Ochratoxin A promotes porcine circovirus type 2 replication in vitro and in vivo.

Ochratoxin A (OTA), a worldwide mycotoxin found in food and feeds, is a potent nephrotoxin in animals and humans. Porcine circovirus-associated disease (PCVAD), including porcine dermatitis and nephropathy syndrome, is a worldwide swine disease. To date, little is known concerning the relationship between OTA and porcine circovirus type 2 (PCV2), the primary causative agent of PCVAD. The effects of OTA on PCV2 replication and their mechanisms were investigated in vitro and in vivo. The results in vitro showed that low doses of OTA significantly increased PCV2 DNA copies and the number of infected cells. Maximum effects were observed at 0.05 µg/ml OTA. The results in vivo showed that PCV2 replication was significantly increased in serum and tissues of pigs fed 75 µg/kg OTA compared with the control group and pigs fed 150 µg/kg OTA. In addition, low doses of OTA significantly depleted reduced glutathione and mRNA expression of NF-E2-related factor 2 and γ-glutamylcysteine synthetase; increased reactive oxygen species, oxidants, and malondialdehyde; and induced p38 and ERK1/2 phosphorylation in PK15 cells. Adding N-acetyl-L-cysteine reversed the changes induced by OTA. Knockdown of p38 and ERK1/2 by their respective specific siRNAs or inhibition of p38 and ERK1/2 phosphorylation by their respective inhibitors (SB203580 and U0126) eliminated the increase in PCV2 replication induced by OTA. These data indicate that low doses of OTA promoted PCV2 replication in vitro and in vivo via the oxidative stress-mediated p38/ERK1/2 MAPK signaling pathway. This suggests that low doses of OTA are potentially harmful to animals, as they enhance virus replication, and partly explains why the morbidity and severity of PCVAD vary significantly in different pig farms.

Glutathione peroxidase 4 has a major role in protecting mitochondria from oxidative damage and maintaining oxidative phosphorylation complexes in gut epithelial cells.

Intake of the micronutrient selenium, which is incorporated into 25 selenoproteins in humans, has been implicated in affecting risk of colorectal cancer. A genetic variant in the gene encoding the selenoprotein glutathione peroxidase 4 (GPX4) has been reported to influence colorectal cancer risk. In this study GPX4 expression was knocked down by 60% using RNA silencing and the effects were investigated using an unbiased transcriptomic analysis. Microarray analysis of the total Caco-2 cell transcriptome was carried out using Illumina HumanHT-12v3 beadchips and the data were validated by real-time PCR. Ingenuity Pathway Analysis showed that the major canonical pathways affected by GPX4 knockdown were oxidative phosphorylation, ubiquinone biosynthesis, and mitochondrial dysfunction and the top two toxicological lists were mitochondrial dysfunction and oxidative stress. Western blotting and real-time PCR confirmed that knockdown affected target genes encoding components of respiratory complexes I, IV, and V as well as the protein apoptosis-inducing factor (AIF). GPX4 knockdown increased levels of mitochondrial reactive oxygen species and oxidized lipid and decreased mitochondrial adenosine triphosphate levels and mitochondrial membrane potential. Time-course experiments showed that changes in AIF expression preceded those in the respiratory complexes. We conclude that in Caco-2 gut epithelial cells GPx4, through effects on AIF, plays a major role in maintaining the oxidative phosphorylation system and protecting mitochondria from oxidative damage.