Interaction of aflatoxin B₁ and fumonisin B₁ in mice causes immunotoxicity and oxidative stress: Possible protective role using lactic acid bacteria

Figures & data

Table 1. Effect of AFB₁ and FB₁ on yield of L. paracasei BEJ01.

	Log (n/n0) after 24 h incubation
Strain	MRS	AFB1 (50 µg/ml)	FB1 (50 µg/ml)	AFB1 + FB1 (50 µg/ml)
L. paracasei  BEJ01	4.68 ± 0.09	4.53 ± 0.06	4.61 ± 0.02	4.64 ± 0.05
E. coli ATCC  10536	3.61 ± 0.05	1.65 ± 0.06*	1.37 ± 0.04*	1.01 ± 0.03*

n, number of bacteria after 24 h; n₀, number of bacteria at 0 h; MRS, MRS medium.

Total toxin level in broth (from individual of combined toxins) was 50 µg/ml.

Results shown are mean ± SD from three experiments.

*Value significantly different from control (p < 0.05).

Table 2. Percentage AFB₁ and FB₁ removal from PBS by live and killed LAB in PBS.

Treatment	AFB1 (20 µg/ml)			FB1 (20 µg/ml)			AFB1 + FB1 (20 µg/ml)
	0 h	12 h	24 h	0 h	12 h	24 h	0 h	12 h	24 h
PBS only	2.2 ± 0.2	2.4 ± 0.2	2.2 ± 0.3	3.0 ± 0.2	2.4 ± 0.2	2.3 ± 0.2	3.0 ± 0.2	2.4 ± 0.2	2.3 ± 0.2
Live LAB	27.3 ± 2.3*‡	63.4 ± 6.1*‡	83.4 ± 7.1*‡	26.2 ± 4.2*‡	67.5 ± 7.2*‡	88.9 ± 5.9*‡	29.9 ± 6.1*‡	70.2 ± 4.2*‡	91.2 ± 7.5*‡
Killed LAB	7.3 ± 0.2	22.1 ± 2.1*	21. 8 ± 3.3*	3.9 ± 0.2	23.4 ± 4.2*	25.5 ± 1.3*	7.3 ± 1.4	24.4 ± 5.2*	26.3 ± 4.1*

To determine the effect of bacteria viability on the binding affinity to AFB₁ and FB₁, “killed” bacteria (10⁸ CFU/ml) were heated at 90 °C for 15 min before being used in the assay.

Values shown are mean ± SD from three determinations per experimental scenario tested.

*Values significantly different from control (PBS only) and heated bacteria at p ≤ 0.05.

‡Values significantly different from that of the heated bacteria (p ≤ 0.05).

Figure 1. Splenic indices for mice after treatments. Mice were orally exposed daily for 2 weeks to LAB (2 × 10⁹ CFU/L, ∼2 mg/kg BW), AFB₁ (80 µg/kg), FB₁ (100 µg/kg), AFB₁ + FB₁, AFB₁ + LAB, FB₁ + LAB, or AFB₁ + FB₁ + LAB. Measures were made after 12 h fasting (i.e. on day after the final treatment in each group). Data shown are mean ± SD. In each histogram, values (bars) with superscripts bearing differ significantly (p < 0.05).

Table 3. Effect of treatments on splenic mRNA expression of select inflammatory cytokines.

Parameter	Control	LAB	AFB1	FB1	AFB1 + FB1	LAB + AFB1	LAB + FB1	LAB + AFB1 + FB1
IL-10	1.30 ± 0.06	1.31 ± 0.07	1.59 ± 0.01*	1.61 ± 0.02*	1.82 ± 0.09*	1.37 ± 0.07	1.33 ± 0.05	1.35 ± 0.06
IL-4	0.92 ± 0.03	0.90 ± 0.03	1.08 ± 0.04*	1.07 ± 0.04*	1.11 ± 0.06*	0.94 ± 0.05	0.91 ± 0.04	0.93 ± 0.05
IFN-γ	1.38 ± 0.08	1.40 ± 0.05	0.96 ± 0.07*	0.94 ± 0.03*	0.90 ± 0.08*	1.31 ± 0.05	1.33 ± 0.06	1.35 ± 0.06
TNF-α	1.32 ± 0.08	1.31 ± 0.03	1.00 ± 0.01*	0.98 ± 0.04*	0.93 ± 0.03*	1.30 ± 0.05	1.32 ± 0.04	1.30 ± 0.06

Balb/c mice were orally exposed daily (for 2 wk) to saline, to LAB (2 × 10⁹ CFU/L, ∼2 mg/kg), AFB₁ (80 µg/kg), FB₁ (100 µg/kg), mixture of AFB₁ + FB₁ made with respective same doses, AFB₁ + LAB, FB₁ + LAB, or and a mixture of AFB₁ + FB₁ + LAB.

Values shown are mean [±SD] ratios of the cytokine-specific RT-PCR product versus the corresponding cyclophilin band intensity (expressed in arbitrary units [AU]) from six determinations per experimental scenario tested.

*Value significantly differs from control, LAB alone, or corresponding co-treatment value (p < 0.05).

Table 4. Effect of treatments on splenic oxidative stress enzymes.

Parameter	Control	LAB	AFB1	FB1	AFB1 + FB1	LAB + AFB1	LAB + FB1	LAB + AFB1 + FB1
MDA (nmol/mg prot)	62.1 ± 1.6	61.5 ± 2.7	81.9 ± 5.1*	81.6 ± 5.2*	82.7 ± 6.9*	61.3 ± 6.7	61.4 ± 5.5	60.5 ± 6.6
SOD (U/mg prot)	104.1 ± 8.3	103.9 ± 9.3	68.1 ± 5.3*	70.2 ± 5.4*	59.9 ± 5.6*	101.4 ± 6.5	99.8 ± 6.04	100.9 ± 4.3
GSH (U/mg prot)	128.8 ± 7.8	129.4 ± 6.4	78.9 ± 5.1*	75.4 ± 5.3*	74.9 ± 3.8*	121.3 ± 7.5	129.3 ± 0.06	126.9 ± 7.6
T-AOC (U/mg prot)	27.8 ± 3.5	28.1 ± 0.03	18.5 ± 3.2*	19.8 ± 3.8*	16.3 ± 4.1*	26.9 ± 3.5	27.2 ± 4.4	26.3 ± 3.8

Balb/c mice were treated as outlined in footnotes to Table 3.

*Value significantly differs from control, LAB alone, or corresponding co-treatment value (p < 0.05).

Figure 2. Caspase-3 activity in splenocytes from mice after treatment. Mice treated as indicated in the legend for Figure 1. Cells were harvested from spleens isolated after hosts were fasted for 12 h (i.e. on day after the final treatment in each group). Data shown are mean ± SD. In each histogram, bars with (*) differ significantly to other treatments (p < 0.05).

Figure 3. Effect of LAB on AFB₁/FB₁ induced apoptotic DNA. Freshly-isolated thymocytes (1.5 × 10⁶) from each mouse treated as indicated in the legend for Figure 1. Propidium iodide fluorescence was measured using a flow cytometer with an FL-2 filter. Results are expressed in each histogram as the percentage of sub-G₁ population.