Effects of Mycotoxins on Mitogen-stimulated Proliferation of Bovine Peripheral Blood Mononuclear Cells

Abstract

ABSTRACT. The effects of mycotoxins on mitogen-stimulated proliferation of bovine peripheral blood mononuclear cells (PBMCs) were investigated. Aflatoxin B 1 (AFB 1 ), deoxynivalenol (DON) and zearalenone (ZEN) were added to cultures of PBMCs, and the proliferation responses were measured using MTT bioassays. Suppression of the proliferation of calf PBMCs by AFB 1 and DON was significantly stronger than that of cow PBMCs, whereas there were no differences in suppressive effects on PBMCs from Holstein and Japanese Black calves and cows. The suppressive effect was greatest in the order of DON, AFB 1 and ZEN, and the effects of DON and AFB 1 seemed to be dose-dependent. The results suggest that some mycotoxins directly suppress proliferation of bovine PBMCs. KEY WORDS: bovine, mononuclear cell, mycotoxin.J. Vet. Med. Sci. 70(2): 193-196, 2008 Mycotoxin is a general term for harmful substances produced by certain fungi. In Japan, there are permissible concentrations of the mycotoxins aflatoxin B 1 (AFB 1 ), deoxynivalenol (DON) and zearalenone (ZEN) in feed for animals. There have been many studies on the effects of mycotoxins on the immune functions of various animals [1][2][3][4][5][6][7][8][9][10][11][12]15]. We previously conducted blood profile tests for cows on dairy farms that were given feed contaminated with mycotoxins (mainly AFB 1 ) and found reduced levels of T cells in many cows [14]. These findings indicate that mycotoxins may induce immune suppression, particularly suppression of bovine T-lymphocyte function. However, the relationships between the direct effect of a mycotoxin on bovine peripheral blood mononuclear cells (PBMCs) and age or breed has not been elucidated. Moreover, there have been only a few studies in which the effects of various mycotoxins were examined [4,5]. The aim of this study was therefore to clarify the effects of AFB1, DON and ZEN on proliferation of bovine PBMCs.The cattle tested were clinically healthy Holstein cows (n=6), Japanese Black cows (n=6), Holstein calves (n=6, =1, =5) and Japanese Black calves (n=6, =3, =3). The cows were between 3 and 7 years old, and the calves were between 1 and 2 months old. Aflatoxin B 1 from Aspergillus flavus (AFB 1 ), deoxynivalenol (DON) and zearalenone (ZEN; Sigma-Aldrich Co., St. Louis, MO, U.S.A.) were used as mycotoxin reagents.Blood was collected from each animal into sampling tubes containing heparin. Each 5 ml blood sample was diluted with 5 ml 1/15M PBS (pH #7.4) and laid on 4.5 ml Lymphocepar I (Immuno-Biological Laboratories Co., Ltd. Gunma, Japan). PBMCs were isolated by centrifugation (1,500 rpm, 60 min) and washed several times with PBS. The samples were seeded into 96-well microplates at 1 × 10 6 cells/well and diluted with 10% FCS-containing RPMI Medium 1640 (Gibco BRL, Gaithersburg, MD, U.S.A.) at a final volume of 200 µl/well. Ten µg/ml of phytohaemagglutin-P (PHA-P; Sigma-Aldrich Co.) was added to each PBMC culture, and then each culture was mixed with AFB 1 , DON, ZEN, AFB 1 +DON, AFB 1 +ZEN or DON+ZEN and incubated f...

Effects of deoxynivalenol (DON) and related compounds on bovine peripheral blood mononuclear cells (PBMC) in vitro and in vivo

“…For the latter substance no IC 50 could be (Charoenpornsook et al 1998), suggesting the DNA synthesis to be more vulnerable to the effects of DON than the metabolic activity (MTT assay). The age of ruminants was shown as another factor influencing the susceptibility of bovine PBMC as this cell type was more sensitive to DON when isolated from calves compared with the adult cows, while the genetic background of cows appeared to have no influence (Wada et al 2008). Using the BrdU (5-bromo-2'-deoxyuridine) assay, which is a measure for DNA synthesis, the IC 50 values for DON, 15AcDON, 3AcDON, and de-epoxy DON were 510 ng/ml (1.5 μM), 444 ng/ml (1.5 μM), 4,888 ng/ml (14.5 μM) and 23,266 ng/ml (83.1 μM) when tested on Swiss mouse fibroblasts (3T3 cells) (Eriksen et al 2004).…”

Aflatoxin B1 affects apoptosis and expression of Bax, Bcl-2, and Caspase-3 in thymus and bursa of fabricius in broiler chickens

“…Primarily being a hepatotoxin, AFB1 also exerts negative impact on immune system. It has been proved that AFB1 develops its immunotoxicity by inhibiting lymphocyte proliferation (Meissonier et al, ; Wada et al, ), reducing the number of circulating lymphocytes (Han et al, ), altering the activities of natural killer(NK) cells and disturbing the expression of cytokines (Methenitou et al, ). A recent research also showed that severe lymphocyte depletion in the thymus and bursa of Fabricius was due to apoptosis (Solcan et al, ).…”

Comparison of the Immunosuppressive Effects of Dexamethasone, Flunixin Meglumine and Meloxicam on the In Vitro Response of Calf Peripheral Blood Mononuclear Cells

“…Each sample was cultured with 10 g/ml phytohemagglutinin (PHA; Sigma, St. Louis, MO, U.S.A.) [24] alone, PHA and DEX (Sigma; 10, 15 or 20 μg/ml) [13,20], PHA and FLU (Sigma; 10, 25 or 50 μg/ml) [2,12] or PHA and MEL (Nippon Boehringer Ingelheim Co., Ltd., Tokyo, Japan; 10, 25 or 50 μg/ml) [14]. The mitogen PHA and three AIDs were used at concentrations based on those reported for previous in vitro experiments with cattle [11,12,19] and other animal species [2,12,14].…”

“…The concentrations of PHA and the three AIDs were as described above. PHA (10 μg/ml) [20,24] was added to each PBMC culture, which was then mixed with DEX (10 or 20 μg/ml) [13], FLU (10 or 50 μg/ml) [2] or MEL (10 or 50 μg/ml) [12] and incubated for 12 hr at 37C. PBMCs were then washed and re-suspended in TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.) to extract RNA.…”