Cytotoxicity Of Aflatoxin B1 Research Articles

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.

Screening of the phytochemical constituents of Teucrium polium extract and evaluation of their prophylactic role against the oxidative damage and cytotoxicity of Aflatoxin B1 in rats

Aflatoxin B1 (AFB1) is common carcinogen causing acute and chronic hepatocyte injuries. This study aimed to determine the bioactive components of Teucrium polium methanolic extract (TPE) and to evaluate their protective role against AFB1-induced oxidative damage, cytotoxicity, and genotoxicity in rats. Six groups of male albino rats were treated orally for 4 weeks including the control group, the ِAFB1-treated group (80 μg/kg b.w.), the groups treated with low (LD) or high (HD) dose TPE (50 or 100 mg/kg b.w.), and the groups treated with AFB1 plus TEP (LD) or TPE (HD). Blood and serum samples were collected for different assays. The GC-MS identified 34 compounds, the major compounds were pinene, germacrene D, α-cadinol, α-thujene, epi-bicyclosesquiphellandrene, and limonene. Animals that received AFB1 showed significant changes in all indicators of oxidative stress, biochemistry, cytokines, MNPCEs, comet tail formation in bone marrow, mRNA expression of inflammatory-related genes, Nrf2, and iNOS beside histological changes in the liver. TPE at the two doses tested showed insignificant changes in all tested parameters. The extract could normalize most of these parameters and the hepatic structure in AFB1-treated animals in a dose-dependent fashion. therefore, we concluded that TPE supplementation is effective for protection against AFB1 in endemic areas.

Degradation of Aflatoxin B1 in Moldy Maize by Pseudomonas aeruginosa and Safety Evaluation of the Degradation Products

Aflatoxin B1 (AFB1) is the most harmful mycotoxin commonly found in food and feed. Pollution from AFB1 causes serious economic and health issues worldwide because it causes strong mutagenicity and carcinogenicity in humans and animals. In this study, Pseudomonas aeruginosa was used to degrade AFB1 in moldy maize, and the safety of this biological method was investigated using genotoxicity and cytotoxicity tests. Using response surface methodology, we established the optimal conditions for degrading AFB1 by the fermentation supernatant of P. aeruginosa. Under these conditions, the degradation rate of AFB1 reached 99.67%. Furthermore, the Ames mutagenicity test showed that AFB1 treated with P. aeruginosa fermentation supernatant for 72 h was not mutagenic. CCK-8 cell assay showed that AFB1 cytotoxicity was significantly reduced after degradation. Overall, our findings show that the fermentation supernatant of P. aeruginosa may be a good candidate for biodegradation of AFB1.

Integrated Profiles of Transcriptome and mRNA m6A Modification Reveal the Intestinal Cytotoxicity of Aflatoxin B1 on HCT116 Cells.

Aflatoxin B1 (AFB1) is widely prevalent in foods and animal feeds and is one of the most toxic and carcinogenic aflatoxin subtypes. Existing studies have proved that the intestine is targeted by AFB1, and adverse organic effects have been observed. This study aimed to investigate the relationship between AFB1-induced intestinal toxicity and N6-methyladenosine (m6A) RNA methylation, which involves the post-transcriptional regulation of mRNA expression. The transcriptome-wide m6A methylome and transcriptome profiles in human intestinal cells treated with AFB1 are presented. Methylated RNA immunoprecipitation sequencing and mRNA sequencing were carried out to determine the distinctions in m6A methylation and different genes expressed in AFB1-induced intestinal toxicity. The results showed that there were 2289 overlapping genes of the differentially expressed mRNAs and differentially m6A-methylation-modified mRNAs. After enrichment of the signaling pathways and biological processes, these genes participated in the terms of the cell cycle, endoplasmic reticulum, tight junction, and mitophagy. In conclusion, the study demonstrated that AFB1-induced HCT116 injury was related to the disruptions to the levels of m6A methylation modifications of target genes and the abnormal expression of m6A regulators.

Does Bentonite Cause Cytotoxic and Whole-Transcriptomic Adverse Effects in Enterocytes When Used to Reduce Aflatoxin B1 Exposure?

Aflatoxin B1 (AFB1) is a major food safety concern, threatening the health of humans and animals. Bentonite (BEN) is an aluminosilicate clay used as a feed additive to reduce AFB1 presence in contaminated feedstuff. So far, few studies have characterized BEN toxicity and efficacy in vitro. In this study, cytotoxicity (WST-1 test), the effects on cell permeability (trans-epithelial electrical resistance and lucifer yellow dye incorporation), and transcriptional changes (RNA-seq) caused by BEN, AFB1 and their combination (AFB1 + BEN) were investigated in Caco-2 cells. Up to 0.1 mg/mL, BEN did not affect cell viability and permeability, but it reduced AFB1 cytotoxicity; however, at higher concentrations, BEN was cytotoxic. As to RNA-seq, 0.1 mg/mL BEN did not show effects on cell transcriptome, confirming that the interaction between BEN and AFB1 occurs in the medium. Data from AFB1 and AFB1 + BEN suggested AFB1 provoked most of the transcriptional changes, whereas BEN was preventive. The most interesting AFB1-targeted pathways for which BEN was effective were cell integrity, xenobiotic metabolism and transporters, basal metabolism, inflammation and immune response, p53 biological network, apoptosis and carcinogenesis. To our knowledge, this is the first study assessing the in vitro toxicity and whole-transcriptomic effects of BEN, alone or in the presence of AFB1.

Cytotoxicity assessment of Aflatoxin B1 after high voltage atmospheric cold plasma treatment

Aflatoxin B1 (AFB1) is a secondary metabolite produced by Aspergillus flavus and A. parasiticus, and is a known carcinogen in humans and animals. High voltage atmospheric cold plasma (HVACP) technology has already shown promise to decontaminate AFB1 in food and feed. This study aimed to investigate the cytotoxicity of AFB1 after HVACP treatment. AFB1 (100 μM) was treated at 85 kV with HVACP for 0, 2, 5, 10, and 20 min. HepG2 cells were exposed to HVACP-treated AFB1 for 72 h and assessed for cell viability, caspase-3 activity, DNA fragmentation, and protein carbonyls for each treatment time. Cell viability, caspase-3 activity, DNA fragmentation levels, and protein carbonyls contents of HepG2 cells exposed to HVACP-treated AFB1 after 20 min was not significantly different compared to non-exposed HepG2 cells (P > 0.05). However, their contents were significantly higher in non-exposed cells compared to the other HVACP treatment times (P < 0.01). Twenty minutes of HVACP treatment for AFB1 significantly reduced AFB1 cytotoxicity and oxidative damage and showed potential as a safe aflatoxin decontamination technology.

Transcriptomics and flow cytometry reveals the cytotoxicity of aflatoxin B1 and aflatoxin M1 in bovine mammary epithelial cells

Aflatoxin is a known mycotoxin that pollutes various grains widely in the environment. Aflatoxin B1 (AFB1) and Aflatoxin M1 (AFM1) have been shown to induce cytotoxicity in many cells, yet their effects on mammary epithelial cells remain unclear. In this study, we examined the toxicity and the effects of AFB1 and AFM1 on bovine mammary epithelial cells (BME cells). The cells were treated with AFB1 or AFM1 at a concentration of 0–10 mg/L for 24 or 48 h, followed by cytotoxicity assays, flow cytometry, and transcriptomics. Our results demonstrated that AFB1 and AFM1 induced cell proliferation inhibition, apoptosis and cell cycle arrest. However, the level of intracellular reactive oxygen species has no significant difference. The RNA-Seq results also showed that AFB1 and AFM1 changed many related gene expressions like apoptosis and oxidative stress, cycle, junction, and signaling pathway. Taken together, AFB1 and AFM1 were found to affect cytotoxicity and related gene changes in BME cells. Notably, this study reported that 2 mg/L of AFB1 and AFM1 affected the expression of methylation-related genes, and ultimately altered the rate of m6A methylation in RNA. It may provide a potential direction for toxins to indirectly regulate gene expression by affecting RNA methylation modification. Our research provides some novel insights and data about AFB1 and AFM1 toxicity in BME cells.

Detection of acute toxicity of aflatoxin B1 to human hepatocytes in vitro and in vivo using chimeric mice with humanized livers.

Aflatoxin B1 (AFB1), a mycotoxin, is acutely hepatotoxic to many animals including humans. However, there are marked interspecies differences in sensitivity to AFB1-induced toxicity depending on bioactivation by cytochrome P450s (CYPs). In the present study, we examined the applicability of chimeric mice with humanized livers and derived fresh human hepatocytes for in vivo and vitro studies on AFB1 cytotoxicity to human hepatocytes. Chimeric mice with highly humanized livers and SCID mice received daily injections of vehicle (corn oil), AFB1 (3 mg/kg), and carbon tetrachloride (50 mg/kg) for 2 days. Histological analysis revealed that AFB1 promoted hepatocyte vacuolation and inflammatory cell infiltration in the area containing human hepatocytes. A novel human alanine aminotransferase 1 specific enzyme-linked immunosorbent assay demonstrated the acute toxicity of AFB1 to human hepatocytes in the chimeric mouse livers. The sensitivity of cultured fresh human hepatocytes isolated from the humanized liver mice for AFB1 cytotoxicity was comparable to that of primary human hepatocytes. Long-term exposure to AFB1 (6 or 14 days) produced a more severe cytotoxicity. The half-maximal lethal concentration was 10 times lower in the 2-week treatment than after 2 days of exposure. Lastly, the significant reduction of AFB1 cytotoxicity by a pan-CYP inhibitor or transfection with CYP3A4 specific siRNA clearly suggested that bioactivation of AFB1 catalyzed by CYPs was essential for AFB1 cytotoxicity to the human hepatocytes in our mouse model. Collectively, our results implicate the humanized liver mice and derived fresh human hepatocytes are useful models for studies of AFB1 cytotoxicity to human hepatocytes.

Secondary metabolites from Bacillus sp. MERNA97 extract attenuates the oxidative stress, genotoxicity and cytotoxicity of aflatoxin B1 in rats

This study aimed to determine the bioactive compounds of Bacillus sp. MERNA97 extract and to evaluate their efficacy against the oxidative damage, genotoxicity, chromosomal aberration and DNA fragmentation in rats treated with AFB1. Sixty male Sprague-Dawley rats were divided into 6 groups and treated for 6 weeks and included the control group, AFB1-treated group (80 μg/kg b. w), the groups treated with Bacillus extract (BE) at low (2 mg/kg b.w) or high (4 mg/kg b.w) dose and the groups treated with AFB1 plus BE at the two doses. Blood and tissues samples were collected for different assays. The GC-MS results revealed the isolation of 44 compounds belong to different classes. The in vivo results showed that AFB1 disturbs all the biochemical parameters, oxidative stress markers, cytokines gene expression chromosomal aberration and DNA fragmentation along with the histological changes in the liver tissue. BE at the two tested doses induced a significant improvement in all parameters tested and the histological picture in a dose dependent manner. It could be concluded that the extract of Bacillus sp. MERNA97 isolated from the marine environment in the Red Sea is a promise as a source of novel compounds with therapeutically benefits.

Quercetin protects the buffalo rat liver (BRL-3A) cells from aflatoxin B1-induced cytotoxicity via activation of Nrf2-ARE pathway

Aflatoxin B1 (AFB1) is the most toxic mycotoxin widely presented in agricultural products, and the protective effect of quercetin (QUE), a natural antioxidant, against AFB1-induced cytotoxicity to the buffalo rat liver (BRL-3A) cells was investigated. With an IC50 of 23 μM, AFB1 induced a significant oxidative stress to BRL-3A cells evidenced by a dose-dependent reduction of mitochondria membrane potential (MMP), ATP content, and activities of endogenous antioxidant enzymes along with increased levels of reactive oxygen species (ROS) and lipid peroxidation biomarker of malondialdehyde (MDA). The activity of CYP1A2, the key enzyme to convert AFB1 to reactive AFB1 exo-8,9- epoxide, was also increased, which, probably in together with ROS, led to cell apoptosis with DNA fragmentation, chromatin condensation and increased lactate dehydrogenase release. After the BRL cells were pre-treated by low level QUE (2.5 and/or 5 μM) for 24 h and then exposed to AFB1, the activities of antioxidant enzymes including haeme oxygenase-1, glutathione S-transferase, superoxide dismutase, and the ratio of reduced to oxidised glutathione were significantly increased whereas the levels of intracellular ROS and MDA were reduced. The QUE pre-treatment also increased the levels of MMP, ATP and DNA integrity, and reduced the expression of apoptosis related genes ofBax andCaspase-3. The Western blotting study revealed increased content of phosphorylated Akt and nuclear NF-E2-related factor 2 (Nrf2), indicating an activation of Nrf2-ARE pathway in counteracting oxidative stress and cytotoxicity of AFB1. Thus, the QUE pre-treatment enhanced the anti-stress capacity of the cells through the activation of the Nrf2-ARE pathway, and QUE-based measures could be developed to ameliorate the toxicity caused by AFB1.

Development of an Escherichia coli-based electrochemical biosensor for mycotoxin toxicity detection

Mycotoxin contamination in food and feed is a global concern because mycotoxin contamination can cause both acute and chronic health effects in humans and animals. In the present work, an Escherichia coli-based biosensor is described for the toxicity assessment of aflatoxin B1 (AFB1) and zearalenone (ZEN). In this electrochemical biosensor, E. coli is used as the signal recognition element, p-benzoquinone is used as the mediator, and a two-step reaction procedure has been developed to separate the mediator from the mycotoxins. The current value of the as-prepared microbial biosensor exhibits a linear decrease with concentrations of AFB1 and ZEN in the range of 0.01–0.3 and 0.05–0.5 μg/mL, with detection limits reaching 1 and 6 ng/mL, respectively. The IC25 values obtained by the present method are 0.25 and 0.40 μg/mL for AFB1 and ZEN, which shows that the cytotoxicity of AFB1 to E. coli is more severe than the cytotoxicity of ZEN to E. coli. The combined toxic effect of these two mycotoxins has also been explored, and synergistic biotoxicity has been observed. Moreover, the biosensor is successfully applied to the toxicity evaluation of mycotoxins in real samples, including peanut and corn oils. This work could provide new insight into mycotoxin and microorganism interactions and could establish a new approach for future mycotoxin detection.

Modulation of aflatoxin B1 cytotoxicity and aflatoxin M1 synthesis by natural antioxidants in a bovine mammary epithelial cell line

Aflatoxin (AF) B1, a widespread food and feed contaminant, is bioactivated by drug metabolizing enzymes (DME) to cytotoxic and carcinogenic metabolites like AFB1-epoxide and AFM1, a dairy milk contaminant. A number of natural antioxidants have been reported to afford a certain degree of protection against AFB1 (cyto)toxicity. As the mammary gland potentially participates in the generation of AFB1 metabolites, we evaluated the role of selected natural antioxidants (i.e. curcumin, quercetin and resveratrol) in the modulation of AFB1 toxicity and metabolism using a bovine mammary epithelial cell line (BME-UV1). Quercetin and, to a lesser extent, resveratrol and curcumin from Curcuma longa (all at 5 μM) significantly counteracted the AFB1-mediated impairment of cell viability (concentration range: 96–750 nM). Moreover, quercetin was able to significantly reduce the synthesis of AFM1. The quantitative PCR analysis on genes encoding for DME (phase I and II) and antioxidant enzymes showed that AFB1 caused an overall downregulation of the detoxifying systems, and mainly of GSTA1, which mediates the GSH conjugation of the AFB1-epoxide. The negative modulation of GSTA1 was efficiently reversed in the presence of quercetin, which significantly increased GSH levels as well. It is suggested that quercetin exerts its beneficial effects by depressing the bio-transformation of AFB1 and counterbalancing its pro-oxidant effects.

Lactoferrin inhibits aflatoxin B1- and aflatoxin M1-induced cytotoxicity and DNA damage in Caco-2, HEK, Hep-G2, and SK-N-SH cells

Aflatoxins, including aflatoxin B1 (AFB1) and M1 (AFM1), are natural potent carcinogens produced by Aspergillus spp. These compounds, which can often be detected in dairy foods, can cause diseases in human beings. However, the molecular mechanisms involved in cytotoxicity, as well as methods for intervention, remain largely unexplored. For example, it is unclear whether lactoferrin (LF), a major antioxidant in milk, can inhibit the cytotoxicity of AFB1 and AFM1. In this study, we assessed AFB1- and AFM1-induced cell toxicity by measuring cell viability, membrane permeability, and genotoxicity, and then investigated the ability of LF to protect cells against AFB1 and AFM1. In Caco-2, HEK, Hep-G2, and SK-N-SH cells, 4 μg/mL AFB1 or AFM1 significantly inhibited cell growth, increased the level of lactate dehydrogenase, induced genetic damage, and increased the levels of signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) (p < 0.05). AFB1 was more genotoxic than AFM1 in all four cell lines, especially in Hep-G2. In Caco-2, Hep-G2, and SK-N-SH, incubation of AF-treated cells with 1000 μg/mL LF significantly decreased cytotoxicity, oxidation level, DNA damage, and levels of ERK1/2 and JNK (p < 0.05). Our data demonstrate that AFB1 or AFM1 induced cytotoxicity and DNA damage in these four cell lines, and that LF alleviated toxicity by decreasing oxidative stress mediated by mitogen-activated protein kinase pathways.

Combined cytotoxicity of aflatoxin B1 and deoxynivalenol to hepatoma HepG2/C3A cells

Co-occurrence of aflatoxin B1 (AFB1) and deoxynivalenol (DON) is one of the most common mycotoxin contaminations in cereal crops and food ingredients. However, the mechanism of their combined toxicity is poorly understood. In the current investigation, the hepatoma HepG2/C3A cell line was used to explore the combined cytotoxicity of AFB1 and DON. The values of IC50, based on a sulforhodamine B (SRB) assay, were 4.5 mM and 18.7 mM for DON and AFB1, respectively. The analysis of cytotoxicity endpoints using the combination index (CI) theory revealed that the changes of mitochondrial membrane permeability and ATP resulted from an additive cytotoxic effect (CI≈1) of AFB1 and DON. However, the endpoints of double strand DNA (ds-DNA), reactive oxygen species (ROS) and cell viability (SRB) were synergistically (CI&lt;1) affected in a dose-dependent manner. RNA-seq analysis demonstrated a number of uniquely expressed genes in their combination (AFB1: 2.5 mM, DON: 0.56 mM), indicating a synergistic interaction between AFB1 and DON at a molecular level. Additional transcriptomics analysis showed that the endoplasmic reticulum stress-associated JNK/p38/MAPK pathway was induced by DON, whereas the p53 signalling pathway was activated by AFB1. The expression profiles of apoptosis-related genes caspase-3, Bax and Bcl-2 suggested a mitochondria-mediated apoptosis pathway that was shared between AFB1 and DON. Thus, different cytotoxicity pathways and their converging at the apoptotic process might be the mechanism of the additive/synergistic cytotoxicity of AFB1 and DON to HepG2/C3A cells.

Cytoprotective effect of palm kernel cake phenolics against aflatoxin B1-induced cell damage and its underlying mechanism of action.

BackgroundPalm kernel cake (PKC), a by-product of the palm oil industry is abundantly available in many tropical and subtropical countries. The product is known to contain high levels of phenolic compounds that may impede the deleterious effects of fungal mycotoxins. This study focused on the evaluation of PKC phenolics as a potential cytoprotective agent towards aflatoxin B1 (AFB1)-induced cell damage.MethodsThe phenolic compounds of PKC were obtained by solvent extraction and the product rich in phenolic compounds was labeled as phenolic-enriched fraction (PEF). This fraction was evaluated for its phenolic compounds composition. The antioxidant activity of PEF was determined by using 1,1-diphenyl-2-picryl-hydrazil scavenging activity, ferric reducing antioxidant power, inhibition of ß-carotene bleaching, and thiobarbituric acid reactive substances assays. The cytotoxicity assay and molecular biomarkers analyses were performed to evaluate the cytoprotective effects of PEF towards aflatoxin B1 (AFB1)-induced cell damage.ResultsThe results showed that PEF contained gallic acid, pyrogallol, vanillic acid, caffeic acid, syringic acid, epicatechin, catechin and ferulic acid. The PEF exhibited free radical scavenging activity, ferric reducing antioxidant power, ß-carotene bleaching inhibition and thiobarbituric acid reactive substances inhibition. The PEF demonstrated cytoprotective effects in AFB1-treated chicken hepatocytes by reducing the cellular lipid peroxidation and enhancing antioxidant enzymes production. The viability of AFB1-treated hepatocytes was improved by PEF through up-regulation of oxidative stress tolerance genes and down-regulation of pro-inflammatory and apoptosis associated genes.ConclusionsThe present findings supported the proposition that the phenolic compounds present in PKC could be a potential cytoprotective agent towards AFB1 cytotoxicity.

Aqueous extract of Corchorus olitorius decreases cytotoxicity of aflatoxin B1and fumonisin B1in H4IIE-luc cells

75 ABSTRACT Aim: Afl atoxin B1 (AFB1) and fumonisin B1 (FB1) are important food-borne mycotoxins. Co-contamination of foodstuffs with these two mycotoxins is well-known and has been implicated in a possible development of hepatocellular carcinoma in humans living in regions of the world where exposures to these mycotoxins in grain are greatest. The aim of the current study was to evaluate the potential protective effects of an aqueous extract of Cochorus olitorius (C. olitorius, moroheiya) against cytotoxicity of AFB1 and/or FB1 in H4IIE-luc rat hepatoma cells, using assays to measure cell viability and disruption of DNA integrity. Although this transactivation assay was originally developed to specifi cally respond to aryl hydrocarbon agonists, this cell line was used because of its hepatic origin. Methods: H4IIE-luc cells were incubated with different concentrations of AFB1 and/or FB1 for 24 and 48 h with or without aqueous extract of C. olitorius. Results: Both mycotoxins decreased cell viability and increased DNA damage. Cytotoxicity was more pronounced when cells were exposed simultaneously to AFB1 and FB1. Conclusion: Aqueous extract of C. olitorius protected cells against cytotoxicity of mycotoxins. C. olitorius contains a water-soluble, natural chemo-preventative agent for cancer that should be isolated and identifi ed.

In-vitro cytotoxicity of aflatoxin B1 to broiler lymphocytes of broiler chickens

The aim of the present work was to study the in-vitro cytotoxic effects of different concentrations of aflatoxin B1 (AFB1) on broiler lymphocytes. Lymphocyte-rich mononuclear cells were separated by Ficoll-Histopaque density and cultured in 96-wellplates containing the evaluated AFB1 concentrations in 5% CO2 atmosphere at 39°C. Thereafter, MTT, PicoGreen, and reactive oxygen species assays were performed. Cell viability decreased in the presence of 10 µg/mL AFB1 at 48 h (p < 0.05) and of 10 and 20 µg/mL AFB1 at 72 h (p < 0.01 and p < 0.001, respectively) when compared to the control (0 µg/mL). However, a dose-dependent increase in the cell-free DNA at 24 h was observed at 1, 10 and 20 µg/mL (p < 0.001). ROS formation significantly increased at 24 h at all concentrations (p < 0.001). The in-vitro results demonstrate that AFB1 is cytotoxic and causes biomolecular oxidative damage in broiler lymphocytes.

The cytotoxicity of aflatoxin b1 in human lymphocytes.

Aflatoxin B1 (AFB1) is a naturally occurring carcinogenic and immunosuppressive compound. This study was designed to measure its toxic effects on human peripheral blood mononuclear cells (PBMC). The study recruited 7 healthy volunteers. PBMC were isolated and cellular respiration was monitored using a phosphorescence oxygen analyser. The intracellular caspase activity was measured by the caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin. Phosphatidylserine exposure and membrane permeability to propidium iodide (PI) were measured by flow cytometry. Cellular oxygen consumption was inhibited by 2.5 μM and 25 μM of AFB1. Intracellular caspase activity was noted after two hours of incubation with 100 μM of AFB1. The number of Annexin V-positive cells increased as a function of AFB1 concentration and incubation time. At 50 μM, a significant number of cells became necrotic after 24 hours (Annexin V-positive and PI-positive). The results show AFB1 is toxic to human lymphocytes and that its cytotoxicity is mediated by apoptosis and necrosis.

Generation of proliferating human hepatocytes using upcyte® technology: characterisation and applications in induction and cytotoxicity assays

We have developed a novel technique which causes primary human hepatocytes to proliferate by transducing them with genes that upregulate their proliferation.Upcyte® hepatocytes did not form colonies in soft agar and are not immortalised anchorage-independent cells. Confluent cultures expressed liver-specific proteins, produced urea and stored glycogen.CYP activities were low but similar to that in 5-day cultures of primary human hepatocytes. CYP1A2 and CYP3A4 were inducible; moreover, upcyte® hepatocytes predicted the in vivo induction potencies of known CYP3A4 inducers using the “relative induction score” prediction model. Placing cells into 3D culture increased their basal CYP2B6 and CYP3A4 basal activities and induction responses.Phase 2 activities (UGTs, SULTs and GSTs) were comparable to activities in freshly isolated hepatocytes.Upcyte® hepatocytes were markedly more sensitive to the hepatotoxin, α-amanitin, than HepG2 cells, indicating functional OATP1B3 uptake. The cytotoxicity of aflatoxin B1, was decreased in upcyte® hepatocytes by co-incubation with the CYP3A4 inhibitor, ketoconazole. Upcyte® hepatocytes also differentiated between ten hepatotoxic and eight non-hepatotoxic compounds.In conclusion, upcyte® hepatocyte cultures have a differentiated phenotype and exhibit functional phase 1 and 2 activities. These data support the use of upcyte® hepatocytes for CYP induction and cytotoxicity screening.

PROTECTIVE ACTION OF DIFFERENT NATURAL FLAVAN-3-OLS AGAINST AFLATOXIN B1-RELATED CYTOTOXICITY

ABSTRACT The aim of this study is to investigate the ability of flavan-3-ols: catechin, epigallocatechin, epigallocatechin gallate, and epicatechin gallate to prevent aflatoxin B1 (AB1)-induced cytotoxicity in A2780 cell line. The treatment with AB1 (0.35 µM) gives an increased level of cytotoxicity versus control (30–50%). Flavan-3-ols are more efficient against AB1 cytotoxicity at 24 h after treatment than after 48 h. AB1-induced dichlorofluorescin diacetate fluorescence intensity was significantly reduced even after 1 h incubation with flavan-3-ols. Concerning the tumor necrosis factor-alpha (TNF-α) release, comparing with the control, AB1 induces a down-expression of this factor. All the flavan-3-ols induced a higher level of TNF-αrelease in the cellular supernatant comparing with the AB1 group, but much lower than the control group. The present study demonstrates the potent protective effect of flavan-3-ols against AB1 cytotoxicity in A2780 epithelial cell line. This protective effect may be due to the inhibitory effect of flavan-3-ols on AB1-induced reactive oxygen species formation and oxidative damage. PRACTICAL APPLICATIONS There is a delicate balance between antioxidants and pro-oxidants, generally and specifically, in the cell. They are responsible for regulation of various metabolic pathways, immunosuppression, growth and development and protection against stress. Mycotoxins have negative impact on this antioxidant/pro-oxidant balance. This balance can be regulated by dietary antioxidants like flavan-3-ols. There are various approaches to control or combat mycotoxin problems; one is by developing new oxidant strategies by improving the natural defense mechanism against mycotoxins at cellular level. Considering the role of reactive oxygen species in chemically induced carcinogenesis, the ability of aflatoxins to induce cytotoxicity to cells may play an important role in aflatoxin-induced carcinogenicity. Chemopreventive strategies designed to limit the several toxic effects, caused by these mycotoxins, are important public health goals for reducing the incidence of aflatoxin-induced diseases. Flavan-3-ols, a class of phenolic compounds ubiquitous in plants and widely found in a number of fruits, vegetables and beverages, can be a choice of preventing the mycotoxin-related toxicity as we reported in this study.