Naturally occurring level of mixed aflatoxins B and G stimulate toll-like receptor-4 in bovine mononuclear cells

Abstract

Background: As common contaminants of agricultural commodities, aflatoxins (AFs) are highly hazardous carcinogenic mycotoxins originating from very common fungi present in the environment. Their effect on bovine key immune-surveillance molecules is unclear.Objective: To determine the in vitro effects of naturally occurring level of AFs on toll-like receptor-4 (TLR4) and viability in bovine peripheral blood mononuclear cells (PBMCs) as a model system for immunotoxicity.Methods: Ten healthy dairy cows were selected as a source of PBMCs. The mRNA expression of TLR4 was quantitatively evaluated in PBMCs treated with a mix of 1.0, 0.5, 0.25 and 0.25 ng/ml of AFB₁, AFB₂, AFG₁ and AFG₂, respectively, and 10 ng/ml of the well-known TLR4 agonist lipopolysaccharide (LPS) for 0.5, 2, 5 and 18 hours. We also analyzed the viability of PBMCs exposed to AFs by means of the trypan blue exclusion method and the tetrazolium bromide assay.Results: Compared with control PBMCs, transcription of TLR4 in AFs-treated PBMCs was significantly up-regulated at post-exposure hours (PEH) 2 and 5, but was unchanged at PEH 0.5 and 18. Unsurprisingly, much higher up-regulation of TLR4 transcript was observed in LPS-treated PBMCs at PEH 0.5, 2, 5 and 18. Conversely, the viability of post-AFs and/or LPS-exposed PBMCs revealed no significant changes.Conclusion: Naturally occurring levels of AFs result in increased expression of TLR4 mRNA in bovine PBMCs.Clinical importance: The increased expression of TLR4 mRNA in bovine PBMCs by AFs also emphasizes the need to reduce admissible levels of AFs in feed for preventive measures.

Keywords:

• cattle
• cow
• bovine
• aflatoxins
• leukocyte
• monocyte
• innate immunity
• toll-like receptor-4
• qPCR

Acknowledgements

This work was financially supported by a grant from Ferdowsi University of Mashhad, Iran. The authors thank Mr A. Bahari for technical support.