Methodical aspects of in vitro proliferation of porcine blood lymphocytes when exposed to deoxynivalenol (DON)

Trichothecene mycotoxins such as deoxynivalenol (DON) are known to modulate the immune function, but to this date only few studies have been conducted with farm animals, especially pigs, which are regarded as most susceptible to this mycotoxin. Mitogen-stimulated proliferation of porcine blood lympho­ cytes was measured using two different assays, the MTT cleav­ age (metabolic activity) and the BrdU incorporation (DNA-synthesis). Furthermore, immunoglobulin (IgA, IgG and IgM) con­ centrations of culture supernatants were determined using an indirect ELISA. Various conditions were investigated before establishment of the appropriate method: the grade of stimula­ tion of the T-cell mitogens concanavalin A (ConA) and phythaemagglutinin (PHA) were compared to the B-cell mitogen lipopolysaccharide (LPS) from the cell wall of E. coli. Further­ more, the effect of input of 1 x 105, 2 x 105, and 4 x 105 lym­ phocytes per well was studied in the view of sensitivity to the mycotoxin DON. In addition, the effects of DON dose (0, 70, 140, 280, and 560 ng/ml) were tested in lymphocytes of one par­ ticular pig, which were either frozen or freshly processed. ConA appeared to be the most potent mitogen tested and was applied at 2.5 μg/ml in lymphocyte cultures. 1 x 105 cells/well seemed to be more sensitive to DON compared to 2 x 105 and 4 x 105 cells/well, as the inhibiting concentrations of 50 % (IC50) increased with the cell number from 249, 325 to 337 ng DON/ml, respectively. Addition of 70 to 560 ng DON/ml to ConA-stimulated lymphocytes dose-dependently inhibited the metabolic activity (MTT assay) and DNA-synthesis (BrdU assay). No obvious difference in the toxic action of DON was observed when lymphocytes were frozen or freshly processed since the IC50 values were comparable (286 versus 309 ng DON/ml for the MTT and 201 versus 200 ng DON/ml for the BrdU assay). Supernatant Ig concentrations also showed a dose­ dependent reduction with IC50 values of 108 or 121 ng DON/ml for IgA, 84 or 86 ng DON/ml for IgM, and none or 72 ng DON/ml for IgG when lymphocytes were frozen or freshly processed, respectively.