Evaluation of immunotoxicity induced by aflatoxin M1 and its relation to microRNA 155 expression in mice

Objective: Aflatoxin M1 (AFM1) is the hydroxylated metabolite of aflatoxin B1 (AFB1), one of the mycotoxins produced by the Aspergillus fungus. Although, to date, there have been several in vitro and in vivo studies of immunomodulatory effects of AFB1, there have been no studies of immunotoxicity of AFM1. The aim of this study was to investigate in vivo immunotoxicity of AFM1 and the role of miR-155 and its protein targets after repeated intraperitoneal administration for 28 days. Material and methods: Mice were haphazardly divided into 5 groups for experiments (n= 100). Each group was further subdivided into four subsets (n= 5) and treated as follows: (1) IP injection with 25 μg AFM1/kg, 5 days/week for 28 days; (2) IP injection with 50 μg AFM1/kg, 5 days/week for 28 days; (3) IP injection with 20 mg cyclophosphamide (CTX)/kg for 5 days as the positive control; (4) IP injection with normal saline and methanol, 5 days/week for 28 days, as the negative control. Histopathological examinations, body mass, organ masses, and organ/body mass ratios, quantification of hematological parameters, total serum hemolytic activity (CH50), phagocytic activity, and concentrations of antibodies were evaluated. Also, humoral immunity was assessed by measuring the antibody titres by hemagglutination (HA) and cellular immune function using delayed delayed response (DTH) response. In the next step, the cellularty of the spleen was evaluated using a trypan blue assay and the percentage of cell subtypes was evaluated using FACSCalibur TM flow cytometry. The lymphocyte proliferation assay was performed using MTT assay. Finally, the level of interferon gamma (IFNγ), interleukin-4 (IL-4) and interleukin-10 (IL-10) were measured. At the next step, T cells were isolated from spleens of BALB/c mice and amounts of microRNA-155 (miR-155) were measured by real-time quantitative PCR. Finally, Western protein was done for various miR-155 protein targets including phosphatidylinositol-3, 4, 5-trisphosphate 5-phosphatase 1 (Ship1), suppressor of cytokine signaling 1 (Socs1) and macrophage activating factor (c-MAF). Results: Several parameters related to immune function were suppressed: organ mass, cellularity of spleen, proliferation response to LPS and PHA, HA, DTH response, spleen cell subtypes, CH50, serum IgG level and cytokine production. AFM1 did not cause any changes in body mass, hematological parameters, and concentration of IgM in blood serum. Exposure to AFM1 resulted in significantly lesser expression of miR-155 in mice. Western blot results revealed that AFM1 increased the Ship1, Socs1, but not c-MAF. Conclussion: In conclusion, intraperitoneal administration of AFM1 suppressed innate and acquired immunity. These results suggest that miR-155 and targeted proteins might be involved in immunotoxicity observed in mice exposed to AFM1.

Keywords:

Aflatoxin M1 , MicroRNA , Immunotoxicity , Acquired immunity , innate immunity