Estrogenic, androgenic, and genotoxic activities of zearalenone and deoxynivalenol in in vitro bioassays including exogenous metabolic activation

Abstract

Zearalenone (ZEN) and deoxynivalenol (DON) and their derivatives are well-known mycotoxins, which can occur not only in crops but also in water bodies, including drinking water sources. In vitro bioassays can be used to detect biological effects of hazardous compounds in water. To this, when studying biological effects and toxicity in vitro, metabolism is important to consider. In this study, ZEN, α-zearalenol (α-ZEL), DON, 3-acetyl DON, and 15-acetyl DON were evaluated in vitro for hormone receptor-mediated effects (estrogen receptor [ER] and androgen receptor [AR]) and genotoxicity (micronucleus assay) in the presence of an exogenous metabolic activation system (MAS). The ER bioassay proved to be a highly sensitive method to detect low concentrations of the ZEN compounds (EC₁₀ values of 31.4 pM for ZEN, 3.59 pM for α-ZEL) in aqueous solutions. In the presence of the MAS, reduced estrogenic effects were observed for both ZEN compounds (EC₁₀ values of 6.47 × 10³ pM for ZEN, 1.55 × 10² pM for α-ZEL). Of the DON compounds, only 3-acetyl DON was estrogenic (EC₁₀ of 0.31 µM), and the effect was removed in the presence of the MAS. Anti-androgenic effects of the ZEN compounds and androgenic effects of the DON compounds were detected in the micromolar range. No induction of genotoxicity was detected for ZEN or DON in the presence of the MAS. Our study highlighted that inclusion of exogenous MAS is a useful tool to detect biological effects of metabolites in in vitro bioassays.

Keywords: In vitro bioassays; Deoxynivalenol; Effect-based methods; Endocrine receptors; Metabolism; Micronuclei formation; Zearalenone.

Fig. 1

CECs of the ER agonistic and antagonistic effects of ZEN (A, C) and α-ZEL (B, D) in the presence of exogenous MAS. Each test compound (n = 4) was assayed in the absence of MAS (purple, circles), in the presence of S9 alone (light blue, squares), S9 with PHI cofactors (green, triangles), or S9 with PHI and PHII cofactors (light orange, inverted triangles). Data presented as mean ± SD

Fig. 2

CECs of the AR antagonistic effects of ZEN (A) and α-ZEL (B) in the presence of exogenous MAS. Each test compound (n = 4) was assayed in the absence of MAS (black, circles), in the presence of S9 alone (dark purple, squares), S9 with PHI cofactors (purple, triangles), or S9 with PHI and PHII cofactors (pink, inverted triangles). Data presented as mean ± SD

Fig. 3

CECs of the ER agonistic and antagonistic effects of DON (A, D), 3-Ac-DON (B, E), and 15-Ac-DON (C, F) in the presence of exogenous MAS. Each test compound (n = 4) was assayed in the absence of MAS (black, circles), in the presence of S9 alone (pink, squares), S9 with PHI and PHII cofactors (teal, inverted triangles), or S9 with PHII cofactors (dark purple, diamonds). Data presented as mean ± SD

Fig. 4

CECs of the AR agonistic effects of DON (A), 3-Ac-DON (B), and 15-Ac-DON (C) in the presence of exogenous MAS. Each test compound (n = 4) was assayed in the absence of MAS (dark purple, circles), in the presence of S9 alone (light blue, squares), S9 with PHI and PHII cofactors (teal, inverted triangles), or S9 with PHII cofactors (orange, diamonds). Data presented as mean ± SD

Fig. 5

Micronuclei formations (left y-axis) and cytotoxicity (right y-axis) in TK6 cells following continuous long-term exposure (24 h) to ZEN (A), and DON (B). For micronuclei formations, data bars for each test concentration presented as mean ± SD (n = 3). Treatment groups were compared to the vehicle control via pair-wise comparisons. Asterisk (*) represents the level of significant difference in the % micronucleus events from the vehicle control (p ≤ 0.001). For cytotoxicity, the scoring criteria was set as 4-fold increase in % ethidium monoazide (EMA)-Positive over the vehicle control. Blue square symbols of the %EMA-Positive results for each test concentration presented as mean ± SD

Fig. 6

Micronuclei formations (left y-axis) and cytotoxicity (right y-axis) in TK6 cells following short-term exposure (5 h) to ZEN (A), and DON (B) in the absence of MAS (dark grey bars), in the presence of S9 only (dark purple bars), and in the presence of S9 with cofactors (light purple bars). For micronuclei formations, data bars for each test concentration presented as mean ± SD (n = 3). Within each treatment group, the means of each test concentration were compared to the mean vehicle control of that treatment group via pair-wise comparisons. For cytotoxicity, the scoring criteria was set as 4-fold increase in %EMA-Positive over the vehicle control of each treatment group. Blue square symbols of the %EMA-Positive results for each test concentration are presented as mean ± SD. Note that the blue square symbols have been intentionally off-set from the center of each data bars to minimize overlap of the respective SD bars