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.

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.

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.

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.

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.

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.

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.

1α,25-Dihydroxyvitamin D3 inhibits aflatoxin B1-induced proliferation and dedifferentiation of hepatic progenitor cells by regulating PI3K/Akt and Hippo pathways.

Hepatic progenitor cells (HPCs) might be the origin of hepatocellular carcinoma. 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) (VD3) has been documented as an anticancer agent for various cancers. However, the potential effect of VD3 on the proliferation and malignant transformation of HPCs induced by aflatoxin B1 (AFB1) has not been determined. In this study, we found that AFB1 exhibited the stimulative effects on the proliferation, dedifferentiation and invasion of HPCs via activating AKT pathway but turning off Hippo pathway, which were terminated when VD3 was used in combination with AFB1. Furthermore, in AFB1-induced liver damage mouse model, VD3 also showed protective effect by reducing HPCs population. Together, these preclinical data not only provide a newly identified mechanism by which AFB1 affects HPCs but also strengthen the idea of developing VD3 as an anticancer agent.

Detoxification and immunoprotection of Zn(II)-curcumin in juvenile Pacific white shrimp (Litopenaeus vannamei) feed with aflatoxin B1.

Aflatoxins, which was produced by Aspergillus flavus or Aspergillus parasiticus fungi during grain and feed processing or storage, could cause severe health problems and reduction of yield during shrimp cultures. To evaluate toxic effects of aflatoxin B1 (AFB1) in juvenile Pacific white shrimp (Litopenaeus vannamei) and potential protective effect of Zn(II)-curcumin (Zn-CM), four experimental diets (control, 500 µg/kg AFB1, 500 µg/kg AFB1+100 mg/kg Zn-CM, 500 µg/kg AFB1+200 mg/kg Zn-CM) were formulated in quadruplicate to feed the shrimp for 8 weeks. The results revealed that AFB1 could induce significant decrease in final body weight (FBW), weight gain (WG, %) and visible variations of the hepatopancreas structures in L.vannamei. Compared with AFB1 group, AFB1+100 mg/kg Zn-CM group significantly ameliorated the toxic effects of AFB1 on growth performance, while AFB1+100 mg/kg Zn-CM group had no effect on growth performance. Dietary AFB1+100 mg/kg Zn-CM enhanced phenoloxidase (PO) (P < 0.05) activity. Both dietary AFB1+100 mg/kg Zn-CM and AFB1+200 mg/kg Zn-CM reduced inducible nitric oxide synthase (iNOS) activity and glutathione (GSH) level, decreased the content of malondialdehyde (MDA) (P < 0.05) in hepatopancreas compared with AFB1 group. Transmission electron microscopy (TEM) analysis demonstrated that Zn-CM could relieve the microvilli transformation and mitochondria accumulation reduction caused by AFB1. Consequently, the results demonstrated that suitable Zn-CM could mitigate the AFB1-induced hepatotoxicity and immunotoxicity effects on L.vannamei.

Changes in the regulation and activity of glutathione redox system, and lipid peroxidation processes in short-term aflatoxin B1 exposure in liver of laying hens.

The purpose of this study was to investigate the short-term (48 hr) effects of feeding aflatoxin contaminated diet (170.3 µg/kg AFB1) in 49-week-old laying hens. Liver samples were taken at 12-hr intervals. Feed intake, body weight, absolute and relative liver weight were the same in groups. However, there was no feed intake during both dark periods (between 12nd to 24th and 36th to 48th hours of the experiment); therefore, aflatoxin intake was also negligible. Markers of initial phase of lipid peroxidation, conjugated dienes and trienes did not change as effect of aflatoxin, but terminal marker, malondialdehyde content was significantly higher at 12 hr as effect of aflatoxin. No significant difference was found in reduced glutathione concentration and glutathione peroxidase activity between the groups. Expression of glutathione peroxidase 4 gene (GPX4) was significantly reduced due to aflatoxin treatment at 12 and 24 hr, but induced later, while glutathione reductase gene (GSR) expression was significantly lower at 24 hr and glutathione synthetase gene (GSS) in aflatoxin-treated group at 12 hr. The results suggest that aflatoxin induced oxygen-free radical formation, but it did not reach critical level during this short period of time to cause activation of the expression of glutathione system.

Interaction Effects of AFB1 and MC-LR Co-exposure with Polymorphism of Metabolic Genes on Liver Damage: focusing on SLCO1B1 and GSTP1.

AFB1 and MC-LR are two major environmental risk factors for liver damage worldwide, especially in warm and humid areas, but there are individual differences in health response of the toxin-exposed populations. Therefore, we intended to identify the susceptible genes in transport and metabolic process of AFB1 and MC-LR and find their effects on liver damage. We selected eight related SNPs that may affect liver damage outcomes in AFB1 and MC-LR exposed persons, and enrolled 475 cases with liver damage and 475 controls of healthy people in rural areas of China. The eight SNPs were genotyped by PCR and restriction fragment length polymorphism. We found that SLCO1B1 (T521C) is a risk factor for liver damage among people exposed to high AFB1 levels alone or combined with MC-LR, and that GSTP1 (A1578G) could indicate the risk of liver damage among those exposed to high MC-LR levels alone or combined with high AFB1 levels. However, GSTP1 (A1578G) could reduce the risk of liver damage in populations exposed to low MC-LR levels alone or combined with high AFB1 levels. In conclusion, SLCO1B1 (T521C) and GSTP1 (A1578G) are susceptible genes for liver damage in humans exposed to AFB1 and/or MC-LR in rural areas of China.

The comparison of CHCA solvent compositions for improving LC-MALDI performance and its application to study the impact of aflatoxin B1 on the liver proteome of diabetes mellitus type 1 mice.

In nanoflow liquid chromatography-matrix-assisted laser desorption/ionization tandem time-of-flight (nanoLC-MALDI-TOF/TOF) approaches, it is critical to directly apply small amounts of the sample elutes on the sample target using a nanoLC system due to its low flow rate of 200 ~ 300 nl/min. It is recommended to apply a sheath liquid containing a matrix with a several µL/min flow rate at the end of the nanoLC column to ensure a larger co-eluted droplet for more reproducible sample spotting and avoid the laborious task of post-manual matrix spotting. In this study, to achieve a better nanoLC-MALDI performance on sample spotting, we first compared α-Cyano-4-hydroxycinnamic acid (CHCA) solvent composition for efficiently concentrating nanoLC elutes on an anchor chip. The solvent composition of isopropanol (IPA): acetonitrile (ACN):acetone:0.1% Trifluoroacetic acid (TFA) (2:7:7:2) provided strong and homogeneous signals with higher peptide ion yields than the other solvent compositions. Then, nanoLC-MALDI-TOF/TOF was applied to study the impact of aflatoxin B1 on the liver proteome from diabetes mellitus type 1 mice. Aflatoxin B1 (AFB1), produced by Aspergillus flavus and Aspergillus parasiticus is a carcinogen and a known causative agent of liver cancer. To evaluate the effects of long-term exposure to AFB1 on type 1 diabetes mellitus (TIDM), the livers of T1DM control mice and mice treated with AFB1 were analyzed using isotope-coded protein labeling (ICPL)-based quantitative proteomics. Our results showed that gluconeogenesis, lipid, and oxidative phosphorylation mechanisms, normally elevated in T1DM, were disordered following AFB1 treatment. In addition, major urinary protein 1 (MUP1), an indicator of increased insulin sensitivity, was significantly decreased in the T1DM/AFB1 group and may have resulted in higher blood glucose levels compared to the T1DM group. These results indicate that T1DM patients should avoid the AFB1 intake, as they could lead to increased blood glucose levels and disorders of energy-producing mechanisms.

Saffron (Crocus sativus L.) Tea Intake Prevents Learning/Memory Defects and Neurobiochemical Alterations Induced by Aflatoxin B1 Exposure in Adult Mice.

This study aimed to investigate the potential neurotoxic effects of aflatoxin B1 (AFB1) and the preventive effects of saffron. Male Balb-c mice received AFB1 (0.6 mg/kg/day intraperitoneally for 4 days), saffron infusion (90 mg styles/200 mL, ad libitum access for 2 weeks) or saffron infusion plus AFB1 (saffron treatment as previously plus 0.6 mg AFB1/kg/day intraperitoneally for the last 4 days). Control mice were intraperitoneally injected with DMSO:saline (1:1, v/v) during AFB1 treatment. Learning/memory was assessed by passive avoidance task. The activity of acetylcholinesterase [AChE, salt-(SS)/detergent-soluble(DS) isoforms], butyrylcholinesterase (BuChE, SS/DS isoforms), monoamine oxidase (MAO-A, MAO-B), the levels of lipid peroxidation (malondialdehyde, MDA) and reduced glutathione (GSH), were determined in whole brain (minus cerebellum) and cerebellum. We demonstrate for the first time that AFB1 administration impaired the memory of adult mice and decreased significantly whole brain AChE and BuChE activity, cerebellar AChE activity and cerebral GSH content. Moreover, MAO isoforms activity in whole brain, MAO-B activity in cerebellum and MDA levels of both tissues were significantly higher after AFB1 treatment. Pre-treatment with saffron prevented memory decline, activation of MAO-A and MAO-B in whole brain and cerebellum, respectively, and lipid peroxidation triggered by AFB1. Interestingly, the activity of AChE isoforms in whole brain, DS-AChE in cerebellum and GSH levels of both tissues were further significantly decreased in saffron +AFB1-treated mice compared with AFB1 group. Our findings support the neuroprotective efficacy of saffron against AFB1 in adult mice.

[Docosahexaenoic acid inhibits aflatoxin B1-induced migration and invasion in hepatocellular carcinoma cells in vitro].

OBJECTIVE: To investigate the effect of docosahexaenoic acid (DHA) on invasiveness of aflatoxin B1 (AFB1)-induced hepatocellular carcinoma cells in vitro. METHODS: HepG2.2.15 cells were exposed to different concentrations of AFB1 and DHA plus AFB1. The cell migration and invasion were assessed using wound-healing and Transwell assay, and flow cytometry was used to analyze the cell cycle changes. The ultrastructural changes of the cells were observed by transmission electron microscopy. RESULTS: Compared with the control group, the cells exposed to2 µmol/L AFB1 showed obviously enhanced migration and invasion with decreased cell ratio in G1/G1 phase and increased cell ratio in G2/M phase but no changes in S phase cells; transmission electron microscopy revealed the presence of multiple nucleoli and significantly increased mitochondria and Golgi apparatus in the exposed cells. Compared with AFB1-exposed cells, the cells treated with DHA and AFB1 showed decreased migration and invasion abilities, and the G1/G1 phase cells increased and G2/M phase cells decreased significantly; ultrastructurally, the cells contained single nucleoli with decreased mitochondria and vacuolization occurred in the cytoplasm. CONCLUSION: DHA can significantly inhibit AFB1-induced enhancement of cell migration and invasion in hepatocellular carcinoma cells in vitro.

Chemically induced hepatotoxicity in human stem cell-induced hepatocytes compared with primary hepatocytes and HepG2.

Stem cell-induced hepatocytes (SC-iHeps) have been suggested as a valuable model for evaluating drug toxicology. Here, human-induced pluripotent stem cells (QIA7) and embryonic stem cells (WA01) were differentiated into hepatocytes, and the hepatotoxic effects of acetaminophen (AAP) and aflatoxin B1 (AFB1) were compared with primary hepatocytes (p-Heps) and HepG2. In a cytotoxicity assay, the IC50 of SC-iHeps was similar to that in p-Heps and HepG2 in the AAP groups but different from that in p-Heps of the AFB1 groups. In a multi-parameter assay, phenotypic changes in mitochondrial membrane potential, calcium influx and oxidative stress were similar between QIA7-iHeps and p-Heps following AAP and AFB1 treatment but relatively low in WA01-iHeps and HepG2. Most hepatic functional markers (hepatocyte-specific genes, albumin/urea secretion, and the CYP450 enzyme activity) were decreased in a dose-dependent manner following AAP and AFB1 treatment in SC-iHeps and p-Heps but not in HepG2. Regarding CYP450 inhibition, the cell viability of SC-iHeps and p-Heps was increased by ketoconazole, a CYP3A4 inhibitor. Collectively, SC-iHeps and p-Heps showed similar cytotoxicity and hepatocyte functional effects for AAP and AFB1 compared with HepG2. Therefore, SC-iHeps have phenotypic characteristics and sensitivity to cytotoxic chemicals that are more similar to p-Heps than to HepG2 cells.