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Partial least squares-discriminant analysis (PLS-DA) (A) and variable importance in projection (VIP) (B) plots related to the polar fraction of the HepG2 cell line treated with AFM1 compared to untreated cells.
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Hepatoblastoma incidence has been associated with different environmental factors even if no data are reported about a correlation between aflatoxin exposure and hepatoblastoma initiation. Considering that hepatoblastoma develops in infants and children and aflatoxin M1 (AFM1), the aflatoxin B1 (AFB1) hydroxylated metabolite, can be present in moth...
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... It aims to monitor changes in metabolites so as to provide critical targets to study mechanisms at the metabolic level. Previous research has used metabolomics to explore the mechanisms of aflatoxicosis, with a focus on the kidneys and liver [36][37][38]. Lipidomics is a rapidly growing method derived from metabolomics, attempting to construct an all-embracing cellular liposome with lipid biology, technology, and medicine [39]. Recent research has shown that lipidomics can precisely identify markers of aflatoxin cytotoxicity [40]. ...
Aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) are universally found as environmental pollutants. AFB1 and AFM1 are group 1 human carcinogens. Previous sufficient toxicological data show that they pose a health risk. The intestine is vital for resistance to foreign pollutants. The enterotoxic mechanisms of AFB1 and AFM1 have not been clarified at the metabolism levels. In the present study, cytotoxicity evaluations of AFB1 and AFM1 were conducted in NCM 460 cells by obtaining their half-maximal inhibitory concentration (IC50). The toxic effects of 2.5 μM AFB1 and AFM1 were determined by comprehensive metabolomics and lipidomics analyses on NCM460 cells. A combination of AFB1 and AFM1 induced more extensive metabolic disturbances in NCM460 cells than either aflatoxin alone. AFB1 exerted a greater effect in the combination group. Metabolomics pathway analysis showed that glycerophospholipid metabolism, fatty acid degradation, and propanoate metabolism were dominant pathways that were interfered with by AFB1, AFM1, and AFB1+AFM1. Those results suggest that attention should be paid to lipid metabolism after AFB1 and AFM1 exposure. Further, lipidomics was used to explore the fluctuation of AFB1 and AFM1 in lipid metabolism. The 34 specific lipids that were differentially induced by AFB1 were mainly attributed to 14 species, of which cardiolipin (CL) and triacylglycerol (TAG) accounted for 41%. AFM1 mainly affected CL and phosphatidylglycerol, approximately 70% based on 11 specific lipids, while 30 specific lipids were found in AFB1+AFM1, mainly reflected in TAG up to 77%. This research found for the first time that the lipid metabolism disorder caused by AFB1 and AFM1 was one of the main causes contributing to enterotoxicity, which could provide new insights into the toxic mechanisms of AFB1 and AFM1 in animals and humans.
... A common clinical feature of HBL is the elevated level of α-fetoprotein (AFP) in serum [20,21], and abdominal mass and distention. As far as we know, no data have been yet reported about HR-MAS 1 H-NMR of HBL tissue samples except 1 H-NMR metabolomics analysis in vitro of hepatoblastoma cell lines (HepG2) treated with aflatoxin AFM1 and compared with untreated HepG2 cells [22]. The metabolomics study of HBL was explored by LC-MS to determine the differences in metabolite profiles between HBL cells with overexpression and normal expression of sodium-taurocholate co-transporting polypeptide NTCP (SLC10A1) [2]. ...
... A further three samples were taken from three patients, but it was unknown from which part of the HBL tissues. Another fifteen samples were taken as non-cancer liver (NCL) as control samples, of which nine samples (16)(17)(18)(19)(20)(21)(22)(23)(24) were obtained from the healthy part of the liver from patients diagnosed with HBL (adjacent non-HBL samples), and six samples (25)(26)(27)(28)(29)(30) were taken from children with healthy livers. ...
... It was found that AFM1 affects the reprogramming of lipidic, glycolytic, and amino acid metabolism and causes inhibition of hepatoblastoma HepG2 cells. Reported data revealed that HepG2 untreated cells compared with treated cells with AFM1 showed increased concentrations of formate and decreased concentrations of acyl groups of fatty acids, cholesterol, pyruvate/lactate, glycine, choline, phosphorylcholine (PC), glycerophosphorylcholine (GPC), branched-chain amino acids (BCAA), and glutamate [22]. Our results are in agreement with this in terms of decreased concentrations of pyruvate/lactate, glucose, and triglyceride lipids and increased concentrations of formate. ...
Cancer is one of the leading causes of death in children and adolescents worldwide; among the types of liver cancer, hepatoblastoma (HBL) is the most common in childhood. Although it affects only two to three individuals in a million, it is mostly asymptomatic at diagnosis, so by the time it is detected it has already advanced. There are specific recommendations regarding HBL treatment, and ongoing studies to stratify the risks of HBL, understand the pathology, and predict prognostics and survival rates. Although magnetic resonance imaging spectroscopy is frequently used in diagnostics of HBL, high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy of HBL tissues is scarce. Using this technique, we studied the alterations among tissue metabolites of ex vivo samples from (a) HBL and non-cancer liver tissues (NCL), (b) HBL and adjacent non-tumor samples, and (c) two regions of the same HBL samples, one more centralized and the other at the edge of the tumor. It was possible to identify metabolites in HBL, then metabolites from the HBL center and the border samples, and link them to altered metabolisms in tumor tissues, highlighting their potential as biochemical markers. Metabolites closely related to liver metabolisms such as some phospholipids, triacylglycerides, fatty acids, glucose, and amino acids showed differences between the tissues.
... Although AFM1 is less mutagenic and genotoxic than AFB1, it possesses cytotoxicity in hepatocytes in vitro and it has been demonstrated that its acute toxicity in several species is similar to that of AFB1, 3 in addition this AF also possesses hepatotoxic effects and is relatively stable in the processes of pasteurization, storage and processing of milk. 88 Marchese et al., 89 highlights damage in HepG2 cells (human hepatoma cell line), carrying out the analysis of the effects of AFM1 on the metabolomic and cytochemical profile in a hepatoblastoma cell line, it was found that AFM1 is able to block the cell cycle in the G0/G1 phase, it also induces the modulation of lipid, glycolytic and amino acid metabolism, increases the levels of proinflammatory cytokines such as IL-6, IL-8 and TNF-and decreases the levels of IL-4. ...
Milk is the most traded, processed and consumed dairy product in the world, approximately 113.7 to 150 kg per capita. However, one of the food safety and human health problems worldwide is aflatoxin M1 (AFM1), present exclusively in milk and its derivatives, because this mycotoxin is heat-resistant to common food industrialization processes and is capable of producing hepatotoxicity, carcinogenicity, genotoxicity and immunosuppression in humans, so there are international regulations that establish parameters ranging from 0.03 to 0.250 μg/kg depending on each country. The present review aims at compiling information on mycotoxins and specifically on AFM1 from its biotransformation from dairy cattle until the pathologies in humans associated with its consumption.
... A similar study which was conducted on rats also showed a reduction in the concentration of GSH and a high level of thiobarbituric acid reactive substances (TBARS) (Rotimi et al. 2018). Additionally, AF-induced poisoning can also potentiate inflammatory responses by increasing the levels of interleukine-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukine-8 (IL-8) designated as proinflammatory cytokines, and decreasing the level of antiinflammatory cytokine such as IL-4 (Marchese et al. 2018). Exposure to AFs induces a significant increase in the concentration of glucose that exhibits a strong association with metabolic impairments (Cheng et al. 2017). ...
Recently, aflatoxin M1 (AFM1) has emerged as a major health concern owing to its exposure to human being via consumption of milk, dairy products, and food commodities, and this has a strong association with risk factors that may lead to the onset of type 2 diabetes mellitus (T2DM) and various other associated metabolic disorders. This study was conducted to investigate the exposure to AFM1 and its association with sociodemographic features and risk factors of T2DM. Urine and blood samples from 672 participants were collected to investigate the concentration of AFM1 in urine and glucose, glycosylated hemoglobin (HbA1c), insulin, α-amylase, dipeptidyl peptidase-IV (DPP-IV), free fatty acids (FFAs), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-chol), interleukine-6 (IL-6), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), creatinine, uric acid, blood urea nitrogen (BUN), aspartate aminotransferase (AST), and alanine transaminase (ALT) from the blood of study participants. Association of exposure to AFM1 with sociodemographic features and risk factors of T2DM was determined using person correlation coefficient (r), coefficient of determination (R2), and 95% confidence interval, and the level of significance (P<0.05) was measured by Student’s unpaired t-test. Among the participants in which AFM1 was detected, 62.91% of participants were found to be diabetic and 37.09% of participants were found to be non-diabetic. Further to this, it was also found that concentration of AFM1 in the urine of diabetic participants was found to be higher (P<0.05) as compared to that in non-diabetic participants. Association of AFM1 exposure with risk factors of T2MD exhibits that exposure to AFM1 was responsible for the induction of inflammatory responses and oxidative stress that may lead to the onset of impaired insulin secretion and metabolism of carbohydrates and ultimately the onset of T2DM and associated metabolic disorders. Hence, it can be summarized that exposure to AFM1 is one of the causative factors that may lead to potentiate the several risk factors notably inflammatory responses and oxidative stress that ultimately induce the pathogenesis of T2DM and associated metabolic disorders. The key findings of this study suggest that human population who are at greater risk of AFM1 exposure can develop T2DM and other associated metabolic risk factors.
... As demonstrated with 1 H NMR spectroscopy, AFB1 exposure of rats elevated hepatic BCAA levels (324). In accordance, AFM1 exposure of HepG2 cells enhanced cellular BCAA levels (325). Consumption of alcohol (2,000 kcal added to the diet) in human alcoholics for 2 to 4 weeks as well increased BCAA levels (326). ...
This review provides epidemiological and translational evidence for milk and dairy intake as critical risk factors in the pathogenesis of hepatocellular carcinoma (HCC). Large epidemiological studies in the United States and Europe identified total dairy, milk and butter intake with the exception of yogurt as independent risk factors of HCC. Enhanced activity of mechanistic target of rapamycin complex 1 (mTORC1) is a hallmark of HCC promoted by hepatitis B virus (HBV) and hepatitis C virus (HCV). mTORC1 is also activated by milk protein-induced synthesis of hepatic insulin-like growth factor 1 (IGF-1) and branched-chain amino acids (BCAAs), abundant constituents of milk proteins. Over the last decades, annual milk protein-derived BCAA intake increased 3 to 5 times in Western countries. In synergy with HBV- and HCV-induced secretion of hepatocyte-derived exosomes enriched in microRNA-21 (miR-21) and miR-155, exosomes of pasteurized milk as well deliver these oncogenic miRs to the human liver. Thus, milk exosomes operate in a comparable fashion to HBV- or HCV- induced exosomes. Milk-derived miRs synergistically enhance IGF-1-AKT-mTORC1 signaling and promote mTORC1-dependent translation, a meaningful mechanism during the postnatal growth phase, but a long-term adverse effect promoting the development of HCC. Both, dietary BCAA abundance combined with oncogenic milk exosome exposure persistently overstimulate hepatic mTORC1. Chronic alcohol consumption as well as type 2 diabetes mellitus (T2DM), two HCC-related conditions, increase BCAA plasma levels. In HCC, mTORC1 is further hyperactivated due to RAB1 mutations as well as impaired hepatic BCAA catabolism, a metabolic hallmark of T2DM. The potential HCC-preventive effect of yogurt may be caused by lactobacilli-mediated degradation of BCAAs, inhibition of branched-chain α-ketoacid dehydrogenase kinase via production of intestinal medium-chain fatty acids as well as degradation of milk exosomes including their oncogenic miRs. A restriction of total animal protein intake realized by a vegetable-based diet is recommended for the prevention of HCC.
... A further AFB1 microsomal biotransformation is the CYP1A2-dependent hydroxylation to AFM1, a metabolite commonly detected in humans and animals exposed to AFB1. This derivative is the most carcinogenic AFB1-hydroxylated derivatives because, likewise to AFBO, it is able to form DNA adducts [25]. Accordingly, AFM1 has been classified as a group 1 carcinogen by IARC [26]. ...
... A great number of epidemiological studies on HCC patients exposed to high levels of aflatoxins proved that some genes (e.g., P53 tumor suppressor gene, c-KRAS oncogene, and HRAS proto-oncogene) are particularly subjected to these mutations [32][33][34][35], strengthening the association between HCC and aflatoxin exposure. Additional mechanisms have been suggested to contribute to AFB1 carcinogenesis: AFB1 exposure has been associated with rising oxidative stress and the resulting generation of reactive oxygen species (ROS) [25,36] and lipid peroxidation. The former may react with DNA, causing strand breaks and mutations, which could initiate carcinogenesis [37]. ...
... The nuclear factor kappa B gene (NF-κB), as a key regulator of cellular stress in hepatocytes, controls inflammation, apoptosis, and cell injury [101]. A recent study performed in HepG2 cells demonstrated that treatment with the IC50 (i.e., 9 μM) of AFM1 for 48 h increased the amount of both IL6 and IL8 [25]. Likewise, IL6 mRNA levels were significantly induced in the liver of broilers exposed to AFB1 compared to controls [102]. ...
Aflatoxins, and particularly aflatoxin B1 (AFB1), are toxic mycotoxins to humans and farm animal species, resulting in acute and chronic toxicities. At present, AFB1 is still considered a global concern with negative impacts on health, the economy, and social life. In farm animals, exposure to AFB1-contaminated feed may cause several untoward effects, liver damage being one of the most devastating ones. In the present study, we assessed in vitro the transcriptional changes caused by AFB1 in a bovine fetal hepatocyte-derived cell line (BFH12). To boost the cellular response to AFB1, cells were pre-treated with the co-planar PCB 3,3′,4,4′,5-pentachlorobiphenyl (PCB126), a known aryl hydrocarbon receptor agonist. Three experimental groups were considered: cells exposed to the vehicle only, to PCB126, and to PCB126 and AFB1. A total of nine RNA-seq libraries (three replicates/group) were constructed and sequenced. The differential expression analysis showed that PCB126 induced only small transcriptional changes. On the contrary, AFB1 deeply affected the cell transcriptome, the majority of significant genes being associated with cancer, cellular damage and apoptosis, inflammation, bioactivation, and detoxification pathways. Investigating mRNA perturbations induced by AFB1 in cattle BFH12 cells will help us to better understand AFB1 toxicodynamics in this susceptible and economically important food-producing species.
... Only minor effects were noted on IL-2 and IFN-c cytokines mRNA expression in stimulated cells that had been preincubated with AFM1 (Luongo et al., 2014). Another in vitro study from the same team, performed on the human hepatoblastoma HepG2 cell line, demonstrated a decreased cell viability, an increase in the concentration of three pro-inflammatory cytokines, IL-6, IL-8 and TNF-a, and a decrease of the antiinflammatory interleukin, IL-4 (Marchese et al., 2018). An in vivo study performed with an i.p. administration of AFM1 (25 and 50 lg/kg bw) for 28 days also demonstrated an effect on some immune parameters including proliferative response to lipopolysaccharide and phytohemagglutinin-A, hemagglutination titre, delayed type of hypersensitivity, serum haemolytic activity, serum immunoglobulin G level and cytokine production (Shirani et al., 2018). ...
Abstract EFSA was asked to deliver a scientific opinion on the risks to public health related to the presence of aflatoxins in food. The risk assessment was confined to aflatoxin B1 (AFB1), AFB2, AFG1, AFG2 and AFM1. More than 200,000 analytical results on the occurrence of aflatoxins were used in the evaluation. Grains and grain‐based products made the largest contribution to the mean chronic dietary exposure to AFB1 in all age classes, while ‘liquid milk’ and ‘fermented milk products’ were the main contributors to the AFM1 mean exposure. Aflatoxins are genotoxic and AFB1 can cause hepatocellular carcinomas (HCCs) in humans. The CONTAM Panel selected a benchmark dose lower confidence limit (BMDL) for a benchmark response of 10% of 0.4 μg/kg body weight (bw) per day for the incidence of HCC in male rats following AFB1 exposure to be used in a margin of exposure (MOE) approach. The calculation of a BMDL from the human data was not appropriate; instead, the cancer potencies estimated by the Joint FAO/WHO Expert Committee on Food Additives in 2016 were used. For AFM1, a potency factor of 0.1 relative to AFB1 was used. For AFG1, AFB2 and AFG2, the in vivo data are not sufficient to derive potency factors and equal potency to AFB1 was assumed as in previous assessments. MOE values for AFB1 exposure ranged from 5,000 to 29 and for AFM1 from 100,000 to 508. The calculated MOEs are below 10,000 for AFB1 and also for AFM1 where some surveys, particularly for the younger age groups, have an MOE below 10,000. This raises a health concern. The estimated cancer risks in humans following exposure to AFB1 and AFM1 are in‐line with the conclusion drawn from the MOEs. The conclusions also apply to the combined exposure to all five aflatoxins.
... Carcinogenesis, harmful effects on HepG2 cells, abnormalities in cytokinomic cell profiling of the cells, and apoptosis (Marchese et al., 2018) Milk and dairy products (Kos et al., 2014) Codex Alimentarius and the EC have set the maximum upper limit of 0.05 µg/kg AFM1 in milk or milk-based products, whereas, the US and some Latin America countries have set 0.5 µg/kg as permissible limits in milk and products thereof (Quevedo-Garza, Gustavo Amador-Espejo, ...
Fungal contamination of food and animal feed, especially by mycotoxigenic fungi, is not only a global food quality concern for food manufacturers, but it also poses serious health concerns because of the production of a variety of mycotoxins, some of which present considerable food safety challenges. In today’s mega-scale food and feed productions which involve a number of processing steps and the use of a variety of ingredients, fungal contamination is regarded as unavoidable, even good manufacturing practices are followed. Chemical preservatives, to some extent, are successful in retarding microbial growth and achieving considerably longer shelf-life. However, the increasing demand for clean label products requires manufacturers to find natural alternatives to replace chemically-derived ingredients to guarantee the clean label. Lactic acid bacteria (LAB), with the status generally recognized as safe (GRAS), are apprehended as an apt choice to be used as natural preservatives in food and animal feed to control fungal growth and subsequent mycotoxin production. LAB species produce a vast spectrum of anti-fungal metabolites to inhibit fungal growth; and also have the capacity to adsorb, degrade or detoxify fungal mycotoxins including ochratoxins, aflatoxins and Fusarium toxins. The potential of many LAB species to circumvent spoilage associated with fungi has been exploited in a variety of human food and animal feed stuff. This review provides the most recent updates on the ability of LAB to serve as anti-fungal and anti-mycotoxigenic agents. In addition, some recent trends of the use of LAB as bio-preservative agents against fungal growth and mycotoxin production are highlighted.
Aflatoxin M1 (AFM1) and ochratoxin A (OTA) are common mycotoxins in cereal foods and milk products, and may cause serious negative impacts on human health. The intestine is crucial for immune regulation as it protects host homeostatic health from external contaminants; however, the underlying mechanisms of AFM1 and OTA mediated intestinal immunotoxicity remain unclear. In this study, whole transcriptome analysis was used to characterize BALB/c mouse intestines exposed to individual and combined AFM1 and OTA [3.0 mg/kg body weight (BW)] for 28 days to screen for key intestinal immunotoxicity-related differentially expressed mRNAs (DEmRNAs), differentially expressed microRNAs (DEmiRNAs), differentially expressed long non-coding RNAs (DElncRNAs), and associated enriched signaling pathways. Functional validation was then conducted in intestinal differentiated Caco-2 cells using different inhibitor assays to verify the accuracy of transcriptome and the importance of the key screened regulatory factors. In vivo data revealed that AFM1 and OTA exposure disrupted the intestines and exerted intestinal immunosuppression effects. When compared with AFM1, OTA had stronger intestinal toxicity in combined treatments. Further analyses of competitive endogenous RNA (ceRNA) regulatory networks in mice showed that AFM1 and OTA mediated-intestinal immunosuppression was putatively explained as follows: (i) toxins affected DEmRNAs regarding transfer and transduction mechanisms between cells (Csf1, Csf1r, Cxcl10, Cx3cr1, and Irf1), which were regulated by key DEmiRNAs (miR-106-x, miR-107-y, and miR-124-y) and the DElncRNA Rian, and (ii) toxins inhibited transforming growth factor-β-activated kinase 1 (TAK1)/I-kappaB kinase (IKK)/inhibitor of kappa Bα (IκBα)/p65 nuclear factor-κB (NF-κB) signaling phosphorylation levels, which was validated in differentiated Caco-2 cells using the TAK1 inhibitor (5Z-7-oxozeaenol). In conclusion, we evaluated the risk of co-exposure to AFM1 and OTA and associated health hazards from a whole transcriptome perspective.
To reveal the interaction between aflatoxin M1 (AFM1) and DNA, and then explore a new method to remove AFM1, spectroscopy, electrophoresis, and fluorescence microscopy were used. Fluorescence spectra assay showed that AFM1 and ethidium bromide (EB) competed for the intercalation sites in DNA, and the binding constant between AFM1 and DNA was 3.81±0.1×10⁷ L mol⁻¹. The comparison of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in the fluorescence enhancement of AFM1 further proved that the binding sites were between the base pairs of DNA. In the presence of AFM1, the melting temperature of DNA increased by 6.2 ℃, and the circular dichroism of DNA changed. These events were closely related to the intercalation between AFM1 and DNA. The binding between AFM1 and DNA was firm. AFM1 and DNA did not separate in agarose gel electrophoresis. Based on the intercalation between AFM1 and DNA, with DNA as an intermediate medium, AFM1 can be indirectly adsorbed onto magnetic beads. Then, AFM1 could be removed by magnetic separation. At an initial concentration of 10 μg/L, the removal efficiencies were 95.5%±3.7 and 85.5%±3.3 for aqueous solution and milk, respectively. This research result provided a direction in the detoxification of AFM1, which deserves to be developed further.
