Characterization of deoxynivalenol-induced anorexia using mouse bioassay

Abstract

A short-term mouse model was devised to investigate induction of food refusal by the common foodborne trichothecene deoxynivalenol (DON). DON dose-dependently induced anorexia within 2 h of exposure when administered either by intraperitoneal (ip.) injection or by oral gavage. The no observed adverse effect and lowest observed adverse effect levels in this assay were 0.5 and 1 mg/kg bw for ip. exposure and 1 and 2.5 mg/kg bw for oral exposure, respectively. DON's effects on food intake were transient, lasting up to 3h at 1 mg/kg bw and up to 6 h at 5 mg/kg bw. Interestingly, a dose-dependent orexigenic response was observed in the 14 h following the initial 2h food intake measurement. Toxin-treated mice exhibited partial resistance to feed refusal when exposed to DON subsequently after 2 d, but not after 7 d suggesting that this modest tolerance was reversible. The short-term mouse bioassay described here was useful in characterizing DON-induced anorexia and should be applicable to elucidating mechanisms underlying this adverse nutritional effect.

Figure 1. Experimental design for mouse feed refusal assay

Mice are fasted from 10:00 h on Day 1. At 18:00 h, the onset of the dark cycle, mice are treated DON or PBS vehicle and then and then provided with food. At 20:00 h lights were turned on for 30 m and food intake was measured and measured again at 10:00 h on Day 2.

Figure 2. Intraperitoneal DON exposure in mice induces an early anorexigenic response followed by a delayed orexigenic response

Mice were given an ip. injection of DON immediately before the dark cycle. Food intake was measured at 2 h and 16 h post injection time and graphically depicted. Data are mean ± SEM (n = 13/gp). A One-way ANOVA using Dunnett’s Test was used to assess significant differences in food intake between doses and the control. Asterisks indicate statistically significant differences in food consumption as compared to the control (p < 0.05).

Figure 3. Oral DON exposure in mice induces an early anorexigenic response followed by a delayed orexigenic response

Mice were orally gavaged with DON immediately before the dark cycle. Food intake was measured at 2 h and 16 h post DON exposure and graphically depicted. Data are mean ± SEM (n = 8/gp). One-way ANOVA and Dunnett’s test were used to assess significant differences in food intake between doses and the control. Asterisks indicate statistically significant differences in food consumption as compared to the control (p > 0.05).

Figure 4. DON-induced feed refusal is transient

(A) Experimental design for determining the duration of DON-induced feed refusal. Mice were given an ip. injection of DON either 0, 1, 2 or 4 hours before the dark cycle as indicated by gray inverted arrows. Food intake was measured 2 hr after the injection for a total exposure time of 2, 3, 4 and 6 h. (B) At 1 mg/kg bw DON feed refusal is attenuated within 3 h and at 5 mg/kg bw DON, feed refusal is attenuated within 6 h after exposure. Data are mean ± SEM (n = 4–5/gp). Two-Way ANOVA and Holm-Sidak post hoc tests were employed to determine the effects of dose and time on food intake. Symbols: *Food consumption for a given treatment is significantly different from treatment control at specified time (p < 0.05); ^ŧFood consumption for a given time is significantly different from 2 hr exposure time at specified dose (p < 0.05).

Figure 4. DON-induced feed refusal is transient

(A) Experimental design for determining the duration of DON-induced feed refusal. Mice were given an ip. injection of DON either 0, 1, 2 or 4 hours before the dark cycle as indicated by gray inverted arrows. Food intake was measured 2 hr after the injection for a total exposure time of 2, 3, 4 and 6 h. (B) At 1 mg/kg bw DON feed refusal is attenuated within 3 h and at 5 mg/kg bw DON, feed refusal is attenuated within 6 h after exposure. Data are mean ± SEM (n = 4–5/gp). Two-Way ANOVA and Holm-Sidak post hoc tests were employed to determine the effects of dose and time on food intake. Symbols: *Food consumption for a given treatment is significantly different from treatment control at specified time (p < 0.05); ^ŧFood consumption for a given time is significantly different from 2 hr exposure time at specified dose (p < 0.05).

Figure 5. Short-term repeated DON exposure results in partial tolerance to DON-induced feed refusal

Mice were given an ip. injection of DON immediately before the dark cycle. Food intake was measured 2 h after injection time. This was repeated twice, 48 h apart. Data are mean ± SEM (n = 5/gp). Two-Way Repeated Measures (one factor) ANOVA and Holm-Sidak post hoc tests were employed to determine the effects of dose and time on food intake. Symbols: ^*Food consumption for a given treatment is significantly different from treatment control at specified exposure number (p < 0.05). ^ŧFood consumption for a given exposure number is significantly different from the first exposure at the specified dose (p < 0.05).

Figure 6. DON-induced feed refusal tolerance is short-lived

Mice were treated with DON via (A) ip. injection and (B) oral gavage and food intake measured after 2 h. After randomization, mice were exposed to DON, 7 d later. Data are mean ± SEM (n = 8/gp). Two-Way ANOVA with Holm-Sidak post hoc tests were employed to determine the effects of dose and time on food intake. Symbols: ^*Food consumption for a given treatment is significantly different from treatment control at specified exposure number (p < 0.05). ^ŧFood consumption for a given exposure number is significantly different from the first exposure at the specified dose (p < 0.05).

Figure 6. DON-induced feed refusal tolerance is short-lived

Mice were treated with DON via (A) ip. injection and (B) oral gavage and food intake measured after 2 h. After randomization, mice were exposed to DON, 7 d later. Data are mean ± SEM (n = 8/gp). Two-Way ANOVA with Holm-Sidak post hoc tests were employed to determine the effects of dose and time on food intake. Symbols: ^*Food consumption for a given treatment is significantly different from treatment control at specified exposure number (p < 0.05). ^ŧFood consumption for a given exposure number is significantly different from the first exposure at the specified dose (p < 0.05).

Figure 7. Comparison of feed refusal induction by ip. injection and oral gavage of DON

To determine the dose at which mice exhibit 50% reduction in food intake relative to the control, a linear regression was calculated for ip. injection (Fig. 2) (y = 94.852 – 26.961x; r² = 0.94) and for gavage (Fig. 3) (y = 94.741 – 24.496x; r² = 0.88) with×being dose and y being percent food consumption relative to the control.