Institute of Occupational Medicine and Industrial Hygiene College of Public Health
National Taiwan University Master Thesis
Effects of oropharyngeal aspiration to diesel exhaust particles on central nervous system toxicity
in 3xTg-AD mice
Chen-Huan Fu
TJC Lab
Royal
MB
108 7
(Alzheimer’s disease, AD)
(diesel exhaust particles, DEPs) C57BL/6
Tau
— (3xTg-AD mice)
(amyloid-beta, Aβ)
(amyloid plaque) Tau
(phosphorylated Tau protein) (Neurofibrillary Tangles, NFTs) 3xTg-AD
6-8 34 3xTg-AD
(Western Blot) (malondialdehyde, MDA) (Iba-
1) (LC3b) (Aβ42, t-Tau)
H&E
MDA (p<0.05)
Abstract
Air pollution has been known as a major health threat for decades. Many epidemiological and toxicological studies of air pollution have confirmed that ambient particulate matter can cause adverse effects on the central nervous system (CNS), including neuroinflammation, oxidative stress, autophagy dysfunction and contribute to neurodegenerative diseases, such as Alzheimer’s disease (AD).
In the modern city, the most important source of pollution is traffic-related air pollutants. Diesel exhaust particles (DEPs) emitted by diesel engine combustion is the most important component of near-road and urban air pollution. In our previous study, C57BL/6 mice were acutely exposed to DEPs and found that pro-inflammatory cytokines, lipid peroxidation, and tau tauopathies increased significantly in the brain. However, the mechanisms are still unclear. Therefore, the objective of this study was to investigate the central nervous system toxicity caused by DEPs in Alzheimer’s disease mouse model.
6 to 8-week-old 3xTg-AD female mice were exposed to 300µg of DEPs SRM 1650b by oropharyngeal aspiration (50 µg/time, 2 times/week and for 3 weeks). SRM 1650b was purchased from National Institute of Standards and Technology, and it represents particulate from the combustion of heavy-duty diesel engines. After the last exposure, all
examination.
The results showed that MDA concentration significantly increased in the hippocampus (p<0.05), but not in cerebral cortex and cerebellum. Expression of LC3b, Iba-1, Aβ42, and total tau protein was not significant between the control and the exposure group in 3 brain region. The DEPs exposure did not cause histopathological changes in the brain, but there was slight inflammation in the lungs.
In all, the data support that the hippocampus may be more sensitive to the oxidative stress from the exposure to DEPs. However, further studies should include the aged animals for prolonged period of time to elucidate the underlying mechanisms of DEPs induced neurotoxicity.
Keywords: diesel exhaust particulates, oropharyngeal aspiration, central nervous system toxicity, microglia activation, oxidative stress, autophagy
... 1
... 2
2.1 ... 2
2.1.1 ... 3
2.2 ... 4
2.2.1 ... 5
2.2.2 ... 7
2.2.3 ... 9
2.3 (Alzheimer's disease, AD) ... 11
2.3.1 ... 13
2.3.2 ... 14
2.3.3 ... 15
2.4 (3xTg-AD mice) ... 17
2.5 (Oropharyngeal Aspiration, OA) ... 19
... 20
3.1 ... 20
3.5.2 ... 24
3.5.3 MDA ... 25
3.5.4 LC-MS/MS ... 26
3.6 ... 28
3.6.1 ... 28
3.6.2 ... 29
3.6.3 ... 31
3.7 ... 32
3.8 ... 32
... 33
4.1 MDA ... 33
4.1.1 ... 33
4.1.2 MDA ... 33
4.2 ... 34
4.2.1 Iba-1 ... 34
4.2.2 LC3b ... 34
4.2.3 Aβ42 ... 34
4.2.4 t-Tau ... 34
4.3 ... 35
... 36
5.1 ... 36
5.2 3xTg-AD ... 39
5.3 ... 41
5.3.1 ... 41
5.3.2 ... 43
5.3.3 ... 45
5.3.4 ... 47
5.3.5 ... 48
... 49
... 50
1 ... 66
2 SRM1650b PAH (NIST 2013) ... 67
3 LC-MS/MS ... 69
4 HESI ... 69
5 SRM ... 69
6 ... 70
7 MDA ... 71
8 Iba-1 (Fold of control) ... 72
9 LC3b (Fold of control) ... 72
10 Aβ42 (Fold of control) ... 73
11 t-Tau (Fold of control) ... 73
12 ... 74
1 ... 75
2 MDA-DNPH ... 76
3 MDA ... 77
4 Iba-1 ... 78
5 Iba-1 ... 79
6 Iba-1 ... 80
7 LC3b ... 81
8 LC3b ... 82
9 LC3b ... 83
10 Aβ42 ... 84
11 Aβ42 ... 85
12 Aβ42 ... 86
13 t-Tau ... 87
14 t-Tau ... 88
15 t-Tau ... 89
16 ... 90
(International Agency for Research on Cancer ,
IARC) 2013 PM2.5
92%
PM2.5
Tau
3xTg-AD
1. MDA
2. LC3b
3. Iba-1
4. (amyloid-β, Aβ) Tau
(total Tau protein, t-Tau)
2.1
90% (Kurt, Zhang et al. 2016)
(Akimoto 2003)
(Akimoto 2003) (Craig, Brook et al. 2008)
(Cui, Huang et al. 2014) (Kurt, Zhang et al. 2016) (Kaufman, Adar et al. 2016)
(CALDERon-GARCIDUEnas, Reed et al. 2004, Calderón- Garcidueñas, Mora-Tiscareño et al. 2008, Mateen and Brook 2011)
(Dementia) (MCI) (Alzheimer’s disease)
(Parkinson’s disease) (Gatto, Henderson et al. 2014, Tonne, Elbaz et al. 2014, Zanobetti, Dominici et al. 2014, Tzivian, Dlugaj et al. 2016)
2.1.1
(Fagundes,
Fleck et al. 2015) (Blood–brain barrier, BBB)
(Heusinkveld, Wahle et al. 2016, Kreyling 2016)
100 (Calderón-Garcidueñas, Solt et al. 2008, Wang, Xiong et al. 2017)
(Cheng, Saffari et al. 2016)
(IL-1β, IL-6, TNF-α) (Wang, Xiong et al. 2017) (Chiu, Von Hehn et al. 2012)
2.2
(Hesterberg,
Long et al. 2012) 2012
(diesel engine exhaust, DEE) DEE
(Ris 2007)
(diesel exhaust particulates, DEPs) 30~500 nm
(aromatic hydrocarbons)
(Taxell and Santonen 2017)
(Suwannasual, Lucero et al. 2018)
(Chuang, Wu et al. 2018)
(Levesque, Surace et al. 2011)
( 1)
2.2.1
20
(superoxide anion, O2 −) (hydroxyl radical, OH) (hydrogen peroxide, H2O2) (reactive oxygen species, ROS)
(Cobley, Fiorello et al. 2018) ATP(adenosine triphosphate) (reactive nitrogen species, RNS)
ROS ROS
ROS
(oxidative stress) DNA
(Salim 2017)
(Borza, Muntean et al. 2013)
(Hulbert, Pamplona et al.
2007)
(Wang and Michaelis 2010)
(polyunsaturated
fatty acids, PUFAs) (Sultana,
Perluigi et al. 2006) PUFAs
(Anzai, Ogawa et al. 1999, Yehuda, Rabinovitz et al. 2002)
(Farooqui and Horrocks 1998) PUFAs
(malondialdehyde, MDA) 4- -2- (4-hydroxy-2-nonenal, HNE) (acrolein) MDA
(Giera, Lingeman et al. 2012)
(Block, Wu et al. 2004, van Berlo, Albrecht et al. 2010)
(Guerra, Vera-Aguilar et al. 2013)
MDA (Cole, Coburn et al. 2016)
2.2.2
5~12
(ramified) (activated,
amoeboid)
(Bolton, Smith et al. 2012)
M1 M2 M1
IL-1β, IL-6, IL-12, TNF-α NO
ROS (neurotrophic factors)
M2
IL-10, TGF-β (Cherry, Olschowka
et al. 2014)
(Block and Calderón- Garcidueñas 2009)
Iba-1(Ionized calcium-binding adaptor protein-1)
Iba-1
(Block, Wu et al. 2004)
Iba-1 (Bolton, Smith et al. 2012, Win-Shwe, Fujimaki
et al. 2012, Cole, Coburn et al. 2016) (Cole, Coburn et al. 2016)
(Levesque, Taetzsch et al.
2011, Bolton, Marinero et al. 2017)
Iba-1
2.2.3
(Cuervo 2004)
(Mizushima, Yamamoto et al. 2004) (Scherz-Shouval, Shvets et al. 2007)
(Mazure and Pouysségur 2010) (Song, Zhao et al. 2013)
(Liu, Gao et al. 2010) (Macroautophagy)
(Microautophagy) (Chaperone-mediated
autophagy, CMA) (macroautophagy)
(phagophore)
(elongation) (autophagosome)
(lysosome) (autolysosome)
(Zare-shahabadi, Masliah et al. 2015, Pellacani and Costa 2018)
(Lee, Giordano et al.
2012, Tung, Wang et al. 2012)
(autophagy-related (ATG) proteins) LC3(microtubule-associated protein 1-light chain 3)
(Bernard and
Klionsky 2014) LC3 LC3a, LC3b, LC3c
LC3a LC3b LC3b
(Koukourakis, Kalamida et al. 2015)
LC3 (Pro LC3) LC3-I
LC3-I (phosphatidylethanolamine, PE) LC3-II
LC3-II
LC3II/LC3I
(Orrenius, Kaminskyy et al. 2013)
(Numan, Brown et al. 2015)
(Bai, Chuang et al. 2018)
LC3-II/LC3-I
LC3b
2.3
(Alzheimer's disease, AD)
(Förstl and Kurz 1999)
(amyloid precursor protein, APP) (β-secretase, γ-secretase)
(amyloid-β, Aβ) 42 Aβ(Aβ42)
(Selkoe 2001) Aβ
(monomers) (oligomers) (fibrils)
(amyloid-β plaques)(Carvajal and Inestrosa 2011, Selkoe 2011) (microtubule) Tau
(neurofibrillary tangles, NFTs)
(Duyckaerts, Potier et al.
2008)
(Braak and Braak 1991, Nelson, Alafuzoff et al. 2012)
Aβ (Zhao and Zhao 2013,
Heneka, Carson et al. 2015)
Aβ42 (Levesque, Surace et al. 2011)
PM2.5 Aβ40 (Bhatt, Puig et al. 2015)
Aβ
(Hullmann, Albrecht et al. 2017) Tau
(1 mg/m3)
Tau(phosphorylated tau, p-Tau) (Levesque, Surace et al. 2011)
p-Tau 3 4
(Iqbal, Liu et al. 2010) tau(t-Tau)
(Liazoghli, Perreault et al. 2005, Chesser, Pritchard et al. 2013) t-Tau p-Tau
2.3.1
(Butterfield, Lange et al. 2010) (Ruipérez, Darios et al. 2010)
(Reuter, Gupta et al. 2010)
(Lee, Giordano et al. 2012) (Scherz-Shouval, Shvets et al. 2007, Mizushima, Levine et al. 2008) Aβ (Behl 1997, Abramov and Duchen 2005, Shelat, Chalimoniuk et al. 2008) Tau
(Mondragón-Rodríguez, Perry et al. 2013, Alavi Naini and Soussi-
Yanicostas 2015) (Sultana, Perluigi
et al. 2013)
(Montine, Reich et al. 1998, Calingasan, Uchida et al. 1999)
Aβ (Smith,
Hirai et al. 1998, Praticò, Uryu et al. 2001) (Liu, Smith et al. 2005)
2.3.2
M2
M1 M1
M2 (Wang, Tan et al. 2015)
(Olmos-Alonso, Schetters et al. 2016, Martin, Boucher et al. 2017)
Aβ Tau
Aβ 50% (Kamphuis, Kooijman et al.
2016) Aβ
(Neniskyte, Neher et al. 2011)
(Yoshiyama, Higuchi et al. 2007) Tau (Gorlovoy, Larionov
et al. 2009) Tau
Tau Tau
(Asai, Ikezu et al. 2015)
2.3.3
(Nixon, Wegiel et al. 2005, Nixon and Cataldo 2006)
(Wolfe, Lee et al. 2013, Chuang, Wu et al. 2018)
(Maday, Wallace et al.
2012) (Ashrafi, Schlehe et al. 2014)
(autophagic vacuoles, AV) (Li, Liu et al. 2017)
(Sanchez-Varo, Trujillo-Estrada et al. 2012)
Aβ Tau
Aβ
(Mizushima 2005, Nixon, Wegiel et al. 2005) Tau
(Hamano, Gendron et al. 2008, Congdon, Wu
et al. 2012) tau
p-Tau (Inoue, Rispoli et al. 2012)
Tau Tau
(Li, Liu et al. 2017)
Beclin-1
(Boland, Kumar et al. 2008, Lipinski, Zheng et al. 2010)
(Mizushima, Levine et al. 2008, Moreno, Villar et al. 2018)
2.4
(3xTg-AD mice)
(3xTg-AD mice) C57BL/6
APPSwedish TauP301L PSEN1 M146V (Oddo,
Caccamo et al. 2003) 3xTg-AD 3 Aβ
6 Aβ Tau
18 26 Tau (Billings,
Oddo et al. 2005, Mastrangelo and Bowers 2008)
(Villamil-Ortiz and Cardona-Gomez 2015)
3xTg-AD
(Giménez-Llort, Arranz et al. 2008, Sterniczuk, Antle et al. 2010, Stover, Campbell et al. 2015)
LC3
Beclin-1 (Villamil-Ortiz and Cardona-Gomez 2015)
(GFAP) (Iba-1)
(Chen, Liang et al. 2013)
Tau
Aβ (Huber, Yee et al. 2018) (8-arm radial maze, RAM) 3xTg-AD
2 (Stevens and Brown 2015) (Morris water maze, MWM)
6 (Billings, Green et al. 2007, McKee, Carreras et al.
2008, Stover, Campbell et al. 2015) (novel
object recognition task, NOR) 6
(Blanchard, Wanka et al. 2010, Chen, Liang et al. 2013) 3xTg-AD
2.5
(Oropharyngeal Aspiration, OA)
(whole-body inhalation) (nose-only inhalation)
(intratracheal instillation) (nose instillation) (oropharyngeal aspiration)
(phosphate-buffered saline,
PBS) (Shang and Sun 2018)
60~80%
32%
24 57%
80% (Foster, Walters et al. 2001)
(De Vooght, Vanoirbeek et al. 2009)
(Lakatos, Burgess et al. 2006)
(Kinaret, Ilves et al. 2017)
3.1
3xTg-AD
(In vitro fertilization, IVF) IVF 3xTg-AD
3xTg-AD 9
22±2℃ 55±10% 12-12
(Institutional Animal Care and Use Committee,
IACUC) ( 20160545)
3.2
6-8 3xTg-AD
DEP
(PBS) 24
1
CO2
80 ℃
MDA Western blot CO2
(Heparin) (Lactated ringer's B injection)
Bouin 10%
3.3
(National Institute of Standards and Technology, NIST, USA) Standard Reference Material®
1650b SRM1650b 200
0.18 µm
108 m2/g DEP (Phosphate
buffered saline, PBS) DEP PBS
SRM 1650b DEP 2 mg/mL PBS
620nm (Bengalli, Zerboni et al. 2019) PAH
2 PAH 202.1 ng/mg phenanthrene fluoranthene
pyrene 2.011 ng/µg
(Bengalli, Zerboni et al. 2019)
DEP PBS 1 µg/µL
parafilm 20 ℃ DEP
(Jiang, Oberdörster et al.
2009) 50 µL DEP
50 µL PBS
3.4
4 % (isoflurane)
isoflurane
50 µL
3.5
—MDA
DNA
(malondialdehyde, MDA) (Kesavulu, Rao et al. 2001) LC-MS/MS MDA
3.5.1 MDA DNPH
2 µL TEP(1,1,3,3 tetraethoxypropane) 8 mL
1mM MDA stocking solution 100 µL MDA stocking solution 5 mL
1% 2 20nmol/ml MDA working
solution
0.016 g DNPH(2,4-Dinitrophenylhydrazine, C6H3(NO2)2NHNH2) ( 33%) 12 mL 2 M
25 ℃ 2 5 mM pH0.09 DNPH
MDA working solution DNPH 3.5.2
(Acetonitrile, ACN)
2, 1, 0.5, 0.25, 0.125, 0.0625 nmol/mL 25 µL DNPH
25 ℃ 10
R2 0.995
(Matrix matched calibration curve, MCC)
8 mg 6 mg 6 mg 20 mg
MDA MDA
2, 1, 0.5, 0.25, 0.125, 0.0625, 0 nmol/mL 25 µL
DNPH 25 ℃ 10
R2 0.995
(%)=MCC #SCC
SCC ×100%
±15%
±15%
3.5.3 MDA
Tukozkan (Tukozkan, Erdamar et al. 2006)
10 mg 250 µL
1.15% (Potassium chloride, KCl)
100µL 6M (Sodium hydroxide, NaOH)
MDA 60 ℃ 45
250 µL 100%ACN
12200 rpm 10
MDA 25 µL
DNPH 25 µL 25 ℃ 10
LC-MS/MS MDA-DNPH
3.5.4 LC-MS/MS
(Liquid chromatography-tandem mass spectrometry, LC-MS/MS) MDA-DNPH
(Autosampler, San Joes, CA, USA) (Thermo Scientific Accela 1250 quaternary pump)
4.6 mm 15 cm 5 µm Syncronis C18 (Thermo Fisher Scientific
Inc., Waltham, WA, USA) 0.1% (Acetic acid, AA) ACN
0.1% AA 25 µL 15
0 50%
300 µL/min 5 95% 300 µL/min
11 50% 300 µL/min 11.10
50% 500 µL/min 14.10 50%
(Capillary temperature) 200 ℃ (Vaporizer temperature) 200 ℃ (Aux gas pressure) 5 arb (Sheath gas pressure) 35 psi
SRM HESI 4 5 Xcalibur 2.2(Thermo Fisher Scientific
Inc.)
3.6
Iba-1
LC3b t-Tau Aβ42
(running) (transfer) (blocking)
3.6.1
15 mg 60 µL
(Protease inhibitor cocktail, EDTA-Free, 100X in DMSO, MCE) (Phosphatase inhibitor Cocktail I, 100X in DMSO, MCE) (Ethylenediaminetetraacetic acid, EDTA, 0.5M, pH8.0)
RIPA (Radioimmunoprecipitation assay buffer, RIPA Buffer, PH7.4, 2X
(Bovine Serum Albumin Standards, BSA, Thermo Fisher Scientific) 1, 0.5, 0.25, 0.125, 0.0625, 0 mg/mL
20 µL 980 µL Bradford (Bio-rad
Protein Assay Dye Reagent Concentrate, BIO-RAD)
1 µL 999 µL Bradford
150 µL 96
(BioTek™ Epoch™ Microplate Spectrophotometer)
595 nm R2 0.995
(T-Pro
Laemmli SDS sample Reagent reducing 4X) 95 ℃ 5
4 µg/µL −20 ℃
3.6.2
(Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)
SDS-PAGE t-Tau(55 kDa) 10% LC3b(14, 16
kDa) Iba1(17 kDa) Aβ42 oligomer(37 kDa) 15% 4%
β-actin internal control
30% Acrylamide (30% Acrylamide/Bis Solution, 37.5:1, BIO- RAD) 1.5 M Tris-HCl (Resolving Buffer, 1.5 M Tris-HCl pH8.8, BIO-RAD) 10% SDS
(10% (w/v) sodium dodecyl sulfate (SDS) solution, BIO-RAD) 10% APS (Ammonium persulfate solution) TEMED (Tetramethylethylenediamine)
75%
10 Well SDS-PAGE
well (Prestained Protein Ladders)
5 µL Well 20 µg
running buffer 60 V 30
100 V 120
0.45 0.2 µm (Polyvinylidene fluoride
membranes, PVDF transfer membrane) 100%
PVDF PVDF
PVDF
20%(v/v) transfer buffer 100V 60
0.1% Tween-20
TBS(tris-buffered saline) ( TBS-T) 5%
(blocking)
actin 1:10000 TBS-T 1:1000 TBS-T TBS-T
90 Rabbit IgG antibody (HRP)(GTX213110-01,
GeneTex) Mouse IgG antibody (HRP)(GTX213111-01, GeneTex) 1:10000
TBS-T TBS-T PVDF
ECL(Clarity Western ECL Substrate, BIO-RAD) (UVP BioSpectrum 810 Imaging System)
t-Tau β-actin
t-Tau β-actin t-Tau
(RapidStrip Western Blot Stripping Buffer, Omics Bio)
20 TBS-T
TBS-T ECL β-actin
3.6.3
ImageJ (National Institutes of Health, Bethesda,
MD, USA) β-actin
LC3b LC3b-II/ LC3b-I
3.7
- (hematoxylin and eosin stain, H&E stain)
3.8
SAS 9.4
Wilcoxon rank sum test
0.05 ±
4.1
MDA
MDA
4.1.1
6 (SCC) (MCC)
R2 0.995 −4.83%
±15% (SCC)
MDA 4.1.2 MDA
MDA-DNPH (Chromatogram) 2 (Retention time)
10.02 MDA
(p=0.0051) (p=0.1282) (p=0.0927)
7 3
4.2
internal control LC3b LC3b-II/
LC3b-I
4.2.1 Iba-1
Iba-1 4 5 6
( p=0.4712 p=0.0927
p=0.2980) 8
4.2.2 LC3b
LC3b-II/LC3b-I LC3b 7
8 9 (
p=0.2298 p=0.4712 p=0.2298) 9
4.2.3 Aβ42
Aβ42 10 11 12
( p=0.0927 p=0.9362
4.3
(Shackelford, Long et al.
2002) (slight)
(minimal)
12 16 17 18 19
24
MDA Iba-1 LC3b Aβ42
Tau
MDA
5.1
15-30 nm
300 2000 µg/m3
SRM 2975 SRM 1650 SRM 2975
SRM 1650
DEP
SRM 2975 81.69 ng/mg 9454 ng/µg
SRM 1650b SRM 2975 2~3 (202.1 ng/mg)
(2011 ng/µg)(Bengalli, Zerboni et al. 2019)
SRM 2975 SRM 1650b ROS RNS
(Bonvallot, Baeza-Squiban et al. 2001, Totlandsdal, Herseth et al. 2012, Totlandsdal, Låg et al. 2015) PAH
SRM 1650b
PBS
PBS
SRM 1650b
5.2
3xTg-AD
6-8
C57BL/6 C57BL/6
BALB/c Sprague Dawley
Maja Hullmann 5XFAD
0.95 mg/m3
Aβ42 (Hullmann, Albrecht et al. 2017)
3xTg-AD
3xTg-AD
C57BL/6
6 12 18
3xTg-AD
6 3xTg-AD
(Villamil-Ortiz and Cardona-Gomez 2015) 3xTg-AD
3xTg-AD 2
(Belfiore, Rodin et al. 2019) 3xTg-AD
3xTg-AD 12
3xTg-AD
744 450 3xTg-AD
(Rae and Brown 2015)
5.3 5.3.1
MDA HNE MDA
MDA MDA
Cole C57BL/6
250-300 µg/m3 6 MDA
(Cole, Coburn et al. 2016)
MDA C57BL/6
MDA (2-
thiobarbituric acid reacting substances test, TBARS assay)
(Thiobarbituric acid, TBA)
MDA MDA-TBA (Spectrophotometric
assay)
TBA MDA-TBA
(Meagher and FitzGerald 2000) TBARS
MDA DNPH
high-performance liquid chromatography (HPLC)
MDA(Cordis, Das et al. 1998, Pilz, Meineke et al. 2000)
TBA DNPH MDA TBA
(Ceconi, Cargnoni et al. 1991, Pilz, Meineke et al. 2000, Tukozkan,
Erdamar et al. 2006) HPLC-MS/MS DNPH MDA
MDA-DNPH
(
Dideuterated MDA, D2-MDA) MDA (Mendonça, Gning et al.
2017)
5.3.2
Iba-1
Iba-1
IL-1α, IL- 1β, TNFα, IL-6
DEPs Iba-1
50 µg 100 µg
150 µg 300 µg IL-1β, TNFα, IL-6
50 µg
M1 M2
M1 IL-1β,
IL-6, IL-12, TNF-α
M1 M2
M1 M2
M2 M1 (Jimenez, Baglietto-Vargas et al. 2008) Iba-1
M1 CD36, CD14, CD11c, MHC-II
M1 iNOS, TNFα, NADPH
MHC-II
(IHC) Iba-1 CD68
5.3.3
LC3b
(lipopolysaccharide, LPS)
(François, Terro et al. 2014)
LC3
LC3 16 kDa LC3-I 14
kDa LC3-II LC3b-1 LC3b-11
(Mizushima 2004)
LC3-II/LC3-I LC3 LC3-II
LC3
(autophagic flux) (Klionsky,
Abdelmohsen et al. 2016) LC3
Beclin-1 p62
Western blot LC3-II LC3-I LC3-II LC3-I
LC3 LC3-
I LC3-II
LC3-II(Chu, Plowey et al. 2009) LC3-I LC3-II SDS
LC3-I LC3-II
(Mizushima and Yoshimori 2007) LC3-I
LC3-II ( tubulin β-actin)
LC3 (Klionsky, Abdelmohsen et al. 2016, Yoshii and Mizushima 2017)
5.3.4
42 Aβ42 (monomers)
(oligomers) (fibrils) (plaques)
Aβ Aβ (Walsh, Klyubin et al.
2002, Kayed, Head et al. 2003) Aβ42
Tau (p-tau)
Tau (Liazoghli, Perreault et al. 2005)
Tau (Chesser, Pritchard et al. 2013) Tau
Tau Tau
Aβ Tau
Aβ Tau
1000µg/m3
Aβ42 Tau
5XFAD
Aβ C57BL6
t-Tau p-Tau 3xTg-AD
5.3.5
H&E
( 2016, 2017, 2018)
(Kim, Kabir et al.
2015)
(Levesque, Surace et al. 2011)
(Hartz, Bauer et al. 2008, Levesque,
Taetzsch et al. 2011) (Kim, Lee et al.
2016)
Aβ Tau 3xTg-AD
C57BL/6
12 3xTg-AD
24
(Bovine Serum Albumin, BSA) 20 (Tween-20)
Tau
(Immunohistochemistry, IHC) (Congo Red)
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1
, 2018 , 2017 , 2016
C57BL/6 C57BL/6 C57BL/6
DEPs (SRM1650b) DEPs (SRM2975) DEPs (SRM2975)
(OA) (IT) (IT)
50ug/ 2 / 150ug/ 1 /
50ug/ 1 /
150ug/ 1 /
IL-6
2 SRM1650b PAH (NIST 2013)
PAH Mass Fraction(a)
(mg/kg) Phenanthrene(b,c,d,e,f,g,h,i,j,k) 65.6 ± 3.6 1-Methylphenanthrene(b,c,d,e,k,l,m) 32.1 ± 1.4 2-Methylphenanthrene(b,c,d,e,k,l,m) 72.3 ± 1.2 3-Methylphenanthrene(b,c,d,e,k,l,m) 56.7 ± 1.9 9-Methylphenanthrene(b,c,d,e,k,l,m) 36.6 ± 1.6
Fluoranthene(b,c,d,e,f,g,h,i,j,k)
48.1 ± 1.1(n) Pyrene(b,c,d,e,f,g,h,i,j,k) 44.1 ± 1.2 Benzo[ghi]fluoranthene(c,e,f,g,h,i,j,k) 11.1 ± 0.7 Benzo[c]phenanthrene(b,c,d,e,f,g,h,i,k) 2.65 ± 0.24
Benz[a]anthracene(b,c,d,e,f,g,h,i,j,k) 6.45 ± 0.39
Chrysene(b,d,g,o) 13.4 ± 0.6
Triphenylene(b,d,g,o) 9.49 ± 0.63
Benzo[a]fluoranthene(b,c,d,e,f,h,k) 0.384 ± 0.023 Benzo[b]fluoranthene(c,e,f,g,h,i) 6.77 ± 0.92
Benzo[j]fluoranthene(c,e,f,h) 3.24 ± 0.50(n) Benzo[k]fluoranthene(b,c,d,e,f,g,h,i,j,k)
2.30 ± 0.18(n) Benzo[e]pyrene(b,c,d,e,f,g,h,i,j,k) 6.36 ± 0.37 Benzo[a]pyrene(b,c,d,e,f,g,h,i,j,k) 1.25 ± 0.12
Perylene(c,d,e,k) 0.167 ± 0.019(n)
Indeno[1,2,3-cd]pyrene(b,c,d,e,f,g,h,i,j,k,o) 4.48 ± 0.21 Benzo[ghi]perylene(b,c,d,e,f,g,h,i,j,k,o) 6.04 ± 0.30 Dibenz[a,c]anthracene(c,e,f,g,h,i) 0.439 ± 0.048
Dibenz[a,h]anthracene(c,e,g,i) 0.365 ± 0.082 Dibenz[a,j]anthracene(c,e,g,i) 0.387 ± 0.068(n) Benzo[b]chrysene(b,c,d,e,f,g,h,i,k)
0.301 ± 0.019(n) Picene(b,c,d,e,f,g,h,i,k)
0.506 ± 0.058(n)
(a) The certified mass fraction values, unless otherwise footnoted, are weighted means of the mass fractions from multiple analytical methods. The uncertainty listed with each value is an expanded uncertainty about the mean with coverage factor, k = 2, calculated by combining within method variances with a between method variance following the ISO/JCGM Guide.
(b) GC/MS (Ia) on a proprietary relatively nonpolar phase after PFE at 100 °C and 13.8 MPa with toluene.
(d) GC/MS (IIa) on a proprietary relatively nonpolar phase after PFE at 200 °C and 13.8 MPa with toluene.
(e) GC/MS (IIb) on 50 % phenyl-substituted methylpolysiloxane phase same extract as GC/MS (IIa).
(f) GC/MS (IIIa) on 50 % phenyl-substituted methylpolysiloxane phase after PFE at 100 °C and 13.8 MPa with DCM. (g) GC/MS (IIIb) on dimethyl 50 % polysiloxane liquid crystalline stationary phase with same extract as GC/MS (IIIa). (h) GC/MS (IVa) on 50
% phenyl-substituted methylpolysiloxane phase after PFE at 200 °C and 13.8 MPa with DCM. (i) GC/MS (IVb) on dimethyl 50 % polysiloxane liquid crystalline stationary phase with same extract as GC/MS (IVa). (j) GC/MS (Environment Canada) on a proprietary relatively nonpolar phase.
(k) GC/MS (VIII) on non-polar extra low bleed proprietary phase after PFE with toluene at 100 °C with 13.8 MPa; 100 °C with 20.7 MPa; 200 °C with 13.8 MPa; and 200 °C with 20.7 MPa.
(l) GC/MS (Va) on 50 % phenyl-substituted methylpolysiloxane phase after PFE at 100 °C and 13.8 MPa with DCM.
(m) GC/MS (VIa) on 50 % phenyl-substituted methylpolysiloxane phase after PFE at 200 °C and 13.8 MPa with DCM.
(n) The certified value is a weighted mean of average mass fractions, with one average from each of two or more analytical methods. The expanded uncertainty is the half width of a symmetric 95 % parametric bootstrap confidence interval which is consistent with the ISO/JCGM Guide with an effective coverage factor, k, equals 2.
(o) GC/MS (VII) on 50 % phenyl-substituted methylpolysiloxane phase after PFE at 100 °C with DCM.
3 LC-MS/MS Time
(min)
Mobile phase A (%) 0.1% AA in ACN
Mobile phase B (%) 0.1% AA in H2O
Flow rate (µL/min)
0.00 50 50 300
1.00 50 50 300
5.00 95 5 300
10.50 95 5 300
11.00 50 50 300
11.10 50 50 500
14.00 50 50 500
14.10 50 50 300
15.00 50 50 300
4 HESI
Device Value
Spray voltage (V) 3000
Vaporizer temperature (℃) 200
Sheath gas pressure (psi) 35
Auxiliary gas pressure (arb) 5
Capillary temperature (℃) 200
5 SRM
Analyte Precursor ion (m/z)
Product ion (m/z)
Collison Energy (V)
Tube Lens (V)
MDA-DNPH [M+H]+235.0 116.2 32 46
131.2 28 46
6
Analyte Standard calibration curve r2 Matrix matched calibration curve r2 Matrix effect (%)
MDA-DNPH y=5884086x+39606 r2=0.9991 y=5599679x+1185836 r2=0.9972 -4.83 %
7 MDA
Brain region Control (nmol/mL) Mean ± SE(Median)
Exposure (nmol/mL)
Mean ± SE (Median) p-value Cerebral cortex
0.013 ± 0.002 (0.012) 0.025 ± 0.007 (0.020) 0.1282 Cerebellum
0.013 ± 0.001 (0.013) 0.020 ± 0.004 (0.018) 0.0927 Hippocampus
0.014 ± 0.002 (0.016) 0.058 ± 0.011 (0.060) 0.0051*
Data are represented as mean ± SE for cerebral cortex, cerebellum and hippocampus (ncontrol=6;
nexposure=6). Wilcoxon rank sum test was used to compare control and exposure group. *p<0.05 compare to control group.
8 Iba-1 (Fold of control)
Brain region Control
Mean ± SE (Median)
Exposure
Mean ± SE (Median) p-value Cerebral cortex
1.00 ± 0.22 (0.99) 0.73 ± 0.15 (0.77) 0.4712 Cerebellum
1.00 ± 0.20 (0.86) 1.54 ± 0.15 (1.48) 0.0927 Hippocampus
1.00 ± 0.13 (0.93) 0.79 ± 0.17 (0.70) 0.2980
Data are represented as mean ± SE for cerebral cortex, cerebellum and hippocampus (ncontrol=6;
nexposure=6). Wilcoxon rank sum test was used to compare control and exposure group. *p<0.05 compare to control group.
9 LC3b (Fold of control)
Brain region Control
Mean ± SE (Median)
Exposure
Mean ± SE (Median) p-value Cerebral cortex
1.00 ± 0.21 (0.79) 0.68 ± 0.09 (0.71) 0.2298 Cerebellum
1.00 ± 0.17 (0.97) 0.76 ± 0.08 (0.79) 0.4712 Hippocampus
1.00 ± 0.15 (0.88) 1.27 ± 0.26 (1.16) 0.2298
10 Aβ42 (Fold of control)
Brain region Control
Mean ± SE (Median)
Exposure
Mean ± SE (Median) p-value Cerebral cortex
1.00 ± 0.10 (1.00) 0.68 ± 0.09 (0.71) 0.0927 Cerebellum
1.00 ± 0.07 (1.03) 0.99 ± 0.09 (0.97) 0.9362 Hippocampus
1.00 ± 0.08 (1.02) 1.08 ± 0.06 (1.05) 0.8102
Data are represented as mean ± SE for cerebral cortex, cerebellum and hippocampus (ncontrol=6;
nexposure=6). Wilcoxon rank sum test was used to compare control and exposure group. *p<0.05 compare to control group.
11 t-Tau (Fold of control)
Brain region Control
Mean ± SE (Median)
Exposure
Mean ± SE (Median) p-value Cerebral cortex
1.00 ± 0.12 (1.03) 0.99± 0.10 (0.90) 0.2980 Cerebellum
1.00 ± 0.11 (0.93) 0.99 ± 0.08 (0.99) 0.9362 Hippocampus
1.00 ± 0.12 (1.00) 1.06 ± 0.10 (1.15) 0.9362
Data are represented as mean ± SE for cerebral cortex, cerebellum and hippocampus (ncontrol=6;
nexposure=6). Wilcoxon rank sum test was used to compare control and exposure group. *p<0.05 compare to control group.
