The importance of transcriptomics and proteomics for studying molecular mechanisms of mycotoxin exposure: A review.

This review aims to highlight recent advances where transcriptomics and proteomics have been used as a key tool to understand molecular toxicity of mycotoxins. The most studied mycotoxin by using transcriptomic approach is deoxynivalenol (DON), followed by aflatoxins (AFs) and zearalenone (ZEA). Instead, proteomics mostly focuses on AFs but also in this case, mildly to ZEA and DON. However, in both omics approaches, fewer studies investigated the toxicological effect of emerging mycotoxins, patulin, ochratoxin A, T-2 toxin, alternariol and amino-14,16-dimethyloctadecan-3-ol. The study of changes in the expression of genes involved in immune system are the most common purposes for transcriptomics whereas cellular processes in proteomics field. Concerning the techniques used to perform the experiments, RT-qPCR is the most employed in gene expression analysis whereas liquid chromatography coupled with mass spectrometry is the master technique for proteomics assays. The gathered data have reported that the interest in using these omic approaches has increased in the last five years. However, in vitro models take precedence over the in vivo and ex vivo ones. Therefore, there is a need to enhance the use of in vivo models and alternative methods to better understand mycotoxins mode of action on animal and human health.

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.

Proteomics evaluation of enniatins acute toxicity in rat liver.

Enniatins (ENs) are emerging mycotoxins produced by Fusarium fungi which are cytotoxic also at low concentrations due to its ionophoric properties. The aim of this study was to evaluate the hepatic toxicity of ENs exposure at different concentrations in Wistar rats through a proteomic approach. Animals were intoxicated by oral gavage with medium (EN A 256, ENA1 353, ENB 540, ENB1 296 µg/mL) and high concentrations (ENA 513, ENA1 706, ENB 1021, ENB1 593 µg/mL) of an ENs mixture and sacrificed after 8 h. Protein extraction was performed using powdered liver. Peptides were analyzed using a liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer. Proteins were filtered by abundance using Mass Professional Profiler software (Agilent Technologies) and 57 were differentially expressed when compared to the control. In terms of abundance, the liver biomarker Carboamoyl-phosphate synthase showed the highest levels in all conditions employed while actin-1 had the lowest. Bioinformatic analysis using DAVID platform reported acetylation, nucleotide phosphate-binding region:NAD and catalytic activity as the most represented terms. Furthermore, metabolism was the most significant and enriched pathway in Reactome overrepresentation. In conclusion, ENs acute exposure caused protein expression changes related to major cellular processes in rats, hinting its involvement in liver disturbance.

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)