Assessment of single-nucleotide variant discovery protocols in RNA-seq data from human cells exposed to mycotoxins.

Food and feed contamination by nonlegislated mycotoxins beauvericin (BEA) and enniatin B (ENB) is a worldwide health concern in the present. The principal objective of this work is to assess some of the existing protocols to discover the single nucleotide variants (SNVs) in transcriptomic data obtained by RNA-seq from Jurkat cells in vitro samples individually exposed to BEA and ENB at three concentration levels (1.5, 3 and 5 µM). Moreover, previous transcriptomic results will be compared with new findings obtained using a different protocol. SNVs rs201003509 in BEA exposed cells and the rs36045790 in ENB were found in the differentially expressed genes in all doses compared to controls by means of the Genome Analysis Toolkit (GATK) Best Practices workflow. SNV-RNA-seq complementary pipeline did not show any SNV. Concerning gene expression, discrepant results were found for 1.5 µM BEA exposed cells compared with previous findings. However, 354 overlapped differentially expressed genes (DEGs) were identified in the three ENB concentrations used, with 147 matches with respect to the 245 DEGs found in the previous results. In conclusion, the two discovery SNVs protocols based on variant calling from RNA-seq used in this work displayed very different results and there were SNVs found manually not identified by any pipeline. Additionally, the new gene expression analysis reported comparable but non identical DEGs to the previous transcriptomic results obtained from these RNA-seq data.

In vitro blood brain barrier exposure to mycotoxins and carotenoids pumpkin extract alters mitochondrial gene expression and oxidative stress.

Food and feed are daily exposed to mycotoxin contamination which effects may be counteracted by antioxidants like carotenoids. Some mycotoxins as well as carotenoids penetrate the blood brain barrier (BBB) inducing alterations related to redox balance in the mitochondria. Therefore, the in vitro BBB model ECV304 was subcultured for 7 days and exposed to beauvericine, enniatins, ochratoxin A, zearalenone (100 nM each), individually and combined, and pumpkin extract (500 nM). Reactive oxygen species were measured by fluorescence using the dichlorofluorescein diacetate probe at 0 h, 2 h and 4 h. Intracellular ROS generation reported was condition dependent. RNA extraction was performed and gene expression was analyzed by qPCR after 2 h exposure. The selected genes were related to the Electron Transport Chain (ETC) and mitochondrial activity. Gene expression reported upregulation for exposures including mycotoxins plus pumpkin extract versus individual mycotoxins. Beauvericin and Beauvericin-Enniatins exposure significantly downregulated Complex I and pumpkin addition reverted the effect upregulating Complex I. Complex IV was the most downregulated structure of the ETC. Thioredoxin Interacting Protein was the most upregulated gene. These data confirm that mitochondrial processes in the BBB could be compromised by mycotoxin exposure and damage could be modulated by dietary antioxidants like carotenoids.

In vitro exposure to pumpkin extract induces a protective transcriptomic profile in blood brain barrier electron transport chain

C. maxima (var. Delica) is a variety of pumpkin known for its beneficial effects and its high content in carotenoids (violaxanthin, astaxanthin, antheraxanthin, zeaxanthin, lutein, lycopene and β-carotene), which are natural antioxidants bioavailable to humans through food consumption. Numerous biological effects have been attributed to carotenoids due to their antioxidant activity: improved immune response, anti-inflammatory and anti-tumor properties and reduced risk of cardiovascular and chronic degenerative diseases. They are capable of accumulating in the brain after crossing the blood-brain barrier. Therefore, the aim of this study is to analyze changes in mitochondrial gene expression using an in vitro cell model (ECV304) of the blood brain barrier, after exposure to pumpkin extract. Cells were treated during 24 h at 5 different β-carotene concentrations, as reference extract compound: 1.72×10-4 – 1.72×10-3– 1.72×10-2 – 0.172 – 1.72 nM in DMSO 0.5%. The extracted RNA was used to perform qPCR analysis on 15 mitochondrial related genes: MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-CO1, MT-CO3, MT-ATP6, MT-ATP8, MT-RNR2, MRPL12, OSGIN1, SRXN1, TXNIP, UCP2, and S18 as reference gene. Results demonstrate that dietary carotenoids act at transcriptional level, especially on the genes belonging to the electron transport chain, reporting an overall protective pattern. The findings show a dose dependent differential gene expression pattern by carotenoids exposure, even at low concentrations. (AU)

Mitochondrial proteomics profile points oxidative phosphorylation as main target for beauvericin and enniatin B mixture.

Beauvericin (BEA) and enniatin B (EN B) are non-legislated Fusarium mycotoxins usually found in cereal and cereal-based products all around the world. By the proteomic analysis of mitochondria enriched extracts from Jurkat cells exposed for 24 h to three concentrations of BEA:EN B (0.01-0.1-0.5 µM), a number of 1821 proteins (202 mitochondrial) were identified and relatively quantified. 340 proteins (59 mitochondrial) were statistically significant altered in our samples (Anova p-value ≤ 0.05 and fold change (FC) ≥1.5). The protein mitochondrial translational release factor 1 like (MTRF1L) was the most abundant protein in the three mycotoxin exposures studied. The mycotoxins mixture exposure induced concentration dependent changes at mitochondrial proteins levels that mainly involve inner and outer membrane complexes, Electron Transport Chain (ETC) and ribosomes. These results showed alteration of proteins levels related to oxidative phosphorylation, metabolic and neurodegenerative diseases related pathways.

Toxicity of mycotoxins in vivo on vertebrate organisms: A review.

Mycotoxins are considered to be a major risk factor affecting human and animal health as they are one of the most dangerous contaminants of food and feed. This review aims to compile the research developed up to date on the toxicological effects that mycotoxins can induce on human health, through the examination of a selected number of studies in vivo. AFB1 shows to be currently the most studied mycotoxin in vivo, followed by DON, ZEA and OTA. Scarce data was found for FBs, PAT, CIT, AOH and Fusarium emerging mycotoxins. The majority of them concerned the investigation of immunotoxicity, whereas the rest consisted in the study of genotoxicity, oxidative stress, hepatotoxicity, cytotoxicity, teratogenicity and neurotoxicity. In order to assess the risk, a wide range of different techniques have been employed across the reviewed studies: qPCR, ELISA, IHC, WB, LC-MS/MS, microscopy, enzymatic assays, microarray and RNA-Seq. In the last decade, the attention has been drawn to immunologic and transcriptomic aspects of mycotoxins' action, confirming their toxicity at molecular level. Even though, more in vivo studies are needed to further investigate their mechanism of action on human health.

Mitochondrial transcriptional study of the effect of aflatoxins, enniatins and carotenoids in vitro in a blood brain barrier model.

C. maxima (var. Delica), a variety of pumpkin, is well known for its high concentration on carotenoids, possessing dietary benefits and antioxidant properties. Aflatoxins and enniatins are common mycotoxins present in food and feed with an extended toxicity profile in humans and animals. Both types of substances reach a wide range of tissues and organs and have the capability to penetrate the blood brain barrier. Since carotenoids and mycotoxins have been reported to modify diverse mitochondrial processes individually, transcriptional in vitro studies on human epithelial cells ECV 304 were conducted to analyze the relative expression of 13 mitochondria related genes. ECV 304 cells were differentiated for 9 days and treated for 2 h with: a) pumpkin (500 nM); b) aflatoxins (100 nM); c) enniatins (100 nM); d) aflatoxins (100 nM) and pumpkin (500 nM); e) enniatins (100 nM) and pumpkin (500 nM). Even at low concentrations, dietary carotenoids activity on mitochondrial genes expression reported a beneficial effect and, for most of the genes studied across the Electron Transport Chain (ETC), developed a protective effect when mixed with aflatoxins (AFs) or enniatins (ENs).

Enniatin B induces expression changes in the electron transport chain pathway related genes in lymphoblastic T-cell line.

Enniatin B is a ionophoric and lipophilic mycotoxin which reaches the bloodstream and has the ability to penetrate into cellular membranes. The purpose of this study was to reveal changes in the gene expression profile caused by enniatin B in human Jurkat lymphoblastic T-cells after 24 h of exposure at 1.5, 3 and 5 µM by next generation sequencing. It was found that up to 27% of human genome expression levels were significantly altered (5750 genes for both down-regulation and up-regulation). In the three enniatin B concentrations studied 245 differentially expressed genes were found to be overlapped, 83 were down and 162 up-regulated. ConsensusPathDB analysis of over-representation of differentially expressed genes provided a list of gene ontology terms in which several biological processes related to nucleoside monophosphate metabolic process, respiratory chain complex, electron transport chain, oxidative phosphorylation and cellular respiration were the most altered. Also, an interesting correlation was found between enniatin B toxicity and the up-regulation of the UCP protein complex. In summary, the transcriptomic analysis revealed that mitochondria are the organelles showing more related differentially expressed genes. Consequently, differentially expressed genes involved in biological processes, molecular functions and pathways related to mitochondrial metabolism and respiration were significantly changed.