The soluble nickel component of residual oil fly ash alters pulmonary host defense in rats

Figures & data

Table 1.  Concentrations for individual metals and metal combinations (μg/rat).

Metal	Treatment Groups
	Saline	Fe	Ni	Al	Zn	Mix, No Fe	Mix, No Ni	Mix, No Al	Mix, No Zn	Total Mixture
Soluble Fe	0	37.2μg	0	0	0	0	37.2μg	37.2μg	37.2μg	37.2μg
Soluble Ni	0	0	55.7μg	0	0	55.7μg	0	55.7μg	55.7μg	55.7μg
Soluble Al	0	0	0	46.6μg	0	46.6μg	46.6μg	0	46.6μg	46.6μg
Soluble Zn	0	0	0	0	8.69μg	8.69μg	8.69μg	8.69μg	0	8.69μg

Figure 1.  Percent (%) change in body weight post-infection of rats that were pre-exposed to individual soluble metals (A) or to soluble metal mixtures (B) 3 days prior to intratracheal inoculation with L. monocytogenes. Values are means ± SE (p < 0.05). Figure 1A: *significantly different from all groups; ^$significantly different from Soluble Fe and Saline groups. Figure 1B: ^asignificantly different from Saline and Mix - No Ni groups; ^bsignificantly different from Saline group; ^csignificantly different from Mix - No Ni group; ^dsignificantly different from Saline, Mix - No Ni, and Mix - No Zn groups.

Table 2.  Bacterial clearance after pre-exposure to individual metals.

Treatment	CFUs LOG^10 Units
	Day 3 Initial Infection	Day 6	Day 8	Day 10
Saline	4.7	5.9 ± 0.10	4.3 ± 0.38	2.2 ± 0.73
Soluble Fe	4.7	5.6 ± 0.03	4.1 ± 0.12	3.1 ± 0.16
Soluble Ni	4.7	7.3 ± 0.05 ^+	5.7 ± 0.63	3.2 ± 0.22
Soluble Al	4.7	5.9 ± 0.07	4.3 ± 0.26	2.2 ± 0.80
Soluble Zn	4.7	6.1 ± 0.12	4.4 ± 0.34	2.4 ± 0.84
Total Mixture	4.7	7.4 ± 0.17 ^+	4.4 ± 0.21	2.2 ± 0.75

Number of bacterial CFUs in the left lungs of rats that were pre-exposed to individual soluble metals, the metal mixture, or saline 3 days prior to intratracheal inoculation with L. monocytogenes. Values are means ± SE, ⁺significantly different from Saline, Soluble Fe, Soluble Al, and Soluble Zn groups (p < 0.05).

Table 3.  Bacterial clearance after pre-exposure to metal mixtures.

Treatment	CFUs LOG^10 Units
	Day 3 Initial Infection	Day 6	Day 8	Day 10
	4.8 ± 0.01	6.1 ± 0.09	4.3 ± 0.31	3.0 ± 0.18
Mix - No Fe	4.8 ± 0.01	7.2 ± 0.14^a	4.7 ± 0.20	3.1 ± 0.17
Mix - No Ni	4.8 ± 0.01	6.1 ± 0.08	4.0 ± 0.14	2.2 ± 0.52
Mix - No Al	4.8 ± 0.01	7.2 ± 0.08^a	5.3 ± 0.48	3.2 ± 0.59
Mix - No Zn	4.8 ± 0.01	7.1 ± 0.09^a	4.8 ± 0.30	3.0 ± 0.13
Total Mixture	4.8 ± 0.01	6.9 ± 0.19^a	4.3 ± 0.16	3.3 ± 0.23

Number of bacterial CFUs in the left lungs of rats that were pre-exposed to soluble metal mixtures, the total metal mixture, or saline 3 days prior to intratracheal inoculation with L. monocytogenes. Values are means ± SE; ^asignificantly different from Saline and Mix - No Ni groups (p < 0.05).

Figure 2.  Lactate dehydrogenase (LDH) (A and B) and albumin (C and D) in the BAL fluid of rats that were pre-exposed to individual soluble metals or to soluble metal mixtures 3 days prior to intratracheal inoculation with L. monocytogenes. Values are means ± SE (p < 0.05). Figures 2A and 2C: *significantly different from all groups; ⁺significantly different from Saline, Soluble Fe, Soluble Al, and Soluble Zn groups. Figures 2B and 2D:^asignificantly different from Saline and Mix - No Ni groups (p < 0.05).

Figure 3.  Totals AM (A and B) and total PMN (C and D) present in the BAL fluid of rats that were pre-exposed to individual soluble metals or to soluble metal mixtures 3 days prior to intratracheal inoculation with L. monocytogenes. Values are means ± SE (p < 0.05). Figures 3A and 3C: ⁺significantly different from Saline, Soluble Fe, Soluble Al, and Soluble Zn groups; ^#significantly different from Saline group; ^$significantly different from Soluble Fe and Saline groups; ^&significantly different from Saline, Soluble Fe, and Soluble Al groups. Figures 3B and 3D: ^asignificantly different from Saline and Mix - No Ni groups; ^csignificantly different from Mix - No Ni group; ^esignificantly different from Saline, Mix – No Fe, and Mix – No Ni groups.

Table 4.  BAL cell phenotype on Day 10 after pre-exposure to individual metals as determined by flow cytometry (3 10⁴ Cells).

Treatment	NK Cells	B-Cells	Total T-Cells	CD4^+ T-Cells	CD8^+ T-Cells
Saline	7.95 ± 3.30	8.62 ± 1.98	34.7 ± 10.3	21.7 ± 5.97	13.0 ± 4.38
Soluble Fe	5.23 ± 1.67	12.9 ± 4.04	35.7 ± 14.1	19.0 ± 7.49	16.6 ± 7.45
Soluble Ni	5.66 ± 2.28	19.6 ± 3.61	191 ± 7.30 ^*	85.6 ± 20.5 ^*	105 ± 53.1 ^*
Soluble Al	3.69 ± 0.590	10.1 ± 3.47	31.0 ± 2.68	19.0 ± 1.93	12.0 ± 0.90
Soluble Zn	3.88 ± 1.03	6.93 ± 0.450	25.5 ± 3.76	16.8 ± 2.95	8.69 ± 1.07
Total Mixture	1.62 ± 0.049	9.83 ± 5.47	37.3 ± 18.6	23.1 ± 13.0	14.2 ± 5.63

Note: Values are means ± SE, ^*significantly different from all groups (p < 0.05).

Table 5.  BAL cell phenotype on Day 10 after pre-exposure to metal combinations as determined by flow cytometry (3 10⁴ Cells).

Treatment	NK Cells	B-Cells	Total T-Cells	CD4^+ T-Cells	CD8^+ T-Cells
Saline	7.29 ± 1.12	9.50 ± 1.36	65.4 ± 12.8	41.0 ± 9.90	24.4 ± 4.11
Mix – No Fe	9.21 ± 2.20	20.1 ± 2.31^a	110 ± 15.0^a	57.5 ± 9.07	52.9 ± 8.93
Mix – No Ni	6.01 ± 0.815	8.06 ± 0.975	49.0 ± 9.68	27.3 ± 5.72	21.7 ± 6.28
Mix – No Al	8.06 ± 1.68	21.2 ± 3.53^a	161 ± 52.6^a	81.8 ± 21.6^a	78.7 ± 32.0
Mix – No Zn	9.12 ± 1.83	21.1 ± 4.37^a	123 ± 22.3^a	77.8 ± 12.6^a	45.2 ± 9.91
Total Mixture	4.99 ± 1.17	18.8 ± 2.74^a	119 ± 27.5^a	65.6 ± 23.8	52.9 ± 13.0

Note: Values are means ± SE, ^asignificantly different from Saline and Mix – No Ni groups (p < 0.05).

Figure 4.  Nitrate and nitrite (NO_x) in the BAL fluid (BAL) of rats that were pre-exposed to individual soluble metals (A) or to soluble metal mixtures (B) 3 days prior to intratracheal inoculation with L. monocytogenes. Values are means ± SE (p < 0.05). Figure 4A: *significantly different from all groups; ⁺significantly different from Saline, Soluble Fe, Soluble Al, and Soluble Zn groups. Figure 4B: ^asignificantly different from Saline and Mix - No Ni groups; ^csignificantly different from Mix - No Ni group.

Figure 5.  BAL cellular chemiluminescence (CL) depicted in total counts per 15 min for total BAL AM after stimulation with zymosan in rats that were pre-exposed to individual soluble metals (A) or to soluble metal mixtures (B) 3 days prior to intratracheal inoculation with L. monocytogenes. Values are means ± SE (p < 0.05). Figure 5A: *significantly different from all groups; ⁺significantly different from Saline, Soluble Fe, Soluble Al, and Soluble Zn groups. Figure 5B: ^asignificantly different from Saline and Mix - No Ni groups; ^bsignificantly different from Saline group; ^csignificantly different from Mix - No Ni group; ^fsignificantly different from all groups (p < 0.05).

Figure 6.  Cytokines involved in inflammation and the adaptive immune responses measured in the BAL fluid 3 days after intratracheal instillation with individual soluble metals (A) or soluble metal mixtures (B). Cytokines measured in the BAL fluid on Day 6 (3 days post-infection with L. monocytogenes) after intratracheal instillation with soluble metals (C) or soluble metal mixtures (D). Values are means ± SE (p < 0.05). Figures 6A and 6C: *significantly different from all groups; ⁺significantly different from Saline, Soluble Fe, Soluble Al, and Soluble Zn groups. Figures 6B and 6D: ^asignificantly different from Saline and Mix - No Ni groups; ^bsignificantly different from Saline group; ^csignificantly different from Mix - No Ni group; ^esignificantly different from Saline, Mix – No Fe, and Mix – No Ni groups; ^gsignificantly different from Saline, Mix - No Fe, and Mix - No Al groups.