Vinclozolin Modulates Splenic Natural Killer Cell Activity, Antibody-Forming Cell Response and Phenotypic Marker Expression in Sprague Dawley Rats: A Two-Generation Feeding Study

Figures & data

Effect of vinclozolin on the body and organ weights of Sprague Dawley rats

			Spleen weight		Thymus weight
Vinclozolin (ppm)		Body weight (g)	mg	% Body	mg	% Body
F0	Control	297.9 ± 5.6	561 ± 21	0.189 ± 0.008	397 ± 33	0.133 ± 0.012
	10	305.8 ± 8.5	571 ± 18	0.189 ± 0.008	354 ± 23	0.116 ± 0.007
	150	301.1 ± 3.4	597 ± 19	0.198 ± 0.007	355 ± 15	0.118 ± 0.005
	750	286.8 ± 5.5	608 ± 34	0.212 ± 0.009	400 ± 23	0.140 ± 0.007
F1M	Control	289.2 ± 10.0	630 ± 26	0.219 ± 0.009	649 ± 39	0.223 ± 0.009
	10	297.2 ± 6.2	657 ± 19	0.223 ± 0.005	612 ± 16	0.208 ± 0.005
	150	302.6 ± 5.6	649 ± 35	0.213 ± 0.013	597 ± 29	0.198 ± 0.010
	750	278.3 ± 11.4	674 ± 25	0.244 ± 0.011	608 ± 23	0.220 ± 0.009
F1F	Control	199.6 ± 4.7	515 ± 23	0.261 ± 0.009	576 ± 25	0.293 ± 0.009
	10	192.3 ± 5.5	455 ± 20	0.243 ± 0.011	575 ± 31	0.308 ± 0.010
	150	191.0 ± 5.1	477 ± 17	0.253 ± 0.013	581 ± 25	0.314 ± 0.020
	750	189.6 ± 3.9	437 ± 17	0.234 ± 0.009	521 ± 19	0.283 ± 0.019

Note. Animals were exposed in feed to casein NIH-31C (as a control) or vinclozolin as described. At the end of exposure, the animals were sacrificed; spleen and thymus were removed and weighed. Values represent the mean ± SE derived from ten animals. All the data were homogeneous.

*, p ≤ 0.05. F₁M = F₁ male rats; F₁F = F₁ female rats.

Effect of vinclozolin on the number and percentage of splenocyte subpopulations in F₀ female Sprague Dawley rats

			Vinclozolin (ppm)
Values	Splenocyte subsets	Control	10	150	750	H/NH
Absolute	B Cell (OX-33^+)	271.3 ± 18.8	308.5 ± 13.0	304.1 ± 19.9	344.7 ± 10.8	H
(×10^6)	T Cell (OX-19^+)	203.8 ± 16.2	206.8 ± 10.6	222.5± 11.3	261.1 ± 12.0	H
	TH (OX-38^+OX-19^+)	115.0 ± 9.2	113.1 ± 5.3	124.5 ± 6.2	144.4 ± 6.7	H
	TC (OX-8^+OX-19^+)	77.5 ± 5.6	85.2 ± 6.3	87.3 ± 5.6	105.1 ± 4.9	H
	NK (NKR-P1A^+ OX-8^+)	20.2 ± 1.0	23.6 ± 1.6	22.3 ± 1.3	20.4 ± 1.2	H
	Mϕ (HIS36^+)	10.3 ± 1.0	8.9 ± 0.7	11.2 ± 0.9	12.4 ± 1.0	H
Percent	B Cell (OX-33^+)	47.4 ± 1.1	49.5 ± 1.3	47.1 ± 1.2	47.3 ± 1.1	H
	T Cell (OX-19^+)	35.5 ± 1.5	33.1 ± 1.2	34.8 ± 1.0	35.7 ± 1.1	H
	TH (OX-38^+OX-19^+)	20.0 ± 0.9	18.1 ± 0.6	19.4 ± 0.5	19.7 ± 0.5	H
	TC (OX-8^+OX-19^+)	13.5 ± 0.5	13.6 ± 0.8	13.6 ± 0.6	14.4 ± 0.6	H
	NK (NKR-P1A^+ OX-8^+)	3.6± 0.3	3.8 ± 0.3	3.5 ± 0.2	2.8 ± 0.1	H
	Mϕ (HIS36^+)	1.9 ± 0.3	1.5 ± 0.2	1.8 ± 0.2	1.7 ± 0.1	H
	Spleen cell number (×10^7)	57.1 ± 3.3	62.3 ± 1.9	64.2 ± 3.2	73.0 ± 2.1	H

Note. Splenocytes were labeled with appropriate fluorochrome-conjugated antibodies and analyze using flow cytometric analysis. Irrelevant isotype matched antibodies were used as a control. Values representing the mean ± SE derived from ten animals are expressed as the absolute cell number per spleen (× 10⁶) and percentage of total spleen cells. T Cells, CD5⁺; T Helper cells (T_H), CD4⁺CD5⁺; Cytotoxic T Cell (T_C), CD8⁺CD5⁺; B cell, CD45RA or A/B⁺; NK Cell, NKR-P1A⁺CD8⁺; Macrophages (Mϕ), HIS36⁺. H = homogeneous; NH = non-homogeneous.

*, p ≤ 0.05.

1 Effect of vinclozolin on IgM antibody-forming cell (AFC) response in F₀ (A, D), and F₁ male (B, E) and female (C, F) rats. Rats were exposed to vinclozolin, and the number of IgM antibody-forming cells to sRBC in splenocytes determined as described. Data are presented as AFC/10⁶ cells (the specific activity) and AFC/Spleen. All data are homogeneous except for the AFC/Spleen in F₁ generation male rats. Values represent the mean ± SE derived from ten animals. *, p ≤ 0.05.

2 Effect of vinclozolin on NK cell activity in F₀ (A), and F₁ male (B) and female (C) rats. Rats were exposed to vinclozolin, and the activity of NK cells determined as described. All the data in F₀ are homogeneous except for those at the E:T ratios of 25:1 and 50:1. The data in F₁ male rats are homogeneous at the E:T ratios of 12.5:1, 25:1 and 200:1, and non-homogeneous at the E:T ratios of 6.25:1, 50:1 and 100:1. All the data in F₁ female rats are homogeneous. Values represent the mean ± SE derived from ten animals. *, p ≤ 0.05.

Effect of vinclozolin on the number and percentage of splenocyte subpopulations in F₁ generation male Sprague Dawley rats

			Vinclozolin (ppm)
Values	Splenocyte subsets	Control	10	150	750	H/NH
Absolute	B Cell (OX-33^+)	327.2 ± 26.0	335.6 ± 22.2	326.7 ± 40.3	309.6 ± 31.7	H
(×10^6)	T Cell (OX-19^+)	210.6 ± 9.7	196.6 ± 11.9	222.2 ± 12.7	204.6 ± 11.8	H
	TH (OX-38^+OX-19^+)	116.9 ± 5.8	110.2 ± 6.8	128.5 ± 8.0	109.5 ± 6.4	H
	TC (OX-8^+OX-19^+)	78.6 ± 4.1	71.2 ± 4.4	81.7 ± 5.5	77.8 ± 4.4	H
	NK (NKR-P1A^+ OX-8^+)	18.6 ± 1.1	23.3 ± 1.3	21.0 ± 1.4	19.8 ± 0.8	H
	Mϕ (HIS36^+)	17.1 ± 1.2	13.0 ± 1.0	14.0 ± 1.8	15.6 ± 1.2	H
Percent	B Cell (OX-33^+)	50.3 ± 1.4	52.3 ± 0.8	52.7 ± 1.1	53.6 ± 0.8	H
	T Cell (OX-19^+)	33.1 ± 1.2	30.8 ± 0.6	32.2 ± 1.0	31.1 ± 0.9	H
	TH (OX-38^+OX-19^+)	18.3 ± 0.6	17.3 ± 0.4	18.6 ± 0.8	16.7 ± 0.6	H
	TC (OX-8^+OX-19^+)	12.3 ± 0.4	11.2 ± 0.3	11.7 ± 0.3	11.8 ± 0.3	H
	NK (NKR-P1A^+ OX-8^+)	2.9 ± 0.1	3.7 ± 0.3	3.1 ± 0.2	3.1 ± 0.1	NH
	Mϕ (HIS36^+)	2.7 ± 0.2	2.1 ± 0.2	2.1 ± 0.3	2.4 ± 0.1	H
	Spleen cell number (× 10^7)	64.5 ± 3.9	63.9 ± 3.6	69.8 ± 4.6	65.7 ± 3.3	H

Note. Splenocytes were labeled with appropriate fluorochrome-conjugated antibodies and analyze using flow cytometric analysis. Irrelevant isotype matched antibodies were used as a control. Values representing the mean ± SE derived from ten animals are expressed as the absolute cell number per spleen (×10⁶) and percentage of total spleen cells. T Cells, CD5⁺; T Helper cells (T_H), CD4⁺CD5⁺; Cytotoxic T Cell (T_C), CD8⁺CD5⁺; B cell, CD45RA or A/B⁺; NK Cell, NKR-P1A⁺CD8⁺; Macrophages (Mϕ), HIS36⁺.

*, p ≤ 0.05. H = homogeneous; NH = non-homogeneous.

Effect of vinclozolin on the number and percentage of splenocyte subpopulations in F₁ generation female Sprague Dawley rats

			Vinclozolin (ppm)
Values	Splenocyte subsets	Control	10	150	750	H/NH
Absolute	B Cell (OX-33^+)	269.8 ± 13.9	233.9 ± 19.0	251.8 ± 15.8	282.9 ± 17.0	H
(×10^6)	T Cell (OX-19^+)	158.2 ± 8.2	140.1 ± 13.0	133.2 ± 7.1	157.7 ± 7.6	H
	TH (OX-38^+OX-19^+)	83.5 ± 3.0	76.7 ± 6.3	74.4 ± 3.8	83.9 ± 4.1	H
	TC (OX-8^+OX-19^+)	67.3 ± 3.6	54.0 ± 6.0	49.7 ± 2.8	66.1 ± 4.4	H
	NK (NKR-P1A^+ OX-8^+)	11.7 ± 0.6	13.4 ± 1.0	12.2 ± 1.2	10.8 ± 0.8	H
	Mϕ (HIS36^+)	7.6 ± 0.5	8.1 ± 0.8	8.1 ± 1.2	7.6 ± 0.8	H
Percent	B Cell (OX-33^+)	54.0 ± 0.9	53.4 ± 0.5	56.2 ± 0.8	56.1 ± 0.5	H
	T Cell (OX-19^+)	32.3 ± 0.7	31.9 ± 0.8	29.9 ± 0.7	31.4 ± 0.5	H
	TH (OX-38^+OX-19^+)	16.9 ± 0.5	17.6 ± 0.5	16.7 ± 0.4	16.8 ± 0.3	H
	TC (OX-8^+OX-19^+)	13.5 ± 0.4	12.2 ± 0.5	11.2 ± 0.4	13.1 ± 0.4	H
	NK (NKR-P1A^+ OX-8^+)	2.4 ± 0.1	3.1 ± 0.1	2.7 ± 0.2	2.2 ± 0.1	H
	M ϕ (HIS36^+)	1.5 ± 0.1	1.9 ± 0.1	1.9 ± 0.2	1.5 ± 0.1	H
	Spleen cell number (× 10^7)	49.9 ± 2.1	43.8 ± 3.6	44.7 ± 2.6	50.4 ± 2.8	H

Note. Splenocytes were labeled with appropriate fluorochrome-conjugated antibodies and analyze using flow cytometric analysis. Irrelevant isotype matched antibodies were used as a control. Values representing the mean ± SE derived from ten animals are expressed as the absolute cell number per spleen (×10⁶) and percentage of total spleen cells. T Cells, CD5⁺; T Helper cells (T_H), CD4⁺CD5⁺; Cytotoxic T Cell (T_C), CD8⁺CD5⁺; B cell, CD45RA or A/B⁺; NK Cell, NKR-P1A⁺CD8⁺; Macrophages (Mϕ), HIS36⁺.

*, p ≤ 0.05. H = homogeneous; NH = non-homogeneous.