Evaluation of Acute Immunotoxicity of Aerosolized Aflatoxin B₁ in Female C57BL/6N Mice

Figures & data

TABLE 1 Experimental design

		Number of mice
Treatment day	Exposure group^1	Aerosol exposed	Untreated (Control)	Cyclophosphamide treated (Positive control)
1	0 ug AFB1/L	21	21	21
2	2.86 μg AFB1/L	21	21	21
3	6.59 μg AFB1/L	21	21	21
4	10.0 μg AFB1/L	21	21	21

¹Values indicate aerosol exposure concentration in μg AFB/L air. Each aerosol exposure was for 90 min

TABLE 2 Exposure concentration of AFB₁ aerosol

Exposure group	Estimated ethanol exposure concentration (mg/L)^1	Exposure duration (min)	AFB1 C × T (μg × min/L air)	Estimated amount of AFB1 Inhaled (μg)^2
0 μg AFB1/L	67.8	90	0	0
2.86 μg AFB1/L	61.4	90	257	5.1
6.59 μg AFB1/L	50.4	90	593	11.9
10.1 μg AFB1/L	58.2	90	900	18.0

¹Calculated based on the amount of ethanol used/volume of air passed through chamber.

²Calculated based on exposure concentration (μg/L) × estimated minute volume (0.02 L/min) × exposure duration (90 min).

TABLE 3 Summary of organ weights

	Liver weight (g)^1			Lung weight (g)^1
Exposure group	Untreated on same day	Aerosol exposed	Cyclophosphamide treated on same day	Untreated on same day	Aerosol exposed	Cyclophosphamide treated on same day
0 μg AFB1/L	1.06 ± 0.08	0.99 ± 0.10 (0.93) ^2	1.02 ± 0.10 (0.97)	0.152 ± 0.020	0.148 ± 0.017 (0.97)	0.1 77 ± 0.024 (1.17)
2.86 μg AFB1/L	1.10 ± 0.10	1.19 ± 0.08 (1.09)	1.14 ± 0.11 (1.04)	0.168 ± 0.041	0.179 ± 0.028 (1.06)	0.199 ± 0.036 (1.18)
6.59 μg AFB1/L	1.06 ± 0.13	1.08 ± 0.06 (1.02)	1.19 ± 0.12 (1.12)	0.155 ± 0.015	0.184 ± 0.042 (1.18)	0.198 ± 0.038 (1.27)
10.1 μg AFB1/L	1.08 ± 0.09	1.14 ± 0.10 (1.05)	1.17 ± 0.10 (1.08)	0.154 ± 0.014	0.154 ± 0.012 (1.00)	0.211 ± 0.053 (1.37)
	Thymus weight (g)^1			Spleen weight (g)^1
0 μg AFB1/L	0.078 ± 0.026	0.055 ± 0.017 (0.70)	0.025 ± 0.005 (0.32)	0.072 ± 0.017	0.057 ± 0.010 (0.78)	0.032 ± 0.006 (0.44)
2.86 μg AFB1/L	0.055 ± 0.016	0.043 ± 0.009 (0.77)	0.026 ± 0.005 (0.46)	0.061 ± 0.010	0.054 ± 0.006 (0.89)	0.032 ± 0.005 (0.53)
6.59 μg AFB1/L	0.069 ± 0.017	0.048 ± 0.011 (0.69)	0.028 ± 0.005 (0.41)	0.071 ± 0.009	0.062 ± 0.031 (0.87)	0.035 ± 0.007 (0.49)
10.1 μg AFB1/L	0.057 ± 0.013	0.047 ± 0.008 (0.82)	0.024 ± 0.005 (0.42)	0.068 ± 0.022	0.056 ± 0.006 (0.83)	0.030 ± 0.004 (0.45)

¹Values are mean ± standard deviation of 14 individual mice.

²Values in parentheses are the ratios of the organ weight of the aerosol or cyclophosphamide treated mice to the organ weight of the untreated mice from the same exposure day.

*Significantly different from untreated mice analyzed on the same day at p ≤ 0.05 using students 2-tailed t-test.

TABLE 4 Summary of organ/body weight ratios

	Liver weight (g)^1			Lung weight (g)^1
Exposure group	Untreated on same day	Aerosol exposed	Cyclophosphamide treated on same day	Untreated on same day	Aerosol exposed	Cyclophosphamide treated on same day
0 μg AFB1/L	0.054 ± 0.004	0.052 ± 0.003 (0.95)^b	0.054 ± 0.004 (0.99)	0.0078 ± 0.0011	0.0078 ± 0.0012 (1.00)	0.0094 ± 0.0012 (1.20)
2.86 μg AFB1/L	0.053 ± 0.004	0.057 ± 0.004 (1.08)	0.056 ± 0.005 (1.07)	0.008^a ± 0.002^a	0.0086 ± 0.0013 (1.06)	0.0098 ± 0.001 8 (1.21)
6.59 μg AFB1/L	0.053 ± 0.007	0.054 ± 0.003 (1.03)	0.060 ± 0.005 (1.13)	0.0078 ± 0.0008	0.0093 ± 0.0022 (1.20)	0.0099 ± 0.0018 (1.28)
10.1 μg AFB1/L	0.053 ± 0.004	0.056 ± 0.005 (1.07)	0.059 ± 0.004 (1.12)	0.0075 ± 0.0006	0.0077 ± 0.0006 (1.02)	0.0 106 ± 0.0026 (1.41)
	Thymus weight (g)^1			Spleen weight (g)^1
0 μg AFB1/L	0.0040 ± 0.0013	0.0028 ± 0.0008 (0.71)	rr 0.0013 ± 0.0003 (0.33)	0.0037 ± 0.0008	0.0030 ± 0.0005 (0.80)	0.0017 ± 0.0003 (0.46)
2.86 μg AFB1/L	0.003^a ± 0.001^a	0.0020 ± 0.0004 (0.77)	0.0013 ± 0.0003 (0.48)	0.0029 ± 0.0005	0.0026 ± 0.0003 (0.88)	0.0016 ± 0.0003 (0.55)
6.59 μg AFB1/L	0.0035 ± 0.0009	0.0024 ± 0.0006 (0.70)	0.0014 ± 0.0003 (0.41)	0.0035 ± 0.0005	0.0031 ± 0.0017 (0.88)	0.0017 ± 0.0003 (0.49)
10.1 μg AFB1/L	0.0028 ± 0.0006	0.0023 ± 0.0004 (0.83)	0.0012 ± 0.0002 (0.43)	0.0033 ± 0.00 10	0.0028 ± 0.0003 (0.84)	0.001 5 ± 0.0002 (0.46)

^aValues are mean ± standard deviation of 14 individual mice.

^bValues in parentheses are the ratios of the liver/body weight ratio of the aerosol or cyclophosphamide treated mice to the liver/body weight ratio of the untreated mice from the same exposure day.

*Significantly different from untreated mice analyzed on the same day at p ≤ 0.05 using students 2-tailed t-test.

TABLE 5 White blood cell count

	White Blood Cell Count (× 10^3/μL)
Exposure group	Untreated on same day	Aerosol exposed	Cyclophosphamide treated on same day
0 μg AFB1/L	5.97 ± 1.50	3.48 ± 0.78	0.69 ± 0.25
2.86 μg AFB1/L	6.59 ± 1.37	4.43 ± 1.48	1.23 ± 0.57
6.59 μg AFB1/L	6.31 ± 1.76	5.90 ± 1.69	1.17 ± 0.59
10.0 μg AFB1/L	7.41 ± 2.06	6.25 ± 1.90	0.77 ± 0.37

Values are the mean ± standard deviation.

* Significantly different from untreated mice analyzed on the same day at p ≤ 0.05 using students 2-tailed t-test

FIG. 1 Splenocytes were isolated from mouse spleen three days following exposure to aerosol containing 0 μ g AFB₁/L (aerosolized vehicle, ethanol; A), 2.86 μ g AFB₁/L (B), 6.59 μ g AFB₁/L (C) or 10.0 μ g AFB₁/L (D). The quantities of different lymphocyte subpopulations for aerosol-exposed animals is shown by the solid bar. Lymphocyte subpopulations of splenocytes isolated from untreated mice and cyclophosphamide-treated mice on the same day are illustrated by the open and dotted bars, respectively. Antigenic markers were used to identify and quantitate T-cells (CD3⁺), helper T-cells (CD3⁺/CD4⁺), cytotoxic T-cells (CD3⁺/CD8⁺), B cells (CD19⁺) and natural killer cells (NK1.1⁺) from the isolated splenocytes by fluorescent-activated cell sorting (FACS) as described in the Methods. An asterisk indicates that the value is significantly different from the untreated mice (open bar) using a Student's 2-tailed t-test at p ≤ 0.05.

TABLE 6 Effect of AFB₁ on CD4⁺/CD8⁺ ratio of mouse splenocytes

	CD4^+/CD8^+ Ratio^1
Exposure group	Untreated on same day	Aerosol exposed	Cyclophosphamide treated on same day
0 μg AFB1/L	1.60 ± 0.08	1.53 ± 0.11	1.20 ± 0.14
2.86 μg AFB1/L	1.42 ± 0.24	1.28 ± 0.11	1.14 ± 0.08
6.59 μg AFB1/L	1.79 ± 0.17	1.59 ± 0.15	1.38 ± 0.17
10.0 μg AFB1/L	1.51 ± 0.11	1.67 ± 0.15	1.36 ± 0.09

Values are mean ± standard deviation.

*Significantly different from untreated mice analyzed on the same day at p ≤ 0.05 using students 2-tailed t-test.

TABLE 7 IgM response to keyhole limpet hemocyanin (KLH)

	Serum Anti-KLH IgM (ng/mL)^a
Exposure group	Untreated on same day	Aerosol exposed	Cyclophosphamide treated on same day
0 μ AFB1/L	846 ± 563	761 ± 347 (90.0%)^b	366 ± 138 (43.2%)
2.86 μ AFB1/L	1960 ± 2010	1908 ± 672 (97.3%)	1155 ± 1525^c (58.9%)
6.59 μ AFB1/L	1016 ± 606	1281 ± 891 (126.1%)	431 ± 453 (42.4%)
10.0 μ AFB1/L	1325 ± 225	1299 ± 382 (98.1%)	274 ± 111 (20.7%)

^aValues are mean ± standard deviation of seven mice.

^bValues in parenthesis indicate the percent of the untreated value on the same day.

^cThe large variation was due to variability in individual values, which were 211, 284, 285, 346, 421, 2429, and 4109 ng/mL.

* Significantly different from untreated mice analyzed on the same day at p ≤ 0.05 using students 2-tailed t-test.