Natural Antioxidant By-Product Mixture Counteracts the Effects of Aflatoxin B1 and Ochratoxin A Exposure of Piglets after Weaning: A Proteomic Survey on Liver Microsomal Fraction

Abstract

Mycotoxins are toxic compounds produced by certain strains of fungi that can contaminate raw feed materials. Once ingested, even in small doses, they cause multiple health issues for animals and, downstream, for people consuming meat. It was proposed that inclusion of antioxidant-rich plant-derived feed might diminish the harmful effects of mycotoxins, maintaining the farm animals’ health and meat quality for human consumption. This work investigates the large scale proteomic effects on piglets’ liver of aflatoxin B1 and ochratoxin A mycotoxins and the potential compensatory effects of grapeseed and sea buckthorn meal administration as dietary byproduct antioxidants against mycotoxins’ damage. Forty cross-bred TOPIGS-40 hybrid piglets after weaning were assigned to three (n = 10) experimental groups (A, M, AM) and one control group (C) and fed with experimental diets for 30 days. After 4 weeks, liver samples were collected, and the microsomal fraction was isolated. Unbiased label-free, library-free, data-independent acquisition (DIA) mass spectrometry SWATH methods were able to relatively quantify 1878 proteins from piglets’ liver microsomes, confirming previously reported effects on metabolism of xenobiotics by cytochrome P450, TCA cycle, glutathione synthesis and use, and oxidative phosphorylation. Pathways enrichment revealed that fatty acid metabolism, steroid biosynthesis, regulation of actin cytoskeleton, regulation of gene expression by spliceosomes, membrane trafficking, peroxisome, thermogenesis, retinol, pyruvate, and amino acids metabolism pathways are also affected by the mycotoxins. Antioxidants restored expression level of proteins PRDX3, AGL, PYGL, fatty acids biosynthesis, endoplasmic reticulum, peroxisome, amino acid synthesis pathways, and, partially, OXPHOS mitochondrial subunits. However, excess of antioxidants might cause significant changes in CYP2C301, PPP4R4, COL18A1, UBASH3A, and other proteins expression levels. Future analysis of proteomics data corelated to animals growing performance and meat quality studies are necessary.

Proteomic Changes in the Cytoplasmatic Fraction of Weaned Piglets Liver and Kidney Under Antioxidants and Mycotoxins Diets

“…During this phase, mycotoxins are initially transformed into more water-soluble chemical form that can be metabolized by the enzymes involved in phase II (Croom 2012; Gerdemann et al 2023). As we previously observed by proteomic analysis of the microsomal fraction from the liver (Popescu et al 2023), cytochromes P450 isoforms have a central role in catalysing phase I reactions.…”

“…In a previous study (Popescu et al 2023), the microsomal fraction extracted from weaned piglets exposed to a mycotoxin-contaminated diet, along with an antioxidant additive diet was investigated, to highlight the impact level of AFB1 and OTA on the expression of specific proteins involved in biotransformation metabolism. Since xenobiotic biotransformation involves not only microsomal but also cytoplasmatic proteins related to glycolysis, amino acid metabolism, glycogen metabolism, fatty acid synthesis, and others, we extended our research to include the cytoplasmatic fraction extracted from liver and kidney tissues.…”

“…Ionization was done using electro spray ionization in positive ion mode at a voltage of 5500 V and 200 ⁰C source temperature. The TRIPLE TOF 5600+ operated in Data Independent Acquisition (DIA) mode, with 64 variable windows (SWATH 64 vw) as described by Popescu et al (2023). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al 2022) partner repository with the dataset identifier PXD050835 ( Reviewer account details : Username: reviewer_pxd050835@ebi.ac.uk; Password: YD4S7bda).…”

“…In our previous investigation about the effects of feed mycotoxin contamination with AFB1 and OTA, and the potential of addition of by-product mixture of antioxidants to mitigate these effects on microsomal fraction of weaned piglets’ liver, we found that some proteins expressions have been affected by antioxidants (Popescu et al 2023). However, these observations only apply to the microsomal fraction that contains mostly proteins involved in phase I biotransformation, while phase II or conjugation proteins are located within the cytoplasmatic fraction.…”

“…To date, most studies have tried to confirm the beneficial effects of adding food by-products rich in antioxidants to the animal diet. However, in recent years, with the development of proteomics and metabolomics methods, new studies have highlighted their side or even negative effects (Petcu et al 2023; Popescu et al 2023). Antioxidants are believed to counteract some of the effects of mycotoxins due to their ability to scavenge free radicals, reduce oxidative stress, and have anti-inflammatory and immunomodulatory properties (Chaudhary et al 2023).…”