MATERIALS AND METHODS
B. Markers with 250 nm ZnO dose-response
4.2 UPLC-TQMS analysis on systemic effect of ZnO on rat PC profiling
Further UPLC-TQMS analysis focusing on glycerophosphocholine structure and semi-quantifications can strength our findings from UPLC- TQMS results. Metabolites selected from UPLC-Qtof/MS need to be confirmed their structure either by comparing the LC-MS results of standards or by using Qtof/MS.
According to UPLC- TQMS lipids screening results, glycrophosphocholine take the most proportion than other lipids species, furthermore, PC take even more proportion than lyso PC. And the potential ZnO particle exposure biomarkers of 35 nm
31
and 250 nm analysis results from UPLC- TQMS all are PCs. Moreover, TQMS provides more ideal quantification ability compared with Qtof/MS.
Systemic effect triggered by ZnO exposure has been studied in previous studies.
Gordon T. et al have reported that ZnO NPs could induce lung inflammation in animals.
Human studies also found that inhalation of ZnO particles can cause metal fume fever, a flu-like symptom such as myalgias, cough, fatigue, and induce lung and systemic inflammation responses. But there are few studies that demonstrate interaction between lipids and ZnO particle exposure.
In this study,PC (31:0), PC (18:0/ 18:0) and PC(P-20:5/18:0) are commonly suggested metabolites from PC profiling OPLS-DA results of 35 nm treatments; and fold-changes with regulation was observed in PC(P-20:5/18:0). PC (42:6), PC(37:1)and PC(18:0/18:0) ( m/z 790.78) are lipids suggested based on OPLS-DA results of 3 various dosed of 250 nm ZnO exposure treatments. PCs and other glycerophospholipids are the major structural lipids in eukaryotic membranes. PC has been pointed out own over 50% of the phospholipids in most eukaryotic membranes (Van et al., 2008). In addition to serve as a primary component of cellular membranes and binding sites for intracellular and intercellular proteins, some glycerophospholipids in eukaryotic cells are either precursors of, or are themselves, membrane-derived second messengers. PC is storage form for choline within cytosol. Choline ubiquitously distributes in all cells, mostly in the form of the, lysophosphatidylcholine, choline plasmalogens, and sphingomyeline essential components of all membranes (Zeisel et al., 1990). Rohlfs et al. have discovered the interaction with enzymes (phospholipases A1, phospholipases A2, PC desterase were discovered to play a crucial role in cellular systems (Zeisel et al., 1996; Kanfer et al., 1988). Gland, such as mammary gland has the ability to synthesis
32
and secrete PC (Rohlfs et al., 1993). Systemic effects such as chronic inflammation, atherosclerosis, aging and cancer are related to hypochlorous acid-mediation generated of PC from fatty acyl residues of phospholipids (Lessig et al., 2007). PC up-regulation was reported to induces membrane rupture, nuclear expansion, cell lysis, and enhance intracellular Ca+ level, furthermore, the production of ROS results in cytotoxic injuries and necrosis (Zhou et al., 2006). All the advances prove that the perturbations of PC are important when conduct systemic effects. Platelet-activating factor (PAF), lipid mediator has been implicated in cutaneous inflammation. Findings in literature have proved PC species can activate PAF agonistic activity (Travers et al., 1998). PC species is the product of phospholipase-B mediated de-acylation of phosphatidylcholine and phosphatidylinositol. And studies have evaluated the toxicity of ZnO nano-particles on normal primary human cells and their potential immune-modulatory effects. ZnO nanoparticles trigger and induce the production of the pro-inflammatory cytokines, interferon (IFN)- c , TNF-alpha, and interleukin-12, at concentrations below causing cell death. Consequently, concentration variation of PCs represents systemic effects turbulence occurs in organism (Almaguer et al., 2006) and have potential relationship with ZnO nano-particle exposure (Hanley et al., 2009). However, Oberdorster G. et al.
summarized the effects on the body of inhalation nano-scaled ultrafine particle, which that translocate particles then can induce various damages in different parts of the body.
This study takes a leading role in conducting ZnO particle exposure using lipidomics research approach that has proved PCs demonstrate dose-response potential biomarkers.
33
34
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40
Figures
41
42
1 Fatty Acyls 2 Glycerolipids 3 Glycerophospholipids 4 Sphingolipids
5 Sterol Lipids 6 Prenol Lipids 7 Saccharolipids 8 Polyketides
Figure 1. Primary categories of lipid.
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44
Figure 2 Schematic representation of the nanoparticle generation and exposure system
Figure 3 PCA scores plot from UPLC-QtofMS spectra of rats’ serum samples from various doses of 35 nm ZnO treatment. Each point on PCA scores plot represents one sample run.
t[1] (23%)
t[2] (13%)
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Figure 4 OPLS-DA score plot (high dose/ high dose control) generated from UPLC-Qtof/MS spectra of high dose 35 nm ZnO exposure group. (Q2=0.68)
46
Figure 5 OPLS-DA score plot (moderate dose/ moderate dose control) generated from UPLC-Qtof/MS spectra of moderate dose 35 nm ZnO exposure group. (Q2=0.73)
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Figure 6 OPLS-DA score plot (low dose/ low dose control) generated from UPLC-Qtof/MS spectra of low dose 35 nm ZnO exposure group. (Q2=0.80)
48
Figure 7 UPLC-QtofMS analysis result. 16 common lipids selected from (high-, moderate- and dose/ high-, moderate- and low-dose control) OPLS-DA top 50 VIP in 35 nm ZnO exposure groups.
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Figure 8 PCA scores plot from UPLC-Qtof/MS spectra of rats’ serum samples from various doses of 250 nm ZnO treatment. Each point on PCA scores plot represents one sample run.
50
Figure 9 OPLS-DA score plot (high dose/ high dose control) generated from UPLC-Qtof/MS spectra of high dose 250 nm ZnO exposure group (Q2=0.69).
51
Figure 10 OPLS-DA score plot (moderate dose/control) generated from UPLC-Qtof/MS spectra of moderate dose 250 nm ZnO exposure group. (Q2=0.62)
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Figure 11 OPLS-DA score plot (low dose/ low dose control) generated from UPLC-Qtof/MS spectra of low dose 250 nm ZnO exposure group. (Q2=0.83)
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Figure 12 UPLC-Qtof/MS analysis result. 14 common lipids selected from (high-, moderate- and low-dose/ high-, moderate- and low-dose control) OPLS-DA top 50 VIP in 250 nm ZnO exposure groups.
54
Figure 13 PCA scores plot from UPLC-TQMS spectra of rats’ serum samples from various doses of 35 nm ZnO treatment. Each point on PCA scores plot represents one sample run. (Q2=0.09)
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Figure 14 OPLS-DA score plot (high dose/ high dose control) generated from UPLC-TQMS spectra of high dose 35 nm ZnO exposure group. (Q2=0.32)
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Figure 15 OPLS-DA score plot (moderate dose/ moderate dose control) generated from UPLC-TQMS spectra of moderate dose 35 nm ZnO exposure group. (Q2=0.32)
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Figure 16 OPLS-DA score plot (low dose/ low dose control) generated from UPLC-TQMS spectra of low dose 35 nm ZnO exposure group. (Q2=0.31)
58
.
Figure 17 UPLC-TQMS analysis result. 4 common lipids selected from (high-, moderate- and dose/ high-, moderate- and low-dose control) OPLS-DA top 50 VIP in 35 nm ZnO exposure groups.
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Figure 18 Collision-induced fragmentation spectra of the [M+Na]+ PC (31:0) molecular ion was detected by UPLC-TQMS.
60
Figure 19 Collision-induced fragmentation spectra of the [M+Na]+ m/z PC(36:4) molecular ion was detected by UPLC-TQMS.
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Figure 20 PCA scores plot from UPLC-TQMS spectra of rats’ serum samples from various doses of 250 nm ZnO treatment. Each point on PCA scores plot represents one sample run.
62
Figure 21 OPLS-DA score plot (high dose/ high dose control) generated from UPLC-TQMS spectra of low dose 250 nm ZnO exposure group.
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Figure 22 OPLS-DA score plot (moderate dose/control) generated from UPLC-TQMS spectra of high dose 250 nm ZnO exposure group.
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Figure 23 OPLS-DA score plot (low dose/ low dose control) generated from UPLC-TQMS spectra of high dose 250 nm ZnO exposure group.
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Figure 24 UPLC-TQMS analysis result. 3 common lipids selected from (high-, moderate- and dose/ high-, moderate- and low-dose control) OPLS-DA top 50 VIP in 250 nm ZnO exposure groups.
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Figure 25 Collision-induced fragmentation spectra of the [M+Na]+ PC(37:1) molecular ion was detected by UPLC-TQMS.
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Figure 26 PCA scores plot from UPLC-Qtof/MS spectra of rats’ serum samples from various doses of 250 nm ZnO and 35 nm treatment. Each point on PCA scores plot represents one sample run.
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Figure 27 Total ion current (TIC) chromatogram of UPLC-Qtof/MS separation of rat’s serum sample after extraction.
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Figure 28 Total ion current (TIC) chromatogram of UPLC-TQMS separation of rat’s serum sample after extraction.
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TABLES
71 Table 1 Operation parameters in ZnO exposure experiment.
Low dose Moderate dose High dose 35 nm
Furnace temp. (°C) 5775 5775 5775 N2 (L/min) 0.75 1 1 Reacting air (L/min) 7.5 10 10
Diluting air (L/min) 120 150 50 250 nm
Furnace temp. (°C) 650 650 650
N2 (L/min) 0.2 0.3 0.7 Reacting air (L/min) 2 3 7
Diluting air (L/min) 120 100 50
72 Table 2 Average exposure condition in each experiment.
Low dose Moderate dose High dose 35 nm
Diameter ( nm) (Count Geo. Mean) 37.5 37.9 35.6 Geo. Std. dev. 2 1.9 2 Estimated surface area conc. (mm2/m3) 1.70E+04 2.50E+04 1.00E+04
Number conc. (#/cm3) 1.50E+04 2.10E+04 7.90E+04 Mass conc. (mg/m3) 2.4 3.7 12.1
250 nm
Diameter ( nm) (Count Geo. Mean) 262 242.4 250 Geo. Std. dev. 1.7 1.6 1.6 Estimated surface area conc. (mm2/m3) 2.00E+04 4.20E+04 1.20E+04
Number conc. (#/cm3) 6.20E+04 1.50E+04 4.00E+04 Mass conc. (mg/m3) 7.2 11.5 45.2
73
Table 3 ZnO particle exposure date, number of SD rat used and sample number for MS analysis of each group.
ZnO particle exposure experiment Exposure date # of SD rat # of sample taken for MS analysis
74
Table 4 223 markers selected from the results of PCA from the analysis of UPLC-Qtof/MS spectra of serum of rats exposed to 35 nm ZnO.
75
76
77
78
79
80
81
82
83
84
85
86
PC(18:0/22:5) 5.81 836.62
PC(18:0/22:4) 6.00 838.63
PC(18:0/22:4) 6.23 838.64
PC(20:4/22:6) 4.31 854.57
PC(20:3/22:6) 5.32 856.59
unknown 3.48 876.56
unknown 3.63 876.57
PI(16:0/22:4) 4.21 887.57
unknown 4.10 904.60
unknown 3.86 904.60
unknown 8.88 940.75
unknown 8.97 966.76
87
Table 5 The metabolites with top 50 VIPs values selected from the results of OPLS-DA (high dose vs. high dose control) of UPLC-Qtof/MS spectra from serum of rats exposed to high dose of 35 nm ZnO.
VIP
score Lipid m/z Mean ± SD (Control) Mean ± SD (Treatment) t-test
6.01 PC(34:1) 760.59 447.28±66.43 348.2461±35.06 0.014 a 5.19 LysoPC(18:0) 524.37 550.23±53.12 635.14±53.93 0.05 a 4.98 LysoPC(18:2) 520.34 491.93±42.57 421.15±29.42 0.01 b 3.52 LysoPC(18:1) 522.35 194.5391±14.58714 159.0461±20.77115 0.02b
3.49 LysoPC(16:0) 496.34 722.06±14.32 776.61±67.84 0.16 2.77 Anhydroeschscholtzxanthin 531.41 35.47±43.48 5.35±9.84 0.131
2.47 PC(36:4) 782.57 469.61±55.51 507.17±46.42 0.32 2.46 Lactosylceramide (30:1) 806.57 217.23±19.25 19.25±18.82 0.07
2.44 PC(36:1) 788.61 273.31±41.82 245.80±20.65 0.2 2.22 PC(38:3) 812.62 159.27± 17.85 128.04±20.33 0.038 b
2.2 PC(36:2) 786.60 1097.6±71.17 1066.12±24.51 0.336 2.18 LysoPC(22:6) 568.34 44.68±4.04 33.54±2.65 0.005 b 2.11 PC(32:0) 734.57 54.6146±4.42769 67.8793±9.04513 0.03 a
2.1 CerP(44:1) 799.67 11.81±9.16 27.88±6.63 0.012a
2.02 PC(34:0) 762.60 69.71±7.41 82.2±7.80 0.39
88
2 PC(34:2) 758.57 1086.48±67.58 1117.68±44.89 0.4 1.91 PC(36:4) 782.57 73.14±9.74 89.62±12.14 0.054 1.68 PC(36:3) 784.59 27.24±11.07 15.32±5.56 0.05 1.56 TG(49:3) 815.70 105.58±27.09 123.93±15.91 0.21 1.55 PC(32:1) 732.56 24.4922±9.25880 15.4260±5.37342 0.08 1.52 PC(38:3) 812.62 10.6799±6.00374 2.7716±1.89446 0.02b
1.48 LysoPC(16:1) 494.32 19.31±5.09 13.041.72 0.02 b 1.36 PC(19:0) 553.39 8.62±8.34 1.78±3.51 0.106 1.32 Tetracosanoic acid 369.35 6.94±3.23 14.42±4.69 0.025a
1.31 PC(38:2) 814.64 44.22±4.98 37.98±4.32 0.07 1.3 PC(40:6) 834.60 214.66±31.61 200.92±9.49 0.34 1.3 N-Lignoceroylsphingosine 650.64 42.69±5.42 36.28±3.21 0.045b
1.23 PC(14:0/22:5) 780.55 33.2550±5.95650 26.9832±6.21458 0.15 1.21 PE(42:4) 824.62 16.57±2.54 20.94±2.23 0.02 a 1.15 MG(24:0/0:0/0:0) 521.42 2.55±1.71 7.46±4.89 0.094 1.14 PC(37:4) 796.59 20.04±1.95 23.7±1.30 0.007a
1.13 TG(48:3) 801.69 80.61±14.75 89.45±8.47 0.26 1.1 LysoPC(22:5) 570.36 6.76±0.46 3.86±1.04 0.001 b
89
1.05 PC(33:1) 746.57 9.19±1.71 5.54±2.10 0.02 b 1 PC(35:1) 774.60 14.95±7.45 10.78±2.74 0.07 0.98 LysoPC(20:3) 546.36 11.43±9.07 4.83±3.25 0.13 0.91 SM(40:2) 785.66 0±0 4.69±4.34 0.067
0.9 PE(38:5) 752.56 8.59±1.64 11.58±2.80 0.093 0.88 PC(40:5) 836.62 14.6±7.45 7.96±8.80 0.25 0.86 Huperzine B 257.18 9.42±1.40 12.11±1.26 0.013 a 0.85 PC(33:0) 746.61 17.15±3.00 20.41±3.71 0.182 0.84 Traumatic acid 474.31 1.14±2.10 5.41±8.23 0.347 0.81 LysoPE(20:0) 510.36 15.43±1.05 18.2±3.29 0.147 0.81 Cholesteryl acetate 429.37 1.17±1.68 5.65±9.22 0.37 0.76 LysoPC(20:1) 550.39 5.49±0.46 7.22±1.10 0.018 a 0.76 Iloprost 479.26 0.97±1.81 4.34±4.34 0.332
a increased average concentration compared to control with significant p value with t-test.
b decreased average concentration compared to control with significant p value with t-test.
90
Table 6 The metabolites with top 50 VIP values selected from the results of OPLS-DA (moderate dose vs. moderate dose control) of UPLC-Qtof/MS spectra from serum of rats exposed to high dose of 35 nm ZnO.
VIP score Lipid m/z Mean ± SD (Control) Mean ± SD (Treatment) t-test
5.42 PC(38:3) 812.62 177.6±26.52 74±22.37 <0.0001 b 5.27 PC(38:4) 810.60 584.25±105.59 712.14±110.07 0.14 4.79 PC(34:1) 760.58 485.2±53.69 390±38.89 0.018 b 3.97 PC(34:2) 758.57 1065.33±68.95 1156.92±116.18 0.257 3.77 LysoPC(18:0) 524.37 639.17±54.92 718.47±100.76 0.253 3.20 PC(36:3) 784.59 31.84±7.26 5.1±3.21 <0.0001 b 3.13 PC(36:1) 788.61 274.29±29.18 240.36±11.63 0.035 b 3.06 Goyaglycoside 663.45 152.42±13.83 183.19±14.19 0.033 a 2.53 PC(40:6) 834.60 223.8±25.04 182.63±65.57 0.341 2.28 LysoPC(18:1) 522.35 199.02±5.24 176.02±21.60 0.122 2.26 PC(32:1) 732.56 25.95±7.52 10.8±5.34 0.009 b 2.14 PC(36:5) 780.55 31.62±5.74 18.28±5.31 0.03 b 1.94 trans-4-Decenoic acid 171.14 0±0 21.9±31.84 0.288 1.93 PC(32:0) 734.57 70.54±4.10 58.64±6.24 0.22 1.83 Lactosylceramide (30:1) 806.57 226.87±28.04 201.03±46.92 0.417
91
1.79 PC(38:2) 814.64 40.01±2.29 30.83.63 0.006 b 1.70 LysoPC(16:1) 494.32 19.86±2.87 11.86±2.45 0.019 b 1.61 LysoPC(16:0) 496.34 841.27±71.65 871.7±79.85 0.596 1.53 32,35-anhydrobacteriohopaneterol 529.46 29.11±2.56 36.12±2.78 0.008 a
1.38 PC(36:4) 782.57 430.93±57.07 455.83±74.53 0.63 1.37 LysoPC(18:2) 520.34 437.85±38.15 418.76±42.33 0.533
1.37
1alpha,25-dihydroxy-2alpha-(3-hydroxypropoxy)-19-norvitamin
D3
479.37 27.23±2.42 33.07±2.75 0.025 a
1.33 TG(49:3) 815.70 144±27.51 123.80±25.89 0.35 1.32 GPSer(28:0) 680.48 28.53±3.04 34.49±2.78 0.04 a 1.32 PC(33:1) 746.57 9.65±0.83 4.47±1.97 0.004 b 1.30 Malvidin 3-(6''-acetylglucoside) 536.16 62.21±41.16 83.03±32.57 0.431
1.25 LysoPC(20:4) 544.34 207.59±33.94 220.52±14.05 0.427 1.22 SM(40:1) 787.67 99.16±10.17 88.6±9.84 0.177 1.21 LysoPC(22:6) 568.34 44.12±9.76 36.5±7.29 0.224 1.21 TG(48:3) 801.69 93.31±8.78 104.334±8.07 0.146 1.15 LysoPC(20:3) 546.35 12.48±8.01 19.2±2.40 0.085 1.13 Palmitoyl glucuronide 419.32 7.55±9.03 1.66±2.14 0.15
92
1.11 Cholesteryl acetate 429.37 1±0.87 8.03±8.41 0.206 1.09 PC(16:0/22:5) 808.59 100.4±25.83 86.39±21.07 0.408 1.04 Hericene A 557.42 5.46±1.80 8.804±21.08 0.009 a 1.00 PE(42:4) 824.62 10.00±2.95 15.55±5.94 0.18 0.99 LysoPC(20:5) 542.32 7.32±7.74 11.75±1.41 0.06 0.97 PC(18:0/22:5) 836.62 8.41±2.35 11.34±1.41 0.055 0.95 PC(40:7) 832.59 15.83±1.06 11.93±3.37 0.015b
0.94 PC(35:1) 774.60 0.12±0.10 6.4±11.53 0.099
0.94 1beta,3alpha,7alpha,12alpha-Tetrahydroxy-5beta-cholan-24-oic
Acid
425.29 1.81±0.75 6.84±7.30 0.392
0.92 MG(18:3) 353.27 1.23±0.31 4.52±2.73 0.287 0.91 DG(36:4) 634.54 19.08±2.21 23.38±2.95 0.085 0.89 PC(20:3/22:6) 856.59 10.22±1.02 12.99±1.42 0.009a
0.87 Sabadelin 548.50 10.23±1.02 13±1.42 0.021 a 0.85 PC(34:3) 756.55 19.14±2.32 15.34±3.62 0.148 0.85 PC(34:0) 762.60 70.21±5.14 73.48±2.67 0.166
a increased average concentration compared to control with significant p value (p<0.05).
b decreased average concentration compared to control with significant p value (p<0.05).
93
Table 7 The metabolites with top 50 VIP values selected from the results of OPLS- DA (low dose vs. low dose control) of UPLC-Qtof/MS spectra from serum of rats exposed to high dose of 35 nm ZnO.
VIP score Lipid m/z Mean+±SD (Control) Mean ± SD (Treatment) t-test
7.63 PC(38:4) 810.60 733.34±25.79 998.36±54.37 <0.0001 a 5.65 PC(34:2) 758.57 1127.15±74.76 950.26±78.82 0.009 b 5.23 PC(36:2) 786.60 1101.8±42.58 943.71±93.90 0.014 a 4.27 LysoPC(18:2) 520.34 475.52±290. 377.3±45.78 0.006 b 3.20 PC(34:1) 760.58 326.96±10.40 276.45±18.44 0.001 b 2.54 LysoPC(16:0) 496.34 836.8±84.75 768.98±85.58 0.259 2.53 PC(40:6) 834.60 190.62±18.30 234.6±35.97 0.056 2.53 LysoPC(18:1) 522.35 170.24±17.38 136.03±11.84 0.006 b 1.98 PC(36:3) 784.58 182.58±24.40 130.88±42.00 0.059 1.97 PC(16:0/22:5) 808.59 70.08±27.80 110.59±51.14 0.19 1.91 PC(36:1) 788.61 231.93±6.52 210.93±11.83 0.013 b 1.82 Lactosylceramide (30:1) 806.57 180.6±23.94 212.9±41.83 0.204 1.71 LysoPC(18:0) 524.37 717.39±60.29 760.38±85.79 0.414 1.63 TG(48:3) 801.69 76.15±11.46 97.4±9.76 0.014 b 1.45 LysoPC(20:4) 544.34 253.87±32.00 277.87±32.29 0.287
94
1.32 SM(40:1) 787.67 89.57±4.12 101.51±8.05 0.027 a 1.30 PC(37:4) 796.59 17.14±4.05 27.51±6.84 0.027 a
1.25 PC(38:2) 814.64 38.29±5.21 28.47±6.12 0.031 b 1.16 Anhydroeschscholtzxanthin 531.41 26.49±7.92 14.75±13.91 0.169
1.08 2-demethylmenaquinone-8 703.57 99.2±9.51 113.94±25.22 0.304
1.05 SM(40:0) 811.66 78.13±9.00 88.87±13.29 0.199 1.05 PE(42:4) 824.62 13.63±5.57 21.97±6.20 0.062 0.99 Goyaglycoside 663.45 191.88±25.78 174.51±44.15 0.502 0.95 PC(40:7) 832.59 6.73±3.16 12.88±2.27 0.007 a 0.95
1-tetradecanyl-2-(8-[3]-ladderane-octanyl)-sn-glycerophosphoethanolamine 684.55 6.96±3.33 1.75±1.64 0.01 b
0.95 PC(36:3) 768.59 18.29±2.50 24.96±6.61 0.094 0.92 TG(49:3) 813.68 247.1±19.57 266.4±58.38 0.548 0.92 LysoPC(22:6) 568.34 43.38±4.56 37.16±4.76 0.074 0.89 TG(38:4) 794.61 13.54±2.71 19.47±5.36 0.078 0.87 Tetracosanoic acid 369.35 3.27±1.76 10.33±7.44 0.105 0.85 PE(28:0) 636.55 10.92±4.68 5.22±5.04 0.11 0.84 PC(32:0) 734.57 54.02±8.37 62.71±15.41 0.338 0.80 PC(36:4) 766.58 3.75±2.73 8.11±2.94 0.048 a
95
0.76 32,35-anhydrobacteriohopaneterol 529.46 40±5.95 34.3±6.59 0.199 0.76 Petunidin 3-glucoside-5-(6''-acetylglucoside) 684.20 43.8±5.84 36.53±11.59 0.229
0.75 PC(33:0) 746.60 16±2.19 20.81±5.29 0.128 0.75 PC(19:0) 553.39 9.63±2.09 5.13±4.84 0.122 0.73 Ubiquinol 731.60 5.46±1.82 9.62±3.53 0.064 0.72 PC(38:3) 796.62 11.21±0.68 14.47±2.41 0.04 a 0.70 PC(33:2) 744.55 12.74±2.74 9.33±1.61 0.032 b 0.67 PC(36:0) 790.63 20.45±3.14 24.85±4.93 0.036 a 0.65 PC(35:2) 772.59 35.01±3.32 30.23±7.78 0.287 0.64 DG(36:2) 610.54 4.51±3.41 1.39±12.30 0.071 0.64 Ceramide (42:2) 648.63 5.64±1.01 9.5±3.56 0.072 0.63 PC(40:8) 830.57 3.16±2.60 7.5±5.98 0.214 0.61 MG(18:3) 353.27 8.86±11.68 16.3±22.67 0.567
a increased average concentration compared to control with significant p value (p<0.05).
b decreased average concentration compared to control with significant p value (p<0.05).
96
Table 8 Fold-changes (high-, moderate- and low-dose/ high-, moderate- and low-dose control) of 17 common lipids selected from high-, moderate- and low-dose of 35 nm ZnO treatment from UPLC-Qtof/MS results.
Lipid High dose / High dose control Moderate dose/ Moderate
dose control Low Dose/ Low dose control
PC(38:5) 0.61 0.86 1.58
97
PC(34:1) b 0.78 * 0.80 * 0.85 *
PC(34:2) 1.03 1.09 0.84 PC(36:3) 0.56 0.16 0.72 PC(36:1) 0.90 0.88 0.91 PC(40:6) 0.94 0.82 1.23 LysoPC(18:1) 0.82 0.88 0.80
PC(32:0) 1.24 0.83 1.16 Lactosylceramide (30:1) 0.09 0.89 1.18
PC(36:4) 1.08 1.06 1.28 LysoPC(18:2) 0.86 0.96 0.79
* significant p value with t-test (p<0.05).
a increased average concentration compared to control with significant p value with t-test(p<0.05).
b decreased average concentration compared to control with significant p value with t-test (p<0.05).
98
Table 9 270 markers selected from the results of PCA from the analysis of UPLC-Qtof/MS spectra of serum of rats exposed to 250 nm ZnO. 2-Methylglutaric acid 3.80 147.06 Citramalic acid 4.36 149.02
Unkonwn 0.49 158.97
99
3-(3-Hydroxyphenyl)propanoic acid 5.79 167.07
Pyridoxamine 0.43 169.09
4-Pyridoxic acid 1.88 184.07 cis-stilbene oxide 1.85 197.12
L-Kynurenine 5.79 209.08
Prilocaine 1.87 221.15
Unkonwn 0.49 226.95
Traumatic acid 0.56 229.14
Talbutal 2.19 253.18
Huper ine B 0.69 257.18 Tetradecanedioic acid 3.80 259.19
Acetyl-N-formyl-5-methoxykynurenamine 1.84 265.14
Atenolol 2.28 267.16
Diethylstilbestrol 0.56 269.14 3-Hydroxytetradecanedioic acid 4.31 275.16
Alpha-Linolenic acid 2.74 279.23 Sulfametopyra ine 6.76 281.05
octadecanamide 3.03 284.29
100
Unkonwn 2.73 284.33
17a-Ethynylestradiol 0.69 297.17
Unkonwn 2.84 298.35
PC(O-2:0/O-1:0) 2.21 301.14
9-cis-Retinoic acid 2.74 301.21 Retinoic acid 2.60 303.23 Arachidonic acid 2.86 305.25
Unkonwn 3.54 312.32
Escitalopram 1.89 325.20
Diampromide 2.60 325.21
Ajmaline 2.86 327.23
Docosahexaenoic acid 3.20 329.24 Docosahexaenoic acid 2.74 329.25
Unkonwn 2.45 336.33
N-Dodecyl-N,N-Dimethyl-3-Ammonio-1-Propanesulfonate 6.05 337.27
Unkonwn 3.51 338.34
3b,15b,17a-Trihydroxy-pregnenone 2.58 349.21 Tetrahydrocorticosterone 2.51 351.23
101
MG(18:3) 2.64 353.27
5beta-Chola-3,8(14),11-trien-24-oic Acid 2.88 355.26 N-butyl arachidonoyl amine 3.51 360.32
Unkonwn 0.49 362.93
3-Oxochola-1,4,6-trien-24-oic Acid 2.80 369.24 Tetracosanoic acid 4.35 369.35 Tetradecenoyl carnitine 3.80 371.32
Biocytin 2.51 373.21
MG(0:0/20:5/0:0) 2.51 377.27 24-Nor-5beta-cholane-3alpha,7alpha,12alpha,23-tetrol 3.07 381.30
Clonita ene 1.34 387.18 1alpha,3alpha-Dihydroxy-5beta-cholan-24-oic Acid 3.80 393.30 Etonita ene 3.49 397.24
2-(2-{2-[2-(2-{2-[2-(2-Ethoxy-Ethoxy)-Ethoxy]-Ethoxy}-Ethoxy)-Ethoxy]-Ethoxy}-Ethoxy)-Ethanol, Polyethyleneglycol Peg400 2.20 399.25
L-Palmitoylcarnitine 1.97 400.34
Unkonwn 4.34 404.39
LysoPC(10:0) 3.81 413.27
Palmitoyl glucuronide 4.31 419.32
102
5beta-Cholest-25-ene-3alpha,7alpha,12alpha-triol 5.69 419.35 Linoelaidyl carnitine 1.88 424.34
1beta,3alpha,7alpha,12alpha-Tetrahydroxy-5beta-cholan-24-oic Acid 3.37 425.30 Elaidic carnitine 2.10 426.36
Stearoylcarnitine 2.33 428.37 Cholesteryl acetate 6.08 429.37
Unkonwn 5.47 430.38
phenyl{bis[(trifluoroacetyl)oxy]}-lambda~3~-iodane 0.49 430.91 27-Nor-5b-cholestane-3a,7a,12a,24,25-pentol 4.95 439.32
L-Alfa-Lysophosphatidylcholine, Lauroyl 4.30 441.30 3-Hydroxy-linoleyl carnitine 4.37 441.33
LysoPE(0:0/16:0) 1.93 454.29 Sulfolithocholic acid 2.09 457.28
LysoPC(14:0) 1.60 468.31
Unkonwn 2.09 474.31
Unkonwn 5.68 475.41
Unkonwn 7.01 475.41
LysoPE(18:2) 1.84 478.29
103
104
LysoPE(0:0/20:0) 2.12 510.36
LysoPC(18:3) 1.71 518.32
Genistein 7-O-glucoside-6''-malonate 6.76 519.14
LysoPC(18:2) 1.85 520.34 Anhydroeschscholt xanthin 4.65 531.41
4-Amino-N-{4-[2-(2,6-Dimethyl-Phenoxy)-Acetylamino]-3-Hydroxy-1-Isobutyl-5-Phenyl-Pentyl}-Ben amide 2.12 532.34
SP2456 2.87 533.19
2-Hexaprenyl-6-methoxyphenol 7.36 533.46 Oleoyl estrone 5.79 535.44
Malvidin 3-(6''-acetylglucoside) 6.76 536.16
LysoPE(20:0) 2.47 538.39
105
2-hexaprenyl-6-methoxy-1,4-ben oquinol 5.85 549.42
LysoPC(20:1) 2.38 550.39
LysoPC(20:0) 2.63 552.40
Unkonwn 5.79 557.42
DG(32:3) 6.29 563.47
octatriacontanoic acid 6.33 565.57
Unkonwn 1.89 566.32
106
LysoPC(24:0) 3.48 608.47
Peonidin 3-rutinoside 7.83 610.18
Unkonwn 6.15 610.50
Unkonwn 6.77 610.54
Unkonwn 7.26 612.56
Unkonwn 7.84 618.62
Ceramide (d18:1/22:0) 7.52 622.61
Cer(d18:0/22:0) 7.66 624.63
Ceramide (d18:1/24:1) 7.59 648.63
N-Lignoceroylsphingosine 8.13 650.64
107
Ceramide (d18:1/25:0) 8.38 664.66
N-(24-hydroxytetracosanyl)sphinganine 7.66 668.65
Unkonwn 3.51 675.68
Ceramide (d18:1/26:0) 8.69 678.68
GPSer(14:0/14:0) 7.57 680.48
Unkonwn 6.05 682.54
Petunidin 3-glucoside-5-(6''-acetylglucoside) 8.72 684.20
1-tetradecanyl-2-(8-[3]-ladderane-octanyl)-sn-glycerophosphoethanolamine 6.54 684.55 PE(dm16:0/dm18:1 ) 6.78 686.57
Etn-1-P-Cer(d14:1/18:0) 3.78 689.56
Unkonwn 8.29 696.60
108
109
110
Glucosylceramide (d18:1/22:0) 6.57 784.67
Unkonwn 5.63 785.66
111
Lactosylceramide (30:1) 4.67 806.57
PC(16:0/22:5) 5.59 808.59
PC(16:0/22:5) 5.11 808.60
Unkonwn 5.13 809.62
PC(38:4) 5.13 810.61
GlucosylCeramide (42:2) 6.62 810.68
Unkonwn 5.69 811.66
112
3-decaprenyl-4,5-dihydroxyben oic acid 6.26 835.67
PC(18:0/22:5) 5.56 836.62
113
PI(16:0/22:4) 3.71 887.57
Unkonwn 3.72 904.60
Unkonwn 8.87 940.75
Unkonwn 8.97 966.76
a increased average concentration compared to control with significant p value with t-test (p<0.05).
b decreased average concentration compared to control with significant p value with t-test(p<0.05).
114
Table 10 The metabolites with top 50 VIP values selected from the results of OPLS-DA (high dose vs. high dose control) of UPLC-Qtof/MS spectra from serum of rats exposed to high dose of 250 nm ZnO.
VIPS
score Lipid m/z Mean ± SD (Control) Mean ± SD
(Treatment) t-test
6.93 PC(36:2) 786.60 767.85±33.31 876.67±62.47 0.013 a 6.03 PC(40:6) 834.60 238.84±24.20 328.73±69.09 0.039 a 5.01 PC(16:0/16:0) 734.57 123.82±61.63 62.55±9.99 0.039 4.11 PC(36:4) 782.57 707.36±57.30 646.81±32.28 0.063 3.57 PC(36:1) 788.61 195.68±10.83 225.05±18.20 0.021 a 3.17 LysoPC(16:0) 496.34 719.1±70.25 666.37±96.03 0.377 2.57 PC(38:4) 810.60 1109.67±27.30 1071.98±66.14 0.319 2.56 Anhydroeschscholtzxanthin 531.41 16.6±7.39 3.61±2.98 0.004 b
2.38 PC(14:0/20:0) 762.60 78.53±15.74 63.8±3.51 0.053 2.34 Lactosylceramide (30:1) 806.57 244.14±37.12 272.87±50.53 0.333
2.23 PC(34:2) 758.57 870.97±1.93 901.38±62.05 0.365 2.15 PC(38:3) 812.62 87.1±4.11 112.01±26.03 0.1 2.11 Huperzine B 257.18 21.07±13.47 9.23±2.78 0.063 2.05 1alpha,3alpha-Dihydroxy-5beta-cholan-24-oic
Acid 393.30 11.38±5.66 2.69±2.12 0.008 b
115
1.96 DG(40:8) 682.54 7.22±1.46 15.78±5.55 0.018 a 1.85 17a-Ethynylestradiol 297.17 9.51±7.61 1.38±0.46 0.027 b 1.74 PE(O-18:1/20:4(5Z) 752.56 22.87±18.94 12.03±2.51 0.191 1.61 Goyaglycoside 663.45 186.16±15.15 173.41±20.80 0.326 1.59 PC(10:0/9:0) 553.39 5.99±2.45 1.15±0.93 0.002 b 1.57 DG(44:1) 940.75 7.8±1.77 13.1±2.66 0.008 a 1.54 PE(28:0) 636.56 1.74±0.42 6.86±2.89 0.009 a 1.53 DG(36:4) 634.54 1.86±0.10 6.43±2.07 0.003 a 1.48 L-Alfa-Lysophosphatidylcholine, Lauroyl 441.30 12.34±9.18 4.83±5.64 0.144
1.47
1-tetradecanyl-2-(8-[3]-ladderane-octanyl)-sn-glycerophosphoethanolamine 684.55 1.92±0.73 6.57±2.53 0.008 a
1.42 PC(34:3) 756.55 11.16±3.28 6.69±1.36 0.016 b 1.41 Thromboxane 338.34 8.55±2.27 16.35±11.90 0.24 1.41 Tetracosanoic acid 369.35 24.49±9.23 17.04±3.77 0.109 1.34 PC(33:0) 746.61 27.92±4.46 23.36±1.48 0.045 b 1.32 PC(32:0) 720.59 10.98±3.45 6.98±0.80 0.023 b
1.29 TG(49:3) 815.70 131.09±16.16 116.25±23.47 0.306 1.28 LysoPC(18:2) 520.34 235.12±24.14 249.01±45.10 0.592 1.25 Palmitoyl glucuronide 419.32 8.11±12.23 1.7±3.46 0.248
116
1.24 PE(38:3) 754.58 7.18±9.05 1.58±1.15 0.161 1.23 LysoPC(22:6) 568.34 25.11±2.30 29.7±4.42 0.096 1.19 3-Hydroxy-linoleyl carnitine 441.33 12.7±12.23 6.65±3.31 0.271
1.17 PC(15:0/22:2) 800.63 30.86±1.50 35.76±6.11 0.161 1.16 Arachidonic acid 305.25 1.6±0.63 4.43±1.26 0.003 b 1.15 PC(38:3) 812.62 2.81±3.52 8.05±4.45 0.074 1.12 LysoPC(18:0) 524.37 612.61±31.44 626.22±52.46 0.657 1.11 Ajmaline 327.23 2.09±0.19 4.8±0.84 <0.0001 a 1.11 Ceramide (42:2) 648.63 17.66±2.32 13.64±3.63 0.088 1.09 DG(36:2) 610.54 0.61±0.20 3.16±1.20 0.003 a 1.06 PC(18:2/18:1) 768.59 23.97±3.69 20.73±1.47 0.084 1.05 Citramalic acid 149.02 3.7±5.83 0 0.147 1.05 PC(24:1/P-16:0) 904.60 20.31±5.95 24.19±1.91 0.167 1.05 DG(38:7) 656.52 1.72±0.50 4.03±0.72 0.001 a 1.04 PC(38:3) 796.62 14.32±1.32 11.87±0.96 0.009 b 1.03 Retinoic acid 303.23 2.55±0.44 4.93±1.33 0.009 a 1.03 DG(32:3) 563.47 12.04±3.63 8.82±2.15 0.113 1.02 TG(48:3) 801.69 106.19±9.20 115.12±12.98 0.271
a increased average concentration compared to control with significant p value with t-test (p<0.05).
117
b decreased average concentration compared to control with significant p value with t-test(p<0.05).
118
Table 11The metabolites with top 50 VIP values selected from the results of OPLS-DA (moderate dose vs. high dose control) of UPLC-Qtof/MS spectra from serum of rats exposed to high dose of 250 nm ZnO.
VIP
Acid 758.57 1055.1±32.45 965.07±60.67 0.049 b
5.26 PC(38:4) 810.60 688.88±36.72 758.15±79.78 0.206 5.20 PC(40:6) 834.60 205.34±27.53 257.16±42.61 0.102 4.20 LysoPC(16:0) 496.34 793.09±26.00 752.98±33.39 0.114 3.66 LysoPC(18:2) 520.34 515.81±52.53 480.82±30.08 0.231 3.40 PC(32:0) 787.67 68.61±13.82 86.44±5.62 0.024 a 3.19 PC(38:3) 734.57 77.67±14.80 61.7±4.01 0.034 b 3.00 PC(36:4) 782.57 443.2±27.30 472.87±43.76 0.326 2.67 PC(36:3) 788.61 218.44±2.70 235.68±20.61 0.205 2.62 LysoPC(22:6) 257.18 15.1±7.57 5.3±2.01 0.016 b 2.55 PC(34:2) 524.37 630.33±50.51 660.69±70.61 0.533 2.55 TG(49:3) 524.37 630.33±50.51 660.69±70.61 0.533 2.49 PC(18:4/18:0) 782.57 84.97±9.42 67.39±9.68 0.036 b 2.27 PC(14:0/20:0) 531.41 14.63±25.33 1.9±2.93 0.232
119
2.12 Lactosylceramide (30:1) 812.62 95.33±12.06 117.61±27.35 0.231
2.07 PC(38:4) 808.59 99.92±20.87 117.7±12.22 0.142 2.01 17a-Ethynylestradiol 762.60 84.67±1.21 76.65±7.24 0.108 1.95 Arachidonic acid 752.56 17.6±1.23 12.4±2.28 0.009 b 1.91 PE(28:0) 744.55 8.05±2.02 3.12±1.34 0.003 b 1.85 Anhydroeschscholtzxanthin 806.57 239.16±14.88 256.09±42.61 0.537
1.66 PC(36:2) 786.60 1042.7±4.55 1024.79±52.46 0.586 1.60 PC(38:3) 801.69 68.13±10.25 76.02±4.93 0.149 1.60 PC(34:3) 429.37 6.08±4.40 1.07±1.30 0.029 b 1.58 Tetracosanoic acid 806.57 17.83±2.34 11.67±3.18 0.022 b 1.36 LysoPC(18:0) 522.35 187.82±19.40 179.39±15.35 0.497 1.30 PC(33:0) 904.60 18.22±2.62 21.13±1.23 0.051 1.30 PC(32:0) 768.55 6.61±1.31 3.35±2.69 0.093 1.24
4alpha-formyl-4beta-methyl-5alpha-cholesta-8-en-3beta-ol 429.37 3.96±6.86 0±0 0.17
1.24 DG(36:2) 610.54 7.56±0.99 4.93±1.80 0.055 1.23 Palmitoyl glucuronide 703.57 93.24±13.27 87.2610.02 0.469 1.22 3-Hydroxy-linoleyl carnitine 441.30 4.38±2.16 1.5±0.62 0.015b
1.19 PC(19:0) 553.39 4.22±7.28 0.64±0.97 0.245
120
1.18 PE(38:3) 627.53 15.53±2.10 17.84±0.75 0.039 a 1.14 L-Alfa-Lysophosphatidylcholine, Lauroyl 563.47 7.1±0.52 10.18±3.75 0.213
1.09 PE(38:5) 338.34 6.92±1.63 11.49±9.64 0.456 1.08 TG(48:3) 634.54 8.78±1.14 11.49±9.64 0.192 1.07 Ajmaline 545.46 0.037±0.63 2.29±2.08 0.114 1.04 Ceramide (42:2) 664.66 11.54±3.45 9.18±1.29 0.163 1.03 Huperzine B 784.59 233.9±21.12 225.15±35.55 0.711 1.02 PC(42:1) 812.70 8.13±0.70 6.16±0.75 0.007 b 1.02 DG(32:3) 830.57 3.04±2.65 7.56±6.63 0.305 1.01 PE- NMe(36:2) 758.57 0±0 2.96±2.70 0.109 1.00 PC(38:3) 646.65 10.51±3.42 8.2±1.45 0.182 0.99 Citramalic acid 887.57 9.58±0.78 11.12±0.53 0.009 a 0.98
1-tetradecanyl-2-(8-[3]-ladderane-octanyl)-sn-glycerophosphoethanolamine 799.67 16.24±7.80 20.71±5.05 0.324
0.98 PC(36:1) 772.59 39.81±6.65 36.33±5.35 0.482 0.98 SM(42:1) 815.70 0.11±0.13 4.76±7.73 0.348 0.96 PC(37:2) 832.59 14.29±0.62 16.67±2.57 0.17 0.94 Retinoic acid 668.65 9.29±2.03 7.3±1.60 0.149 0.93 3-Carboxy-4-methyl-5-pentyl-2-furanpropionic 269.14 1.54±1.30 0.18±0.21 0.031 b
121 acid
a increased average concentration compared to control with significant p value (p<0.05).
b decreased average concentration compared to control with significant p value(p<0.05).
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Table 12 The metabolites with top 50 VIP values selected from the results of OPLS-DA (low dose vs. low dose control) of UPLC-Qtof/MS spectra from serum of rats exposed to high dose of 250 nm ZnO.
Table 12 The metabolites with top 50 VIP values selected from the results of OPLS-DA (low dose vs. low dose control) of UPLC-Qtof/MS spectra from serum of rats exposed to high dose of 250 nm ZnO.