enzyme linked immunosorbent assay (ELISA) tests. RESULTS: As results, GD patients showed no statistical comparable clinical data (hepatic function, lipids metabolism, immune cell count) to those in controls or references. Interestingly, cord blood contents of AFB1 in GD patients were significantly increased when compared to those in non-GD controls, characterized with visibly increased cord blood IL10. CONCLUSIONS: Preliminary clinical data reveal that IL10 may function as a biomarker for immunoregulation in AFB1-exposed GD patients.

Low-Level Aflatoxin B1 Promotes Influenza Infection and Modulates a Switch in Macrophage Polarization from M1 to M2.

BACKGROUND/AIMS: Swine influenza virus (SIV) is a major pathogen of both animals and humans. Afatoxin B1 (AFB1) is one of the most common mycotoxins in feed and food. However, the central contribution of AFB1 to SIV infection remains unclear. METHODS: Here, TCID50 assays, fluorescence-based quantitative real-time PCR, western blotting, immunofluorescence staining, histopathological examination, flow cytometry and scanning electron microscopy were performed to investigate the involvement and underlying mechanism of AFB1 in SIV infection in vivo and in vitro using mouse models and porcine alveolar macrophage (PAM) models, respectively. RESULTS: The in vivo study showed that low levels of AFB1 promoted SIV infection and increased its severity, as demonstrated by the increased mRNA expression of viral matrix protein (M); by the increased protein expression of nucleoprotein (NP), matrix protein 1 and ion channel protein; and by animal weight loss, lung index and lung histologic damage. In addition, the increased occurrence of SIV infection accompanied by increases in the level of IL-10 in sera and lungs, in the spleen index and in the number of CD206-positive mouse alveolar macrophages but decreases in the level of TNF-α in sera and lungs, in the thymus index and in the number of CD80-positive mouse alveolar macrophages was observed in SIV-infected mice after low-level AFB1 exposure. The in vitro study showed that low concentrations of AFB1 promoted SIV infection, as demonstrated by the increases in viral titers and viral M mRNA and NP expression levels in SIV-infected PAMs as well as by the number of cells positive for NP protein expression. Furthermore, AFB1 promoted the polarization of SIV-infected PAMs to the M1 phenotype at 8 hpi and to the M2 phenotype at 24 hpi, as measured by the increases in IL-10 expression and in the number of CD206-positive PAMs as well as by the morphological changes observed by scanning electron microscopy. The administration of the immune stimulant lipopolysaccharide (LPS) reversed the switch in PAM polarization from M2 to M1 and thereby counteracted the promotion of influenza virus infection induced by AFB1. CONCLUSION: Our results are the first to confirm that low-level exposure to AFB1 promotes SIV infection and modulates a switch in macrophage polarization from M1 to M2. The work reported here provides important data that point to a role for AFB1 in SIV infection, and it opens a new field of study.